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In vitro epidermis models are important to evaluate and study disease progression and possible dermal drug delivery. An in vitro epidermis model using floating paper chips as a scaffold for proliferation and differentiation of primary human keratinocytes is reported. The formation of the four main layers of the epidermis (i.e., basal, spinosum, granulose, and cornified layers) is confirmed. The development of a cornified layer and the tight junction formation are evaluated as well as the alterations of organelles during the differentiation process. Further, this in vitro model is used to assess keratinocyte migration. Finally, magnetic micromotors are assembled, and their ability to aid cell migration on paper chips is confirmed when a static magnetic field is present. Taken together, this attempt to combine bottom-up synthetic biology with dermatology offers interesting opportunities for studying skin disease pathologies and evaluate possible treatments.

Abstract Mechanical stimuli play important roles on the growth, development, and behavior of tissue. A simple and novel paper-based in vitro tissue chip was developed that can deliver two types of mechanical stimuli—local compression and shear flow—in a programmed manner. Rat vascular endothelial cells (RVECs) were patterned on collagen-coated nitrocellulose paper to create a tissue chip. Localized compression and shear flow were introduced by simply tapping and bending the paper chip in a programmed manner, utilizing an inexpensive servo motor controlled by an Arduino microcontroller and powered by batteries. All electrical compartments and a paper-based tissue chip were enclosed in a single 3D-printed enclosure, allowing the whole device to be independently placed within an incubator. This simple device effectively simulated in vivo conditions and induced successful RVEC migration in as early as 5 h. The developed device provides an inexpensive and flexible alternative for delivering mechanical stimuli to other in vitro tissue models. Graphical abstract

Microfluidics approaches have gained popularity in the field of directed cell migration, enabling control of the extracellular environment and integration with live-cell microscopy; however, technical hurdles remain. Among the challenges are the stability and predictability of the environment, which are especially critical for the observation of fibroblasts and other slow-moving cells. Such experiments require several hours and are typically plagued by the introduction of bubbles and other disturbances that naturally arise in standard microfluidics protocols. Here, we report on the development of a passive pumping strategy, driven by the high capillary pressure and evaporative capacity of paper, and its application to study fibroblast chemotaxis. The paper pumps—flowvers (flow + clover)—are inexpensive, compact, and scalable, and they allow nearly bubble-free operation, with a predictable volumetric flow rate on the order of μl/min, for several hours. To demonstrate the utility of this approach, we combined the flowver pumping strategy with a Y-junction microfluidic device to generate a chemoattractant gradient landscape that is both stable (6+ h) and predictable (by finite-element modeling calculations). Integrated with fluorescence microscopy, we were able to recapitulate previous, live-cell imaging studies of fibroblast chemotaxis to platelet derived growth factor (PDGF), with an order-of-magnitude gain in throughput. The increased throughput of single-cell analysis allowed us to more precisely define PDGF gradient conditions conducive for chemotaxis; we were also able to interpret how the orientation of signaling through the phosphoinositide 3-kinase pathway affects the cells’ sensing of and response to conducive gradients.

In vitro construction of highly organized three-dimentional (3D) tissues is still a key challenge for tissue engineering. In this study, we fabricated multilayered tissues composed of extracellular matrix (ECM) layer and cell layer by stacking cell-seeded ECM papers. A paperlike scaffold was prepared by simply casting dispersion of microfibered ECM. We showed the paperlike scaffold is a superior material for constructing 3D tissue because of its high permeability and cell migration ability, and our method can control the thickness and component of ECM in multilayered 3D tissues. It can contribute to construction of normal and disease tissue models.

Construction of organized three-dimensional (3D) tissue with extracellular matrix (ECM) and multiple types of cells is important for tissue engineering to enable tissue function and enhance cellular function. However, the concentration of ECM and the thickness of the 3D tissue have been limited in previous methods due to a lack of permeability to nutrients and oxygen. Besides, it is difficult to use matured natural ECM as a cell scaffold without chemical modification due to its insolubility. In this article, we focus on multi-layered structure, which is commonly found in living tissue such as skin, blood vessels, and other organs. Here, we describe the preparation of a paper-like scaffold (ECM paper) from micro-fibered natural ECM and the construction of 3D multi-layered tissue composed of cell layers and ECM layers by stacking cell-seeded ECM papers. The thickness and components of the ECM layers are easily controllable by changing the composition of the ECM papers, and the fibrous structure of ECM paper shows high permeability and permits cell migration. Additionally, the ECM microfiber, which is physically defiberized from natural ECM, has a high ECM concentration equal to that of living tissue and high stability under physiological conditions. Therefore, this set of protocols enables construction of multi-layered 3D tissue composed of precisely controlled ECM layers and cell layers that may contribute to the assembly of tissue models. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of extracellular matrix paper Basic Protocol 2: Evaluation of cellular function of cells on extracellular matrix paper Basic Protocol 3: Construction of multi-layered 3D tissue.

Cellular migration is the movement of cells, cultured as a monolayer; cellular invasion is similar to migration, but requires the cells to move through a three-dimensional material such as basement membrane extract or a synthetic hydrogel. Migration assays, such as the transwell assay, are widely used to study cellular movement because they are amenable to high-throughput screens with minimal experimental setup. These assays offer limited information about cellular responses to gradients in vivo because they oversimplify the threedimensional (3D) environment of a tissue. There are a number of invasion assays that support 3D cultures, some of which provide experimental control over the spatial and temporal gradients imparted on the culture. These assays, in their current form, are difficult to setup and maintain, and often require specialized laboratory equipment or engineering expertise. Here we describe a paper-based invasion assay in which cellular movement can be monitored in real-time with fluorescence microscopy. These assays are easily prepared and utilize materials commonly found in any laboratory: a single sheet of paper. These sheets are wax patterned to contain channels in which cells suspended in a hydrogel are seeded and cultured. Cell-containing sheets of paper are placed in a custom-built holder that allows gradients to form along the length of the channels. In this work, we compare the invasion of cells cultured in the presence and absence of an oxygen gradient. Our result support previous findings that oxygen is a chemoattractant, and selectively directs cellular movement in a 3D culture environment.

Microfluidic chemotaxis platforms have historically been utilized to probe phenomena such as neutrophil migration and are beginning to be developed for diagnostic applications; however, current microfluidic chemotaxis systems require specialized engineering equipment such as syringe pumps and long time frames (hours) to develop a chemokine gradient, and cell chemotaxis typically requires multiple additional hours. The paperfluidic device described in this work is a low-cost, sharp (2 mm wide), quasi-stable (at least 20 min) and rapidly generated (

Angiogenesis plays an important role in tissue development and repair, and how to regulate angiogenesis effectively is a widely studied problem in the biomedical field. In recent years, the role of autophagy in vascular endothelial cells has attracted extensive attention. Icariin (ICA) is a traditional Chinese medicine that has been proven to have outstanding protective effects on the vascular system and to regulate cellular autophagy effectively. However, at present, it has not been reported whether ICA can affect the angiogenic ability of endothelial cells by affecting autophagy. In this study, we aimed to investigate whether ICA affects the angiogenesis capacity of EA.hy926 human vascular endothelial cells through autophagy and explain the underlying potential mechanisms. First, we determined that ICA at appropriate concentrations has the ability to promote cell migration and angiogenesis using wound healing assays and tube formation assays. Then, at the molecular level, we observed the upregulation of VEGFA, VEGFR2, ANGI, ANGII, and Tie2 mRNA and detected the upregulation of TGFβ1 protein by Western blotting. We also demonstrated that angiogenic concentrations of ICA can effectively activate autophagy. The autophagy inhibitor 3-MA significantly suppressed TGFβ1 expression and tube formation in EA.hy926 cells. Overall, we hope that our studies might help to further understand the effect of ICA on vascular endothelial cells and provide a theoretical basis for future angiogenic applications of ICA

Esophageal squamous cell carcinoma (ESCC) is the sixth most common cancer type in East Asian countries. Mounting evidences illustrated that long noncoding RNAs (lncRNAs) play important roles in a variety of human cancers, including ESCC. LncRNA PCAT6 has been identified as a tumor promoter in multiple cancers. However, the roles and underlying mechanism of PCAT6 in ESCC remain largely unclear. In the current study, we discovered that lncRNA PCAT6, which was aberrantly upregulated in ESCC tumor tissues, significantly promoted cell proliferation and migration in ESCC cell lines Eca-109 and Kyse-30 cells. Flow cytometry assays showed that PCAT6 knockdown promoted the apoptosis of ESCC cells. Mechanistically, RNA-seq and Gene Ontology analyses indicated that PCAT6 knockdown influenced the expression of genes that participated in cell proliferation and migration. Furthermore, real-time PCR and western blot assays validated that knockdown of PCAT6 could increase the levels of GDF15 and DUSP4 in Eca-109 and Kyse-30 cells. In brief, our findings reveal that lncRNA PCAT6 plays an oncogenic role in the progression of ESCC by inhibiting the expression of genes related to cell proliferation and migration.

