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Purpose The overall response of cisplatin-based chemotherapy in bladder urothelial carcinoma (BUC) remains unsatisfactory due to the complex pathological subtypes, genomic difference, and drug resistance. The genes that associated with cisplatin resistance remain unclear. Herein, we aimed to identify the cisplatin resistance associated genes in BUC. Experimental design The cytotoxicity of cisplatin was evaluated in six bladder cancer cell lines to compare their responses to cisplatin. The T24 cancer cells exhibited the lowest sensitivity to cisplatin and was therefore selected to explore the mechanisms of drug resistance. We performed genome-wide CRISPR screening in T24 cancer cells in vitro, and identified that the gene heterogeneous nuclear ribonucleoprotein U (HNRNPU) was the top candidate gene related to cisplatin resistance. Epigenetic and transcriptional profiles of HNRNPU-depleted cells after cisplatin treatment were analyzed to investigate the relationship between HNRNPU and cisplatin resistance. In vivo experiments were also performed to demonstrate the function of HNRNPU depletion in cisplatin sensitivity. Results Significant correlation was found between HNRNPU expression level and sensitivity to cisplatin in bladder cancer cell lines. In the high HNRNPU expressing T24 cancer cells, knockout of HNRNPU inhibited cell proliferation, invasion, and migration. In addition, loss of HNRNPU promoted apoptosis and S-phase arrest in the T24 cells treated with cisplatin. Data from The Cancer Genome Atlas (TCGA) demonstrated that HNRNPU expression was significantly higher in tumor tissues than in normal tissues. High HNRNPU level was negatively correlated with patient survival. Transcriptomic profiling analysis showed that knockout of HNRNPU enhanced cisplatin sensitivity by regulating DNA damage repair genes. Furthermore, it was found that HNRNPU regulates chemosensitivity by affecting the expression of neurofibromin 1 (NF1). Conclusions Our study demonstrated that HNRNPU expression is associated with cisplatin sensitivity in bladder urothelial carcinoma cells. Inhibition of HNRNPU could be a potential therapy for cisplatin-resistant bladder cancer.

Background Renal ischemia reperfusion injury (IRI) has become a growing concern in clinical practice with high morbidity and mortality rates. There is currently no effective prophylactic regimen available to prevent its occurrence and to improve its clinical prognosis. Dl-3-n-butylphthalide (NBP) has been used for stroke treatment in China for years. Little is known about its role in preventing kidney injury. Methods The kidneys of male C57BL/6J mice were subjected to 33 min of ischemia followed by 24 h of reperfusion. NBP was administered by gavage prior to surgery. The reno-protective effect of NBP was evaluated by serum creatinine, kidney injury markers and renal pathological changes. Furthermore, the inflammation, oxidative stress, and apoptosis markers in kidney tissue were examined. In vitro, HK2 cells were treated prophylactically with NBP and then exposed to hypoxia/reoxygenation (H/R). Cell viability and apoptosis related protein were quantified to verify the protective effect of NBP. Pro-inflammation genes expression as well as ROS generation were further investigated also. Results NBP pretreatment significantly improved renal dysfunction and alleviated pathological injury, renal inflammation response, oxidative stress and cell apoptosis. Consistently, NBP attenuated H/R induced increases in ROS, pro-inflammatory genes expression, apoptosis and cleaved caspase-3 levels in HK2 cells. Conclusion Our promising results validated for the first time that NBP could ameliorate renal IRI via attenuating inflammation, oxidative stress, and apoptosis, which indicated that NBP might be a good candidate against AKI.

Background Solanum nigrum L. decoction has been used as a folklore medicine in China to prevent the postoperative recurrence of bladder cancer (BC). However, there are no previous pharmacological studies on the protective mechanisms of this activity of the plant. Thus, this study aimed to perform a systematic analysis and to predict the potential action mechanisms underlying S. nigrum activity in BC based on network pharmacology. Methods Based on network pharmacology, the active ingredients of S. nigrum and the corresponding targets were identified using the Traditional Chinese Medicines for Systems Pharmacology Database and Analysis Platform database, and BC-related genes were screened using GeneCards and the Online Mendelian Inheritance in Man database. In addition, ingredient-target (I–T) and protein–protein interaction (PPI) networks were constructed using STRING and Cytoscape, Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted, and then the pathways directly related to BC were integrated manually to reveal the pharmacological mechanism underlying S. nigrum-medicated therapeutic effects in BC. Results Seven active herbal ingredients from 39 components of S. nigrum were identified, which shared 77 common target genes related to BC. I-T network analysis revealed that quercetin was associated with all targets and that NCOA2 was targeted by four ingredients. Besides, interleukin 6 had the highest degree value in the PPI network, indicating a hub role. A subsequent gene enrichment analysis yielded 86 significant GO terms and 89 significant pathways, implying that S. nigrum had therapeutic benefits in BC through multi-pathway effects, including the HIF-1, TNF, P53, MAPK, PI3K/Akt, apoptosis and bladder cancer pathway. Conclusions S. nigrum may mediate pharmacological effects in BC through multi-target and various signaling pathways. Further validation is required experimentally. Network pharmacology approach provides a predicative novel strategy to reveal the holistic mechanism of action of herbs.

