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In recent years, increasing evidence shows that circular RNA (circRNA) disorder is closely related to tumorigenesis and cancer progression. However, the regulatory functions of most circRNAs in bladder cancer (BCa) remain unclear. This study was aimed at exploring the molecular regulatory mechanism of circRNAs in BCa. We obtained four datasets of circRNA, microRNA (miRNA), and messenger (mRNA) expression profiles from the Gene Expression Omnibus and The Cancer Genome Atlas microarray databases and identified 434, 367, and 4799/4841 differentially expressed circRNAs, miRNAs, and mRNAs, respectively. With these differentially expressed RNAs, we established a circRNA-miRNA-mRNA targeted interaction network. A total of 18, 24, and 51 central circRNAs, miRNAs, and mRNAs were identified, respectively. Among them, the top 10 mRNAs that had high connectivity with other circRNAs and miRNAs were regarded as hub genes. We detected the expression levels of these 10 mRNAs in 16 pairs of BCa tissues and adjacent normal tissues through quantitative real-time polymerase chain reaction. The differentially expressed mRNAs and central mRNAs were enriched in the processes and pathways that are associated with the growth, differentiation, proliferation, and apoptosis of tumor cells. The outstanding genes (CDCA4, GATA6, LATS2, RHOB, ZBTB4, and ZFPM2) also interacted with numerous drugs, indicating their potency as biomarkers and drug targets. The findings of this study provide a deep understanding of the circRNA-related competitive endogenous RNA regulatory mechanism in BCa pathogenesis.

Psoriasis is a chronic, immune-mediated inflammatory disease which pathogenesis is closely linked to γδ T cells. Recently, a critical role for butyrophilin 3A1 (BTN3A1) in mediating the activation of Vγ9Vδ2 T cells, which are reported to redistribute from blood to the perturbed skin lesions in psoriasis, has been proposed. Additional molecular partners, including RhoB and periplakin, have also been speculated to interact with BTN3A1 in modulating Vγ9Vδ2 T-cell activation. Immunohistochemical staining was performed to examine the expressions of BTN3A1, RhoB, and the plakin family members, including periplakin, epiplakin, and envoplakin in the psoriasis vulgaris lesions as compared with the normal control. The expressions of BTN3A1 and RhoB were found significantly upregulated in the psoriatic lesions. Besides, a downregulation of periplakin and an upregulation of epiplakin were noticed in the psoriasis vulgaris lesions. Our data suggest that BTN3A1 and RhoB might participate in the pathogenesis of psoriasis through Vγ9Vδ2 T-cell responses. In addition, a potential involvement of the plakin protein family, especially periplakin and epiplakin, in psoriasis pathology was proposed.

Background Evidence indicates that Gα12, Gα13, and its downstream effectors, RhoA and Rac1, regulate neuronal morphology affected by stress. This study was aimed at investigating whether repeated stress influences the expression of proteins related to the Gα12/13 intracellular signaling pathway in selected brain regions sensitive to the effects of stress. Furthermore, the therapeutic impact of β(1)adrenergic receptors (β1AR) blockade was assessed. Methods Restraint stress (RS) model in mice (2 h/14 days) was used to assess prolonged stress effects on the mRNA expression of Gα12, Gα13, RhoA, Rac1 in the prefrontal cortex (PFC), hippocampus (HIP) and amygdala (AMY). In a separate study, applying RS model in rats (3–4 h/1 day or 14 days), we evaluated stress effects on the expression of Gα12, Gα11, Gαq, RhoA, RhoB, RhoC, Rac1/2/3 in the HIP. Betaxolol (BET), a selective β1AR antagonist, was introduced (5 mg/kg/p.o./8–14 days) in the rat RS model to assess the role of β1AR in stress effects. RT-qPCR and Western Blot were used for mRNA and protein assessments, respectively. Results Chronic RS decreased mRNA expression of Gα12 and increased mRNA for Rac1 in the PFC of mice. In the mice AMY, decreased mRNA expression of Gα12, Gα13 and RhoA was observed. Fourteen days of RS exposure increased RhoA protein level in the rats’ HIP in the manner dependent on β1AR activity. Conclusions Together, these results suggest that repeated RS affects the expression of genes and proteins known to be engaged in neural plasticity, providing potential targets for further studies aimed at unraveling the molecular mechanisms of stress-related neuropsychiatric diseases.

An untargeted multi-omics study implicated the potential dysregulation of fatty acid, nucleotide, and energy metabolism in the brainstems of spontaneously hypertensive rats (SHRs). A further quantitative exploration of the alterations in the metabolic pathways is necessary for a deep understanding of the central nervous system in SHRs. Targeted metabolic profiling of 40 fatty acids (PeptideAtlas: PASS01671) and 32 metabolites of nucleotides and energy metabolism (PeptideAtlas: PASS01672) and parallel reaction monitoring analysis of 5 proteins (PeptideAtlas: PASS01673) were performed on the brainstems of SHRs (n = 8, 11 weeks old) and normotensive Wistar rats (n = 8, age-matched) using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem MS. The targeted profiling results of metabolites and proteins revealed decreased polyunsaturated fatty acid (PUFA) synthesis with a significant downregulation of cis-11,14-eicosadienoic acid, cis-13,16-docosadienoic acid, and docosatetraenoate and impaired PUFA oxidation with the accumulation of γ-linolenate induced by the significantly downregulated expression of 2,4-dienoyl-CoA reductase (p < 0.05). Dysregulated GTP and ATP metabolism was observed, with significantly decreased GDP and ADP (p < 0.05) correlated with reduced GTPases of guanine nucleotide-binding protein subunit beta-1 (GNB1), transforming protein RhoA (RHOA), and Rho-related GTP-binding protein RhoB (RHOB) in the brainstem of SHRs. In addition, protein-arginine deiminase type-2 was significantly reduced in the brainstems of SHRs (p < 0.05). The aberrant PUFA and energy metabolism might help to explain the alterations in the brainstem of SHRs. The findings on both metabolites and proteins could provide systemic insights into the pathology basis of altered PUFA and energy metabolism in hypertension, especially in the central nervous system.

Dietary intervention to prevent Helicobacter pylori (H. pylori)-associated gastric diseases seems to be ideal with no risk of bacterial resistance, safe long-term intervention, and correcting pathogenic mechanisms including rejuvenation of precancerous atrophic gastritis and anti-mutagenesis. A transcriptome as set of all RNAs transcribed by certain tissues or cells demonstrates gene functions and reveals the molecular mechanism of specific biological processes against diseases. Here, we have performed RNAseq and bioinformatic analysis to explain proof of concept that walnut intake can rescue from H. pylori infection and explore unidentified mode of actions of walnut polyphenol extract (WPE). As results, BIRC3, SLC25A4, f3 transcription, VEGFA, AZU1, HMOX1, RAB3A, RELBTNIP1, ETFB, INPP5J, PPME1, RHOB, TPI1, FOSL1, JUND.RELB, KLF2, MUC1, NDRG1, ALDOA, ENO1, PFKP, GPI, GDF15, and NRTN genes were newly discovered to be enriched with WPE, whereas CCR4, BLNK, CCR7, CXCR4, CDO1, KLSG1, SELE, RASGRP2, PIK3R3, TSPAN32, HOXC-AS3, HCG8, BTNL8, and CXCL3 genes as inhibitory targets by WPE in H. pylori infection. We identified additional genes what WPE afforded actions of avoiding H. pylori-driven onco-inflammation and rejuvenating precancerous atrophic gastritis. Conclusively, after applying RNAseq analysis in order to document walnut intake for precision medicine against H. pylori infection, significant transcriptomic profiling applicable for validation were drawn.

Background Tumor angiogenesis, an essential process for cancer proliferation and metastasis, has a critical role in prognostic of kidney renal clear cell carcinoma (KIRC), as well as a target in guiding treatment with antiangiogenic agents. However, tumor angiogenesis subtypes and potential epigenetic regulation mechanisms in KIRC patient remains poorly characterized. System evaluation of angiogenesis subtypes in KIRC patient might help to reveal the mechanisms of KIRC and develop more target treatments for patients. Method Ten independent tumor angiogenesis signatures were obtained from molecular signatures database (MSigDB) and gene set variation analysis was performed to calculate the angiogenesis score in silico using the Cancer Genome Atlas (TCGA) KIRC dataset. Tumor angiogenesis subtypes in 539 TCGA-KIRC patients were identified using consensus clustering analysis. The potential regulation mechanisms was studied using gene mutation, copy number variation, and differential methylation analysis (DMA). The master transcription factors (MTF) that cause the difference in tumor angiogenesis signals were completed by transcription factor enrichment analysis. Results The angiogenesis score of a prognosis related angiogenesis signature including 189 genes was significantly correlated with immune score, stroma score, hypoxia score, and vascular endothelial growth factor (VEGF) signal score in 539 TCGA KIRC patients. MMRN2, CLEC14A, ACVRL1, EFNB2, and TEK in candidate gene set showed highest correlation coefficient with angiogenesis score in TCGA-KIRC patients. In addition, all of them were associated with overall survival in both TCGA-KIRC and E-MTAB-1980 KIRC data. Clustering analysis based on 183 genes in angiogenesis signature identified two prognosis related angiogenesis subtypes in TCGA KIRC patients. Two clusters also showed different angiogenesis score, immune score, stroma score, hypoxia score, VEGF signal score, and microenvironment score. DMA identified 59,654 differential methylation sites between two clusters and part of these sites were correlated with tumor angiogenesis genes including CDH13, COL4A3, and RHOB. In addition, RFX2, SOX13, and THRA were identified as top three MTF in regulating angiogenesis signature in KIRC patients. Conclusion Our study indicate that evaluation the angiogenesis subtypes of KIRC based on angiogenesis signature with 183 genes and potential epigenetic mechanisms may help to develop more target treatments for KIRC patients.