Gastric cancer (GC) is a malignant tumor with high mortality, but research on its molecular mechanisms remain limited. This study is the first to explore the biological role of nuclear factor NFE2L3 (nuclear factor, erythroid 2 like 3) in GC. We used Western blot and RT–qPCR to detect gene expression at the protein or mRNA level. Short hairpin RNA (shRNA) transfection was used to inhibit NFE2L3 expression. CCK-8 and colony formation assays were used to detect cell proliferation. Cell migration, invasion, cell cycle and apoptosis were detected by Transwell assays and flow cytometry. The results showed that NFE2L3 was highly expressed in gastric cancer tissues and promoted gastric cancer cell proliferation and metastasis. Inhibiting NFE2L3 expression blocks the cell cycle and increases the proportion of apoptotic cells, whereas NFE2L3 expression promotes the epithelial-mesenchymal transformation (EMT) process. In summary, NFE2L3 is highly expressed in gastric cancer and promotes gastric cancer cell proliferation and metastasis and the EMT process.

LncRNA SNHG7 shows a strong relationship with malignant behavior of cancer cells and poor clinical outcome in cancer. The resistance of ovarian cancer for Paclitaxel seriously limits the clinical efficacy in chemotherapy for ovarian cancer patients. In this study, we investigated whether lncRNA SNHG7 was involved in Paclitaxel sensitivity of ovarian cancer as well as the underlying mechanism regulating the behavior of ovarian cancer cells with Paclitaxel resistance. The experiment results of wound healing and transwell showed that in paclitaxel-resistant ovarian cancer cells, transfection with siRNA-SNHG7 in ovarian cancer cells reduced cell migration and invasion. And cell cycle was observed by means of Flow cytometry. RNA immunoprecipitation assay was performed to analyze the interaction of lncRNA SNHG7 and EIF4G2. Overexpression of EIF4G2 by transfection with Ov- EIF4G2 plasmids efficiently blocked the changes of migration and invasion, as well as G0/1 arrest caused by lncRNA SNHG7 silencing. Taken together, these results demonstrated that lncRNA SNHG7 could affect the degradation of EIF4G2 to regulate the sensitivity of ovarian cancer to Paclitaxel, inhibit cell viability, migration, and invasion. The interaction of lncRNA SNHG7 and EIF4G2 plays an important role in the migrative and invasive activity and Paclitaxel resistance of ovarian cancer cells.

Colorectal cancer (CRC) is a global public health issue with increasing prevalence. MicroRNA-934 (miR-934) is a kind of non-coding RNA involved in the regulation of diverse cancers. Though previous researches have revealed part of association between miR-934 and CRC, the role of miR-934 in CRC pathogenesis has not been completely explored yet. In this study, we aim to investigate the effect of miR-934 on cell proliferation, migration, invasion and angiogenesis in CRC. Accordingly, miR-934 was found to be over-expressed in SW480 and HCT116 cells, two typical CRC cell lines. Meanwhile, miR-934 knockdown significantly inhibited cell proliferation and induced cell cycle arrest in SW480 and HCT116 cells. It was further validated that miR-934 knockdown displayed an inhibitory effect on cell migration and invasion in SW480 and HCT116 cells. Additionally, miR-934 deficiency markedly decreased VEGF expression in SW480 and HCT116 cells and suppressed capability of CRC cells to promote tube formation in vascular endothelial cells, which suggests the pro-angiogenesis role of miR-934 in vitro. Dual luciferase reporter assay further showed that miR-934 directly bound to B-cell translocation gene 2 (BTG2). BTG2 knockdown reversed the inhibitory effect of miR-934 silencing on cell proliferation, migration, invasion, and angiogenesis in SW480 and HCT116 cells. In summary, this study suggests that miR-934 facilitates CRC progression by targeting BTG2, and further highlights the role of miR-934 in pathogenesis of CRC.

Wound healing is related to proliferation, migration, and angiogenesis of keratinocytes. Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) is an important N6-methyladenosine (m6A) reader, which is involved in multiple processes, including wound healing. However, the function and mechanism of IGF2BP2 in keratinocyte processes are largely uncertain. In the present study, expression levels of IGF2BP2 and heparanase (HPSE) were detected by quantitative reverse transcription polymerase chain reaction and western blotting assays. Cell proliferation was investigated by cell counting kit-8 (CCK-8) analysis. Cell migration was determined through wound healing assay. Angiogenesis was measured by tube formation assay and vascular endothelial growth factor (VEGF) level using enzyme linked immunosorbent assay (ELISA). The interaction between IGF2BP2 and HPSE was analyzed by RNA immunoprecipitation, pull-down and luciferase reporter analyses. The results showed that IGF2BP2 expression was enhanced in wound healing. IGF2BP2 downregulation constrained HaCaT cell proliferation, migration, and angiogenesis. IGF2BP2 knockdown decreased HPSE expression. IGF2BP2 could regulate HPSE stability by binding with 3ʹ untranslated region (UTR) of HPSE. HPSE upregulation attenuated silencing IGF2BP2-mediated suppression of proliferation, migration, and angiogenesis. As a conclusion, IGF2BP2 knockdown repressed proliferation, migration, and angiogenesis of HaCaT cells by decreasing HPSE stability.

We aim to evaluate the efficacies of combination therapy with low-frequency ultrasound-stimulated microbubbles (USMB) and radiofrequency ablation (RFA) on suppressing the proliferation of pancreatic cancer cell and treating Panc02 subcutaneous xenograft mice. The proliferation of HPDE6-C7 and Panc02 cells after the treatment of USMB and RFA alone or combination were evaluated by CCK-8 assay. Scratch test was performed to assess the cell migration capability. Panc02-bearing mice were received 14-day treatment of USMB and RFA alone or combination. Tumor size and survival rate were recorded once two days. The serum levels of immune-related factors and changes of apoptosis- and autophagy-related factors were detected by ELISA and western blotting methods. As a result, CKK-8 assays revealed significant inhibition on Panc02 cell proliferation in combination therapy with USMB and RFA relative to other groups (all p

Long non-coding RNA (lncRNA) five prime to Xist (FTX) exerts important functions in human cancer, while its role in retinoblastoma (RB) remains unclear. This study aimed to investigate the role of FTX in RB. The expression levels of FTX were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was evaluated by cell counting kit-8 (CCK-8), 5‐ethynyl‐2′‐deoxyuridine (EdU) staining and colony formation assays. Cell migration and invasion were detected by Transwell assay. The relationship among FTX, microRNA-320a (miR-320a) and with-no-lysine kinase 1 (WNK1) was also investigated. In the present study, we found that the expression levels of FTX were notably elevated in RB tissues and cancer cell lines. Overexpression of FTX exacerbated the aggressive phenotypes (cell proliferation, migration and invasion) of RB cells. Downregulation of miR-320a obviously attenuated the inhibitory effects of knockdown of FTX in RB malignant phenotypes, and knockdown of WNK1 also reversed the impacts of miR-320a inhibitor on malignant phenotypes. In vivo experiments further confirmed that knockdown of FTX efficiently prevents tumor growth in vivo. Our results revealed that FTX promoted RB progression by targeting the miR-320a/WNK1 axis (graphical abstract), suggesting that FTX might be a novel therapeutic target for RB.