Background Microfibril-associated protein 2 (MFAP2) is a protein coding gene that exerts important phenotypic effects on cell motility, and increasing research has indicated that MFAP2 was correlated with many cancers. However, the functional and potential clinical role of MFAP2 in papillary thyroid cancer (PTC) has not yet been verified. Materials and methods We performed whole transcriptome sequencing on 78 paired PTC tissues and corresponding adjacent normal tissues and found that MFAP2 was highly expressed in PTC tissues. Then, we analyzed the expression of MFAP2 and its relation with the clinicopathological features of PTC in The Cancer Genome Atlas (TCGA) PTC genomic dataset. We detected MFAP2 expression in 40 paired PTC tissues and corresponding adjacent normal tissues through RT-qPCR (real time-quantitative polymerase chain reaction) to validate the sequencing data and TCGA cohort. Cell functional assays were performed to elucidate the function of MFAP2 in PTC cells, Western blot assay was performed to explore the correlation between MFAP2 and EMT (epithelial-mesenchymal transition)-related proteins. Results Statistical analysis showed that MFAP2 was obviously upregulated in PTC tissues compared to matched normal tissues, and the expression levels of MFAP2 in PTC tissues were strongly related with lymph node metastasis (p=0.016). The results of RT-qPCR of our own tissue specimens showed the same conclusions as that in TCGA dataset. The results of functional assays in PTC cell lines showed that MFAP2 could promote proliferation, colony formation, migration and invasion abilities and decrease the apoptotic rate in PTC cells. Western Blot assay showed that MFAP2 could regulate the expression of EMT-related proteins. Conclusion MFAP2 increases the proliferation, motility and decreases the apoptosis of PTC cells, and might be a potential therapeutic target for papillary thyroid cancer.

Glycosylation is an important modification of membrane proteins that results in functional changes in many cellular activities, from cell-cell recognition to regulatory signaling. Fucosyltransferase 8 (FUT8) is the sole enzyme responsible for core fucosylation, and aberrant fucosylation by dysregulated expression of fucosyltransferases is responsible for the growth of various types of carcinomas. However, the function of FUT8 in the progress of osteosarcoma (OS) has not been reported. In this study, we found that FUT8 is expressed at lower levels in patients with OS and in human OS cell lines such as MNNG/HOS, U2OS, and 143B, suggesting that attenuated expression of FUT8 is involved in the growth and progression of OS. Mechanistically, FUT8 affects the survival strategy of OS by modifying core-fucosylation levels of TNF receptors (TNFRs). Lower fucosylation of TNFRs activates the non-canonical NF-κB signaling pathway, and in turn, decreases mitochondria-dependent apoptosis in OS cells. Together, our results point to FUT8 being a negative regulator of OS that enhances OS-cell apoptosis and suggests a novel therapeutic strategy for treating OS.

It was known that diabetes may affect the male reproductive function by inhibiting the secretion of male accessory glands including seminal vesicles. Increased cell apoptosis induced by oxidative stress is thought to be an important pathological change in the seminal vesicles in diabetic patients. Quercetin is a potent anti-oxidative bioflavonoid. In this study, we explore the effect of quercetin on cell apoptosis of seminal vesicles and its underlying mechanism. The STZ-induced type 1 diabetic rat model was established. Three doses (low, medium and high) of quercetin were administrated to the STZ-induced type 1 diabetic rats for 4 months. Fasting blood glucose, the fructose in seminal plasma, total antioxidant capacity (T-AOC) and malondialdehyde (MDA) in seminal vesicles were determined by colorimetric method. Nuclear transcription factor- Nrf2 was observed by immunofluorescent staining. Biomarkers related to cell apoptosis, such as Bcl-2, Bax and cleaved -Caspase3 were measured by Western blotting and immumohistochemical staining. The body weight and seminal vesicle weight indexes were also determined. The results showed that T-AOC and Nrf2 were decreased, the levels of MDA were increased, the cleaved Caspase-3 was increased and the ratio of Bax to BCL-2 was decreased in seminal vesicles of diabetic rats, along with the severe hyperglycemia. When diabetic rats were treated by quercetin for 4 months, all the indexes were reversed at different degree except the fasting blood glucose. Our results suggested that quercetin could ameliorate oxidative stress‑induced cell apoptosis of seminal vesicles via inhibiting Nrf2 in type 1 diabetic rats, which indicated that quercetin could be used for preventing lesions of seminal vesicles in type 1 diabetes.

The epigenetic abnormality is believed as a major driver for cancer initiation. Histone modification plays a vital role in tumor formation and progression. Particularly, alteration in histone acetylation has been highly associated with gene expression, cell cycle, as well as carcinogenesis. By analyzing glioblastoma (GBM)-related microarray from the GEO database and conducting chromatin immunoprecipitation-sequencing (ChIP-seq), we discovered that solute carrier family 30 member 3 (SLC30A3), a super enhancer (SE)-regulated factor, was significantly reduced in GBM tissues. Furthermore, histone deacetylase 1 (HDAC1), overexpressed in GBM tissues, could inhibit SLC30A3 expression by promoting histone H3K27ac deacetylation modification of the SE region of SLC30A3. Our functional validation revealed that SLC30A3 can inhibit the growth and metastatic spread of GBM cells in vitro and in vivo, and can activate the MAPK signaling pathway to promote apoptosis of GBM cells. Moreover, overexpression of HDAC1 resulted in a significant increase in DNA replication activity, a significant decline in apoptosis and cell cycle arrest in GBM cells. In a word, these findings indicate that combined epigenetic targeting of SLC30A3 by HDAC1 and SE is potentially therapeutically feasible in GBM.