Background The current study investigates the effect of transcription factor Prox1 on the proliferation, migration, and invasion ability of lung cancer. Methods Lung cancer cell lines (A549 and H446 cells) were transfected with Prox1NAD and siRNA, respectively. Thus, the A549 and H446 cells overexpressed Prox1 after transfection of Prox1NAD plasmids, and A549 and H446 cells have low expression of Prox1 after transfection with siRNA. Reverse transcriptase quantitative PCR and western blot analyses were used to detect Prox1 mRNA and protein expression in cells. Plate clone formation experiments and MTT experiments were used to detect cell proliferation. Western blot was used to detect the expression of Rho family-related proteins in cells. Results Compared to untransfected wild-type A549 and H446 that served as blank controls, the expression level of Prox1mRNA and protein in A549 and H446 cells overexpressing Prox1 after plasmid transfection was high, while the expression level of Prox1mRNA and protein in A549 and H446 cells with low expression of Prox1 after siRNA transfection was low. With the increase of Prox1 expression, the expression of RhoA and RhoC increased, while the expression of RhoB decreased. Conclusion The finding of this study may provide a new approach for the treatment of lung cancer using targeted gene therapy.

Alzheimer's disease (AD) poses a significant threat to human life and health. The intraneuronal accumulation of β-amyloid (Aβ) plaques in the brains of AD patients results in neuronal cell death, which is a key factor that triggers multiple changes in the pathogenesis of AD. The inhibition of Aβ-induced neuronal cell death may potentially help in the intervention and treatment of AD. Our previous study reported that tumor necrosis factor α-induced protein 1 (TNFAIP1) is induced by and promotes Aβ25-35-induced neurotoxicity in mouse neuronal cells, but the roles and regulatory mechanisms of TNFAIP1 are still largely unknown. In this study, our experimental results show that TNFAIP1 and p-TNFAIP1 (phosphorylation of TNFAIP1 at Ser280) are overexpressed in the neurons of the cortex and hippocampus in the brains of APP/PS1 mice, and the transcription factor NF-κB is involved in the Aβ-induced upregulation of TNFAIP1. Moreover, our results suggest that TNFAIP1 contributes to the Aβ-induced reactive oxygen species (ROS) production, decreased mitochondrial membrane potential (∆Ψm), and neuronal cell death in human SH-SY5Y cells. We further revealed that Aβ increases the binding of TNFAIP1 to RhoB, and knockdown of RhoB attenuates the TNFAIP1-induced apoptosis of human SH-SY5Y cells. These data suggest that TNFAIP1 is closely associated with AD pathogenesis, and overexpression of TNFAIP1 in the neurons of the brains of AD patients plays a role in apoptosis, at least in part, via RhoB signaling.

BACKGROUND: Hepatocellular carcinoma (HCC) is the leading cause of mortality worldwide. In recent years, the incidence of HCC induced by NAFLD is growing rapidly. OBJECTIVE: To screen for new pathogenic genes and related pathways both in NAFLD and HCC, and to explore the pathogenesis of progression from NAFLD to HCC. METHODS: Gene expression microarrays (GSE74656, GSE62232) were used for identifying differentially expressed genes (DEGs). Functional enrichment and pathway enrichment analyses indicated that these DEGs were related to cell cycle and extracellular exosome, which were closely related to NAFLD and HCC development. We then used the Search Tool for the Retrieval of Interacting Genes (STRING) to establish the protein-protein interaction (PPI) network and visualized them in Cytoscape. And the overall survival (OS) analysis and gene expression validation in TCGA of hub genes was performed. RESULTS: Seven hub genes, including CDK1, HSP90AA1, MAD2L1, PRKCD, ITGB3BP, CEP192, and RHOB were identified. Finally, we verified the expression level of ITGB3BP and CEP192 by quantitative real-time PCR in vitro. CONCLUSIONS: The present study implied possible DEGs, especially the new gene CEP192, in the progression of NAFLD developing to HCC. Further rigorous experiments are required to verify the above results.

LncRNA LOXL1-AS1 has been reported to be upregulated in several types of cancer and plays oncogenic roles. We analysed TCGA dataset and observed the downregulation of LOXL1-AS1 in cervical squamous cell carcinoma (CSCC). We therefore analysed the roles of LOXL1-AS1 in CSCC. We observed that LOXL1-AS1 was downregulated in CSCC. LOXL1-AS1 was predicted to interact with miR-21, while overexpression experiments showed that LOXL1-AS1 and miR-21 had no significant effects on the expression of each other. However, LOXL1-AS1 overexpression led to the upregulation of RHOB, a direct target of miR-21. Cell invasion and migration analysis showed decreased invasion and migration rates of CSCC cells after LOXL1-AS1 and RHOB overexpression. MiR-21 played an opposite role at reduced the effects of LOXL1-AS1 and RHOB overexpression. Therefore, LOXL1-AS1 may promote CSCC cell invasion and migration by sponging miR-21 to upregulate RHOB.

Introduction Emerging evidence has revealed the importance of long non-coding RNAs (lncRNAs) in carcinogenesis. The aim of this work was to investigate the roles of lncRNA growth arrest specific 5 (GAS5) in osteosarcoma (OS) progression. Methods Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to explore GAS5, microRNA-663a (miR-663a), and ras homolog family member B (RHOB) expression levels in OS tissues and cells. Moreover, cell counting kit-8 assay, wound-healing assay, and transwell invasion assay were conducted to investigate biological roles of GAS5 in OS progression. In addition, mechanisms underlying the functions of GAS5 in OS were investigated by bioinformatic analysis, luciferase activity reporter assay, and rescue experiments. Results The GAS5 expression level was significantly decreased in OS tissues and cells compared with normal tissues and cells, and could negatively regulate miR-663a expression. Moreover, we found RHOB expression can be negatively regulated by miR-663a. Overexpression of GAS5 and RHOB suppresses, while overexpression of miR-663a stimulates, OS cell proliferation, migration, and invasion in vitro. In summary, we revealed lncRNA GAS5 was a downregulated lncRNA in OS and impaired OS malignant behaviors. In addition, this regulation relied on miR-663a and its target gene, RHOB. Discussion To sum up, we showed lncRNA GAS5 regulates OS progression via regulating the miR-663a/RHOB axis.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer with a high fatality rate in men and women worldwide. Recently, microRNAs (miRNAs) have been reported to be diagnostic biomarkers and therapeutic targets in NSCLC. MiR-223-3p was proved to act as a promoter in NSCLC progression. However, the regulatory mechanism of miR-223-3p in NSCLC remains little known. This study aimed to explore the regulatory mechanism between miR-223-3p and its target gene Ras homolog family member B (RHOB) in NSCLC. The mRNA level of miR-223-3p and RHOB was measured by quantitative reverse transcription PCR. Furthermore, cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry was conducted to analyze cell apoptosis. Transwell assays and wound healing assay were employed to examine migration and invasion. The target relationship between miR-223-3p and RHOB was predicted by starBase online database and verified by dual-luciferase assay. The protein level of RHOB was tested by western blot. Our data suggested that miR-223-3p was upregulated in NSCLC tissues and cell lines and high level of miR-223-3p contributed to a poor survival in NSCLC patients. Knockdown of miR-223-3p exerted inhibitory effects on NSCLC cell viability, migration, and invasion and promotion effect on cell apoptosis. Furthermore, RHOB was directly targeted by miR-223-3p and constrained NSCLC progression. Moreover, knockdown of RHOB rescued the effect of anti-miR-223-3p on NSCLC progression. In vivo experiments indicated that miR-223-3p deletion suppressed tumor growth. MiR-223-3p could regulate the NSCLC cellular processes through targeting RHOB.