DVL proteins are central mediators of the Wnt pathway and relay complex input signals into different branches of the Wnt signaling network. However, molecular mechanism(s) that regulate DVL-mediated relay of Wnt signals still remains unclear. Here, for the first time, we elucidate the functional significance of three DVL-1 lysines (K/Lys) which are subject to post-translational acetylation. We demonstrate that K34 Lys residue in the DIX domain regulates subcellular localization of β-catenin, thereby influencing downstream Wnt target gene expression. Additionally, we show that K69 (DIX domain) and K285 (PDZ domain) regulate binding of DVL-1 to Wnt target gene promoters and modulate expression of Wnt target genes including CMYC, OCT4, NANOG, and CCND1, in cell line models and xenograft tumors. Finally, we report that conserved DVL-1 lysines modulate various oncogenic functions such as cell migration, proliferation, cell-cycle progression, 3D-spheroid formation and in-vivo tumor growth in breast cancer models. Collectively, these findings highlight the importance of DVL-1 domain-specific lysines which were recently shown to be acetylated and characterize their influence on Wnt signaling. These site-specific modifications may be subject to regulation by therapeutics already in clinical use (lysine deacetylase inhibitors such as Panobinostat and Vorinostat) or may possibly have prognostic utility in translational efforts that seek to modulate dysfunctional Wnt signaling.

Objective: Angiogenesis is involved in multiple biological processes, including atherosclerosis (AS) and cancer. Dickkopf1 (DKK1) plays many roles in both tumors and AS and has emerged as a potential biomarker of cancer progression and prognosis. Targeting DKK1 is a good choice for oncological treatments. Many anticancer therapies are associated with specific cardiovascular toxicity. However, the effects of DKK1 neutralizing therapy on AS are unclear. We focused on how DKK1 affected angiogenesis in AS and ox-LDL-induced human umbilical vein endothelial cells (HUVECs). Methods: ApoE-/- mice were fed a high-fat diet and then injected with DKK1i or DKK1 lentivirus to study the effects of DKK1. In vitro, promoter assays, protein analysis, database mining, dual-luciferase reporter assay (DLR), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and coimmunoprecipitation (co-IP) were used to study the mechanism of DKK1 biogenesis. Cell migration and angiogenesis assays were performed to investigate the function and regulatory mechanisms of DKK1. Results: DKK1 participated in angiogenesis both in the plaques of ApoE-/- mice by knockdown or overexpression of DKK1 and ox-LDL-induced HUVECs. DKK1 induced angiogenesis (increasing migration and capillary formation, inducing expression of VEGFR-2/VEGF-A/MMP) via the CKAP4/PI3K pathway, independent of Wnt/β-catenin. ox-LDL increased the expression and nuclear transfer of Ets-1 and c-jun, and induced the transcriptional activity of DKK1 in HUVECs. Ets-1, along with c-jun and CBP, could bind to the promoter of DKK1 and enhance DKK1 transcription. MiR33a-5p was downregulated in ox-LDL induced HUVECs and aortic artery of high-fat diet ApoE-/- mice. Ets-1 was a direct target of miR33a-5p. MiR33a-5p/Ets-1/ DKK1 axis contributed to angiogenesis. Conclusions: MiR33a-5p/Ets-1/DKK1 signaling participated in ox-LDL-induced angiogenesis of HUVECs via the CKAP4/PI3K pathway. These new findings provide a rationale and notable method for tumor therapy and cardiovascular protection.

BET bromodomain BRD4 and RAC1 oncogenes are considered important therapeutic targets for cancer and play key roles in tumorigenesis, survival and metastasis. However, combined inhibition of BRD4-RAC1 signaling pathways in different molecular subtypes of breast cancer including luminal-A, HER-2 positive and triple-negative breast (TNBC) largely remains unknown. Here, we demonstrated a new co-targeting strategy by combined inhibition of BRD4-RAC1 oncogenic signaling in different molecular subtypes of breast cancer in a context-dependent manner. We show that combined treatment of JQ1 (inhibitor of BRD4) and NSC23766 (inhibitor of RAC1) suppresses cell growth, clonogenic potential, cell migration and mammary stem cells expansion and induces autophagy and cellular senescence in molecular subtypes of breast cancer cells. Mechanistically, JQ1/NSC23766 combined treatment disrupts MYC/G9a axis and subsequently enhances FTH1 to exert antitumor effects. Furthermore, combined treatment targets HDAC1/Ac-H3K9 axis, thus suggesting a role of this combination in histone modification and chromatin modeling. C-MYC depletion and co-treatment with vitamin-C sensitizes different molecular subtypes of breast cancer cells to JQ1/NSC23766 combination and further reduces cell growth, cell migration and mammosphere formation. Importantly, co-targeting RAC1-BRD4 suppresses breast tumor growth in vivo using xenograft mouse model. Clinically, RAC1 and BRD4 expression positively correlates in breast cancer patient's samples and show high expression patterns across different molecular subtypes of breast cancer. Both RAC1 and BRD4 proteins predict poor survival in breast cancer patients. Taken together, our results suggest that combined inhibition of BRD4-RAC1 pathways represents a novel and potential therapeutic approach in different molecular subtypes of breast cancer and highlights the importance of co-targeting RAC1-BRD4 signaling in breast tumorigenesis via disruption of C-MYC/G9a/FTH1 axis and down regulation of HDAC1.

Background: The extracellular vesicles (EVs) secreted by bone marrow mesenchymal stromal cells (MSCs) have the ability to improve Myocardial infarction (MI). Some microRNAs (miRNAs) including miR-497 and related target genes have been proved to be closely linked with heart diseases. However, EVs could regulate MI process through miR-497, and the mechanisms have not been fully reported. Methods: Ligation of left anterior descending artery was performed to established MI animals model. Hypoxia cell model was established through lowering the level of oxygen. The cell invasion, migration, and proliferation were measured using tanswell, wound heating, and MTT assays. HE, Masson trichrome, and Sirius Red staining were used to investigate the morphological changes. Results: Overexpression of miR-497 reversed the promotion of cell migration, invasion, and proliferation caused by EVs. The improvement of cardiac function induced by EVs could also be reversed by overexpression of miR-497. Direct binding site between Smad7 and miR-497 was identified. Knockdown of Smad7 reversed the improvement of cardiac function induced by EVs. Conclusions: We found that EVs isolated from MSCs might improve the cardiac injury caused by MI through targeting miR497/Smad7. This study provides novel potential therapeutic thought for the prevention and treatment of MI through targeting miR-497/Smad7.

This study aimed to evaluate the biological role of forkhead box N3 (FOXN3) in human glioma and clarify the possible molecular mechanisms. FOXN3 expression patterns in clinical tissue specimens were characterized via qPCR and Western blotting. Kaplan-Meier survival curve was applied to assess the correlation between FOXN3 expression and overall survival. Effects of FOXN3 over-expression and depletion on glioma cell proliferation, apoptosis, migration and invasion were assessed by CCK8, colony formation assay, flow cytometry, scratch wound healing assay and Transwell invasion assay, respectively. Moreover, the involvement of AKT/murine double minute 2 (MDM2)/p53 pathway was evaluated. Additionally, tumor transplantation model assay was performed to determine the effects of FOXN3 over-expression on glioma cell growth in vivo. Results showed that FOXN3 was significantly down-regulated in glioma tissues compared with normal tissues. Patients with lower FOXN3 expression exhibited a shorter overall survival time. Gain- and loss-of-function analyses demonstrated that FOXN3 over-expression significantly suppressed proliferation, survival and motility of glioma cells, whereas FOXN3 knockdown remarkably promoted glioma cell proliferation, survival and motility. Furthermore, FOXN3 over-expression inhibited the activation of AKT/MDM2/p53 signaling pathway in glioma cells, while FOXN3 depletion facilitated its activation. Additionally, tumor xenograft assays revealed that FOXN3 over-expression retarded glioma cell growth in vivo. Collectively, these findings indicate that FOXN3 inhibits cell growth and invasion through inactivating the AKT/MDM2/p53 signaling pathway and that FOXN3-AKT/MDM2/p53 axis may represent a novel therapeutic target for glioma patients.

