MiR‐30c protects diabetic nephropathy by suppressing epithelial‐to‐mesenchymal transition in db/db mice

Epithelial‐to‐mesenchymal transition (EMT) plays a significant role in tubulointerstitial fibrosis, which is a hallmark of diabetic nephropathy. Thus, identifying the mechanisms of EMT activation could be meaningful. In this study, loss of miR‐30c accompanied with increased EMT was observed in renal tubules of db/db mice and cultured HK2 cells exposed to high glucose. To further explore the roles of miR‐30c in EMT and tubulointerstitial fibrosis, recombinant adeno‐associated viral vector was applied to manipulate the expression of miR‐30c. In vivo study showed that overexpression of miR‐30c suppressed EMT, attenuated renal tubulointerstitial fibrosis and reduced proteinuria, serum creatinine, and BUN levels. In addition, Snail1 was identified as a direct target of miR‐30c by Ago2 co‐immunoprecipitation, luciferase reporter, and Western blot assays. Downregulating Snail1 by siRNA reduced high glucose‐induced EMT in HK2 cells, and miR‐30c mimicked the effects. Moreover, miR‐30c inhibited Snail1‐TGF‐β1 axis in tubular epithelial cells undergoing EMT and thereby impeded the release of TGF‐β1; oppositely, knockdown of miR‐30c enhanced the secretion of TGF‐β1 from epitheliums and significantly promoted proliferation of fibroblasts and fibrogenesis of myofibroblasts, aggravated tubulointerstitial fibrosis, and dysfunction of diabetic nephropathy. These results suggest a protective role of miR‐30c against diabetic nephropathy by suppressing EMT via inhibiting Snail1‐TGF‐β1 pathway.     

Class I HDACs specifically regulate E‐cadherin expression in human renal epithelial cells

Epithelial‐mesenchymal transition (EMT) and renal fibrosis are closely involved in chronic kidney disease. Inhibition of histone deacetylase (HDAC) has an anti‐fibrotic effect in various diseases. However, the pathophysiological role of isoform‐specific HDACs or class‐selective HDACs in renal fibrosis remains unknown. Here, we investigated EMT markers and extracellular matrix (ECM) proteins in a human proximal tubular cell line (HK‐2) by using HDAC inhibitors or by knockdown of class I HDACs (HDAC1, 2, 3 and 8). Trichostatin A (TSA), MS275, PCI34051 and LMK235 inhibited ECM proteins such as collagen type I or fibronectin in transforming growth factor β1 (TGF‐β1)‐induced HK2 cells. However, restoration of TGF‐β1‐induced E‐cadherin down‐regulation was only seen in HK‐2 cells treated with TSA or MS275, but not with PCI34051, whereas TGF‐β1‐induced N‐cadherin expression was not affected by the inhibitors. ECM protein and EMT marker levels were prevented or restored by small interfering RNA transfection against HDAC8, but not against other class I HDACs (HDAC1, 2 and 3). E‐cadherin regulation is mediated by HDAC8 expression, but not by HDAC8 enzyme activity. Thus, class I HDACs (HDAC1, 2, 3 and 8) play a major role in regulating ECM and EMT, whereas class IIa HDACs (HDAC4 and 5) are less effective.     

Thrombin stimulates RPE cell motility by PKC‐ζ‐ and NF‐κB‐dependent gene expression of MCP‐1 and CINC‐1/GRO chemokines