Breast cancer (BC), the most frequent cancer in women worldwide, is extremely heterogeneous. For effective and precise treatment and to cope with drug resistance in BC, we need to find more therapeutic molecular targets. In this study, we found that the Proteasome 26S Subunit, Non-ATPase 12 (PSMD12) was upregulated in BC samples, its expression was heterogeneous among different cell lines, and high levels of PSMD12 were related to poor prognosis of BC patients. Notably, the expression of PSMD12 increased in the nucleus. Cytological experiments revealed that PSMD12 knockdown inhibited cell growth and migration, and a genome-wide CRISPR-Cas9 knockout (GeCKO) screen also confirmed that PSMD12 is a crucial gene for the growth of BC cells. Flow cytometry showed that cell apoptosis increased in the PSMD12 knockdown, and RNA-seq indicated that the apoptosis pathway was activated, and the TXNIP, GADD45A, GADD45B, RHOB, and CDKN1A pro-apoptotic genes were highly expressed, a result that was validated by RT-qPCR and Western blot. Furthermore, restoration of PSMD12 expression decreased the expression of pro-apoptotic genes. A tumor-bearing mice assay demonstrated that BC growth was arrested by reduced PSMD12 levels in vivo. Taken together, PSMD12, a subunit of 19S regulator of 26S proteasome, was identified as a potential prognostic and therapeutic molecular target for BC, which provides a new insight for developing anticancer drugs that promote apoptosis based on the targeting of the 26S proteasome complex.

RhoB protein belongs to the Rho GTPase family, which plays an important role in governing cell signaling and tissue morphology. RhoB expression is known to have implications in pathological processes of diseases. Investigation in the regulation and communication of this protein detected by immunohistochemical staining on the microscope is worth exploring to gain insightful information that may lead to identifying optimal disease treatment options. In particular, the role of RhoB in rectal cancer is not well-discovered. Here, we report that methods of deep learning-based image analysis and the decomposition of multiway arrays discover the predictive factor of RhoB in two cohorts of rectal-cancer patients having survival rates of less and more than 5 years. The analysis results show distinctions between the tensor-decomposition factors of the two cohorts.

Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR–Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host–pathogen interactions.

BackgroundIschemia–reperfusion injury (IRI) remains an inevitable and major challenge in renal transplantation. The current study aims to obtain deep insights into underlying mechanisms and seek prognostic genes as potential therapeutic targets for renal IRI (RIRI).MethodsAfter systematically screening the Gene Expression Omnibus (GEO) database, we collected gene expression profiles of over 1,000 specimens from 11 independent cohorts. Differentially expressed genes (DEGs) were identified by comparing allograft kidney biopsies taken before and after reperfusion in the discovery cohort and further validated in another two independent transplant cohorts. Then, graft survival analysis and immune cell analysis of DEGs were performed in another independent renal transplant cohort with long-term follow-ups to further screen out prognostic genes. Cell type and time course analyses were performed for investigating the expression pattern of prognostic genes in more dimensions utilizing a mouse RIRI model. Finally, two novel genes firstly identified in RIRI were verified in the mouse model and comprehensively analyzed to investigate potential mechanisms.ResultsTwenty DEGs upregulated in the process of RIRI throughout different donor types (living donors, cardiac and brain death donors) were successfully identified and validated. Among them, upregulation of 10 genes was associated with poor long-term allograft outcomes and exhibited strong correlations with prognostic immune cells, like macrophages. Furthermore, certain genes were found to be only differentially expressed in specific cell types and remained with high expression levels even months after RIRI in the mouse model, which processed the potential to serve as therapeutic targets. Importantly, two newly identified genes in RIRI, Btg2 and Rhob, were successfully confirmed in the mouse model and found to have strong connections with NF-κB signaling.ConclusionsWe successfully identified and validated 10 IRI-associated prognostic genes in renal transplantation across different donor types, and two novel genes with crucial roles in RIRI were recognized for the first time. Our findings offered promising potential therapeutic targets for RIRI in renal transplantation.

Simple Summary Glioblastoma multiforme (GBM) and metastatic triple-negative breast cancer (TNBC) with PTEN mutations are associated with brain tumor spreading and poor patient outcomes. GBM, and possibly TNBC, migrate on axons and blood vessels to disseminate in the brain; however, the mechanism is unresolved. There is a need for new therapeutic targets to blunt brain tumor spreading. Using 3D aligned printed microfibers mimicking brain structures proved that RHOB, in addition to ROCK and PTEN signaling, were essential for GBM and TNBC 3D cell migration. GBM and TNBC cell lines with PTEN loss of function and high RHOB expression exhibited amoeboid morphology with increased durotaxis, binding and migration speed on 3D microfibers, in contrast to the PTEN wildtype. Depending on the PTEN genotype, RHO-ROCK-PTEN inhibitors or PTEN rescue significantly regulated these properties. Regarding GBM and brain metastasizing TNBC, we conclude that RHOB inhibitors could play a novel role for improved therapy response and patient outcome. Abstract Background: Glioblastoma multiforme (GBM) and metastatic triple-negative breast cancer (TNBC) with PTEN mutations often lead to brain dissemination with poor patient outcome, thus new therapeutic targets are needed. To understand signaling, controlling the dynamics and mechanics of brain tumor cell migration, we implemented GBM and TNBC cell lines and designed 3D aligned microfibers and scaffolds mimicking brain structures. Methods: 3D microfibers and scaffolds were printed using melt electrowriting. GBM and TNBC cell lines with opposing PTEN genotypes were analyzed with RHO-ROCK-PTEN inhibitors and PTEN rescue using live-cell imaging. RNA-sequencing and qPCR of tumor cells in 3D with microfibers were performed, while scanning electron microscopy and confocal microscopy addressed cell morphology. Results: In contrast to the PTEN wildtype, GBM and TNBC cells with PTEN loss of function yielded enhanced durotaxis, topotaxis, adhesion, amoeboid migration on 3D microfibers and significant high RHOB expression. Functional studies concerning RHOB-ROCK-PTEN signaling confirmed the essential role for the above cellular processes. Conclusions: This study demonstrates a significant role of the PTEN genotype and RHOB expression for durotaxis, adhesion and migration dependent on 3D. GBM and TNBC cells with PTEN loss of function have an affinity for stiff brain structures promoting metastasis. 3D microfibers represent an important tool to model brain metastasizing tumor cells, where RHO-inhibitors could play an essential role for improved therapy.

Simple Summary The objective of the present study was to investigate the impact of sex in the development of long-term toxicities affecting quality of life in cancer survivors after systemic paclitaxel treatment and cardiac irradiation. Sex-specific differences may affect tumor biology, drug pharmacokinetics and dynamics, and response to local treatment such as radiation therapy (RT). However, sex is rarely taken into consideration when administering cancer therapies. Interestingly, female mice are protected from paclitaxel-induced neurotoxicity as well as from radiotherapy-induced cardiotoxicity, and deficiency in the small GTPase RhoB reversed the protection in females but not in males. In conclusion, our results are the first to identify sex- and organ-specific responses to systemic paclitaxel administration and localized RT. These results may have important implications for the management of cancer patients and implementation of personalized medicine in oncology. Abstract The impact of sex in the development of long-term toxicities affecting the quality of life of cancer survivors has not been investigated experimentally. To address this issue, a series of neurologic and cardiologic endpoints were used to investigate sex-based differences triggered by paclitaxel treatment and radiotherapy exposure. Male and female wild-type (WT) mice were treated with paclitaxel (150 and 300 mg/kg) administered weekly over 6 weeks or exposed to 19 Gy cardiac irradiation. Cohorts were analyzed for behavioral and neurobiologic endpoints to assess systemic toxicity of paclitaxel or cardiovascular endpoints to assess radiotherapy toxicity. Interestingly, female WT mice exhibited enhanced tolerance compared to male WT mice regardless of the treatment regimen. To provide insight into the possible sex-specific protective mechanisms, rhoB-deficient animals and elderly mice (22 months) were used with a focus on the possible contribution of sex hormones, including estrogen. In females, RhoB deficiency and advanced age had no impact on neurocognitive impairment induced by paclitaxel but enhanced cardiac sensitivity to radiotherapy. Conversely, rhoB-deficiency protected males from radiation toxicity. In sum, RhoB was identified as a molecular determinant driving estrogen-dependent cardioprotection in female mice, whereas neuroprotection was not sex hormone dependent. To our knowledge, this study revealed for the first time sex- and organ-specific responses to paclitaxel and radiotherapy.

Introduction Osteosarcoma is the most common bone tumor with high metastasis and recurrence rate. MicroRNA-19a (miR-19a) has been reported to act as tumor oncogene in multiple cancers. The objective of the study was to explore the molecular mechanisms of miR-19a in osteosarcoma cell migration and invasion. Materials and methods Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting were employed to measure the levels of miR-19a and RhoB in osteosarcoma tissues and cell lines. Transwell assay was employed to analyze the tissues and cell lines' migratory and invasive abilities. Dual luciferase reporter assay was utilized to analyze the association between miR-19a and RhoB. Results MiR-19a was overexpressed in osteosarcoma tissues and cell lines. MiR-19a promoted osteosarcoma cell migration and invasion in vitro. RhoB was thus confirmed as a direct and functional target of miR-19a, and it could partially reverse the function of miR-19a. Knockdown miR-19a inhibited osteosarcoma cell epithelial-mesenchymal transition (EMT) and suppressed osteosarcoma xenograft growth. Conclusion MiR-19a enhanced cell migration, invasion and EMT through RhoB in osteosarcoma. The newly identified miR-19a/RhoB axis provides novel insight into the progression of osteosarcoma and offers a promising target for osteosarcoma therapy.