Cholangiocarcinoma (CCA) has been well known as the second most common primary tumor of hepatobiliary system. PSMC2 (proteasome 26S subunit ATPase 2) is a key member of the 19S regulatory subunit of 26S proteasome, responsible for catalyzing the unfolding and translocation of substrates into the 20S proteasome, whose role in CCA is totally unknown. In this study, the results of immunohistochemistry analysis showed the upregulation of PSMC2 in CCA tissues compared with normal tissues, which was statistically analyzed to be associated with CCA tumor grade. Subsequently, the loss-of-function study suggested that knockdown of PSMC2 significantly suppressed cell proliferation, cell migration, promoted cell apoptosis and arrested cell cycle distribution in vitro. The decreased tumorigenicity of CCA cells with PSMC2 knockdown was confirmed in vivo by using mice xenograft model. In PSMC2 knockdown cells, pro-apoptotic protein Caspase3 was upregulated; anti-apoptotic proteins such as Bcl-2 and IGF-II were downregulated; among EMT markers, E-cadherin was upregulated while N-cadherin and Vimentin were downregulated, by which may PSMC2 regulates cell apoptosis and migration. Furthermore, through RNA-seq and verification by qPCR, western blotting and co-IP assays, CDK1 was identified as the potential downstream of PSMC2 mediated regulation of CCA. PSMC2 and CDK1 showed mutual regulation effects on expression level of each other. Knockdown of PSMC2 could aggregate the influence of CDK1 knockdown on cellular functions of CCA cells. In summary, our findings suggested that PSMC2 possesses oncogene-like functions in the development and progression of CCA through regulating CDK1, which may be used as an effective therapeutic target in CCA treatment.

Cancer is one of the leading causes of death in the world. It is very important to find drugs with high efficiency, low toxicity, and low side effects for the treatment of cancer. Flavonoids and their derivatives with broad biological functions have been recognized as anti-tumor chemicals. 8-Formylophiopogonanone B (8-FOB), a naturally existed homoisoflavonoids with rarely known biological functions, needs pharmacological evaluation. In order to explore the possible anti-tumor action of 8-FOB, we used six types of tumor cells to evaluate in vitro effects of this agent on cell viability and tested the effects on clone formation ability, scratching wound-healing, and apoptosis. In an attempt to elucidate the mechanism of pharmacological action, we examined 8-FOB-induced intracellular oxidative stress and -disrupted mitochondrial function. Results suggested that 8-FOB could suppress tumor cell viability, inhibit cell migration and invasion, induce apoptosis, and elicit intracellular ROS production. Among these six types of tumor cells, the nasopharyngeal carcinoma CNE-1 cells were the most sensitive cancer cells to 8-FOB treatment. Intracellular ROS production played a pivotal role in the anti-tumor action of 8-FOB. Our present study is the first to document that 8-FOB has anti-tumor activity in vitro and increases intracellular ROS production, which might be responsible for its anti-tumor action. The anti-tumor pharmacological effect of 8-FOB is worthy of further investigation.

The present study was aimed to evaluate the expression profile of Zinc finger C3H1 domain-containing protein (ZFC3H1) using bioinformatic analysis of public datasets from The Cancer Genome Atlas database (TCGA). The results showed that the expression levels of ZFC3H1 were notably lower than the corresponding non-cancerous tissues in prostate adenocarcinoma (PRAD), and patients in the high ZFC3H1-expression group showed poor survival. We hypothesized that the low expression of ZFC3H1 in tumor tissue might have be an inhibitory effect on the autoimmune system. We predicted the regulatory target and protein interaction partner network of ZFC3H1, and identified a PPI network composed of 26 node genes in PRAD. Furthermore, we found that the expression levels of MPHOSPH6 (encoding M-phase phosphoprotein 6) and MRPS31 (encoding mitochondrial ribosomal protein S31) were lower in PRAD tissues than in non-cancerous tissues, and the survival time of patients with high MPHOSPH6 and MRPS31 expression was poor. To further demonstrate the role of ZC3H1 in PRAD, we knocked-down the ZFC3H1 expression and found that the inhibition of ZFC3H1 significantly inhibited PRAD cell migration and invasion. Furthermore, ZFC3H1 siRNA treatment could reduce cell viability and increase the number of apoptotic cells in PRAD cells. Taken together, ZFC3H1 could represent a new marker for PRAD prognosis and provide a reference for the development of new therapies to treat PRAD.

Aberrant expression of P68 RNA helicase (p68), a prototypical member of the DEAD box family of RNA helicases, contributes to tumor development and progression. P68 tyrosine phosphorylation induced by PDGF signaling facilitates cancer metastasis by promoting EMT. In this report, we show that p68 promotes breast cancer cell EMT and cell migration by upregulation of PDGF receptor β (PDGFR-β). Knockdown of p68 in MDA-MB-231 and BT549 cells significantly decreases PDGFR-β both in mRNA and protein levels. P68 promotes EMT and cell migration in response to PDGF-BB stimulation via upregulation of PDGFR-β, suggesting that p68 enhances PDGF signaling by a positive feedback loop in cancer cells. Furthermore, our study reveals that p68 mediates the effects of PDGFR-β in regulation of androgen receptor (AR) in breast cancer cells. We demonstrate that p68 and PDGFR-β co-regulate AR expression and promote androgen-mediated proliferation in breast cancer cells. Our studies uncover an important pathway of p68-PDGFR-β axis in promoting breast cancer progression.

Gastric cancer is one of the most frequently diagnosed malignant tumors, with rapid progression and poor prognosis. The role of chondroitin sulfate synthase 1 (CHSY1) in the development and progression of gastric cancer was explored and clarified in this study. The immunohistochemistry analysis of clinical tissue samples as well as data mining of public database showed that CHSY1 was significantly upregulated in gastric cancer and associated with more advanced tumor stage and poorer prognosis. In vitro loss-of-function experiments demonstrated the inhibited cell proliferation, colony formation, cell migration, as well as the promoted cell apoptosis by CHSY1 knockdown. Moreover, recovery of CHSY1 expression could attenuate the regulatory effects induced by CHSY1 knockdown. Correspondingly, gastric cancer cells with CHSY1 knockdown showed reduced tumorigenicity and slower tumor growth in vivo. In conclusion, this study identified CHSY1 as a tumor promotor in gastric cancer, which may be utilized as a novel indicator of patients’ prognosis and therapeutic target for developing more effective drug for GC treatment.