Retinal pigment epithelial cells (RPE) are the major cell type involved in the pathogenesis of proliferative vitreoretinopathy (PVR), which involves the epithelial‐mesenchymal transition, proliferation, and directional migration of transformed RPE cells to the vitreous upon RPE exposure to serum components, thrombin among them. Although the aqueous humor and vitreous of PVR patients contain high levels of chemokines, their possible involvement in PVR development has not been explored. We here analyzed the effect of thrombin on chemokine gene expression and its correlation with RPE cell migration using rat RPE cells in culture as a model system. We demonstrated that thrombin induces RPE cell migration through the dose‐dependent stimulation of MCP1 and GRO expression/release, and the autocrine activation of CXCR‐2 and CCR‐2 chemokine receptors. Whereas inhibition of CXCR2 by Sb‐225002 and of CCR2 by Rs‐504393 partially prevented hirudin‐sensitive cell migration, the joint inhibition of these receptors abolished thrombin effect, suggesting the contribution of distinct but coincident mechanisms. Thrombin effects were not modified by Ro‐32‐0432 inhibition of conventional/novel PKC isoenzymes or by the MAPkinase pathway inhibitor U0126. MCP1 and GRO expression/secretion, and cell migration were completely prevented by the inhibitory PKC‐ζ pseudosubstrate and by the nuclear factor‐kappa B (NF‐κB) inhibitor BAY11‐7082, but not by wortmannin inhibition of PI3K. Results show that signaling pathways leading to RPE cell migration differ from the MEK–ERK–PI3K‐mediated promotion RPE of cell proliferation, both of which concur at the activation of PKC‐ζ. J. Cell. Biochem. © 2010 Wiley‐Liss, Inc.     

miR‐655 suppresses epithelial‐to‐mesenchymal transition by targeting Prrx1 in triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial‐to‐mesenchymal transition (EMT) is a key contributor in the metastatic process. In this study, we found that miR‐655 was down‐regulated in TNBC, and its expression levels were associated with molecular‐based classification and lymph node metastasis in breast cancer. These findings led us to hypothesize that miR‐655 overexpression may inhibit EMT and its associated traits of TNBC. Ectopic expression of miR‐655 not only induced the up‐regulation of cytokeratin and decreased vimentin expression but also suppressed migration and invasion of mesenchymal‐like cancer cells accompanied by a morphological shift towards the epithelial phenotype. In addition, we found that miR‐655 was negatively correlated with Prrx1 in cell lines and clinical samples. Overexpression of miR‐655 significantly suppressed Prrx1, as demonstrated by Prrx1 3′‐untranslated region luciferase report assay. Our study demonstrated that miR‐655 inhibits the acquisition of the EMT phenotype in TNBC by down‐regulating Prrx1, thereby inhibiting cell migration and invasion during cancer progression.     

Bortezomib attenuates renal interstitial fibrosis in kidney transplantation via regulating the EMT induced by TNF‐α‐Smurf1‐Akt‐mTOR‐P70S6K pathway

Allograft interstitial fibrosis was characterized by massive extracellular matrix deposition caused by activated fibroblasts and myofibroblasts. Epithelial‐mesenchymal transition (EMT) is recognized as an important source of myofibroblasts contributing to the pathogenesis of allograft interstitial fibrosis. Smad ubiquitination regulatory factor 1 (Smurf1) has been recently reported to be involved in the progression of EMT. Our study was to detect the effect of Bortezomib and Smurf1 in the EMT and allograft interstitial fibrosis. Biomarkers of EMT, as well as Smurf1, were examined in human proximal tubular epithelial cells (HK‐2) treated with tumour necrosis factor‐alpha (TNF‐α) in various doses or at various time points by Western Blotting or qRT‐PCR. We knockdown or overexpressed Smurf1 in HK‐2 cells. Furthermore, rat renal transplant model was established and intervened by Bortezomib. Allograft tissues from human and rats were also collected and prepared for HE, Masson's trichrome, immunohistochemical staining and western blotting assays. As a result, we found that TNF‐α significantly promoted the development of EMT in a time‐dependent and dose‐dependent manner through Smurf1/Akt/mTOR/P70S6K signalling pathway. More importantly, Bortezomib alleviated the progression of EMT and allograft interstitial fibrosis in vivo and in vitro by inhibiting the production of TNF‐α and expression of Smurf1. In conclusion, Smurf1 plays a critical role in the development of EMT induced by TNF‐α. Bortezomib can attenuate the Sumrf1‐mediated progression of EMT and renal allograft interstitial fibrosis, which could be suggested as a novel choice for the prevention and treatment of renal allograft interstitial fibrosis.     