Purpose To explore whether the Rho protein is involved in the radioresistance of colorectal cancer and investigate the underlying mechanisms. Methods and Materials Rho GTPase expression was measured after radiation treatment in colon cancer cells. RhoB knockout cell lines were established using the CRISPR/Cas9 system. In vitro assays and zebrafish embryos were used for analyzing radiosensitivity and invasive ability. Mass cytometry was used to detect RhoB downstream signaling factors. RhoB and Forkhead box M1 (FOXM1) expression were detected by immunohistochemistry in rectal cancer patients who participated in a radiation therapy trial. Results RhoB expression was related to radiation resistance. Complete depletion of the RhoB protein increased radiosensitivity and impaired radiation-enhanced metastatic potential in vitro and in zebrafish models. Probing signaling using mass cytometry–based single-cell analysis showed that the Akt phosphorylation level was inhibited by RhoB depletion after radiation. FOXM1 was downregulated in RhoB knockout cells, and the inhibition of FOXM1 led to lower survival rates and attenuated migration and invasion abilities of the cells after radiation. In the patients who underwent radiation therapy, RhoB overexpression was related to high FOXM1, late Tumor, Node, Metastasis stage, high distant recurrence, and poor survival independent of other clinical factors. Conclusions RhoB plays a critical role in radioresistance of colorectal cancer through Akt and FOXM1 pathways.

Synaptic cell adhesion molecules, including neurexins and neuroligins, mediate the formation and maintenance of connections between neuronal cells. Although neurexins and neuroligins are known to interact with each other in a calcium-dependent manner and several neuropeptides have been shown to act through G protein-coupled receptors to increase intracellular calcium levels, no studies have examined the role of the neuropeptide oxytocin in association with adhesion molecules. Given that oxytocin receptors are located on presynaptic and postsynaptic membranes and that oxytocin exerts direct effects on neuronal excitability, it could be hypothesized that oxytocin affects the expression of cell surface adhesion molecules. In the present study, we show that incubation in the presence of oxytocin (1 μM, 48 h) exerted cell-specific effects on the levels of neurexin 2α, neurexin 2β, and neuroligin 3. Oxytocin significantly increased the mRNA expression levels of neurexin 2α, neurexin 2β, and neuroligin 3 in SH-SY5Y, U-87MG, and primary cerebellar cells. The effect of inhibiting oxytocin receptors on the expression of neurexin 2β was more dramatic in U-87MG cells than in SH-SY5Y cells. Oxytocin did not exert effects in primary corticohippocampal cells. Additionally, we measured the expression of selected GTPases to determine whether they could mediate the effects of oxytocin. Oxytocin induced a decrease in the mRNA level of Rac1 in U-87MG and primary cerebellar cells and exerted a stimulatory effect on the expression of RhoB at the gene and protein level in SH-SY5Y cells. These results suggest that the regulation of neurexins and neuroligins involves the activation of oxytocin receptors. These effects are likely mediated by the stimulation of RhoB GTPase, at least in certain types of cells.

Background. Breast cancer (BC) is one of the most common malignant tumors in women around the world. Atorvastatin (ATO) was found to be associated with a decreased risk of recurrence and mortality in cancer. But the exact mechanism of its carcinostatic effects is unclear. The expression level of Ras homolog family member B (RhoB) in breast cancer cells was found to be upregulated after being treated with ATO. Thus, we conjecture that altered expression of RhoB induced by ATO may be decisive for the migration and progression of breast cancer. Methods. The effects of ATO on breast tumor cells in vivo and in vitro were detected by clone formation assay, CCK-8 assay, flow cytometry, wound healing, transwell assays, tumor xenograft model, and immunohistochemistry. Distribution of RhoB in different breast cancer tissues and its influence on prognosis were analyzed using the data from TCGA or GEO databases. The relationship between RhoB and PTEN/AKT pathway was detected by Western blotting and RT-qPCR. Results. ATO inhibits proliferation, invasion, EMT, and PTEN/AKT pathway and promotes apoptosis in breast tumor cells. In addition, ATO inhibits the volume and weight of breast tumor in tumor-bearing mice and upregulated RhoB in tumor tissues. The expression of RhoB in mRNA and protein level was upregulated in statin-treated breast cancer cells and downregulated in cancer tissues. Low expression of RhoB links with poor prognosis in patients with breast cancer (HR = 0.74[0.66–0.83], p =7e−8, log-rank test). Further research found that RhoB inhibits the proliferation, invasion, EMT, and PTEN/AKT signal pathway in breast tumor cells. Conclusions. The exact mechanism of ATO’s carcinostatic effects in breast cancer is related to downregulating PTEN/AKT pathway via promoting RhoB. Our study also demonstrates the potential applicability of RhoB as a therapeutic target for breast cancer.

Overexpression of HER2 in breast cancer is correlated with poor prognosis. HER2-targeted drugs, such as trastuzumab and lapatinib, have been successful to treat HER2-positive breast cancers, however, the acquisition of the drug resistance of the cells is recognized. Here we suggest the novel molecular targets to cure HER2-positive breast cancers. The oncogenic roles of Rac1, a Rho family small GTPase, in a variety of cancers have been demonstrated. For example, the elevated expression or hyperactivation of Rac1 is frequently observed in human cancers, correlating with their aggressiveness and poor prognosis. In breast cancers, upregulation of Rac1 GEF (GTP exchange factor) and downregulation of Rac1 GAP (GTPase activating protein) have been reported. Moreover, activation of Rac1 contributed to trastuzumab resistance, which poses a serious problem during chemotherapy. In the present study, we first investigated in detail in which subtypes of breast cancers mRNA expression of Rac1 is correlated with their poor prognosis. Using the METABRIC database, we found that high mRNA expression of Rac1 significantly correlated with the poor prognosis of HER2-positive breast cancer (p=0.0012, High: n=49, Low: n=171). On the other hands, other three types (basal, claudin-low, or luminal-B type) did not show significant correlation between the expression levels of Rac1 mRNA and their prognosis (p=0.15, High: n=97, Low: n=102; p=0.052, High: n=110, Low: n=89; p=0.17, High: n=70, Low: n=391; respectively). In luminal-A type breast cancer, low mRNA expression of Rac1 significantly correlated with poor prognosis (p=0.0046, High: n=492, Low: n=187). We next investigated the molecular mechanism underlying Rac1 activation in HER2-positive breast cancer cells, SKBR-3 cells. We found that Cullin-3 (CUL3, a subunit of a RING ubiquitin E3 ligase complex) and its adaptor protein KCTD10 are essential for Rac1 activation. Mechanistically, CUL3/KCTD10 ubiquitinate RhoB, a Rho family small GTPase that suppresses the activation of Rac1, leading to the degradation RhoB. We also found that HER2 signaling is essential for the activation of Rac1. Conclusions: This study reveals that the novel molecular axis CUL3/KCTD10/RhoB regulates the Rac1 activation in HER2-positive breast cancer cells. The interference of CUL3/KCTD10 complex formation may be a new strategy to the treatment of HER2- and Rac1-positive breast cancers. Citation Format: Murakami A, Maekawa M, Kusakabe E, Yamasawa H, Aoki R, Komatsu S, Taguchi K, Nishiyama K, Yamashita M, Sugimori W, Kawai K, Nakayama J, Araki N, Semba K, Taguchi T, Kamei Y, Takada Y, Higashiyama S. Novel mechanisms of Rac1 activation by the Cullin-3/KCTD10 ubiquitin E3 complex in HER2-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-02.

The high mortality rate of cancer is strongly correlated with the development of distant metastases at secondary sites. Although Rho GTPases, such as RhoA, RhoB, RhoC, and RhoE, promote tumor metastasis, the main roles of Rho GTPases remain unidentified. It is also unclear whether rhosin, a Rho inhibitor, acts by suppressing metastasis by a downstream inhibition of Rho. In this study, we investigated this mechanism of metastasis in highly metastatic melanoma and breast cancer cells, and the mechanism of inhibition of metastasis by rhosin. We found that rhosin suppressed the RhoA and RhoC activation, the nuclear localization of YAP, but did not affect ERK1/2, Akt, or NF-&kappa, B activation in the highly metastatic cell lines B16BL6 and 4T1. High expression of YAP was associated with poor overall and recurrence-free survival in patients with breast cancer or melanoma. Treatment with rhosin inhibited lung metastasis in vivo. Moreover, rhosin inhibited tumor cell adhesion to the extracellular matrix via suppression of RHAMM expression, and inhibited SDF-1-induced cell migration and invasion by decreasing CXCR4 expression in B16BL6 and 4T1 cells. These results suggest that the inhibition of RhoA/C-YAP pathway by rhosin could be an extremely useful therapeutic approach in patients with melanoma and breast cancer.