Atherosclerosis (AS) is one of the leading causes of vascular disease, producing high morbidity and mortality in many countries. Autophagy plays an important role when cells are facing serious circumstances, such as oxidative stress induced by Ox-LDL (oxidized low-density lipoprotein). Recent studies have revealed that DEX (dexamethasone acetate) and RAPA (rapamycin) exhibit efficient AS therapeutic ability by protecting endothelial cells and killing foam cells, respectively. Herein, we hypothesize that combining DEX and RAPA together in a specific nanocarrier system can achieve better AS therapy while limiting harmful effects. As a proof of concept, DEX and RAPA coloaded mPEG2k-DSPE calcium phosphate (CaP) nanoparticles (DR-NPs) were prepared by using a biomineralization method. DR-NPs increased HUVEC survival and induced foam cell apoptosis in vitro, which were correlated with autophagy activity. DR-NPs efficiently aggregated at AS plaques in the carotid artery and abdominal artery in ApoE– / – mice 24 h after i.v. injection. Moreover, DR-NPs exhibited excellent plaque regression ability, with smaller necrotic cores and lipid core areas observed after in vivo treatment. Furthermore, the function of vascular endothelial cells was largely promoted, as evidenced by the dramatically decreased expression levels of adhesion factors, such as MMP-2, MMP-9 and ICAM-1. Consequently, DR-NPs can act as an effective AS therapeutic agent and broaden the AS therapeutic approach by inducing autophagy.

Objective: MicroRNA (miRNA) is implicated in the pathogenic mechanism of pneumonia. Role of miR-874-3p in pediatric pneumonia was therefore evaluated in this study. Methods: Expression levels of miR-874-3p in the serum samples from pediatric patients with pneumonia and LPS-treated HPAEpiC were determined by RT-qPCR (reverse transcription quantitative real-time PCR). Secretion of inflammatory factors in LPS-treated HPAEpiC were determined by qRT-PCR and ELISA. Cell viability and apoptosis were evaluated by CCK8 and flow cytometry, respectively. HPAEpiC was used for the validation of binding target of miR-874-3p. Mechanism was determined by NF-κB promoter activity assay. Results: MiR-874-3p was reduced in serum samples of pediatric patients with pneumonia, and LPS treatment dose-dependently decreased miR-874-3p expression in HPAEpiC. TNF-α and IL-1β expression levels were increased in HPAEpiC post LPS treatment. Over-expression of miR-874-3p attenuated LPS-induced increase of TNF-α and IL-1β and reversed LPS-induced decrease of cell viability and increase of cell apoptosis in HPAEpiC. EGR3 (early growth response 3), increased in LPS-induced HPAEpiC, was a target gene of miR-874-3p. EGR3 over-expression reversed miR-874-3p over-expression-induced increase of cell viability, decrease of cell apoptosis, TNF-α and IL-1β in LPS-induced HPAEpiC. Over-expression of miR-874-3p reduced p65 expression and NF-κB promoter activity in LPS-induced HPAEpiC, while EGR3 over-expression reversed these suppressive effects. Conclusion: MiR-874-3p negatively regulates EGR3 expression to promote cell viability and inhibit apoptosis as well as inflammation in LPS-treated HPAEpiC via suppression of NF-κB pathway, suggesting a potential therapeutic strategy for pneumonia.

Background Microfibril-associated protein 2 (MFAP2) is a protein coding gene that exerts important phenotypic effects on cell motility, and increasing research has indicated that MFAP2 was correlated with many cancers. However, the functional and potential clinical role of MFAP2 in papillary thyroid cancer (PTC) has not yet been verified. Materials and Methods We performed whole transcriptome sequencing on 78 paired PTC tissues and corresponding adjacent normal tissues and found that MFAP2 was highly expressed in PTC tissues. Then, we analyzed the expression of MFAP2 and its relation with the clinicopathological features of PTC in The Cancer Genome Atlas (TCGA) PTC genomic dataset. We detected MFAP2 expression in 40 paired PTC tissues and corresponding adjacent normal tissues through RT-qPCR (real time-quantitative polymerase chain reaction) to validate the sequencing data and TCGA cohort. Cell functional assays were performed to elucidate the function of MFAP2 in PTC cells, Western blot assay was performed to explore the correlation between MFAP2 and EMT (epithelial-mesenchymal transition)-related proteins. Results Statistical analysis showed that MFAP2 was obviously upregulated in PTC tissues compared to matched normal tissues, and the expression levels of MFAP2 in PTC tissues were strongly related with lymph node metastasis (p=0.016). The results of RT-qPCR of our own tissue specimens showed the same conclusions as that in TCGA dataset. The results of functional assays in PTC cell lines showed that MFAP2 could promote proliferation, colony formation, migration and invasion abilities and decrease the apoptotic rate in PTC cells. Western Blot assay showed that MFAP2 could regulate the expression of EMT-related proteins. Conclusion MFAP2 increases the proliferation, motility and decreases the apoptosis of PTC cells, and might be a potential therapeutic target for papillary thyroid cancer.