uPA and MMP‐2 were involved in self‐assembled network formation in a two dimensional co‐culture model of bone marrow stromal cells and endothelial cells

Two dimensional (2D) co‐cultures of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) stimulate osteoblastic differentiation of HBMSCs, induce the formation of self‐assembled network and cell interactions between the two cell types involving many vascular molecules. Because of their strong activities on angiogenesis and tissue remodeling, urokinase plasminogen activator (uPA), plasminogen activator inhibitor‐1 (PAI‐1), matrix metalloproteinase‐2 (MMP‐2) as well tissue inhibitors of matrix metalloproteinase‐2 (TIMP‐2) were investigated in this 2D co‐culture model. We found that the expression of uPA, MMP‐2 in the co‐cultured cells was significantly higher than those in mono‐cultured cells. In opposite, PAI‐1, expressed only by HUVECs is not regulated in the co‐culture. Inhibition assays confirm that uPA played a critical role in the formation of self‐assembled network as neutralization of uPA disturbed this network. In the same context, inhibition of MMP‐2 prevented the formation of self‐assembled network, while the inhibition of uPA abolished the over expression and the activity of MMP‐2. This upregulation could initiate the uPA expression and proteolysis processes through the MMP‐2 activity, and may contribute to endothelial cell migration and the formation of this self‐assembled network observed in these 2D co‐cultured cells. J. Cell. Biochem. 114: 650–657, 2013. © 2012 Wiley Periodicals, Inc.     

TIPE1 suppresses invasion and migration through down‐regulating Wnt/β‐catenin pathway in gastric cancer

Epithelial–mesenchymal transition (EMT) plays an important role in the invasiveness and metastasis of gastric cancer. Therefore, identifying key molecules involved in EMT will provide new therapeutic strategy for treating patients with gastric cancer. TIPE1 is a newly identified member of the TIPE (TNFAIP8) family, and its contributions to progression and metastasis have not been evaluated. In this study, we found that the levels of TIPE1 were significantly reduced and inversely correlated with differentiation status and distant metastasis in primary gastric cancer tissues. We further observed overexpression of TIPE1 in aggressive gastric cancer cell lines decreased their metastatic properties both in vitro and in vivo as demonstrated by markedly inhibiting EMT and metastasis of gastric cancer cells in nude mice. Consistently, gene silencing of TIPE1 in well‐differentiated gastric cancer cell line (AGS) inhibited these processes. Mechanistically, we found that TIPE1‐medicated Wnt/β‐catenin signalling was one of the critical signal transduction pathways that link TIPE1 to EMT inhibition. Importantly, TIPE1 dramatically restrained the expression and activities of MMP2 and MMP9 which are demonstrated to promote tumour progression and are implicated in EMT. Collectively, these findings provide new evidence for a better understanding of the biological activities of TIPE1 in progression and metastasis of gastric cancer and suggest that TIPE1 may be an innovative diagnostic and therapeutic target of gastric cancer.     

LncRNA HOXA11‐AS regulates calcium oxalate crystal–induced renal inflammation via miR‐124‐3p/MCP‐1

Long noncoding RNA (lncRNA) has been suggested to play an important role in a variety of diseases over the past decade. In a previous study, we identified a novel lncRNA, termed HOXA11‐AS, which was significantly up‐regulated in calcium oxalate (CaOx) nephrolithiasis. However, the biological function of HOXA11‐AS in CaOx nephrolithiasis remains poorly defined. Here, we demonstrated that HOXA11‐AS was significantly up‐regulated in CaOx nephrolithiasis both in vivo and in vitro. Gain‐/loss‐of‐function studies revealed that HOXA11‐AS inhibited proliferation, promoted apoptosis and aggravated cellular damage in HK‐2 cells exposed to calcium oxalate monohydrate (COM). Further investigations showed that HOXA11‐AS regulated monocyte chemotactic protein 1 (MCP‐1) expression in HK‐2 cell model of CaOx nephrolithiasis. In addition, online bioinformatics analysis and dual‐luciferase reporter assay results showed that miR‐124‐3p directly bound to HOXA11‐AS and the 3'UTR of MCP‐1. Furthermore, rescue experiment results revealed that HOXA11‐AS functioned as a competing endogenous RNA to regulate MCP‐1 expression through sponging miR‐124‐3p and that overexpression of miR‐124‐3p restored the inhibitory effect of proliferation, promotion effects of apoptosis and cell damage induced by HOXA11‐AS overexpression. Taken together, HOXA11‐AS mediated CaOx crystal–induced renal inflammation via the miR‐124‐3p/MCP‐1 axis, and this outcome may provide a good potential therapeutic target for nephrolithiasis.     