Metastasis is the most prevalent cause of cancer-associated deaths amongst patients with cervical cancer. Epithelial–mesenchymal transition (EMT) is essential for carcinogenesis, and it confers metastatic properties to cancer cells. Gossypol is a natural polyphenolic compound with anti-inflammation, anti-oxidant, and anticancer activities. In this study, we investigated the antimetastatic and antitumour effects of gossypol on human cervical cancer cells (HeLa and SiHa cells). Gossypol exerted a strong inhibition effect on the migration and invasion of human cervical cancer cells. It reduced the focal adhesion kinase (FAK) pathway-mediated expression of matrix metalloproteinase-2 and urokinase-type plasminogen activator, subsequently inhibiting the invasion of SiHa cells. In addition, gossypol reversed EMT induced by transforming growth factor beta 1 (TGF-[Formula: see text]1) and up-regulated epithelial markers, such as E-cadherin but significantly suppressed Ras homolog family member (Rho)A, RhoB, and p-Samd3. The tail vein injection model showed that gossypol treatment via oral gavage reduced lung metastasis. Gossypol also decreased tumour growth in vivo in the nude mouse xenograft model. All these findings suggest that gossypol suppressed the invasion and migration of human cervical cancer cells by targeting the FAK signaling pathway and reversing TGF-[Formula: see text]1-induced EMT. Hence, gossypol warrants further attention for basic mechanistic studies and drug development.

Estrogen is a commonly used hormone in the adjuvant treatment of intrauterine adhesion (IUA), which can promote endometrial growth. Stem cell transplantation has also been reported to promote endometrial regeneration in IUA due to its potential differentiative capacity. Human Wharton's jelly mesenchymal stem cells (WJ‑MSCs) are isolated from the umbilical cord, possess strong self‑renewal and proliferative abilities, and are hypo‑immunogenic and non‑tumorigenic. Therefore, the present study aimed to investigate the therapeutic effects and underlying mechanism of WJ‑MSCs transplantation with estrogen treatment, separately or as a combined therapy, on IUA. The IUA model was established using the ethanol damage method. A total of 50 Sprague‑Dawley female rats were randomly divided into the control, IUA model, WJ‑MSCs treatment, estrogen treatment and WJ‑MSCs+ estrogen treatment groups (n=10/group). WJ‑MSCs were injected three times at 5‑day intervals. IUA rats in the estrogen group received 0.2 mg/kg estrogen through intragastric administration, once every 2 days for 8 weeks. Morphological changes were evaluated by hematoxylin‑eosin staining. Immunohistochemical evaluations of pan‑keratin, vimentin, transforming growth factor (TGF)‑β1, RhoA, RhoB, RhoC, Rho‑associated coiled‑coil‑containing protein kinase (ROCK)I, and ROCKII expression were performed in uterine tissue. After treatment, the uterine specimens were observed to have increased uterine thickness and gland numbers in all treatment groups compared with the IUA group; however, the degree of restoration in the independent WJ‑MSCs and estrogen treatment groups was better than in the combined treatment group. Immunohistochemical analysis demonstrated that pan‑keratin expression was increased, and RhoA, ROCKI and TGF‑β1 expression was significantly inhibited in the WJ‑MSCs and WJ‑MSCs + estrogen treatment groups compared with the IUA group; however, the expression levels of these proteins were similar among all treatment groups. No change in vimentin expression was detected in any treatment group. The expression levels of RhoB, RhoC and ROCKII were clearly not affected by WJ‑MSCs intervention alone. In conclusion, transplantation of WJ‑MSCs may repair endometrial damage in IUA rats via TGF‑β1‑mediated inhibition of RhoA/ROCKI signaling.

In addition to commonly associated environmental factors, genomic factors may cause cerebral palsy. We performed whole-exome sequencing of 250 parent-offspring trios, and observed enrichment of damaging de novo mutations in cerebral palsy cases. Eight genes had multiple damaging de novo mutations; of these, two (TUBA1A and CTNNB1) met genome-wide significance. We identified two novel monogenic etiologies, FBXO31 and RHOB, and showed that the RHOB mutation enhances active-state Rho effector binding while the FBXO31 mutation diminishes cyclin D levels. Candidate cerebral palsy risk genes overlapped with neurodevelopmental disorder genes. Network analyses identified enrichment of Rho GTPase, extracellular matrix, focal adhesion and cytoskeleton pathways. Cerebral palsy risk genes in enriched pathways were shown to regulate neuromotor function in a Drosophila reverse genetics screen. We estimate that 14% of cases could be attributed to an excess of damaging de novo or recessive variants. These findings provide evidence for genetically mediated dysregulation of early neuronal connectivity in cerebral palsy.

To interfere with cell function, many scientists rely on methods that target DNA or RNA due to the ease with which they can be applied. Proteins are usually the final executors of function but are targeted only indirectly by these methods. Recent advances in targeted degradation of proteins based on proteolysis-targeting chimaeras (PROTACs), ubiquibodies, deGradFP (degrade Green Fluorescent Protein) and other approaches have demonstrated the potential of interfering directly at the protein level for research and therapy. Proteins can be targeted directly and very specifically by antibodies, but using antibodies inside cells has so far been considered to be challenging. However, it is possible to deliver antibodies or other proteins into the cytosol using standard laboratory equipment. Physical methods such as electroporation have been demonstrated to be efficient and validated thoroughly over time. The expression of intracellular antibodies (intrabodies) inside cells is another way to interfere with intracellular targets at the protein level. Methodological strategies to target the inside of cells with antibodies, including delivered antibodies and expressed antibodies, as well as applications in the research areas of neurobiology, viral infections and oncology, are reviewed here. Antibodies have already been used to interfere with a wide range of intracellular targets. Disease-related targets included proteins associated with neurodegenerative diseases such as Parkinson’s disease (α-synuclein), Alzheimer’s disease (amyloid-β) or Huntington’s disease (mutant huntingtin [mHtt]). The applications of intrabodies in the context of viral infections include targeting proteins associated with HIV (e.g. HIV1-TAT, Rev, Vif, gp41, gp120, gp160) and different oncoviruses such as human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Epstein-Barr virus, and they have been used to interfere with various targets related to different processes in cancer, including oncogenic pathways, proliferation, cell cycle, apoptosis, metastasis, angiogenesis or neo-antigens (e.g. p53, human epidermal growth factor receptor-2 [HER2], signal transducer and activator of transcription 3 [STAT3], RAS-related RHO-GTPase B (RHOB), cortactin, vascular endothelial growth factor receptor 2 [VEGFR2], Ras, Bcr-Abl). Interfering at the protein level allows questions to be addressed that may remain unanswered using alternative methods. This review addresses why direct targeting of proteins allows unique insights, what is currently feasible in vitro, and how this relates to potential therapeutic applications.

Rationale: Glioma stem-like cells (GSCs) contribute to temozolomide (TMZ) resistance in gliomas, although the mechanisms have not been delineated. Methods: In vitro functional experiments (colony formation assay, flow cytometric analysis, TUNEL assay) were used to assess the ability of extracellular vesicles (EVs) from hypoxic GSCs to promote TMZ resistance in glioblastoma (GBM) cells. RNA sequencing and quantitative Reverse Transcription-PCR were employed to identify the functional miRNA in hypoxic EVs. Chromatin immunoprecipitation assays were performed to analyze the transcriptional regulation of miRNAs by HIF1α and STAT3. RIP and RNA pull-down assays were used to validate the hnRNPA2B1-mediated packaging of miRNA into EVs. The function of EV miR-30b-3p from hypoxic GSCs was verified by in vivo experiments and analysis of clinical samples. Results: Hypoxic GSC-derived EVs exerted a greater effect on GBM chemoresistance than those from normoxic GSCs. The miRNA profiling revealed that miR-30b-3p was significantly upregulated in the EVs from hypoxic GSCs. Further, HIF1α and STAT3 transcriptionally induced miR-30b-3p expression. RNA immunoprecipitation and RNA-pull down assays revealed that binding of miR-30b-3p with hnRNPA2B1 facilitated its transfer into EVs. EV-packaged miR-30b-3p (EV-miR-30b-3p) directly targeted RHOB, resulting in decreased apoptosis and increased proliferation in vitro and in vivo. Our results provided evidence that miR-30b-3p in CSF could be a potential biomarker predicting resistance to TMZ. Conclusion: Our findings indicated that targeting EV-miR-30b-3p could provide a potential treatment strategy for GBM.

