MicroRNA‐302c modulates peritoneal dialysis‐associated fibrosis by targeting connective tissue growth factor

Long‐term peritoneal dialysis (PD) can lead to the induction of mesothelial/epithelial‐mesenchymal transition (MMT/EMT) and fibrosis; these effects eventually result in ultrafiltration failure and the discontinuation of PD. MicroRNA‐302c (miR‐302c) is believed to be involved in regulating tumour cell growth and metastasis by suppressing MMT, but the effect of miR‐302c on MMT in the context of PD is unknown. MiR‐302c levels were measured in mesothelial cells isolated from the PD effluents of continuous ambulatory peritoneal dialysis patients. After miR‐302c overexpression using lentivirus, human peritoneal mesothelial cell line (HMrSV5) and PD mouse peritoneum were treated with TGF‐β1 or high glucose peritoneal dialysate respectively. MiR‐302c expression level and MMT‐related factors alteration were observed. In addition, fibrosis of PD mouse peritoneum was alleviated by miR‐302c overexpression. Furthermore, the expression of connective tissue growth factor (CTGF) was negatively related by miR‐302c, and LV‐miR‐302c reversed the up‐regulation of CTGF induced by TGF‐β1. These data suggest that there is a novel TGF‐β1/miR‐302c/CTGF pathway that plays a significant role in the process of MMT and fibrosis during PD. MiR‐302c might be a potential biomarker for peritoneal fibrosis and a novel therapeutic target for protection against peritoneal fibrosis in PD patients.     

Periostin down‐regulation attenuates the pro‐fibrogenic response of hepatic stellate cells induced by TGF‐β1

Liver fibrosis is characterized by an exacerbated accumulation of deposition of the extracellular matrix (ECM), and the activation of hepatic stellate cells (HSC) plays a pivotal role in the development of liver fibrosis. Periostin has been shown to regulate cell adhesion, proliferation, migration and apoptosis; however, the involvement of periostin and its role in transforming growth factor (TGF)‐β1‐induced HSC activation remains unclear. We used RT‐PCR and Western blot to evaluate the expression level of periostin in hepatic fibrosis tissues and HSCs, respectively. Cell proliferation was determined using the Cell Proliferation ELISA BrdU kit, cell cycle was analysed by flow cytometry. The expression of α‐smooth muscle actin (α‐SMA), collagen I, TGF‐β1, p‐Smad2 and p‐Smad3 were determined by western blot. Our study found that periostin was up‐regulated in liver fibrotic tissues and activated HSCs. In addition, siRNA‐periostin suppressed TGF‐β1‐induced HSC proliferation. The HSC transfected with siRNA‐periostin significantly inhibited TGF‐β1‐induced expression levels of α‐SMA and collagen I. Furthermore, TGF‐β1 stimulated the expression of periostin, and siRNA‐periostin attenuated TGF‐β1‐induced Smad2/3 activation in HSCs. These results suggest that periostin may function as a novel regulator to modulate HSC activation, potentially by promoting the TGF‐β1/Smad signalling pathway, and propose a strategy to target periostin for the treatment of liver fibrosis.     

GP73 promotes invasion and metastasis of bladder cancer by regulating the epithelial–mesenchymal transition through the TGF‐β1/Smad2 signalling pathway