Pancreatic cancer is a highly malignant disease with an extremely poor prognosis. The benefit of chemotherapy treatment for pancreatic cancer is very limited. Therefore, new therapeutic targets and approaches are urgently needed for this deadly disease. Multi-target therapy is a potential and feasible treatment strategy. Given the important roles that histone deacetylases (HDACs) and phosphoinositide-3-kinase (PI3K) play in pancreatic cancer, we investigated the antitumor activity and mechanism of novel HDAC and PI3K dual inhibitor CUDC-907 in pancreatic cancer. MTT assay and flow cytometric analysis were used to examine the in vitro antitumor activity of CUDC-907. A BxPC-3-derived xenograft mouse model was used to determine CUDC-907 in vivo efficacy. The TUNEL assay as used to determine apoptosis in tumors in vivo post CUDC-907 treatment. Western blots were used to determine the effect of CUDC-907 on protein levels. Our results show that CUDC-907 decreased viable cells and induced cell death in a concentration-dependent manner. Furthermore, CUDC-907 showed promising in vivo antitumor activity in the BxPC-3-derived xenograft mouse model while exhibiting tolerable toxicity. Furthermore, long-term treatment with CUDC-907 induced phosphorylation of AKT, S6 (ribosomal protein S6), and ERK (extracellular regulated protein kinase), and inhibition of PI3K (phosphatidylinositol 3-kinase), mTOR (mammalian target of rapamycin), or ERK significantly enhanced CUDC-907-induced cell death in pancreatic cell lines. Taken together, these findings support the clinical development of CUDC-907 for the treatment of pancreatic cancer and identify compensatory activation of mTOR and MEK/ERK as a possible mechanism of resistance to CUDC-907.

Cisplatin (Pt(II)) resistance is an important factor in the high mortality rates of ovarian cancer. Herein, we synthesized multifunctional tumor-targeted poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs-cRGD) for monitoring therapeutic effects by dual-mode imaging and overcoming cisplatin resistance. Uniformly sized NPs-cRGD demonstrated controlled and sustained release of drugs and genes, excellent gene loading and gene protection capacity, good storage stability and no serum-induced aggregation in vitro. NPs-cRGD demonstrated clear, targeting and prolonged ultrasound imaging and magnetic resonance imaging (MRI) in vivo. The targeting of NPs-cRGD combined with ultrasound facilitated nanoparticle penetrattion into cells; entry was time-dependent. NPs-cRGD escaped from lysosomes, thereby preventing siBIRC5 degradation, which enabled siBIRC5 to efficiently inhibit the antiapoptosis effects of BIRC5 in SKO3-DDP to overcome the antiapoptosis properties of resistant cells. Furthermore, Pt(IV) in NPs-cRGD exhausted glutathione (GSH), thereby increasing drug accumulation to effectively increase Pt(II) levels. The subsequent combination of Pt(II) with DNA prevented the expressions of genes and upregulated the expression of p53 to induce the mitochondria apoptosis pathway. The reduced GSH activity and the generation of Pt(II) further promoted high levels of reactive oxygen species (ROS) to induce cell apoptosis. Therefore, NPs-cRGD with ultrasound promoted the apoptosis of resistant ovarian cancer cells by multiple mechanisms, including increased cellular drug accumulation, reversed antiapoptotic effects by siBIRC5, and enhanced ROS levels. In a tumor-bearing nude mice model, NPs-cRGD with US demonstrated excellent tumor-targeting, high efficiency tumor inhibition and low systemic toxicity. Therefore, NPs-cRGD provides a means to monitor treatment processes and can be combined with ultrasound treatment to overcome ovarian cancer resistance in vitro and in vivo.

Introduction In this article, we utilized Ingenuity® Pathway Analysis (IPA®) bioinformatics analysis software and Metascape® bioinformatics analysis website tools to analyse the possible mechanism of ERH affecting tumourigenesis (proliferation and apoptosis) in bladder cancer (BC) T24 cells. Methods The ERH gene was knocked down, and BC T24 cells were divided into ERH normal and knockdown groups. Affymetrix® gene expression microarrays were performed to obtain a differentially expressed gene list (DEGL) between the 2 groups. IPA® data analyses contain five modules: disease and function analysis, upstream analysis, regulator effects analysis, canonical pathway analysis and molecular network analysis. The results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were analysed by Metascape®. Results The results of the gene expression profiling chip and the DEGL showed that 344 genes were upregulated and 254 genes were downregulated. The IPA® and Metascape® pathway analyses showed that the ERH gene may affect proliferation and apoptosis by affecting the apoptosis, cell cycle, Toll-like receptor (TLR), NF-κB or TGF-beta signalling pathways. Upstream analysis determined that the ERH gene may regulate TNF and NK-κB in the BC T24 cell lines. The ERH gene may be involved in the “cell death and survival” molecular network in BC T24 cells. ERH may be a regulator of KITLG through TNF. Conclusions The ERH gene may affect apoptosis through the TLR, NF-κB, TNF or TGF-beta signalling pathways in BC T24 cells, and may be a regulator of KITLG to ultimately activate the growth of malignant tumours.