Ophiopogonin B (OP-B), a kind of saponin compound that exists in Radix Ophiopogonis is frequently adopted for the treatment of lung disease as traditional Chinese medicine. The present work aimed to explore the anti-tumor activity of OP-B on non-small cell lung carcinoma (NSCLC) and its possible mechanism. We found that OP-B-treated cells suppressed the viability and proliferation of cells depending on its concentration, as assayed by MTT and Alamar Blue (IC50 were 14.22 ± 1.94, 12.14 ± 2.01, and 16.11 ± 1.83 μM in A549, NCI-H1299, and NCI-H460 cells, respectively). Then, the suppressive effect of OP-B on the invasion and migration of NSCLC was observed through wound healing and Transwell assays, and the epithelial-mesenchymal transition (EMT) markers was detected by immunofluorescence and western blotting. In addition, a dose-dependent reduction of β-catenin both within cytoplasm and nucleus was observed, and the downstream proteins cyclin D1 and c-Myc of Wnt/β-catenin pathway were also reduced. We further constructed β-catenin-overexpression cell models to reveal the underlying mechanism. The results showed that 10 μM of OP-B notably reduced β-catenin protein levels, as well as cell migration and invasion. In spite of the increasement of β-catenin, activation of Wnt pathway and EMT progression, knockdown of Axin leaded to de-function of OP-B on cell metastasis. Taken together, OP-B reduced NSCLC migration and invasion by strengthening the Axin/β-catenin interaction and reducing β-catenin protein translocation.

Epidermal growth factor receptor (EGFR) signalling and the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) are aberrantly activated in ovarian cancer. However, inhibition of EGFR signalling in ovarian cancer patients resulted in a disappointing clinical benefit. In this study, we found that EGFR could activate IL-6-STAT3 pathway in ovarian cancer cells. However, we also demonstrated that EGFR knockdown could increase STAT3 phosphorylation in HO8910 and OVCAR-3 ovarian cancer cells. Interestingly, we further demonstrated that the non-coding RNA miR-146b could simultaneously block both the EGFR and IL-6-STAT3 pathways. Finally, our data demonstrated that miR-146b overexpression resulted in a greater suppression of cell migration than STAT3 pathway inhibition alone.These results suggest a complex and heterogeneous role of EGFR in ovarian cancer. Combined blockade of EGFR and IL-6-STAT3 pathways by miR-146b might be a strategy for improving the clinical benefit of targeting the EGFR pathway in ovarian cancer patients in the future.

In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously (‘‘cells-in-gels-in-paper’’ or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, ‘‘sections’’ all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures.

It has been well-established that polarized migrating cells exhibit a stable and transient gradient of intracellular calcium concentration ([Ca2+]i), increasing from front-to-rear, that is thought to be responsible for rear retraction. The paradox that arises is how calcium at the front of a cell catalyzes critical high-threshold calcium-dependent processes during cell migration and particularly in decision-making for a cell to turn. In this brief review, we discuss the recent discovery of flickering high-[Ca2+]i microdomains (“calcium flickers”) at the front of migrating fibroblasts and their common role in transducing local membrane mechanical stress (via TRPM7, a stretch-activated calcium-permeating transient receptor potential channel) and chemoattractant-elicited signals (via type 2 inositol 1,4,5-trisphosphate receptor in the endoplasmic reticulum). Furthermore, we present a new model for patterned calcium flicker activity as the mechanism for steering the turning of a migrating cell.

A wide variety of disorders are associated with an imbalance in the plasminogen activator system, including inflammatory diseases, atherosclerosis, intimal hyperplasia, the response mechanism to vascular injury, and restenosis. Urokinase-type plasminogen activator (uPA) is a multifunctional protein that in addition to its fibrinolytic and matrix degradation capabilities also affects growth factor bioavailability, cytokine modulation, receptor shedding, cell migration and proliferation, phenotypic modulation, protein expression, and cascade activation of proteases, inhibitors, receptors, and modulators. uPA is the crucial protein for neointimal growth and vascular remodeling. Moreover, it was recently shown to be implicated in the stimulation of angiogenesis, which makes it a promising multipurpose therapeutic target. This review is focused on the mechanisms by which uPA can regulate arterial remodeling, angiogenesis, and cell migration and proliferation after arterial injury and the means by which it modulates gene expression in vascular cells. The role of domain specificity of urokinase in these processes is also discussed.

Elevated expression of the nucleoporin Nup88, a constituent of the nuclear pore complex, is seen in various types of malignant tumors, but whether this overexpression contributes to the malignant phenotype has yet to be determined. Here, we investigated the effect of the overexpression of Nup88 on the migration and invasion of cervical cancer HeLa cells. The overexpression of Nup88 promoted a slight but significant increase in both migration and invasion, whereas knockdown of Nup88 by RNA interference suppressed these phenotypes. The observed phenotypes in Nup88-overexpressing HeLa cells were not due to the progression of the epithelial-to-mesenchymal transition or activation of NF-κB, which are known to be important for cell migration and invasion. Instead, we identified an upregulation of matrix metalloproteinase-12 (MMP-12) at both the gene and protein levels in Nup88-overexpressing HeLa cells. Upregulation of MMP-12 protein by the overexpression of Nup88 was also observed in one other cervical cancer cell line and two prostate cancer cell lines but not 293 cells. Treatment with a selective inhibitor against MMP-12 enzymatic activity significantly suppressed the invasive ability of HeLa cells induced by Nup88 overexpression. Taken together, our results suggest that overexpression of Nup88 can stimulate malignant phenotypes including invasive ability, which is promoted by MMP-12 expression. Supplementary Information The online version contains supplementary material available at 10.1007/s00418-021-02020-w.

Persistent activation of signal transducer and activator of transcription 3 (STAT3) is frequently reported in cancers and plays important roles in tumor progression. Therefore, directly targeting persistent STAT3 signaling is an attractive cancer therapeutic strategy. The aim of this study is to test the inhibitory efficacy of novel STAT3 small molecule inhibitors, LLY17 and LLL12B, in combination with irradiation in human medulloblastoma cells. Both LLY17 and LLL12B inhibit the IL-6-induced and persistent STAT3 phosphorylation in human medulloblastoma cells. Irradiation using 4 Gy alone exhibits some inhibitory effects on medulloblastoma cell viability, and these effects are further enhanced by combining with either STAT3 inhibitor. Irradiation alone also shows certain inhibitory effects on medulloblastoma cell migration and invasion and the combination of LLY17 or LLL12B with irradiation further demonstrates greater inhibitory effects than monotherapy. STAT3 inhibitor alone or irradiation alone exhibits some suppression of medulloblastoma tumorsphere growth, and the combination of LLY17 or LLL12B and irradiation exhibits greater suppression of tumorsphere growth than monotherapy. Combining either STAT3 inhibitor with irradiation reduces the expression of STAT3 downstream targets, Cyclin D1 and Survivin, and induces apoptosis in medulloblastoma cells. These results support that combination of a potent STAT3 inhibitor such as LLY17 or LLL12B with irradiation is an effective and novel therapeutic approach for medulloblastoma.

Background: Although we previously revealed that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and important for gemcitabine resistance, the role of DNA-PKcs in the progression and metastasis of PDAC remain unclear. To date, the upstream signaling events stimulating DNA-PKcs overexpression in PDAC are still not well characterized. Methods: Expression of DNA-PKcs was measured by western blot. The levels of miRNA-101 and lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) were detected by real-time PCR. Cell viability was determined by CCK-8. Cell migration and cell invasion were measured by transwell assay. The regulatory relationship between NEAT1 and miR-101 was determined by a luciferase assay. Results: DNA-PKcs expression was significantly elevated in human PDAC tissues and cells. DNA-PKcs overexpression was correlated with TNM stage and lymph node metastasis. Higher expression of DNA-PKcs was closely correlated with patients of worse overall survival (OS). DNA-PKcs knockdown suppresses malignant behaviors of PDAC cells. Further study showed that miRNA-101 level was decreased in PDAC tissues and cells, which could be responsible for DNA-PKcs overexpression and DNA-PKcs mediated oncogenic actions in PDAC cells. Moreover, NEAT1 functions as an oncogene influencing cell proliferation, migration and invasion in part by serving as a competing endogenous RNA (ceRNAs) modulating miR-101 expression, leading to up-regulation of DNA-PKcs. Conclusion: These findings suggest that NEAT1/miR-101-dependent up-regulation of DNA-PKcs promotes the malignant behaviors of PDAC cells. The NEAT1/miR-101/DNA-PKcs axis may serve as a viable prognostic marker and therapeutic target for PDAC.