This study investigated the effects of Golgi membrane protein 73 (GP73) on the epithelial–mesenchymal transition (EMT) and on bladder cancer cell invasion and metastasis through the TGF‐β1/Smad2 signalling pathway. Paired bladder cancer and adjacent tissue samples (102) and normal bladder tissue samples (106) were obtained. Bladder cancer cell lines (T24, 5637, RT4, 253J and J82) were selected and assigned to blank, negative control (NC), TGF‐β, thrombospondin‐1 (TSP‐1), TGF‐β1+ TSP‐1, GP73‐siRNA‐1, GP73‐siRNA‐2, GP73‐siRNA‐1+ TSP‐1, GP73‐siRNA‐1+ pcDNA‐GP73, WT1‐siRNA and WT1‐siRNA + GP73‐siRNA‐1 groups. Expressions of GP73, TGF‐β1, Smad2, p‐Smad2, E‐cadherin and vimentin were detected using RT‐qPCR and Western blotting. Cell proliferation, migration and invasion were determined using MTT assay, scratch testing and Transwell assay, respectively. Compared with the blank and NC groups, levels of GP73, TGF‐β1, Smad2, p‐Smad2, N‐cadherin and vimentin decreased, and levels of WT1 and E‐cadherin increased in the GP73‐siRNA‐1 and GP73‐siRNA‐2 groups, while the opposite results were observed in the WT1 siRNA, TGF‐β, TSP‐1 and TGF‐β + TSP‐1 groups. Cell proliferation, migration and invasion notably decreased in the GP73‐siRNA‐1 and GP73‐siRNA‐2 groups in comparison with the blank and NC groups, while in the WT1 siRNA, TGF‐β, TSP‐1 and TGF‐β + TSP‐1 groups, cell migration, invasion and proliferation showed the reduction after the EMT. These results suggest that GP73 promotes bladder cancer invasion and metastasis by inducing the EMT through down‐regulating WT1 levels and activating the TGF‐β1/Smad2 signalling pathway.     

RAR‐Related Orphan Receptor Gamma (ROR‐γ) Mediates Epithelial‐Mesenchymal Transition Of Hepatocytes During Hepatic Fibrosis

The epithelial‐mesenchymal transition (EMT) is involved in many different types of cellular behavior, including liver fibrosis. In this report, we studied a novel function of RAR‐related orphan receptor gamma (ROR‐γ) in hepatocyte EMT during liver fibrosis. To induce EMT in vitro, primary hepatocytes and FL83B cells were treated with TGF‐β1. Expression of ROR‐γ was analyzed by Western blot in the fibrotic mouse livers and human livers with cirrhosis. To verify the role of ROR‐γ in hepatocyte EMT, we silenced ROR‐γ in FL83B cells using a lentiviral short hairpin RNA (shRNA) vector. The therapeutic effect of ROR‐γ silencing was investigated in a mouse model of TAA‐induced fibrosis by hydrodynamic injection of plasmids. ROR‐γ expression was elevated in hepatocyte cells treated with TGF‐β1, and ROR‐γ protein levels were elevated in the fibrotic mouse livers and human livers with cirrhosis. Knockdown of ROR‐γ resulted in the attenuation of TGF‐β1‐induced EMT in hepatocytes. Strikingly, ROR‐γ bound to ROR‐specific DNA response elements (ROREs) in the promoter region of TGF‐β type I receptor (Tgfbr1) and Smad2, resulting in the downregulation of Tgfbr1 and Smad2 after silencing of ROR‐γ. Therapeutic delivery of shRNA against ROR‐γ attenuated hepatocyte EMT and ameliorated liver fibrosis in a mouse model of TAA‐induced liver fibrosis. Overall, our results suggest that ROR‐γ regulates TGF‐β‐induced EMT in hepatocytes during liver fibrosis. We suggest that ROR‐γ may become a potential therapeutic target in treating liver fibrosis. J. Cell. Biochem. 118: 2026–2036, 2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.     

Platelet derived growth factor B and epithelial mesenchymal transition of peritoneal mesothelial cells

Platelet derived growth factor (PDGF) is involved in wound healing in various organ systems. Its potential role in the context of peritoneal injury following long-term peritoneal dialysis is unclear. We used an adenovirus expressing the B chain of PDGF (AdPDGF-B) to assess its effect on pro-fibrotic pathways in the peritoneal membrane. To assess the transforming growth factor (TGF) β independent effects of PDGF, we over-expressed PDGF-B in the peritoneum of either wild-type mice (Smad3^+/+) or those with a deletion of the TGFβ signaling protein Smad3 (Smad3^?/?). PDGF-B induced sustained angiogenesis in both Smad3^+/+ and Smad3^?/? mice. Despite increased collagen gene expression, collagen accumulation was transient and fibrogenesis was associated with induction of collagenase activity. We observed epithelial to mesenchymal transition (EMT) involving the peritoneal mesothelial cells, as shown by increased SNAIL and decreased E-Cadherin expression with evidence of mesothelial cells expressing both epithelial and mesenchymal markers. Unlike TGFβ-induced EMT, PDGF-B exposure did not lead to mobilization of the mesothelial cells; they remained as a single monolayer throughout the observation period. This “non-invasive” EMT phenomenon is a novel finding and may have implications concerning the role of EMT in peritoneal fibrosis and injury to other organ systems. The observed effects were similar in Smad3^?/? and Smad3^+/+ animals, suggesting that the PDGF-B effects were independent of TGFβ or Smad signaling.     