MicroRNA‐1275 induces radiosensitization in oesophageal cancer by regulating epithelial‐to‐mesenchymal transition via Wnt/β‐catenin pathway

Acquired radioresistance is one of the main obstacles for the anti‐tumour efficacy of radiotherapy in oesophageal cancer (EC). Recent studies have proposed microRNAs (miRNAs) as important participators in the development of radioresistance in various cancers. Here, we investigated the role of miR‐1275 in acquired radioresistance and epithelial‐mesenchymal transition (EMT) in EC. Firstly, a radioresistant cell line KYSE‐150R was established, with an interesting discovery was observed that miR‐1275 was down‐regulated in KYSE‐150R cells compared to the parental cells. Functionally, miR‐1275 inhibition elevated radioresistance in KYSE‐150 cells via promoting EMT, whereas enforced expression of miR‐1275 increased radiosensitivity in KYSE‐150R cells by inhibiting EMT. Mechanically, we demonstrated that miR‐1275 directly targeted WNT1 and therefore inactivated Wnt/β‐catenin signalling pathway in EC cells. Furthermore, WNT1 depletion countervailed the promoting effect of miR‐1275 suppression on KYSE‐150 cell radioresistance through hampering EMT, whereas WNT1 overexpression rescued miR‐1275 up‐regulation‐impaired EMT to reduce the sensitivity of KYSE‐150R cells to radiation. Collectively, our findings suggested that miR‐1275 suppressed EMT to encourage radiosensitivity in EC cells via targeting WNT1‐activated Wnt/β‐catenin signalling, providing a new therapeutic outlet for overcoming radioresistance of patients with EC.     

Plantamajoside inhibits the proliferation and epithelial‐to‐mesenchymal transition in hepatocellular carcinoma cells via modulating hypoxia‐inducible factor‐1α‐dependent gene expression

As a potential antitumor herbal medicine, plantamajoside (PMS) benefits the treatment of many human malignances. However, the role of PMS in the progression of hepatocellular carcinoma (HCC) and the related molecular mechanisms is still unknown. Here, we proved that the cell viabilities of HepG2 cells were gradually decreased with the increasing concentrations of CoCl₂ and/or PMS via cell counting kit‐8 assay. Meanwhile, 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) and western blot assays were used to further confirm that PMS inhibited the CoCl₂‐induced cell proliferation in HepG2 cells via suppressing the Ki67 and proliferating cell nuclear antigen expressions. We also performed wound‐healing and transwell assays and demonstrated that PMS inhibited CoCl₂‐induced migration and invasion in HepG2 cells via suppressing the epithelial–mesenchymal transition (EMT) process. In addition, the use of 3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzylindazole further proved that PMS inhibited the malignant biological behaviors of HepG2 cells under hypoxic condition by suppressing the hypoxia‐inducible factor‐1α (HIF‐1α) expression. Besides, we further confirmed that PMS suppressed the growth and metastasis of implanted tumors in vivo. Given that PMS suppressed the proliferation and EMT induced by CoCl₂ in HCC cells via downregulating HIF‐1α signaling pathway, we provided evidence that PMS might be a novel anti‐cancer drug for HCC treatment.     

miR‐200/375 control epithelial plasticity‐associated alternative splicing by repressing the RNA‐binding protein Quaking

Members of the miR‐200 family are critical gatekeepers of the epithelial state, restraining expression of pro‐mesenchymal genes that drive epithelial–mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR‐200c and another epithelial‐enriched miRNA, miR‐375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA‐binding protein Quaking (QKI). During EMT, QKI‐5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI‐5 is both necessary and sufficient to direct EMT‐associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial‐derived cancer types. Importantly, several actin cytoskeleton‐associated genes are directly targeted by both QKI and miR‐200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT. These findings demonstrate the existence of a miR‐200/miR‐375/QKI axis that impacts cancer‐associated epithelial cell plasticity through widespread control of alternative splicing.     

