H19/miR-148a/USP4 axis facilitates liver fibrosis by enhancing TGF-β signaling in both hepatic stellate cells and hepatocytes

Liver fibrosis is a wound-healing response represented by excessive extracellular matrix deposition. Activation of hepatic stellate cell (HSC) is the critical cellular basis for hepatic fibrogenesis, whereas hepatocyte undergoes epithelial-mesenchymal transition (EMT) which is also involved in chronic liver injury. Long noncoding RNA H19 has been found to be associated with cholestatic liver fibrosis lately. However, the role of H19 in liver fibrosis remains largely to be elucidated. In this study, we found that the expression of H19 was significantly upregulated in the liver tissue of CCl₄-induced mice, a toxicant-induced liver fibrogenesis model. Overexpression of H19 significantly aggravated activation of HSC and EMT of hepatocyte both by stimulating transforming growth factor-β (TGF-β) pathway. In terms of mechanism, H19 functioned as a competing endogenous RNA to sponge miR-148a and subsequently sustained the level of ubiquitin-specific protease 4 (USP4), which was an identified target of miR-148a and was able to stabilize TGF-β receptor I. In conclusion, our findings revealed a novel H19/miR-148a/USP4 axis which promoted liver fibrosis via TGF-β pathway in both HSC and hepatocyte, indicating that H19 could become a promising target for the treatment of liver fibrosis.     

Upregulation of microRNA-141 suppresses epithelial-mesenchymal transition and lymph node metastasis in laryngeal cancer through HOXC6-dependent TGF-β signaling pathway

Laryngeal cancer is one of the most malignant cancers among the head and neck malignant tumors. Abnormal expression of microRNAs (miRNAs) contributes to cancer development through regulating proliferation and apoptosis of cancer cells. In this study, we aim to explore the roles of microRNA-141 (miR-141), Homeobox C6 (HOXC6) and TGF-β signaling pathway in epithelial-mesenchymal transition (EMT) and lymph node metastasis in laryngeal cancer. Initially, we identified differentially expressed genes in laryngeal cancer, among which HOXC6 was identified. Then the target miRNA of HOXC6 was predicted and verified. Next, expression of miR-141, HOXC6, TGF-β1, Smad3, Vimentin and Snail in cancer tissues was detected. Then, AMC-HN-8 cells were transfected with miR-141 mimic, miR-141 inhibitor and HOXC6-siRNA to investigate specific role of miR-141, HOXC6 and TGF-β signaling pathway in laryngeal cancer in vivo and in vitro. Our results showed that HOXC6 was a target gene of miR-141, which was downregulated in laryngeal cancer. Besides, overexpression of miR-141 could downregulate HOXC6 and inhibit the TGF-β signaling pathway. Upregulation of miR-141 or silencing of HOXC6 can repress EMT, viability, migration and invasion abilities of laryngeal cancer cells. In addition, upregulation of miR-141 inhibited the tumor growth and lymph node metastasis in vivo. In summary, our findings demonstrated that upregulated miR-141 decreased HOXC6 expression, and inhibited the TGF-β signaling pathway, EMT and lymph node metastasis in laryngeal cancer, which is of clinical significance in the treatment of laryngeal cancer.     

Long Non-Coding RNA H19 Promotes Glioma Cell Invasion by Deriving miR-675

H19 RNA has been characterized as an oncogenic long non-coding RNA (lncRNA) in breast and colon cancer. However, the role and function of lncRNA H19 in glioma development remain unclear. In this study, we identified that H19/miR-675 signaling was critical for glioma progression. By analyzing glioma gene expression data sets, we found increased H19 in high grade gliomas. H19 depletion via siRNA inhibited invasion in glioma cells. Further, we found H19 positively correlated with its derivate miR-675 expression and reduction of H19 inhibited miR-675 expression. Bioinformatics and luciferase reporter assays showed that miR-675 modulated Cadherin 13 expression by directly targeting the binding site within the 3′ UTR. Finally, introduction of miR-675 abrogated H19 knockdown-induced cell invasion inhibition in glioma cells. To our knowledge, it is first time to demonstrate that H19 regulates glioma development by deriving miR-675 and provide important clues for understanding the key roles of lncRNA-miRNA functional network in glioma.     