Gastric cancer (GC) is one of the most common digestive system diseases and yet lacks effective therapeutic regimen. The aim of our present research was to probe the value of hsa_circ_0001017 in GC treatment. qRT-PCR and Western blot were performed to detect gene and protein expressions, respectively. CCK-8 assay and clone formation assay were used to ensure the proliferation of GC cell lines. Transwell assay was performed to measure the migration and invasion of GC cell lines. The relationship between hsa_circ_0001017 and miR-197 and that between miR-197 and RHOB 3′-UTR were ensured using the luciferase reporter assay. Decreased hsa_circ_0001017 was discovered in GC, and upregulation of hsa_circ_0001017 notably repressed proliferation, migration, and invasion of GC cell lines. We further certificated that hsa_circ_0001017 served as miR-197 sponge and suppressed the expression of miR-197. Moreover, hsa_circ_0001017 upregulation meaningfully accelerated RHOB expression in both gene and protein levels, and RHOB was a downstream target of miR-197. Overexpression of miR-197 could markedly restrain hsa_circ_0001017-induced RHOB increasing and stifle inhibition of hsa_circ_0001017 to the malignant phenotype of GC cell lines. Next, our results further confirmed that hsa_circ_0001017 increasing notably inhibited tumor growth, impeded miR-197 production, while it enhanced the expression of RHOB in vivo. Our data demonstrated that upregulation of hsa_circ_0001017 could notably muffle the proliferation as well as the metastasis of GC cell lines and impede the formation of GC tumor via targeting to miR-197/RHOB signaling pathway. Our results evidenced that hsa_circ_0001017 may act as a rising biomarker for GC treatment.

Lowering intraocular pressure (IOP) is the most effective treatment of glaucoma, however most of the current available glaucoma drugs target a single molecule. MicroRNAs (miRNAs) are noncoding RNAs that target a network of molecules. This study aims to investigate the role of miR-21-5p in regulating IOP and the mechanism of function. miR-21-5p mimics was topically applied to C57/BL6 mouse eyes, which significantly increased miR-21-5p expression in the conventional outflow tissue and reduced IOP by a maximum of 17.77% at 24 h after treatment. The conventional outflow facility measured by ex vivo moue eye perfusion of miR-21-5p was significantly increased by 60.14%. Moreover, miR-21-5p overexpression significantly reduced the transendothelial electrical resistance in porcine angular aqueous plexus cells. Transcriptome analysis and further quantification by Western blot and PCR revealed that SMAD7 and FGF18 might be the downstream target of miR-21-5p in regulating aqueous humor outflow. The predicted functional pathways PTEN/eNOS, RhoB/pMLC and TIMP3/MMP9 were significantly altered after miR-21-5p transfection. Dual luciferase assay verified the direct targets of miR-21-5p. In conclusion, miR-21-5p seems to regulate IOP by modulating multiple genes that are associated with aqueous humor outflow, including genes those regulating cell adhesion, cytoskeletal dynamics and extracellular matrix turnover. Thus, miR-21-5p represents a new therapeutic strategy for glaucoma and a viable alternative to existing multidrug regimens.

Chronic hyperlipidemia causes the dysfunction of pancreatic β-cells, such as apoptosis and impaired insulin secretion, which are aggravated in the presence of hyperglycemia. The underlying mechanisms, such as endoplasmic reticulum (ER) stress, oxidative stress and metabolic disorders, have been reported before; however, the time sequence of these molecular events is not fully understood. Here, using isobaric labeling-based mass spectrometry, we investigated the dynamic proteomes of INS-1 cells exposed to high palmitate in the absence and presence of high glucose. Using bioinformatics analysis of differentially expressed proteins, including the time-course expression pattern, protein-protein interaction, gene set enrichment and KEGG pathway analysis, we analyzed the dynamic features of previously reported and newly identified lipotoxicity- and glucolipotoxicity-related molecular events in more detail. Our temporal data highlight cholesterol metabolism occurring at 4 h, earlier than fatty acid metabolism that started at 8 h and likely acting as an early toxic event highly associated with ER stress induced by palmitate. Interestingly, we found that the proliferation of INS-1 cells was significantly increased at 48 h by combined treatment of palmitate and glucose. Moreover, benefit from the time-course quantitative data, we identified and validated two new molecular targets: Setd8 for cell replication and Rhob for apoptosis, demonstrating that our temporal dataset serves as a valuable resource to identify potential candidates for mechanistic studies of lipotoxicity and glucolipotoxicity in pancreatic β-cells.

Osteosarcoma is a primary malignancy that develops in bone, along with serious recurrence and metastasis. As an isoform of Rho family GTPases, RhoB could suppress cell proliferation, invasion, and anti-angiogenesis. But it is not clear how RhoB involves in tumor metastasis. Here we found that expression of RhoB was decreased in osteosarcoma primary samples and cell lines. Ectopic expression of RhoB restrains the migration of osteosarcoma cells in vitro and in vivo, and induces osteosarcoma cell apopotsis. Further study showed that overexpression of RhoB could increase the proportion of B55 in PP2A complex and enhance the dephosphorylation of AKT1 by interacting with B55. Moreover, we demonstrated that miR-19a, which exhibits abnormal expression in highly metastatic osteosarcoma cell lines, could inhibit the expression of RhoB and promote the lung metastasis of osteosarcoma cells in vivo. Our results indicate that miR-19a-mediated RhoB is a critical regulator for the dephosphorylation of AKT1 in osteosarcoma cells. It may have a possible strategy on suppressing osteosarcoma metastasis by miR-19a inhibitory oligonucleotides. The miR-19a/RhoB/AKT1 network may help us to better understand the mechanism of osteosarcoma metastasis.

Natália Rubio Claret Pereira,1,* Rodrigo Azevedo Loiola,1,* Stephen Fernandes Rodrigues,1 Catiuscia P de Oliveira,2 Sabrina L Büttenbender,3 Silvia S Guterres,2 Adriana R Pohlmann,3 Sandra H Farsky1 1Department of Clinical and Toxicological Analysis, Faculty of Pharmaceutical Sciences, University of São Paulo, Sao Paulo, Brazil; 2Faculty of Pharmacy, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil; 3Department of Organic Chemistry, Institute of Chemistry, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil *These authors contributed equally to this work Purpose: The low penetration of drugs across the blood–brain barrier (BBB) compromises the delivery of chemotherapeutic agents to the brain parenchyma and contributes to the poor prognosis of glioblastoma multiforme (GBM). We investigated the efficacy of methotrexate-loaded lipid-core nanocapsules (MTX-LNC) administered by the oral route to treat murine GBM, its ability to cross the BBB, and the mechanisms of MTX-LNC uptake by cultured GL261 glioma and BV2 microglia cells. Materials and methods: Female C57B/6 mice were used in intravital microscopy assays to investigate the penetrance of rhodamine B-label MTX-LNC (RhoB/MTX-LNC) in the brain after oral or IV administration, and to evaluate the BBB integrity. Intracranial implantation of GL261 cells was undertaken to induce a murine GBM model, and the effectiveness of oral MTX or MTX-LNC treatments (started on Day 10 of GBM, every 2 days for 12 days) was quantified by tumor size, body weight, and leukogram. Pharmacological blockade of endocytic pathways was done to investigate the mechanisms of MTX-LNC uptake by cultured GL261 and microglia BV2 cells by using fluorescence microscopy. The effect of MTX-LNC or MTX on GL261 and BV2 proliferation was evaluated to compare the cytotoxicity of such compounds. Results: RhoB/MTX-LNC was detected in brain parenchyma of mice after IV or oral administration, without any damage on BBB. Oral treatment with MTX-LNC reduced tumor volume and prevented weight loss and leukopenia in comparison to MTX-treated mice. MTX-LNC uptake by GL261 is caveolae-dependent, whereas endocytosis of MTX-LNC by BV2 occurs via phagocytosis and macropinocytosis. Both MTX-LNC and MTX reduced GL261 and BV2 proliferation; however, MTX-LNC showed higher efficacy in the inhibition of glioma proliferation. Conclusion: Together, we infer that the higher ability of MTX-LNC to cross the BBB and be captured by cancer and immune brain cells by different mechanisms is responsible for the higher efficacy of oral MTX-LNC treatment in GBM. Keywords: glioma, nanomedicine, blood–brain barrier, microglia, endocytosis

Lung cancer is the leading cause of cancer mortality worldwide, with adenocarcinomas of the non-small cell lung carcinoma (NSCLC) subtype accounting for the majority of cases. Therefore, an urgent need exists for a more detailed dissection of the molecular events driving NSCLC development and the identification of clinically relevant biomarkers. Even though originally identified as a tumour suppressor, recent studies associate the cytoplasmically (mis)localised CDK inhibitor p27Kip1 (p27) with unfavourable responses to chemotherapy and poor outcomes in NSCLC, supporting the hypothesis that the protein can execute oncogenic activities. In a recent issue of The Journal of Pathology, Calvayrac and coworkers uncover a novel molecular mechanism that can explain this oncogenic role of p27. They demonstrate that cytoplasmic p27 binds and inhibits the small GTPase RhoB and thereby relieves a selection pressure for RhoB loss that is frequently observed in NSCLC. This is supported not only by studies with genetically modified mice, but also through identification of a cohort of human lung cancer patients with cytoplasmic p27 and continued RhoB expression, where this signature correlates with decreased survival. This not only establishes a potentially useful biomarker, but also provides yet another facet of the complex roles p27 undertakes in tumourigenesis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

It was reported that statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase that are used to prevent hypercholesterolemia, have antitumor activity in several cancers. In this study, we investigated the cell viability of statins in Cisplatin-resistant HCP4 and PCDP5 cells compared with their parent Hela and PC3 cells, respectively, and found that HCP4 and PCDP5 cells were 37-fold and 18-fold more resistant to Cisplatin but 13-fold and 7-fold more sensitive to Lovastatin by cell proliferation assay. Lovastatin induced the apoptosis of HCP4 cells more rapidly and to greater extent than in Hela cells as assessed by flow cytometry and western blotting analyses. The MVA pathway was not involved in this acquired Cisplatin resistance. To elucidate the mechanism underlying the reduced viability to Lovastatin, we performed cDNA microarray analysis and identified 65 and 54 genes that were induced more than 2-fold by Lovastatin in HCP4 and PCDP5 cells, respectively. Of these, only three genes, KLF2, KLF6, and RHOB, were commonly induced between HCP4 and PCDP5 cells. These mRNAs were strongly induced by Lovastatin with transcriptional regulation in HCP4 cells. Consistent with transcription, the protein expression of RHOB also was induced by Lovastatin. The induction of these genes was associated with cell cycle arrest and apoptosis. Combination treatment with Cisplatin and Lovastatin resulted in an agonistic effect in Hela and PC3 cells and an antagonistic effect in HCP4 and PCDP5 cells. These results suggest that statins might have the potential to overcome Cisplatin resistance as single-agent therapy.