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.     

Regulatory mechanisms of TGF‐β1‐induced fibrogenesis of human alveolar epithelial cells

Pulmonary fibrosis is characterized by an extensive activation of fibrogenic cells and deposition of extracellular matrix (ECM). Transforming growth factor (TGF)‐β1 plays a pivotal role in the pathogenesis of pulmonary fibrosis, probably through the epithelial‐ to‐mesenchymal transition (EMT) and ECM production. The present study investigates potential mechanism by which TGF‐β1 induces EMT and ECM production in the fibrogenesis of human lung epithelial cells during pulmonary fibrosis. The expression of EMT phenotype and other proteins relevant to fibrogenesis were measured and the cell bio‐behaviours were assessed using Cell‐IQ Alive Image Monitoring System. We found that TGF‐β1‐induced EMT was accompanied with increased collagen I deposition, which may be involved in the regulation of connective tissue growth factor (CTGF) and phosphoinositide 3‐kinase (PI3K) signalling pathway. Treatment with PI3K inhibitors significantly attenuated the TGF‐β1‐ induced EMT, CTGF expression and collagen I synthesis in lung epithelial cells. The interference of CTGF expression impaired the basal and TGF‐β1‐stimulated collagen I deposition, but did not affect the process of EMT. Our data indicate that the signal pathway of TGF‐β1/PI3K/CTGF plays an important role in the fibrogenesis of human lung epithelial cells, which may be a novel therapeutic approach to prevent and treat pulmonary fibrosis.     

Acetyl‐11‐keto‐β‐boswellic acid ameliorates renal interstitial fibrosis via Klotho/TGF‐β/Smad signalling pathway

Acetyl‐11‐keto‐β‐boswellic acid (AKBA), an active triterpenoid compound from the extract of Boswellia serrate, has been reported previously in our group to alleviate fibrosis in vascular remodelling. This study aimed to elucidate the in vivo and in vitro efficacy and mechanism of AKBA in renal interstitial fibrosis. The experimental renal fibrosis was produced in C57BL/6 mice via unilateral ureteral obstruction (UUO). Hypoxia‐induced HK‐2 cells were used to imitate the pathological process of renal fibrosis in vitro. Results showed that the treatment of AKBA significantly alleviated UUO‐induced impairment of renal function and improved the renal fibrosis by decreasing the expression of TGF‐β1, α‐SMA, collagen I and collagen IV in UUO kidneys. In hypoxia‐induced HK‐2 cells, AKBA displayed remarkable cell protective effects and anti‐fibrotic properties by increasing the cell viability, decreasing the lactate dehydrogenase (LDH) release and inhibiting fibrotic factor expression. Moreover, in obstructed kidneys and HK‐2 cells, AKBA markedly down‐regulated the expression of TGFβ‐RI, TGFβ‐RII, phosphorylated‐Smad2/3 (p‐Smad2/3) and Smad4 in a dose‐dependent fashion while up‐regulated the expression of Klotho and Smad7 in the same manner. In addition, the effects of AKBA on the Klotho/TGF‐β/Smad signalling were reversed by transfecting with siRNA‐Klotho in HK‐2 cells. In conclusion, our findings provide evidence that AKBA can effectively protect kidney against interstitial fibrosis, and this renoprotective effect involves the Klotho/TGF‐β/Smad signalling pathway. Therefore, AKBA could be considered as a promising candidate drug for renal interstitial fibrosis.     

