miR-21 overexpression enhances TGF-β1-induced epithelial-to-mesenchymal transition by target smad7 and aggravates renal damage in diabetic nephropathy.

Epithelial-to-mesenchymal transition (EMT) plays an important role in renal interstitial fibrosis (RIF) with diabetic nephropathy (DN). Smad7 (a inhibitory smad), a downstream signaling molecules of TGF-β1, represses the EMT. The physiological function of miR-21 is closely linked to EMT and RIF. However, it remained unclear whether miR-21 over-expression affected TGF-β1-induced EMT by regulating smad7 in DN. In this study, real-time RT-PCR, cell transfection, luciferase reporter gene assays, western blot and confocal microscope were used, respectively. Here, we found that miR-21 expression was upregulated by TGF-β1 in time- and concentration -dependent manner. Moreover, miR-21 over-expression enhanced TGF-β1-induced EMT(upregulation of a-SMA and downregulation of E-cadherin) by directly down-regulating smad7/p-smad7 and indirectly up-regulating smad3/p-smad3, accompanied by the decrease of Ccr and the increase of col-IV, FN, the content of collagen fibers, RTBM, RTIAW and ACR. Meantime, the siRNA experiment showed that smad7 can directly regulate a-SMA and E-cadherin expression. More importantly, miR-21 inhibitor can not only inhibit EMT and fibrosis but also ameliorate renal structure and function. In conclusion, our results demonstrated that miR-21 overexpression can contribute to TGF-β1-induced EMT by inhibiting target smad7, and that targeting miR-21 may be a better alternative to directly suppress TGF-β1-mediated fibrosis in DN.

Wang JY ，Gao YB ，Zhang N ，Zou DW ，Wang P ，Zhu ZY ，Li JY ，Zhou SN ，Wang SC ，Wang YY ，Yang JK ... -  《-》

Tongxinluo ameliorates renal structure and function by regulating miR-21-induced epithelial-to-mesenchymal transition in diabetic nephropathy.

Wang JY ，Gao YB ，Zhang N ，Zou DW ，Xu LP ，Zhu ZY ，Li JY ，Zhou SN ，Cui FQ ，Zeng XJ ，Geng JG ，Yang JK ... -  《-》

BMP-7 inhibits renal fibrosis in diabetic nephropathy via miR-21 downregulation.

Epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition in renal tubular epithelial cells are critical to diabetic nephropathy (DN) pathogenesis, but the underlying mechanisms remain undefined. Bone morphogenetic protein 7 (BMP-7) inhibits EMT and ECM accumulation in renal tubular epithelial cells cultured in presence of high glucose. Meanwhile, miRNA-21 (miR-21) downregulates Smad7, promoting EMT and ECM deposition. However, the association of BMP-7 with miR-21/Smad7 in DN is unknown. Here, NRK-52E cells incubated in presence of high glucose and STZ-induced C57BL diabetic mice were considered in vitro and in vivo models of DN, respectively. In both models, BMP-7 (mRNA/protein) amounts were decreased as well as Smad7 protein expression, while miR-21 expression and TGF-β1/Smad3 pathway activation were enhanced, accompanied by enhanced EMT and ECM deposition. Further, addition of BMP-7 human recombinant cytokine (rhBMP-7) and injection of the BMP-7 overexpression plasmid in diabetic mice markedly downregulated miR-21 and upregulated Smad7, reduced Smad3 activation without affecting TGF-β1 amounts, and prevented EMT and ECM accumulation. MiR-21 overexpression in the in vitro model downregulated Smad7, promoted EMT and ECM accumulation without affecting BMP-7 amounts, and miR-21 downregulation reversed it. By interfering with BMP-7 and miR-21 expression in high glucose conditions, miR-21 amounts and Smad3 phosphorylation were further decreased. Smad7 was then upregulated, and EMT and ECM deposition were inhibited; these effects were reversed after miR-21 overexpression. These findings suggest that BMP-7 decreases renal fibrosis in DN by regulating miR-21/Smad7 signaling, providing a theoretical basis for the development of novel and effective therapeutic drugs for DN.