TGF-β1-induced epithelial–mesenchymal transition in lung cancer cells involves upregulation of miR-9 and downregulation of its target,E-cadherin

Background

TGF-β1 plays an important role in the epithelial–mesenchymal transition (EMT) of epithelial cancers, including non-small cell lung cancer (NSCLC). While the full underlying mechanism remains unclear, miR-9 is known to play a critical role in the regulation of NSCLC cell invasion. We tested whether miR-9 targets E-cadherin and thus affects TGF-β1-induced EMT in NSCLC cells by assessing the expression levels of miR-9 and E-cadherin for NSCLC patients and then verifying the targeting of E-cadherin by miR-9 using the dual luciferase reporter system.

Results

MiR-9 was significantly upregulated in NSCLC tissues compared with its level in adjacent normal tissues. The expression of E-cadherin in NSCLC tissues was significantly decreased. In addition, we found that TGF-β1 significantly upregulated the expression of miR-9 and downregulated the expression of E-cadherin. E-cadherin was confirmed as a direct target gene of miR-9. Using an miR-9 inhibitor reversed the TGF-β1-mediated inhibition of E-cadherin expression and upregulation of the mesenchymal marker α-SMA. TGF-β1 significantly induced cell invasion, and this effect was significantly inhibited by miR-9 inhibitors.

Conclusions

TGF-β1 induced EMT in NSCLC cells by upregulating miR-9 and downregulating miR-9’s target, E-cadherin.

     

Celastrol inhibits TGF-β1-induced epithelial–mesenchymal transition by inhibiting Snail and regulating E-cadherin expression

The epithelial–mesenchymal transition (EMT) is a pivotal event in the invasive and metastatic potentials of cancer progression. Celastrol inhibits the proliferation of a variety of tumor cells including leukemia, glioma, prostate, and breast cancer; however, the possible role of celastrol in the EMT is unclear. We investigated the effect of celastrol on the EMT. Transforming growth factor-beta 1 (TGF-β1) induced EMT-like morphologic changes and upregulation of Snail expression. The downregulation of E-cadherin expression and upregulation of Snail in Madin–Darby Canine Kidney (MDCK) and A549 cell lines show that TGF-β1-mediated the EMT in epithelial cells; however, celastrol markedly inhibited TGF-β1-induced morphologic changes, Snail upregulation, and E-cadherin expression. Migration and invasion assays revealed that celastrol completely inhibited TGF-β1-mediated cellular migration in both cell lines. These findings indicate that celastrol downregulates Snail expression, thereby inhibiting TGF-β1-induced EMT in MDCK and A549 cells. Thus, our findings provide new evidence that celastrol suppresses lung cancer invasion and migration by inhibiting TGF-β1-induced EMT.     

miR-32 promotes esophageal squamous cell carcinoma metastasis by targeting CXXC5

MicroRNA-32 (miR-32) functioned as a tumor oncogene in some cancer, which control genes involved in important biological and pathological functions and facilitate the tumor growth and metastasis. However, the role of miR-32 modulates esophageal squamous cell carcinoma (ESCC) malignant transformation has not been clarified. Here, we focused on the function and the underlying molecular mechanism of miR-32 in ESCC. Results discovered a significant increased expression of miR-32 in ESCC tissues and cells. Downregulation of miR-32 inhibited the migration, invasion, adhesion of ESCC cell lines (EC9706 and KYSE450), and the levels of EMT protein in vitro. In vivo, miR-32 inhibitors decrease tumor size, tumor weight, and the number of metastatic nodules. Hematoxylin and eosin (H&E) results revealed that inhibition of miR-32 attenuate lung metastasis. Immunohistochemistry and immunofluorescence assay showed increased level of E-cadherin and decreased level of N-cadherin and Vimentin with treatment of miR-32 inhibitors. Furthermore, miR-32 targeted the 3′-untranslated region (3′-UTR) of CXXC5, and inhibited the level of mRNA and protein of CXXC5. There is a negative correlation between the expressions of CXXC5 and miR-32. Then, after EC9706 and KYSE450 cells cotransfected with si-CXXC5 and miR-32 inhibitors, the ability of cell migration, invasion, and adhesion was significantly reduced. In addition, the protein expression of EMT and TGF-β signaling was also depressed. Collectively, these data supply an insight into the positive role of miR-32 in ESCC progression and metastasis, and its biological effects may attribute the inhibition of TGF-β signaling mediated by CXXC5.     