Astragaloside IV modulates TGF‐β1‐dependent epithelial‐mesenchymal transition in bleomycin‐induced pulmonary fibrosis

Epithelial‐mesenchymal transition (EMT) plays an important role in idiopathic pulmonary fibrosis (IPF). Astragaloside IV (ASV), a natural saponin from astragalus membranaceus, has shown anti‐fibrotic property in bleomycin (BLM)‐induced pulmonary fibrosis. The current study was undertaken to determine whether EMT was involved in the beneficial of ASV against BLM‐induced pulmonary fibrosis and to elucidate its potential mechanism. As expected, in BLM‐induced IPF, ASV exerted protective effects on pulmonary fibrosis and ASV significantly reversed BLM‐induced EMT. Intriguing, transforming growth factor‐β1 (TGF‐β1) was found to be up‐regulated, whereas Forkhead box O3a (FOXO3a) was hyperphosphorylated and less expressed. However, ASV treatment inhibited increased TGF‐β1 and activated FOXO3a in lung tissues. TGF‐β1 was administered to alveolar epithelial cells A549 to induce EMT in vitro. Meanwhile, stimulation with TGF‐β1‐activated phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) pathway and induced FOXO3a hyperphosphorylated and down‐regulated. It was found that overexpression of FOXO3a leading to the suppression of TGF‐β1‐induced EMT. Moreover, ASV treatment, similar with the TGF‐β1 or PI3K/Akt inhibitor, reverted these cellular changes and inhibited EMT in A549 cells. Collectively, the results suggested that ASV significantly inhibited TGF‐β1/PI3K/Akt‐induced FOXO3a hyperphosphorylation and down‐regulation to reverse EMT during the progression of fibrosis.     

PAX3 silencing inhibits prostate cancer progression through the suppression of the TGF‐β/Smad signaling axis

Multiple studies have confirmed the pro‐oncogenic effects of PAX3 in an array of cancers, but its role in prostate cancer (PCa) remains largely undefined. The aim of this study is to investigate the role of PAX3 in PCa. PAX3 expression was compared between PCa tumor tissue and nontumor tissues and PCa cell lines and normal prostate epithelial cells (PNT2) by western blot analysis and immunohistochemistry staining. MTT and immunofluorescence assays were used to detect PCa cell proliferation. Flow cytometry was used to evaluate cell apoptosis in PCa. Transwell assays were used for the determination of cell migration and PCa cell invasion. PAX3 expression was higher in PCa tissues and human PCa cell lines. Moreover, PAX3 silencing inhibited the proliferation, metastasis, and epithelial–mesenchymal transition (EMT) of PCa cells, and increased the rates of apoptosis. PAX3 silencing inhibited transforming growth factor‐β (TGF‐β)/Smad signaling in PCa cells. The effects of si‐PAX3 on the proliferation, apoptosis, metastasis, and EMT of PCa cells were alleviated by TGF‐β1 treatment. PAX3 silencing inhibits PCa progression through the inhibition of TGF‐β/Smad signaling. This reveals PAX3 as a novel biomarker and therapeutic target for future PCa treatments.     

SIRT1 inhibits TGF‐β‐induced endothelial‐mesenchymal transition in human endothelial cells with Smad4 deacetylation

Endothelial‐mesenchymal transition (EndMT) plays a pivotal role in organ fibrosis. This study examined the effect of SIRT1 on transforming growth factor beta (TGF‐β)‐induced EndMT in human endothelial cells (ECs) and its probable molecular mechanism. We assessed EndMT by immunofluorescence staining, quantitative real‐time polymerase chain reaction, Western blotting, and migration and invasion assays. Adenovirus was used to overexpress or knockdown SIRT1 in ECs. The regulatory relationship between SIRT1 and Smad4 was analyzed by coimmunoprecipitation assay. We found that SIRT1 was decreased in TGF‐β‐induced EndMT, and SIRT1 inhibited TGF‐β‐induced EndMT through deacetylating Smad4. Our findings suggest that SIRT1 has an important role in inhibiting EndMT by regulating the TGF‐β/Smad4 pathway in human ECs and, thus, protecting against fibrosis.     

miR‐193a/b‐3p relieves hepatic fibrosis and restrains proliferation and activation of hepatic stellate cells