Expression patterns of estrogen receptors [ERα, ERβ, and G-protein associated ER (GPER)] in melanoma and skin may suggest their differential roles in carcinogenesis. Phytoestrogenic compound cyanidin-3-o-glucoside (C3G) has been shown to inhibit the growth and metastatic potential of melanoma, although the underlying molecular mechanism remains unclear. The aim of this study was to clarify the mechanism of action of C3G in melanoma in vitro and in vivo, as well as to characterize the functional expressions of ERs in melanoma. In normal skin or melanoma (n = 20/each), no ERα protein was detectable, whereas expression of ERβ was high in skin but weak focal or negative in melanoma; and finally high expression of GPER in all skin vs. 50% melanoma tissues (10/20) was found. These results correspond with our analysis of the melanoma survival rates (SRs) from Human Protein Atlas and The Cancer Genome Atlas GDC (362 patients), where low ERβ expression in melanoma correlate with a poor relapse-free survival, and no correlations were observed between SRs and ERα or GPER expression in melanoma. Furthermore, we demonstrated that C3G treatment arrested the cell cycle at the G2/M phase by targeting cyclin B1 (CCNB1) and promoted apoptosis via ERβ in both mouse and human melanoma cell lines, and inhibited melanoma cell growth in vivo. Our study suggested that C3G elicits an agonistic effect toward ERβ signaling enhancement, which may serve as a potential novel therapeutic and preventive approach for melanoma.

The present study explored the effects of disodium cantharidinate (DC) on the peripheral blood-derived dendritic cells of patients with bladder carcinoma. The peripheral blood mononuclear cells from the 15 cases of urinary bladder carcinoma of middle and advanced stage were separated, and dendritic cells were prepared. The morphological changes of dendritic cells were observed. Flow cytometry was used to detect the expression levels of CD1a and CD83 on dendritic cell surface. MTT assay was utilized to measure the proliferation ability of allogeneic lymphocyte stimulated by DC. Annexin V-FITC/propidium iodide (PI) double staining flow cytometry method was carried out to detect cell apoptosis after treatment with DC. The changes in caspase-3 and PARP expression levels were investigated by western blot method. The high-dose DC resulted in a significant increase in the expressions of dendritic cell phenotyptic molecules CDla and CD83 as compared to control group. In addition, the proliferation index of allogenic lymphocyte stimulated by DC was significantly higher than that of control group. Moreover, MTT assay showed significant inhibition of the growth of BIU-87 cells. After 24 h of DC treatment, double staining flow cytometry confirmed the ability of DC to induce cell apoptosis. Further, western blot method showed a significant elevation of caspase-3 and PARP protein expression after DC treatment. In conclusion, DC treatment could induce dendritic cell maturation of patient with carcinoma of urinary bladder and promote its functional changes. Furthermore, DC was able to inhibit the proliferation of cell BIU-87 and also has the ability to induce cell apoptosis.

Aims The effects of PFKFB4 on glycolysis during the cancer progression has been investigated, while its role in glioma remains unclear. The present study evaluated the molecular mechanism of PFKFB4 in glycolysis of glioma progression. Materials and methods The pan-cancer platform SangerBox was inquired to investigate the E2F2 expression in tumors. The E2F2 expression was studied by qRT-PCR and immunohistochemistry in collected glioma and normal brain tissues and by qRT-PCR and western blot in glioma cells. The relationship between the E2F2 expression in glioma tissues and patients' prognosis was analyzed. The cell malignant phenotype, glycolysis, growth and metastasis were examined by CCK-8, EdU, colony formation, flow cytometry, wound healing, Transwell assays, ELISA kits, and tumorigenesis and metastasis assays. Downstream targets of E2F2 were searched in hTFtarget, followed by pathway enrichment analysis. The expression of these targets and their correlation with E2F2 expression in gliomas were investigated through the GEPIA website. After ChIP and luciferase assays, the effect of the target on glioma was investigated. Key findings E2F2 was overexpressed in glioma patients and predicted poor prognoses. E2F2 promoted cell proliferation, colony formation, DNA synthesis, migration, invasion and glycolysis, and inhibited apoptosis. Meanwhile, inhibition of E2F2 suppressed the growth and metastasis of gliomas. E2F2 elevated the PFKFB4 expression transcriptionally by binding to its promoter and activated PI3K/AKT pathway. The promotion of glioma metastasis and glycolysis by E2F2 was mitigated by PFKFB4 knockdown. Significance E2F2-mediated transcriptional enhancement of PFKFB4 expression regulated the phosphorylation of PI3K/AKT to promote glioma malignancy progression.