Many microRNAs participate in the development, differentiation and function preservation of the embryonic and adult inner ear, but many details still need to be elucidated regarding the numerous microRNAs in the inner ear. Based on previous investigations on the microRNA profile in the inner ear, we confirmed that several microRNAs are expressed in the inner ear, and we detected the spatial expression of these microRNAs in the neonatal mouse inner ear. Then we focused on miR-194 for its specific expression with a dynamic spatiotemporal pattern during inner ear development. Overexpression of miR-194 in cultured spiral ganglion cells significantly affected the dendrites of differentiated neurons, with more branching and obviously dispersed nerve fibres. Furthermore, the cytoskeleton of cultured cells was markedly affected, as disordered actin filaments resulting from miR-194 overexpression and enhanced filaments resulting from miR-194 knockdown were observed. Together with the bioinformatic methods, the RT-qPCR and western blot results showed that RhoB is a candidate target of miR-194 in the morphogenesis of spiral ganglion neurons. Additionally, the double luciferase reporter system was used to identify RhoB as a novel target of miR-194. Finally, the inhibition of RhoB activation by Clostridium difficile toxin B disturbed the organization of the actin filament, similar to the effects of miR-194 overexpression. In summary, we investigated microRNA expression in the mouse inner ear, and demonstrated that miR-194 is dynamically expressed during inner ear development; importantly, we found that miR-194 affects neuron morphogenesis positively through Rho B-mediated F-actin rearrangement.

RhoGTPases are known regulators of intracellular actin dynamics that are important for maintaining endothelial barrier function. RhoA is most extensively studied as a key regulator of endothelial barrier function, however the function of the 2 highly homologous family-members (> 88%) RhoB and RhoC in endothelial barrier function is still poorly understood. This study aimed to determine whether RhoA, RhoB and RhoC have overlapping or distinct roles in barrier function and permeability in resting and activated endothelium. By using primary endothelial cells in combination with siRNA transfection to establish individual, double or triple knockdown of the RhoA/B/C RhoGTPases, we found that RhoB, but not RhoA or RhoC, is in resting endothelium a negative regulator of permeability. Loss of RhoB accounted for an accumulation of VE-cadherin at cell-cell contacts. Thrombin-induced loss of endothelial integrity is mediated primarily by RhoA and RhoB. Combined loss of RhoA/B showed decreased phosphorylation of Myosin Light Chain and increased expression of VE-cadherin at cell-cell contacts after thrombin stimulation. RhoC contributes to the Rac1-dependent restoration of endothelial barrier function. In summary, this study shows that these highly homologous RhoGTPases differentially control the dynamics of endothelial barrier function.

The present study aimed to screen potential target genes for the early diagnosis and treatment of early metastatic clear cell renal cell carcinoma (ccRCC) using the microarray data of early metastatic and non-metastatic ccRCC samples. The DNA microarray dataset GSE47352 was downloaded from Gene Expression Omnibus and included 4 early metastatic and 5 non-metastatic ccRCC samples. Differentially expressed genes (DEGs) were screened using the limma package. Then, pheatmap package was used to conduct two-way clustering for the DEGs. Subsequently, MAPPFinder and GenMAPP were employed separately to perform functional and pathway enrichment analysis for the DEGs. Additionally, a protein-protein interaction (PPI) network was constructed using Cytoscape, and small drug molecules were searched using Connectivity map (cmap). In total, 196 upregulated and 163 downregulated genes were identified. DEGs, including JUN, tumor necrosis factor (TNF), Ras homolog family member B (RHOB) and transforming growth factor β2 (TGFβ2) were significantly enriched in the signaling pathway of renal cell carcinoma. Furthermore, nuclear receptor subfamily 4 group A member 1 (NR4A1) was significantly enriched in the mitogen-activated protein kinase signaling pathway; in addition, laminin subunit α (LAMA) 1, LAMA2 and LAMA4 were significantly enriched in extracellular matrix-receptor interaction. JUN (degree=6) had the highest degree in the PPI network. Thapsigargin (score=−0.913) possessed the highest performance in terms of the treatment of early metastatic ccRCC. In the present study, it was discovered that certain DEGs, including JUN, TNF, RHOB, NR4A1, TGFβ2, LAMA1, LAMA2 and LAMA4 were potential target genes associated with early metastatic ccRCC. In addition, thapsigargin could be used as an efficient small drug molecule for the treatment of early metastatic ccRCC.

Pierce et al. describe a pediatric patient with a fatal systemic capillary leak syndrome (Clarkson’s disease). They identify a point mutation in p190BRhoGAP and show that patient-derived microvascular endothelial cells show prolonged activation RhoB that correlates with impaired barrier recovery after treatment with TNF compared with control cultures., We describe a fatal case of pediatric systemic capillary leak (Clarkson’s disease) associated with a point mutation in p190BRhoGAP. Dermal microvascular endothelial cells (ECs) isolated from this patient form monolayers with similar levels and distribution of junctional proteins and transendothelial electrical resistance compared with normal human dermal microvascular ECs. However, patient-derived ECs demonstrate a greater increase in permeability and impaired recovery of barrier function in response to tumor necrosis factor (TNF) compared with normal donor EC cultures. TNF transiently activates RhoB in ECs coincident with developing leak, and inactivation of RhoB correlates with barrier recovery. The mutation in p190BRhoGAP impairs RhoB inactivation, and the mutant phenotype of patient-derived ECs is replicated by siRNA knockdown of p190BRhoGAP in normal ECs. These data suggest a previously unknown function for p190BRhoGAP in control of capillary EC barrier function that may also be important in acquired systemic capillary leak associated with critical illness in humans.

Purpose Chronic lung allograft dysfunction (CLAD) limits long-term survival after lung transplantation. Different immune and non-immune insults contribute to a chronic inflammatory and fibrotic process that ultimately reduces the graft's function and survival. The renin-angiotensin-aldosterone system (RAA) has been shown to play a role in fibrogenesis in the kidney, heart and lung. Notably, 6 RAA signature proteins (RHOB, BST1, LYPA1, GLNA, TSP1, LAMB1) have been identified in urine and kidney tissue of patients with Interstitial Fibrosis and Tubular Atrophy. We hypothesize that RAA is involved in CLAD pathogenesis and that these RAA signature proteins could be used as biomarkers of CLAD in Bronchoalveolar Lavage (BAL). Methods We performed immunofluorescence staining of angiotensin receptors (AGTR1 and AGTR2) and immunohistochemical staining of TSP1 in 10 explanted CLAD lungs (obtained at time of re-transplantation), compared to 5 control lungs (obtained from donors or lobectomies for lung nodule resections). Using mass spectrometry, we quantified two peptides for each RAA signature protein in the BAL of 40 lung transplant recipients undergoing bronchoscopy. Results We observed significantly more AGTR1+ cells in CLAD versus control lungs (p=0.04) (Fig1 a), while the percentage of AGTR2+ cells was not different. We also found a trend towards higher expression of TSP1, particularly in macrophages and fibrotic zones, in CLAD versus controls (Fig1b). Mass spectrometry-based assays were developed for BAL quantification of the RAA proteins (Fig1c). There was a trend towards higher concentration of peptides of RHOB, BST1 and LYPA1 peptides in the BAL of CLAD patients with concomitant acute >10% decline in lung allograft function in comparison to patients with stable lung function (Fig1d). Conclusion Our pilot data support the hypothesis that RAA is involved in CLAD and that RAA-regulated proteins may help identify patients with active CLAD. We are currently validating these findings using a larger cohort and additional endpoints.