MicroRNAs (miRNAs) have been confirmed to participate in liver fibrosis progression and activation of hepatic stellate cells (HSCs). In this study, the role of miR‐193a/b‐3p in concanavalin A (ConA)‐induced liver fibrosis in mice was evaluated. According to the results, the expression of miR‐193a/b‐3p was down‐regulated in liver tissues after exposure to ConA. Lentivirus‐mediated overexpression of miR‐193a/b‐3p reduced ConA‐induced liver injury as demonstrated by decreasing ALT and AST levels. Moreover, ConA‐induced liver fibrosis was restrained by the up‐regulation of miR‐193a/b‐3 through inhibiting collagen deposition, decreasing desmin and proliferating cell nuclear antigen (PCNA) expression and lessening the content of hydroxyproline, transforming growth factor‐β1 (TGF‐β1) and activin A in liver tissues. Furthermore, miR‐193a/b‐3p mimics suppressed the proliferation of human HSCs LX‐2 via inducing the apoptosis of LX‐2 cells and lowering the levels of cell cycle‐related proteins Cyclin D1, Cyclin E1, p‐Rb and CAPRIN1. Finally, TGF‐β1 and activin A‐mediated activation of LX‐2 cells was reversed by miR‐193a/b‐3p mimics via repressing COL1A1 and α‐SMA expression, and restraining the activation of TGF‐β/Smad2/3 signalling pathway. CAPRIN1 and TGF‐β2 were demonstrated to be the direct target genes of miR‐193a/b‐3p. We conclude that miR‐193a/b‐3p overexpression attenuates liver fibrosis through suppressing the proliferation and activation of HSCs. Our data suggest that miR‐193a‐3p and miR‐193b‐3p may be new therapeutic targets for liver fibrosis.     

Tetraspanin 1 as a mediator of fibrosis inhibits EMT process and Smad2/3 and beta‐catenin pathway in human pulmonary fibrosis

Tetraspanin 1(TSPAN1) as a clinically relevant gene target in cancer has been studied, but there is no direct in vivo or vitro evidence for pulmonary fibrosis (PF). Using reanalysing Gene Expression Omnibus data, here, we show for the first time that TSPAN1 was markedly down‐regulated in lung tissue of patient with idiopathic PF (IPF) and verified the reduced protein expression of TSPAN1 in lung tissue samples of patient with IPF and bleomycin‐induced PF mice. The expression of TSPAN1 was decreased and associated with transforming growth factor‐β1 (TGF‐β₁)‐induced molecular characteristics of epithelial‐to‐mesenchymal transition (EMT) in alveolar epithelial cells (AECs). Silencing TSPAN1 promoted cell migration, and the expression of alpha‐smooth muscle actin, vimentin and E‐cadherin in AECs with TGF‐β₁ treatment, while exogenous TSPAN1 has the converse effects. Moreover, silencing TSPAN1 promotes the phosphorylation of Smad2/3 and stabilizes beta‐catenin protein, however, overexpressed TSPAN1 impeded TGF‐β₁‐induced activation of Smad2/3 and beta‐catenin pathway in AECs. Together, our study implicates TSPAN1 as a key regulator in the process of EMT in AECs of IPF.     

Petchiether A attenuates obstructive nephropathy by suppressing TGF‐β/Smad3 and NF‐κB signalling

Obstructive nephropathy is the end result of a variety of diseases that block drainage from the kidney(s). Transforming growth factor‐β1 (TGF‐β1)/Smad3‐driven renal fibrosis is the common pathogenesis of obstructive nephropathy. In this study, we identified petchiether A (petA), a novel small‐molecule meroterpenoid from Ganoderma, as a potential inhibitor of TGF‐β1‐induced Smad3 phosphorylation. The obstructive nephropathy was induced by unilateral ureteral obstruction (UUO) in mice. Mice received an intraperitoneal injection of petA/vehicle before and after UUO or sham operation. An in vivo study revealed that petA protected against renal inflammation and fibrosis by reducing the infiltration of macrophages, inhibiting the expression of proinflammatory cytokines (interleukin‐1β and tumour necrosis factor‐α) and reducing extracellular matrix deposition (α‐smooth muscle actin, collagen I and fibronectin) in the obstructed kidney of UUO mice; these changes were associated with suppression of Smad3 and NF‐κB p65 phosphorylation. Petchiether A inhibited Smad3 phosphorylation in vitro and down‐regulated the expression of the fibrotic marker collagen I in TGF‐β1‐treated renal epithelial cells. Further, we found that petA dose‐dependently suppressed Smad3‐responsive promoter activity, indicating that petA inhibits gene expression downstream of the TGF‐β/Smad3 signalling pathway. In conclusion, our findings suggest that petA protects against renal inflammation and fibrosis by selectively inhibiting TGF‐β/Smad3 signalling.