To study the regulation of miR-148a-3p on CDK6 and its mechanism in the progress of acute myeloid leukemia (AML), differential miRNAs were analyzed by bioinformatics, and the miR-148a-3p levels in AML cell lines were detected. Results showed that miR-148a-3p played a crucial role in AML, and the level was lower in AML cells, especially in J111 and KG-1a cells. In J111 and KG-1a cells, the up-regulation of miR-148a-3p mimics blocked the cell growth by arresting cell cycle at G2/M and enhancing cell apoptosis. Transwell and EMT markers detection indicated that miR-148a-3p reduced the cell migration and invasion. Afterward, through bioinformatics analysis, it showed that the CDK6 is one of the direct target genes of miR-148a-3p. DLR assay confirmed the target regulation. CDK6 overexpression reversed the effects of miR-148a-3p on AML cells. Collectively, miR-148a-3p inhibited the process of AML cells through disturbing the CDK-6 expression, implying that the trageting miR-148a-3p might be regarded as effective therapy of AML., Graphical abstract

miR-17-5p has been proved that play important roles in many kinds of tumors progression. This study aimed at explore the function and mechanism of miR-17-5p in thyroid cancer (TC). RT-qPCR was used to detect miR-17-5p and Early growth response 2 (EGR2) expression in TC tissues and cells. CCK8 and colony formation assay were used to analyze cell proliferation. Cell migration and cell invasion was detected by Wound-healing assay and Transwell assay. Detection of protein expression using Western blot analysis. Dual-Luciferase assay was used to analyze the relationship between miR-17-5p and EGR2. In vivo experiment was performed by establishing Xenograft animal model to observe the function of miR-17-5p. We found that miR-17-5p is significantly increased in thyroid cancer tissues and cells. miR-17-5p inhibition repressed cell proliferation, clonal formation, cell migration, and cell invasion in thyroid carcinoma. Moreover, miR-17-5p inhibition suppressed tumorigenesis in vivo. Dual-Luciferase assay and Western blotting assay further proved that miR-17-5p has a negative regulation to EGR2. EGR2 was decreased in TC tissues and cells. Overexpressed EGR2 inhibited the development of thyroid carcinoma both vivo and in vivo. EGR2 knockdown remarkably decreased the anti-cancer effect of miR-17-5p inhibition. miR-17-5p is a thyroid cancer oncomir by modulation of the EGR2., Graphical abstract

Long noncoding RNAs play key roles in several cancers, but their potential functions in gastroenteropancreatic neuroendocrine neoplasms remain to be investigated. We performed GeneChip assay to explore differentiated lncRNAs in gastric NENs and peri-cancerous tissues. The regulation of HNF1A-AS1 on biological behavior of GEP-NENs cells and in vivo xenograft model was confirmed by CCK8, colony formation assay, transwell, western blot and qRT-PCR. We next detected the potential transcription factors and the binding sites between them with bioinformatic analysis. qRT-PCR was performed to analyze the exact relationship between them. HNF1A-AS1 expression was decreased in gastric NENs tissues (p < 0.01). Over-expression of HNF1A-AS1 suppressed cellular proliferation, migration and invasion. Knockdown of transcription factor 3 inhibited the expression of HNF1A-AS1 and promoted cellular migration and invasion. Oncostatin M was identified as the downstream target of HNF1A-AS1. Inhibition of transforming growth factor-β activity inhibited HNF1A-AS1/Oncostatin M-mediated epithelial-mesenchymal transition. Our data suggest that transcription factor 3/HNF1A-AS1/Oncostatin M axis inhibits the tumorigenesis and metastasis of gastroenteropancreatic neuroendocrine neoplasms via transforming growth factor-β signaling.

ETS1 − an evolutionarily conserved transcription factor involved in the regulation of a number of cellular processes − is overexpressed in several malignancies, including ovarian cancer. Most studies on ETS1 expression have been focused on the transcriptional and RNA levels, with post-translational control mechanisms remaining relatively unexplored in the pathogenesis of malignancies. Here, we show that ETS1 forms a complex with glycogen synthase kinase-3β (GSK3β). Specifically, GSK3β-mediated phosphorylation of ETS1 at threonine 265 and serine 269 promoted protein stability, induced the transcriptional activation of matrix metalloproteinase (MMP)-9, and increased cell migration. In vivo experiments revealed that a GSK3β inhibitor was able to suppress both endogenous ETS1 expression and induction of MMP-9 expression. Upon generation of a specific antibody against phosphorylated ETS1, we demonstrated that phospho-ETS1 immunohistochemical expression in ovarian cancer specimens was correlated with that of MMP-9. Notably, the cumulative overall survival of patients with low phospho-ETS1 histoscores was significantly longer than that of those showing higher scores. We conclude that the GSK3β/ETS1/MMP-9 axis may regulate the biological aggressiveness of ovarian cancer and can serve as a prognostic factor in patients with this malignancy.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited targeted therapeutic options. A defining feature of TNBC is the propensity to metastasize and acquire resistance to cytotoxic agents. Mitogen activated protein kinase (MAPK) and extracellular regulated kinase (ERK) signaling pathways have integral roles in cancer development and progression. While MEK5/ERK5 signaling drives mesenchymal and migratory cell phenotypes in breast cancer, the specific mechanisms underlying these actions remain under-characterized. To elucidate the mechanisms through which MEK5 regulates the mesenchymal and migratory phenotype, we generated stably transfected constitutively active MEK5 (MEK5-ca) TNBC cells. Downstream signaling pathways and candidate targets of MEK5-ca cells were based on RNA sequencing and confirmed using qPCR and Western blot analyses. MEK5 activation drove a mesenchymal cell phenotype independent of cell proliferation effects. Transwell migration assays demonstrated MEK5 activation significantly increased breast cancer cell migration. In this study, we provide supporting evidence that MEK5 functions through FRA-1 to regulate the mesenchymal and migratory phenotype in TNBC.

Circular RNA (circRNA) features prominently in the progression of hepatocellular carcinoma (HCC), of which the biological function and potential mechanism of circ_0008274 in HCC are obscure. The present study aims to explore circ_ 0008274’s biological functions and underlying mechanisms in HCC. The expressions of circ_0008274, miR-140-3p and Granulin (GRN) mRNA in HCC tissues and cells were investigated by quantitative real-time polymerase chain reaction. Besides, GRN protein expression was measured by Western blot. Furthermore, chi-square test was used to probe the interrelation between circ_0008274 expression and clinicopathological parameters. In addition, cell counting kit-8 (CCK-8) and EdU assays were applied to detect cell proliferation. Moreover, transwell assay was used to detect cell migration and invasion. What’s more, bioinformatics prediction, dual-luciferase reporter gene assay and RNA Immunoprecipitation experiments were used to corroborate the targeting interrelations among circ_0008274, miR-140-3p and GRN. Herein we reported that circ_0008274 was highly expressed in HCC, and its high expression enhanced the proliferation, migration, and invasion of HCC cells, while depleting circ_0008274 inhibited the malignant biological behaviors of HCC cells. Mechanistically, circ_0008274 upregulates GRN expressions via adsorbing miR-140-3p to expedite the progression of HCC., GRAPHICAL ABSTRACT

Nasopharyngeal carcinoma (NPC) is known for its potential to progress to the lymph nodes and distant metastases at an early stage. As an important regulator in tumorigenesis biological processes, the functions of lncRNA in NPC tumor development remain largely unclear. In this research, the expression of EPB41L4A-AS2 in NPC tissues and cells was analyzed via real-time quantitative polymerase chain reaction (qRT-PCR). CCK8, colony formation, and EDU experiments were used to determine the viability of NPC cells. Transwell and wound healing assays were performed to test NPC cell migration and invasion. RNA pull-down and mass spectrometry analysis were used to identify potential binding proteins. Then, a popliteal lymph node metastasis model was established to test NPC metastasis. EPB41L4A-AS2 is repressed by transforming growth factor-beta, which is downregulated in NPC cells and tissue. It is associated with the presence of distant metastasis and adverse outcomes. The univariate and multivariate survival assays confirmed that EPB41L4A-AS2 expression was an independent predictor of progression-free survival (PFS) in patients with NPC. Biological analyses showed that overexpression of EPB41L4A-AS2 reduced the metastasis and invasion of NPC in vitro and in vivo, but had no significant effect on cell proliferation. Mechanistically, in the nucleus we identified that EPB41L4A-AS2 relies on binding to YBX1 to reduce the stability of Snail mRNA to enhance the expression of E-cadherin and reverse the progression of epithelial-to-mesenchymal transition (EMT). In the cytoplasm, we found that EPB41L4A-AS2 blocked the invasion and migration of NPC cells by promoting LATS2 expression via sponging miR-107. In a whole, the findings of this study help to further understand the metastasis mechanism of NPC and could help in the prevention and treatment of NPC metastasis.