Backgrounds The incidence of thyroid carcinoma continues to increase every year. The incidence of papillary thyroid carcinoma (PTC) is the largest of all thyroid cancers, and is the most widely seen. Thus, Do some identification and testing of biomarkers related to tumorigenesis and development, which will help understand and treat PTC.Methods The expression of miR-223 and RHOB needs to be detected by polymerase chain reaction and western blot, respectively. Thyroid cell-line BCPAP and K1 cells were cultured, transfected for overexpression of miR-223or RHOB, and evaluated using MTT, Transwell, and apoptosis assays. In order to better understand the relationship between miR-223 and RHOB, dual luciferase reporter gene detection and RHOB rescue experiments were performed. In addition, clinical pathological parameters and correlation analysis of patients with miR-223 were also performed.Results Aberrant expression of miR-223 was detected in PTC samples. We also determined that miR-223 expression was associated with TNM staging and cervical metastasis. Moreover, miR-223 inhibits the increase in cell number and position, as well as defense against cancer cells and accelerates cell death. Furthermore, miR-223 excessive expression was associated with a significant inhibition of RHOB levels. According to the luciferase test report, it can be known miR-223 was able to bind RHOB 3′-UTR, it is possible to change its stability through these, and finally achieve the purpose of reducing RHOB. Finally,excessive expression of RHOB has undergone earth-shaking changes in the effect of miR-223 overexpression on cell number growth, invasion and disappearance.Conclusion Aberrant miR-223/RHOB expression took part in PTC make headway and miR-223 inhibition caused RHOB overexpression and cancer progression. Therefore, miR-223 may be a potential therapeutic target for PTC.

RhoGTPases regulate cytoskeletal dynamics, migration and cell-cell adhesion in endothelial cells. Besides regulation at the level of guanine nucleotide binding, they also undergo post-translational modifications, for example ubiquitination. RhoGTPases are ubiquitinated by Cullin RING ligases which are in turn regulated by neddylation. Previously we showed that inhibition of Cullin RING ligase activity by the neddylation inhibitor MLN4924 is detrimental for endothelial barrier function, due to accumulation of RhoB and the consequent induction of contractility. Here we analyzed the effect of pharmacological activation of Cullin RING ligases on endothelial barrier integrity in vitro and in vivo. CSN5i-3 induced endothelial barrier disruption and increased macromolecule leakage in vitro and in vivo. Mechanistically, CSN5i-3 strongly induced the expression and activation of RhoB and to lesser extent of RhoA in endothelial cells, which enhanced cell contraction. Elevated expression of RhoGTPases was a consequence of activation of the NF-κB pathway. In line with this notion, CSN5i-3 treatment decreased IκBα expression and increased NF-κB-mediated ICAM-1 expression and consequent adhesion of neutrophils to endothelial cells. This study shows that sustained neddylation of Cullin RING-ligases leads to activation the NF-κB pathway in endothelial cells, elevated expression of RhoGTPases, Rho/ROCK-dependent activation of MLC and disruption of the endothelial barrier.

BackgroundWhile Nanos-mediated maintenance of germ cells inDrosophilaand mice has been related to regulation of apoptosis, the relevance of these findings to human physiology is uncertain. Previously we have described the p.[(Pro34Thr);(Ser83del)] doubleNANOS1mutation as associated with an absence of germ cells in the testes of infertile patients. The aim of this study was to identify the mechanism underlying infertility phenotype of patients bearing theNANOS1mutation.MethodsConstructs encoding a wild-type or mutated NANOS1 protein were used for transfection of TCam-2 cell line, representing male germ cells in culture. Influence of this mutation on cell proliferation was performed using MTS assay while apoptosis and cell cycle were measured by flow cytometry. RNA-Seq analysis including quantitative RT-PCR was conducted for selecting pro-apoptotic genes, repressed by the wild-type NANOS1. Influence of the p.[(Pro34Thr);(Ser83del)] NANOS1 mutation on that repression was investigated by quantitative RT-PCR.ResultsWe show here that the p.[(Pro34Thr);(Ser83del)] doubleNANOS1mutation causes NANOS1 to functionally switch from being anti-apoptotic to pro-apoptotic in the human male germ cell line TCam-2. This mutation disrupts repression of mRNAs encoding pro-apoptotic GADG45A, GADD45B, GADD45G and RHOB factors, which could contribute to an increase in apoptosis.ConclusionsThis report underscores the conservation of Nanos from flies to humans as a repressor of pro-apoptotic mRNAs in germ cells, and provides a basis for understanding NANOS1 functions in human reproductive health.

Atrial fibrillation (AF) is an arrhythmia caused by disorganized electrical activity in the atria, and it is an important cause of mortality and morbidity. There is a limited data about Rho/Rho-kinase (ROCK) pathway contribute to AF development. The aim of the present study was to elucidate leukocyte RHO/ROCK gene expressions in patients with non-valvular AF (NVAF). A total of 37 NVAF patients and 47 age and sex-matched controls were included in this study. mRNA was extracted from leukocytes, and real-time polymerase chain reaction was used for gene expression analysis. A marked increase in ROCK1 and ROCK2 gene expressions in patients with NVAF was observed (P

Mirror Image Polydactyly 1 (MIPOL1) is generally associated with congenital anomalies. However, its role in cancer development is poorly understood. Previously, by utilizing the functional complementation approach, microcell-mediated chromosome transfer (MMCT), a tumor suppressor gene, MIPOL1, was identified. MIPOL1 was confirmed to be downregulated in nasopharyngeal carcinoma (NPC) cells and tumor tissues, and re-expression of MIPOL1 induced tumor suppression. The aim of the current study is to further elucidate the functional tumor suppressive role of MIPOL1. In our study, with an expanded sample size of different clinical stages of NPC tumor tissues, we further confirmed the downregulation of MIPOL1 in different cancer stages. MIPOL1 re-expression down-regulated angiogenic factors and reduced phosphorylation of metastasis-associated proteins including AKT, p65, and FAK. In addition, MIPOL1 was confirmed to interact with a tumor suppressor, RhoB, and re-expression of MIPOL1 enhanced RhoB activity. The functional role of MIPOL1 was further validated by utilizing a panel of wild-type (WT) and truncated MIPOL1 expression constructs. The MIPOL1 tumor-suppressive effect can only be observed in the WT MIPOL1-expressing cells. In vitro and nude mice in vivo functional studies further confirmed the critical role of WT MIPOL1 in inhibiting migration, invasion and metastasis in NPC. Overall, our study provides strong evidence about the tumor-suppressive role of MIPOL1 in inhibiting angiogenesis and metastasis in NPC.

Dendritic cells (DCs) can initiate and regulate adaptive immunity depending on their maturation status. Many pharmacological and genetic means have been used in the generation of immature/tolerogenic DCs. However, the key factors controlling DCs tolerogenicity remain obscure. In this work, we demonstrated that AZD8055, an ATP-competitive inhibitor of mammalian target of rapamycin (mTOR), could also lead to a tolerogenic DC phenotype from several lines of evidence, such as suppression of T cell proliferation, promoting the generation of Tregs, and inducing allogeneic T cell apoptosis. Further studies using RNA-seq method identified 430, 1172 and 1436 differentially expressed genes (DEGs) between AZD-DCs vs. Control-DCs, LPS-DCs vs. Control-DCs, and AZD-DCs vs. LPS-DCs, respectively. The 5 most differentially expressed transcripts identified by RNA-seq expression profiles were validated by quantitative RT-PCR assays. NF-κB, p38MAPK, the ribosome and PPAR signaling pathways may be involved in the induction of tolerogenic DCs by AZD8055. Functional annotation showed some genes like MGL2, Cadherin-1, 4-1BB, RhoB and Pdpn, were quite different between AZD-DCs and Control-DCs/LPS-DCs, which might be related to the tolerogenic properties of AZD-DCs. Our work provided the potential underlying molecular mechanisms involved in the generation of tolerogenic DCs. Further functional characterization of individual target gene in DC tolerogenicity will help to develop novel therapeutic modalities in circumstances like transplant tolerance induction and autoimmunity.

Objective: Restenosis is a frequent complication of angioplasty. It consists of a neointimal hyperplasia resulting from progression and migration of vascular smooth muscle cells (VSMC) into the vessel lumen. microRNA miR-223 has recently been shown to be involved in cardiovascular diseases including atherosclerosis, vascular calcification and arterial thrombosis. In this study, our aim was to assess the impact of miR-223 modulation on restenosis in a rat model of carotid artery after balloon injury. Methods: The over and down-expression of miR-223 was induced by adenoviral vectors, containing either a pre-miR-223 sequence allowing artificial miR-223 expression or a sponge sequence, trapping the native microRNA, respectively. Restenosis was quantified on stained rat carotid sections. Results: In vitro, three mRNA (Myocyte Enhancer Factor 2C (MEF2C), Ras homolog gene family, member B (RhoB) and Nuclear factor 1 A-type (NFIA)) reported as miR-223 direct targets and known to be implicated in VSMC differentiation and contractility were studied by RT-qPCR. Our findings showed that down-expression of miR-223 significantly reduced neointimal hyperplasia by 44% in carotids, and was associated with a 2-3-fold overexpression of MEF2C, RhoB and NFIA in a murine monocyte macrophage cell line, RAW 264.7 cells. Conclusions: Down-regulating miR-223 could be a potential therapeutic approach to prevent restenosis after angioplasty.