// Li Wang 1, * , Haifeng Li 1, * , Shiping Yang 1 , Wenqiang Ma 1 , Mei Liu 2 , Shichao Guo 1 , Jun Zhan 3 , Hongquan Zhang 3 , Suk Ying Tsang 4 , Ziding Zhang 1 , Zhaoyi Wang 5 , Xiru Li 2 , Yang-Dong Guo 1 , Xiangdong Li 1 1 State Key Laboratory of the Agro-Biotechnology, College of Horticultural Science, China Agricultural University, Beijing, China 2 Department of General Surgery, The 301th Hospital of PLA, Beijing, China 3 Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing, China 4 School of Life Sciences and State Key Laboratory of Agro-Biotechnology, Chinese University of Hong Kong, Hong Kong, China 5 Beijing Shenogen Pharma Group, Beijing, China * These authors have contributed equally to this work Correspondence to: Yang-Dong Guo, email: yaguo@cau.edu.cn Xiru Li, email: 2468li@sina.com Zhaoyi Wang, email: charlie.wang@shenogen.com Xiangdong Li, email: xiangdongli@cau.edu.cn Keywords: Cy-3-glu, ERα36, EGFR, triple-negative breast cancer, apoptosis Received: December 03, 2015 Accepted: September 02, 2016 Published: September 15, 2016 ABSTRACT Anthocyanins have been shown to inhibit the growth and metastatic potential of breast cancer (BC) cells. However, the effects of individual anthocyanins on triple-negative breast cancer (TNBC) have not yet been studied. In this study, we found that cyanidin-3-o-glucoside (Cy-3-glu) preferentially promotes the apoptosis of TNBC cells, which co-express the estrogen receptor alpha 36 (ERα36) and the epidermal growth factor receptor (EGFR). We demonstrated that Cy-3-glu directly binds to the ligand-binding domain (LBD) of ERα36, inhibits EGFR/AKT signaling, and promotes EGFR degradation. We also confirmed the therapeutic efficacy of Cy-3-glu on TNBC in the xenograft mouse model. Our data indicates that Cy-3-glu could be a novel preventive/therapeutic agent against the TNBC co-expressed ERα36/EGFR.

M. avium subsp. paratuberculosis (MAP) is the causative pathogen of Johne&rsquo, s disease, a chronic granulomatous enteritis that principally affects ruminants and can survive, proliferate and disseminate in macrophages. MicroRNAs (miRNAs) are important regulators of gene expression and can impact the processes of cells. To investigate the role of miRNAs in monocyte-derived macrophages (MDMs) during MAP infection, we used high-throughput sequencing technology to analyze small RNA libraries of MAP-infected and control MDMs. The results showed that a total of 21 miRNAs were differentially expressed in MDMs after MAP infection, and 8864 target genes were predicted. A functional analysis showed that the target genes were mainly involved in the MAPK signaling pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway and apoptosis. In addition, using a dual-luciferase reporter assay, flow cytometry, and a small interfering (si)RNA knockdown assay, the role of miR-150 in regulating macrophage apoptosis by targeting the programmed cell death protein-4 (PDCD4) was demonstrated. These results provide an experimental basis to reveal the regulatory mechanism of MAP infection and suggest the potential of miRNAs as biomarkers for the diagnosis of Johne&rsquo, s disease in bovines.

M. avium subsp. paratuberculosis (MAP) is the causative pathogen of Johne’s disease, a chronic granulomatous enteritis that principally affects ruminants and can survive, proliferate and disseminate in macrophages. MicroRNAs (miRNAs) are important regulators of gene expression and can impact the processes of cells. To investigate the role of miRNAs in monocyte-derived macrophages (MDMs) during MAP infection, we used high-throughput sequencing technology to analyze small RNA libraries of MAP-infected and control MDMs. The results showed that a total of 21 miRNAs were differentially expressed in MDMs after MAP infection, and 8864 target genes were predicted. A functional analysis showed that the target genes were mainly involved in the MAPK signaling pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway and apoptosis. In addition, using a dual-luciferase reporter assay, flow cytometry, and a small interfering (si)RNA knockdown assay, the role of miR-150 in regulating macrophage apoptosis by targeting the programmed cell death protein-4 (PDCD4) was demonstrated. These results provide an experimental basis to reveal the regulatory mechanism of MAP infection and suggest the potential of miRNAs as biomarkers for the diagnosis of Johne’s disease in bovines.

The possibility of micro ribonucleic acid-29b (miR-29b) regulating blood pressure and cardiac function in the rat model of hypertension was investigated. Sixty rat models of hypertension were established and randomly divided into the lentivirus group (n=20), the negative lentivirus group (n=20) and the control group (n=20). Rats in the lentivirus group were injected with the recombinant lentivirus, and those in the negative lentivirus and control groups were injected with the negative control virus and infection enhancement solution, respectively. The systolic pressure of rats was monitored using the tail-cuff method, and changes in the cardiac function of rats were evaluated via high-frequency ultrasound. At 3 weeks after virus infection, rats were weighed and sacrificed, the heart was taken and the left ventricular mass index was calculated. Moreover, the expression of miR-29b in myocardial tissues was detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The systolic pressure in the lentivirus group was significantly decreased compared with those in the negative lentivirus and control groups (P

Ischemia reperfusion injury (IRI) is one of the most common causes of acute kidney injury (AKI). However, the pathogenesis and biomarkers predicting the progression of IRI-induced AKI to chronic kidney disease (CKD) remain unclear. A side-by-side comparison between different IRI animal models with variable ischemic duration and episodes was performed. The dynamic changes of KIM-1 and NGAL continuously from AKI to CKD phases were studied as well. Short-term duration of ischemia induced mild renal tubule-interstitial injury which was completely reversed at acute phase of kidney injury, while long-term duration of ischemia caused severe tubular damage, cell apoptosis and inflammatory infiltration at early disease stage, leading to permanent chronic kidney fibrosis at the late stage. Repeated attacks of moderate IRI accelerated the progression of AKI to CKD. Different from serum and urine levels of KIM-1 that increased at acute phase of IRI then declined gradually in chronic phase, NGAL increased continuously during AKI-to-CKD transition. Severity and frequency of ischemia injury determines the progression and outcome of ischemia-induced AKI. Inflammation, apoptosis and fibrogenesis likely participate in the progression of AKI to CKD. Both KIM-1 and NGAL enable noninvasive and early detection of AKI, but NGAL is associated better with the process of AKI-to-CKD progression.