Background: Hepatocellular carcinoma (HCC) is a major leading cause of cancer mortality worldwide. Thyroid hormone responsive (THRSP) gene is primarily known for regulating responses to thyroid hormones, but its expression has been correlated with differential outcomes in some cancers. To date, however, its role in the progression of HCC remains unknown. Methods: The mRNA and protein expression of THRSP was measured in HCC tissues and cell lines via qPCR and western blot assays. Lentiviral transfection was used to establish stable cell lines overexpressing THRSP and shRNA was used to silence THRSP. The effects of THRSP on cell growth were then determined in vivo and in vitro. Cell migration and invasion of HCC cells were investigated using transwell and wound healing assays. Results: In tissue samples from patients, HCC tissues had decreased THRSP expression relative to adjacent healthy tissues. Further, patients with decreased THRSP protein and mRNA expression had worse outcomes. Knockdown of THRSP led to increased cell growth, migration, and invasion of HCC cells, and THRSP overexpression exerted an anti-tumor effect in vivo and in vitro. We found that increased expression of THRSP inhibited hepatocellular carcinogenesis by inhibiting the process of epithelial-to-mesenchymal transition through acting on the ERK/ZEB1 signaling pathway. Conclusion: THRSP may act as a functional tumor suppressor and was frequently reduced in HCC tissue samples. We identified a novel pathway for the THRSP/ERK/ZEB1-regulated suppression of HCC tumorigenesis and invasion. Restoring THRSP expression may represent a promising approach for HCC therapies.

Tranilast has an ameliorative effect on myocardial fibrosis (MF), but the specific mechanism has not been studied. S100A11 is a key regulator of collagen expression in MF. In this paper, we will study the regulatory roles of Tranilast and S100A11 in MF. After the introduction of angiotensin II (AngII) to Human cardiac fibroblasts (HCF), Tranilast was administered. CCK-8 kit was used to detect cell viability. Wound Healing assay detected cell migration, and Western blot was used to detect the expression of migration-related proteins and proteins related to extracellular matrix synthesis. The expression of α-SMA was detected by immunofluorescence (IF). The expression of S100A11 was detected by qPCR and Western blot, and then S100A11 was overexpressed by cell transfection technology, so as to explore the mechanism by which Tranilast regulated MF. In addition, the expression of TGF-β1/Smad pathway related proteins was detected by Western blot. Tranilast inhibited Ang II–induced over-proliferation, migration and fibrosis of human cardiac fibroblasts (HCF), and simultaneously significantly decreased S100A11 expression was observed. Overexpression of S100A11 reversed the inhibition of Tranilast on AngII–induced over-proliferation, migration, and fibrosis in HCF, accompanied by activation of the TGF-β1/Smad pathway. Overall, Tranilast inhibits angiotensin II-induced myocardial fibrosis through S100A11/TGF-β1/Smad axis.

Mounting evidence has shown that miRNA-150 expression is upregulated in gastric cancer (GC) and is associated with gastric carcinogenesis, but the underlying oncogenic mechanism remains elusive. Here, we discovered that miRNA-150 targets the tumor suppressor SUFU to promote cell proliferation, migration, and the epithelial–mesenchymal transition (EMT) via the dual activation of Hedgehog (Hh) and Wnt signaling. MiRNA-150 was highly expressed in GC tissues and cell lines, and the level of this miRNA was negatively related to that of SUFU. In addition, both the miRNA-150 and SUFU levels were associated with tumor differentiation. Furthermore, miRNA-150 activated GC cell proliferation and migration in vitro. We found that miRNA-150 inhibitors repressed not only Wnt signaling by promoting cytoplasmic β-catenin localization, but also repressed Hh signaling and EMT. MiRNA-150 inhibition also resulted in significant tumor volume reductions in vivo, suggesting the potential application of miRNA-150 inhibitors in GC therapy. The expression of genes downstream of Hh and Wnt signaling was also reduced in tumors treated with miRNA-150 inhibitors. Notably, anti-SUFU siRNAs rescued the inhibitory effects of miRNA-150 inhibitors on Wnt signaling, Hh activation, EMT, cell proliferation, cell migration, and colony formation. Taken together, these findings indicate that miRNA-150 is oncogenic and promotes GC cell proliferation, migration, and EMT by activating Wnt and Hh signaling via the suppression of SUFU expression.

This study is performed to evaluate the role of long noncoding RNA (lncRNA) LINC00619 in osteosarcoma through the PI3K-Akt signalling pathway by binding to HGF. Osteosarcoma and osteochondroma tissues from patients were collected. The relationship between lncRNA LINC00619 and HGF was proved by the dual-luciferase reporter gene assay. The expression patterns of lncRNA LINC00619 as well as the levels of proliferating cell nuclear antigen (PCNA), hepatocyte growth factor (HGF), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), Bax, Bcl-2, alkaline phosphatase (ALP), and osteopontin (OPN) were detected by RT-qPCR and Western blot analysis. In addition, MTT assay, flow cytometry, scratch test, and Transwell assay were performed to assess the cell proliferation, cell cycle distribution, apoptosis, cell migration, and invasion in each group, respectively. Osteosarcoma tissues presented with elevated positive expression rate of HGF, up-regulated expression levels of PCNA, HGF, PI3K, Akt, Bcl-2, ALP and OPN, and down-regulated expressions of Bax and LINC00619. HGF was verified as a target gene of lncRNA LINC00619. LINC00619 was found to down-regulate the expressions of PCNA, HGF, PI3K, Akt, Bcl-2, ALP, and OPN in osteosarcoma cells. Up-regulation of lncRNA LINC00619 decreased cell growth, migration intensity, and invasion ability, but enhanced the apoptosis rate of osteosarcoma cells. Our findings suggest that lncRNA LINC00619 inhibits proliferation, migration and invasion and improves apoptosis of osteosarcoma cells through the inhibition of the activation of the HGF-dependent PI3K-Akt signalling pathway.