Liu L ，Wang Y ，Yan R ，Liang L ，Zhou X ，Liu H ，Zhang X ，Mao Y ，Peng W ，Xiao Y ，Zhang F ，Liu L ，Shi M ，Guo B ... -  《-》

miR-133b and miR-199b knockdown attenuate TGF-β1-induced epithelial to mesenchymal transition and renal fibrosis by targeting SIRT1 in diabetic nephropathy.

Transforming growth factor-β1 (TGF-β1)-induced epithelial to mesenchymal transition (EMT) and renal fibrosis plays critical role in the development and progression of diabetic nephropathy (DN). Our study aimed to determine the detailed roles of miR-133b & miR-199b on TGF-β1-induced EMT & renal fibrosis in DN and its underlying mechanism. The expressions of miR-133b & miR-199b in OLETF rats, LETO rats & TGF-β1-treated human proximal tubule cell line (HK-2) were examined by qRT-PCR. Inhibition of miR-133b or miR-199b was realized in cells by transfection of lentivirus containing miR-133b inhibit or miR-199b inhibitor. The expression levels of collagen I (COL I), fibronectin (FN), α-smooth muscle actin (α-SMA), E-cadherin & sirtuin 1 (SIRT1) were detected by western blot and immunohistochemistry. Masson staining was conducted to estimate the degree of renal fibrosis. The interaction between SIRT1 and miR-133b, miR-199b was explored by luciferase reporter assay and RNA immunoprecipitation (RIP) assay. miR-133b and miR-199b were highly expressed in the renal cortex of diabetic OLETF rats and TGF-β1-treated HK-2 cells. EMT and renal fibrosis were induced in diabetic OLETF rats and TGF-β1-treated HK-2 cells. Inhibition of miR-133b and miR-199b attenuated EMT and renal fibrosis in diabetic OLETF rats and TGF-β1-treated HK-2 cells. In addition, SIRT1 was identified as a target of miR-133b & miR-199b in HK-2 cells. SIRT1 knockdown dramatically reversed the suppression on TGF-β1-induced EMT and renal fibrosis in HK-2 cells mediated by anti-miR-133b or anti-miR-199. Inhibition of miR-133b & miR-199b attenuated TGF-β1-induced EMT & renal fibrosis by upregulating SIRT1 shows that using different miRNAs is a potential strategy for the future treatment of DN.

Sun Z ，Ma Y ，Chen F ，Wang S ，Chen B ，Shi J ... -  《-》

Up-regulation of microRNA-93 inhibits TGF-β1-induced EMT and renal fibrogenesis by down-regulation of Orai1.

TGF-β1-induced excessive deposition of ECM and EMT process of tubular epithelial cells play critical roles in the development and progression of fibrosis in diabetic nephropathy (DN). Orai1 has been demonstrated to be involved in TGF-β1-induced EMT via TGF-β/Smad3 pathway. We are aimed to explore the effects of miR-93 on TGF-β1-induced EMT process in HK2 cells. In this study, our data showed that miR-93 was dramatically decreased in renal tissues of patients with DN and TGF-β1-stimulated HK2 cells. Moreover, the decreased level of miR-93 was closely associated with the increased expression of Orai1. Overexpression of miR-93 decreased Orai1 expression, and then suppressed TGF-β1-mediated EMT and fibrogenesis. Next, we predicted that the Orai1 was a potential target gene of miR-93, and demonstrated that miR-93 could directly target Orai1. SiRNA targeting Orai1 was sufficient to suppress TGF-β1-induced EMT and fibrogenesis in HK2 cells. Furthermore, Overexpression of Orai1 partially reversed the protective effect of miR-93 overexpression on TGF-β1-mediated EMT and fibrogenesis in HK2 cells. Taken together, Orai1 and miR-93 significantly impact on the progression of TGF-β1-mediated EMT and fibrogenesis in HK2 cells, and they may represent novel targets for the prevention strategies of fibrosis in the context of DN.

Ma J ，Zhang L ，Hao J ，Li N ，Tang J ，Hao L ... -  《-》