Atherosclerosis is the leading risk factor for cardiovascular disease, in which vascular smooth muscle cell (VSMC) proliferation and apoptosis play an important role. Research has demonstrated that long non-coding RNAs (lncRNAs) are critical regulatory factors for VSMC function, however, the molecular mechanism leading to this pathology is still not fully understood. To explore this further, an ox-LDL induced VSMC model was established, in which lncRNA MEG8 expression was suppressed, while miR-181a-5p was enhanced in a dose- and time-dependent manner. Enhanced MEG8 expression suppressed the proliferation and migration ability of VSMCs, and induced apoptosis. Mechanically, MEG8 was found to promote the PPARα protein via sponging miR-181a-5p. Rescue experiments demonstrated that MEG8 and PPARα collectively regulate the proliferation, migration and apoptosis of VSMCs. Overall, this research illustrates that MEG8 regulates the proliferation and migration of VSMCs via the MEG8/miR-181a/PPARα axis.

AZD9291 can effectively prolong survival of non‐small cell lung cancer (NSCLC) patients. Unfortunately, the mechanism of its acquired drug resistance is largely unknown. This study shows that autophagy and fibroblast growth factor receptor 1 signaling pathways are both activated in AZD9291 resistant NSCLC, and inhibition of them, respectively, by chloroquine (CQ) and PD173074 can synergistically reverse AZD9291 resistance. Herein, a coloaded CQ and PD173074 pH‐sensitive shell–core nanoparticles CP@NP‐cRGD is developed to reverse AZD9291 resistance in NSCLC. CP@NP‐cRGD has a high encapsulation rate and stability, and can effectively prevent the degradation of drugs in circulation process. CP@NP‐cRGD can target tumor cells by enhanced permeability and retention effect and the cRGD peptide. The pH‐sensitive CaP shell can realize lysosome escape and then release drugs successively. The combination of CP@NP‐cRGD and AZD9291 significantly induces a higher rate of apoptosis, more G0/G1 phase arrest, and reduces proliferation of resistant cell lines by downregulation of p‐ERK1/2 in vitro. CQ in CP@NP‐cRGD can block protective autophagy induced by both AZD9291 and PD173074. CP@NP‐cRGD combined with AZD9291 shows adequate tumor enrichment, low toxicity, and excellent antitumor effect in nude mice. It provides a novel multifunctional nanoparticle to overcome AZD9291 resistance for potential clinical applications., This work develops a coloaded chloroquine and fibroblast growth factor receptor 1 (FGFR1) inhibitor pH‐sensitive shell–core nanoparticles CP@NP‐cRGD, which can significantly dual‐block autophagy and FGFR1 pathway to overcome AZD9291 resistance. CP@NP‐cRGD can ensure excellent tumor targeting ability, realize lysosomal escaping, and release drugs sequentially by pH‐sensitive CaP shell. CP@NP‐cRGD has low toxicity and long circulation time in vivo.

Erlong Zuoci decoction (ELZCD), a typical traditional Chinese medicine (TCM) prescription, has long been clinically used in treatment of deafness and tinnitus with the syndrome of "kidney yin deficiency". However, there are few studies to investigate its pharmacological mechanisms. Until now, there is not report about its effects on the age-related hearing loss (ARHL).The present study was conducted to observe the effects of ELZCD on the ARHL in C57BL/6J mice and explore the mechanisms.ELZCD was fed to C57BL/6J mice from 3 months to 6 months in ELZCD group as a dose of 6g/kg/d. And the same volume of saline was fed to mice in ARHL group. 3-months-old C57BL/6J mice were used as control group. High performance liquid chromatography (HPLC) was used for the quality control of ELZCD. Auditory brainstem response (ABR) was used to assess the hearing function of mice. The morphologic changes were observed by hematoxylin eosin (HE) staining. Apoptosis was tested by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method. Mitochondrial damage was detected by transmission electron microscopy (TEM). Quantitative RT-PCR (qRT-PCR) was used to observe the mRNA expression of p53 and Bak. Fluorescence immunohistochemical technique was used to test the protein expression of p53 and Bak.The hearing threshold of ARHL group was higher than that of control group (P0.001) and ELZCD decreased the rise of hearing threshold levels of ARHL mice (P0.001), which suggested ELZCD inhibited the hearing loss of ARHL mice. HE staining showed that ELZCD decreased the spiral ganglion (SG) cell damage and loss in ARHL. TUNEL test showed that the apoptotic SG cells increased in ARHL group compared to control group and decreased in ELZCD group compared to ARHL group. TEM observation showed that mitochondrial damage was obvious in SG cells of ARHL group and ELZCD inhibited the mitochondrial damage. The qRT-PCR results showed that the mRNA expression of p53 and Bak in ARHL group increased compared to that of control group (P0.05), and ELZCD reduced the elevated mRNA expression levels of p53 and Bak (P0.01, P0.05). In addition, ELZCD inhibited the increased proteins expression (green fluorescence) of p53 and Bak.The results demonstrated that ELZCD prevented ARHL in C57BL/6J mice and p53/Bak-mediated mitochondrial apoptosis of SG cells might be involved in the mechanisms.