Metabolic reprogramming, especially Warburg effect, is a key event in tumor initiation and progression. ZEB1 plays a vital role in metastasis of various cancers. We previously found that ZEB1 was excessively expressed in hepatocellular carcinoma (HCC) and its high expression was closely correlated with metastasis and recurrence of HCC. We want to know whether glycolytic enzymes are regulated by ZEB1 and contribute to carcinogenesis and metastasis of HCC. Methods: To explore whether ZEB1 could enhance glycolysis in HCC, we knocked down ZEB1 by short hairpin RNA (shRNA) in MHCC-97H and HCC-LM3 cells and performed glucose uptake, lactate production, ECAR and OCR assays. To investigate how ZEB1 enhances glycolysis, the protein levels of glycolytic enzymes were detected in the same cell lines using Western blot. The regulatory effect of ZEB1 on PFKM mRNA level was confirmed by RT-qPCR, luciferase report assay and ChIP assay. In order to assess the role of ZEB1-PFKM axis in cell proliferation, cell counting and CCK-8 assays were performed in MHCC-97H and HCC-LM3 cell lines knocked down for ZEB1 and further re-expressed for either ZEB1 or PFKM or not. To explored whether the ZEB1-PFKM axis also functions in HCC cell migration, invasion and metastasis, the same MHCC-97H and HCC-LM3 cell lines were performed for wound healing assays, transwell assays and colony formation assays, meanwhile, MHCC-97H cell lines were performed for orthotopic liver transplantation assays. Finally, the expression of ZEB1 and PFKM were examined in human liver cancer specimens and non-tumorous liver tissues using immunohistochemical and Western blot. Results: We found that ZEB1 transcriptionally upregulates the expression of the muscle isoform of phosphofructokinase-1 (PFKM), a rate-limiting enzyme in glycolysis. Intriguingly, a non-classic ZEB1-binding sequence in the promoter region of PFKM was identified through which ZEB1 directly activates the transcription of PFKM. Silencing of ZEB1 in MHCC-97H and HCC-LM3 cell leads to impaired PFKM expression, glycolysis, proliferation and invasion, and such impairments are rescued by exogenous expression of PFKM. Importantly, in-situ HCC xenograft assays and studies from TCGA database demonstrate that ZEB1-PFKM axis is crucial for carcinogenesis and metastasis of HCC. Conclusions: Our study reveals a novel mechanism of ZEB1 in promoting HCC by activating the transcription of PFKM, establishing the direct link of ZEB1 to the promotion of glycolysis and Warburg effect and suggesting that inhibition of ZEB1 transcriptional activity toward PFKM may be a potential therapeutic strategy for HCC.

Long noncoding RNAs (lncRNAs) are involved in the progression of various cancers, including hepatocellular carcinoma (HCC). However, the biological functions of lncRNA small ubiquitin-like modifier 1 pseudogene 3 (SUMO1P3) and the underlying mechanisms remain unclear. In this study, we revealed that SUMO1P3 expression was enhanced in HCC tissues and cell lines, positively associating with tumor size and number, poor differentiation, lymphatic and distant metastasis, TNM stage, and poor prognosis in HCC patients. In vitro assays showed that SUMO1P3 depletion reduced HCC cell viability and proliferation by hindering cyclin D1 expression and Akt phosphorylation. SUMO1P3 knockdown induced HCC cell apoptosis, as indicated by increased Bax and cleaved caspase-3 expression and the decreased Bcl-2 level. SUMO1P3 silencing suppressed HCC cell migration and invasion by increasing epithelial marker E-cadherin expression and decreasing mesenchymal marker vimentin expression, as well as reducing matrix metalloproteinase (MMP)-2 and MMP-9 levels. Consistently, SUMO1P3 depletion in HCC cells retarded tumor growth and lung metastasis in vivo. Overall, these results supported the applicability of SUMO1P3 as a useful predictor of HCC prognosis and a potential therapeutic target for HCC patients.

Background: Lung cancer is a malignant tumor in mammary gland epithelium with high morbidity and mortality among women worldwide. Long noncoding RNA GAS5 (GAS5) has been proved to be closely related with tumor progression. However, the influence of GAS5 on lung cancer and the specific mechanism remain unclear. Methods: Cell invasion, cell migration, cell apoptosis and cell cycle were investigated after transfection with pcDNA-GAS5 and sh-GAS5. Sizes of tumors were measured by establishing transplanted tumor model in vivo. E-cadherin and N-cadherin expressions were investigated. Results: Cell invasion and migration were inhibited markedly in GAS5 overexpressed cell line. Cell cycle results indicated that the percentage of S-phase cells was increased, and G2-phase was reduced in the GAS5 overexpression cell line. Tumor size was suppressed obviously after GAS5 overexpression treatment. GAS5 markedly inhibited the expression of E-cadherin and induced the expression of N-cadherin. GAS5 overexpression significantly inhibited lung cancer cell proliferation by increasing the E-cadherin and decreasing N-cadherin. Conclusions: These findings provide novel evidence that GAS5 can be viewed as an anti-lung cancer agent through affecting EMT pathway.

Background: Cholangiocarcinoma (CCA) is a variety of biliary epithelial tumors involving intrahepatic, perihilar and distal bile duct. It is the most common malignant bile duct tumor in the liver and the second most common primary liver cancer, whose molecular mechanism not fully understood. Specifically, the relationship between CCA and chondroitin polymerizing factor (CHPF) is still not clear.Methods: In this study, detection of clinical specimens was performed to preliminarily study the role of CHPF in cholangiocarcinoma. Cholangiocarcinoma cells with CHPF knockdown were constructed for in vitro study, which was also used in the construction of mice xenograft model for investigating the role of CHPF in the development of cholangiocarcinoma.Results: The results demonstrated that CHPF was significantly upregulated in cholangiocarcinoma tissues compared with normal tissues. High expression of CHPF was correlated with more advanced tumor grade. Moreover, knockdown of CHPF significantly inhibited cell proliferation, cell migration, promoted cell apoptosis and arrest cell cycle in G2 phase in vitro, as well as suppressed tumor growth in vivo.Conclusions: In conclusion, CHPF was identified as a tumor promotor in the development and metastasis of cholangiocarcinoma, which may provide a novel therapeutic target for the targeted therapy against cholangiocarcinoma.

Vegfc/Vegfr3 signaling is critical for lymphangiogenesis, the sprouting of lymphatic vessels. In zebrafish, cells sprouting from the posterior cardinal vein can either form lymphatic precursor cells or contribute to intersegmental vein formation. Both, the Vegfc-dependent differential induction of Prox1a in sprouting cells as well as a Notch-mediated pre-pattern within intersegmental vessels have been associated with the regulation of secondary sprout behavior. However, how exactly a differential lymphatic versus venous sprout cell behavior is achieved is not fully understood. Here, we characterize a zebrafish mutant in the adaptor protein Grb2b, and demonstrate through genetic interaction studies that Grb2b acts within the Vegfr3 pathway. Mutant embryos exhibit phenotypes that are consistent with reduced Vegfr3 signaling outputs prior to the sprouting of endothelial cells from the vein. During secondary sprouting stages, loss of grb2b leads to defective cell behaviors resulting in a loss of parachordal lymphangioblasts, while only partially affecting the number of intersegmental veins. A second GRB2 zebrafish ortholog, grb2a, contributes to the development of lymphatic structures in the meninges and in the head, but not in the trunk. Our results illustrate an essential role of Grb2b in vivo for cell migration to the horizontal myoseptum and for the correct formation of the lymphatic vasculature, while being less critically required in intersegmental vein formation. Thus, there appear to be higher requirements for Grb2b and therefore Vegfr3 downstream signaling levels in lymphatic versus vein precursor-generating sprouts. Supplementary Information The online version contains supplementary material available at 10.1007/s10456-021-09774-w.

Objective: To investigate the correlation between estrogen-related receptor a (ERRα) expression level and gastric cancer (GC). Methods: We collected GC and adjacent normal tissues from 50 patients. The parameters of the patients were summarized, and correlation with the expression level of ERRα was calculated. Downregulated ERRα using lentivirus was designed and transfected to SGC-7901 and MGC-803 cells. Cell migration, invasion and wound assays were conducted to determine the correlation between ERRα and capacity for cell migration and invasion. The expression level of the genes involved in epithelial–mesenchymal transition, including E-cadherin, γ-catenin, N-cadherin and vimentin, was determined via real-time or quantitative polymerase chain reaction(qPCR) and Western blot analysis. Results: The expression of ERRα tends to be higher in GC tissues than in adjacent normal tissues. Analyses ofthe expression level of ERRα and patient parameters show that the ERRα level is significantly correlated with TNM staging and patient survival (P