Alpha B‐crystallin promotes the invasion and metastasis of gastric cancer via NF‐κB‐induced epithelial‐mesenchymal transition

Alpha B‐crystallin (CRYAB) is overexpressed in a variety of cancers. However, little is known about its specific function and regulatory mechanism in gastric cancer. Here, we first explore the role of CRYAB in gastric cancer progression and metastasis. The expression of CRYAB was determined by western blot and immunohistochemistry in gastric cancer tissues. Besides, methods including stably transfected against CRYAB into gastric cancer cells, western blot, migration and invasion assays in vitro and metastasis assay in vivo were also conducted. The expression of CRYAB is up‐regulated in gastric cancer tissues compared with matched normal tissues. High expression level of CRYAB is closely correlated with cancer metastasis and shorter survival time in patients with gastric cancer. Additionally, CRYAB silencing significantly suppresses epithelial‐mesenchymal transition (EMT), migration and invasion of gastric cancer cells in vitro and in vivo, whereas CRYAB overexpression dramatically reverses these events. Mechanically, CRYAB facilitates gastric cancer cells invasion and metastasis via nuclear factor‐κ‐gene binding (NF‐κB)‐regulated EMT. These findings suggest that CRYAB expression predicts a poor prognosis in patients with gastric cancer. Besides, CRYAB contributes to gastric cancer cells migration and invasion via EMT, mediated by the NF‐κB signalling pathway, thus possibly providing a novel therapeutic target for gastric cancer.     

miR‐516a‐3p inhibits breast cancer cell growth and EMT by blocking the Pygo2/Wnt signalling pathway

miR‐516a‐3p has been reported to play a suppressive role in several types of human tumours. However, the expression level, biological function and fundamental mechanisms of miR‐516a‐3p in breast cancer remain unclear. In the present study, we found that miR‐516a‐3p expression was down‐regulated and Pygopus2 (Pygo2) expression was up‐regulated in human breast cancer tissues and cells. Through analysing the clinicopathological characteristics, we demonstrated that low miR‐516a‐3p expression or positive Pygo2 expression was a predictor of poor prognosis for patients with breast cancer. The results of a dual luciferase reporter assay and Western blot analysis indicated that Pygo2 was a target gene of miR‐516a‐3p. Moreover, overexpression of miR‐516a‐3p inhibited cell growth, migration and invasion as well as epithelial‐mesenchymal transition (EMT) of breast cancer cells, whereas reduced miR‐516a‐3p expression promoted breast cancer cell growth, migration, invasion and EMT. Furthermore, we showed that miR‐516a‐3p suppressed cell proliferation, metastasis and EMT of breast cancer cells by inhibiting Pygo2 expression. We confirmed that miR‐516a‐3p exerted an anti‐tumour effect by inhibiting the activation of the Wnt/β‐catenin pathway. Finally, xenograft tumour models were used to show that miR‐516a‐3p inhibited breast cancer cell growth and EMT via suppressing the Pygo2/Wnt signalling pathway. Taken together, these results show that miR‐516a‐3p inhibits breast cancer cell growth, metastasis and EMT by blocking the Pygo2/ Wnt/β‐catenin pathway.     