Osteoclasts (OCs) play a pivotal role in a variety of lytic bone diseases including osteoporosis, arthritis, bone tumors, Paget's disease and the aseptic loosening of orthopedic implants. The primary focus for the development of bone-protective therapies in these diseases has centered on the suppression of OC formation and function. In this study we report that thonzonium bromide (TB), a monocationic surface-active agent, inhibited RANKL-induced OC formation, the appearance of OC-specific marker genes and bone-resorbing activity in vitro. Mechanistically, TB blocked the RANKL-induced activation of NF-κB, ERK and c-Fos as well as the induction of NFATc1 which is essential for OC formation. TB disrupted F-actin ring formation resulting in disturbances in cytoskeletal structure in mature OCs during bone resorption. Furthermore, TB exhibited protective effects in an in vivo murine model of LPS-induced calvarial osteolysis. Collectively, these data suggest that TB might be a useful alternative therapy in preventing or treating osteolytic diseases.

Emerging evidence shows that microRNAs (miRNAs) act as critical regulators in the progression and chemoresistance of multiple tumors, including osteosarcoma (OS). In this study, we found that the level of miR-24 was increased in OS patients’ serum, tumor tissues and OS cell lines. Furthermore, we found that knockdown of miR-24 by its specific inhibitors significantly increased the therapeutic effect of doxorubicin (DOX) on OS cell lines (MG-63 and HOS). Moreover, miR-24 inhibitors resensitized the doxorubicin-resistant MG-63 cells (MG-63/R) and HOS cells (HOS/R) to DOX. As the gene of Bcl-2 interacting mediator of cell death (BIM) was proved to be a target of miR-24 in MG-63/R cells, we further observed that the miR-24 inhibitors promoted the DOX-induced apoptosis via mitochondrial pathway. In addition, results of immunoprecipitation showed the release of second mitochondria derived activator of caspase/ direct IAP binding protein with low pI (Smac/DIABLO) abolished the biological activity of X-linked inhibitor of apoptosis protein (XIAP) by binding with it, which subsequently induced the activation of caspase 9, 7 and 3. In summary, those results strongly suggest that the miR-24-BIM-Smac/DIABLO axis might be a novel target for the treatment of OS.

The CD44 isoform containing variant exon v6 (CD44v6) plays an important role in the progression, metastasis, and prognosis of colorectal cancer (CRC). Recently, it was found that CD44v6 is involved in acquired drug resistance. This study aimed to investigate the molecular mechanism of CD44v6 in the resistance of CRC cells to chemotherapy. A stable CD44v6 overexpression model in SW480 cells was established via lentiviral transduction. The chemosensitivity of cells to 5-fluorouracil (5-FU) and oxaliplatin (L-OHP) was determined by cell counting kit (CCK)-8, lactate dehydrogenase (LDH) release, and colony formation assays. Immunohistochemical staining of CD44v6 was performed in human CRC tissues. The key components in cell apoptosis, drug efflux and metabolism, mismatch repair, autophagy, epithelial-mesenchymal transition (EMT), and the PI3K-Akt and MAPK-Ras-Erk1/2 pathways were assessed using flow cytometry, quantitative real-time polymerase chain reaction (PCR), and western blot assays. The CD44v6 overexpression cells showed a higher viability, a lower LDH release rate, and an increased clonogenicity than the control cells under drug treatment. Moreover, overexpression of CD44v6 resulted in enhanced autophagy flux, EMT, and phosphorylation of Akt and Erk in the presence of drugs. Furthermore, high CD44v6 expression in the primary tumor was closely associated with an early recurrence in CRC patients who underwent curative surgery and adjuvant chemotherapy. In conclusion, overexpression of CD44v6 contributes to chemoresistance in SW480 cells under cytotoxic stress via the modulation of autophagy, EMT, and activation of the PI3K-Akt and MAPK-Ras-Erk pathways.

Aseptic loosening and periprosthetic infection leading to inflammatory osteolysis is a major complication associated with total joint arthroplasty (TJA). The liberation of bacterial products and/or implant-derived wear particles activates immune cells that produce pro-osteoclastogenic cytokines that enhance osteoclast recruitment and activity, leading to bone destruction and osteolysis. Therefore, agents that prevent the inflammatory response and/or attenuate excessive osteoclast (OC) formation and bone resorption offer therapeutic potential by prolonging the life of TJA implants. Alexidine dihydrochloride (AD) is a bisbiguanide compound commonly used as an oral disinfectant and in contact lens solutions. It possesses antimicrobial, anti-inflammatory and anticancer properties; however, its effects on OC biology are poorly described. Here, we demonstrate that AD inhibits OC formation and bone resorption in vitro and exert prophylatic protection against LPS-induced osteolysis in vivo. Biochemical analysis demonstrated that AD suppressed receptor activator of NF-κB ligand (RANKL)-induced activation of mitogen-activated protein kinases (ERK, p38, and JNK), leading to the downregulation of NFATc1. Furthermore, AD disrupted F-actin ring formation and attenuated the ability of mature OC to resorb bone. Collectively, our findings suggest that AD may be a promising prophylactic anti-osteoclastic/resorptive agent for the treatment of osteolytic diseases caused by excessive OC formation and function.