FMNL2 enhances invasion of colorectal carcinoma by inducing epithelial-mesenchymal transition

FMNL2 is a member of diaphanous-related formins that control actin-dependent processes such as cell motility and invasion. Its overexpression in metastatic cell lines and tissues of colorectal carcinoma has been associated with aggressive tumor development in our previous study. But its specific role in cancer is largely unknown. Here we report that FMNL2 is involved in epithelial-mesenchymal transition (EMT) maintenance in human colorectal carcinoma cells. A positive correlation between FMNL2 and vimentin expression and an inverse correlation between FMNL2 and E-cadherin expression were found in colorectal carcinoma cell lines and cancer tissues. Specific knockdown of FMNL2 led to an epithelial-state transition, confirmed by the cobblestone-like phenotype, upregulation of E-cadherin, α-catenin, and γ-catenin, and downregulation of vimentin, snail, slug. Loss of FMNL2 expression lowered the ability of TGF-β to induce cell invasion and EMT, as shown by morphology and the expression levels. Upregulation of vimentin, slug, snail, downregulation of E-cadherin and activation of receptor-Smad3 phosphorylation were observed in M5 and MDCK cells induced by TGF-β, whereas altered expression of these markers was not obvious in FMNL2-depleting M5 cells. High levels of activation of p-MAPK and p-MEK, but not p-PI3K and p-AKT, were observed in SW480/FMNL2+ cells compared with control cells. Treatment with U0126 could abrogate the activation of p-MAPK and p-MEK, whereas LY294002 treatment had no effect on the PI3K/AKT pathway. In conclusion, these findings identify a novel EMT and tumor promoting function for FMNL2, which is involved in TGF-β-induced EMT and colorectal carcinoma cell invasion via Smad3 effectors, or in collaboration with MAPK/MEK pathway.     

Retracted: MicroRNA-133a inhibits gastric cancer cells growth,migration, and epithelial-mesenchymal transition process by targeting presenilin 1

Gastric cancer (GC) is one of the most common malignancies and a leading cause of cancer-related death worldwide. Accumulating evidence reported that microRNA (miR)-133a was involved in GC. This study aimed to investigate the function and mechanism of miR-133a in the development and progression of GC. The expression of miR-133a and presenilin 1 (PSEN1) in two GC cell lines, SGC-7901 and BGC-823, were inhibited and overexpressed by transient transfections. Thereafter, cell viability, migration, and apoptosis were measured by trypan blue exclusion assay, transwell migration assay, and flow cytometry assay, respectively. Dual-luciferase reporter assay was conducted to verify whether PSEN1 was a direct target of miR-133a. Furthermore, quantitative real-time polymerase chain reaction and Western blot analysis were mainly performed to assess the expression changes of epithelial-mesenchymal transition (EMT)-associated proteins, apoptosis-related proteins, and Notch pathway proteins. MiR-133a inhibitor significantly increased cell viability and migration, while miR-133a mimic decreased cell viability, migration, and induced apoptosis. miR-133a suppression accelerated transforming growth factor-β1 (TGF-β1)-induce EMT, as evidenced by upregulation of E-cadherin, and downregulation of N-cadherin, vimentin, and Slug. Of contrast, miR-133a overexpression blocked TGF-β1-induce EMT by altering these factors. PSEN1 was a direct target of miR-133a, and suppression of PSEN1 abolished the promoting functions of miR-133 suppression on cell growth and metastasis. Moreover, PSEN1 inhibition decreased Notch 1, Notch 2, and Notch 3 protein expressions. This study demonstrates an antigrowth and antimetastasis role of miR-133a in GC cells. Additionally, miR-133a acts as a tumor suppressor may be via targeting PSEN1.