Suppression of MAPK signaling in BRAF‐activated PTEN‐deficient melanoma by blocking β‐catenin signaling in cancer‐associated fibroblasts

Cancer‐associated fibroblasts (CAFs) in the tumor microenvironment have been associated with formation of a dynamic and optimized niche for tumor cells to grow and evade cell death induced by therapeutic agents. We recently reported that ablation of β‐catenin expression in stromal fibroblasts and CAFs disrupted their biological activities in in vitro studies and in an in vivo B16F10 mouse melanoma model. Here, we show that the development of a BRAF‐activated PTEN‐deficient mouse melanoma was significantly suppressed in vivo after blocking β‐catenin signaling in CAFs. Further analysis revealed that expression of phospho‐Erk1/2 and phospho‐Akt was greatly reduced, effectively abrogating the activating effects and abnormal cell cycle progression induced by Braf and Pten mutations. In addition, the epithelial–mesenchymal transition (EMT)‐like process was also suppressed in melanoma cells. Taken together, our data highlight an important crosstalk between CAFs and the RAF‐MEK‐ERK signaling cascade in BRAF‐activated melanoma and may offer a new approach to abrogate host‐dependent drug resistance in targeted therapy.     

lncRNA‐POIR promotes epithelial–mesenchymal transition and suppresses sorafenib sensitivity simultaneously in hepatocellular carcinoma by sponging miR‐182‐5p

Sorafenib (SOR) resistance remains a major obstacle in the effective treatment of hepatocellular carcinoma (HCC). A number of long noncoding RNAs (lncRNAs) are responsible for this chemoresistance. This study aimed to reveal the essential function of a recently defined lncRNA, lncRNA‐POIR, in the epithelial–mesenchymal transition (EMT) and SOR sensitivity of HCC cells. SOR‐induced cytotoxicity was analyzed via cell counting kit‐8 and ethynyl‐2'‐deoxyuridine incorporation assays, whereas immunoblotting and confocal immunofluorescence were used to determine the expression levels of EMT markers. Furthermore, loss‐ or gain‐of‐function approaches were used to demonstrate the role of lncRNA‐POIR/miR‐182‐5p on EMT and SOR sensitivity in HCC. The direct interaction between lncRNA‐POIR and miR‐182‐5p was verified using a luciferase reporter assay. We found that knockdown of lncRNA‐POIR sensitized HCC cells to SOR and simultaneously reversed EMT. As expected, miR‐182‐5p was confirmed as the downstream target of lncRNA‐POIR. Moreover, miR‐182‐5p overexpression clearly reversed EMT and promoted SOR‐induced cytotoxicity in representative HCC cells, whereas miR‐182‐5p downregulation played a contrasting role; miR‐182‐5p knockdown abolished the modulatory effects of lncRNA‐POIR siRNA on EMT and SOR sensitivity. Together, these pieces of data suggest that lncRNA‐POIR promotes EMT progression and suppresses SOR sensitivity simultaneously by sponging miR‐182‐5p. Thus, we proposed a compelling rationale for the use of lncRNA‐POIR as a promising predictor of SOR response and as a potential therapeutic target for HCC treatment in the future.     

Truncated O‐glycans promote epithelial‐to‐mesenchymal transition and stemness properties of pancreatic cancer cells

Aberrant expression of Sialyl‐Tn (STn) antigen correlates with poor prognosis and reduced patient survival. We demonstrated that expression of Tn and STn in pancreatic ductal adenocarcinoma (PDAC) is due to hypermethylation of Co re 1 s ynthase specific m olecular c haperone (COSMC) and enhanced the malignant properties of PDAC cells with an unknown mechanism. To explore the mechanism, we have genetically deleted COSMC in PDAC cells to express truncated O‐glycans (SimpleCells, SC) which enhanced cell migration and invasion. Since epithelial‐to‐mesenchymal transition (EMT) play a vital role in metastasis, we have analysed the induction of EMT in SC cells. Expressions of the mesenchymal markers were significantly high in SC cells as compared to WT cells. Equally, we found reduced expressions of the epithelial markers in SC cells. Re‐expression of COSMC in SC cells reversed the induction of EMT. In addition to this, we also observed an increased cancer stem cell population in SC cells. Furthermore, orthotopic implantation of T3M4 SC cells into athymic nude mice resulted in significantly larger tumours and reduced animal survival. Altogether, these results suggest that aberrant expression of truncated O‐glycans in PDAC cells enhances the tumour aggressiveness through the induction of EMT and stemness properties.