| Hepatic fibrosis,characterized by excessive accumulation and abnormal distribution of extracellular matrix（ECM）components such as collagen and glycoproteins,is a common pathological result of diffuse injury of hepatocytes caused by various chronic liver diseases.Activation of hepatic stellate cells（HSCs）is the most important step in the process of hepatic fibrosis.In general,inhibition of cell proliferation,induction of apoptosis and senescence are potential therapies to block HSCs activation.Spontaneous recovery of liver fibrosis includes degradation of ECM protein,apoptosis of activated HSCs and regeneration of liver cells.Activated HSCs apoptosis plays a key role in the fibrosis reversal process.Therefore,regulating the apoptosis of activated HSCs may help to treat liver fibrosis.Tumor necrosis factor-related apoptosis-inducing ligand（TRAIL）can effectively induce apoptosis of activated HSCs in the recovery stage of liver fibrosis.Normal HSCs were resistant to TRAIL cytotoxicity.Therefore,enhancing the sensitivity of TRAIL induced activation of HSCs apoptosis may be helpful for the treatment of liver fibrosis.Micro RNAs（miRNAs）are a class of endogenous non-coding RNAs with19-25 nucleotides in size,was involved in the occurrence and development of various liver diseases,including liver fibrosis,cirrhosis and liver cancer.As a widely recognized tumor suppressor,miR-145 has also been reported to play an important role in liver fibrosis,hepatitis,and HCC.Previous studies in our group have shown that miR-145 inhibited the activation and proliferation of HSCs by targeting ZEB2,and restricts the further development of liver fibrosis.However,whether miR-145 was involved in TRAIL-induced apoptosis of activated HSCs in the course of liver fibrosis has not been clarified.LncRNAs（long non-coding RNA）are a class of RNA molecules with transcripts of more than 200 nt.They regulate the concentration and biological functions of miRNAs through competitive endogenous RNAs,and participate in a series of biological processes including cell proliferation,apoptosis and differentiation.Through bioinformatics prediction,this study found that miR-145 may directly bind to linc-ROR,and predicted the existence of three possible binding sites.However,the specific role of linc-ROR/miR-145 in TRAIL-induced apoptosis of activated HSCs remained unclear.Therefore,this study aimed to investigate the effect of linc-ROR/miR-145/ZEB2on TRAIL-induced apoptosis of activated HSCs and its possible mechanism.The research contents are as follows:1.Expression of miR-145,ZEB2 and linc-ROR in liver fibrosisqRT-PCR and Western blot were used to detect the expression of miR-145,ZEB2and linc-ROR in liver fibrosis tissues.The results showed that the expression of miR-145 was significantly decreased,while the expression of linc-ROR and ZEB2 was significantly increased in the liver tissues of CCl4-induced hepatic fibrosis mice and LX-2 cells treated with TGF-β1 in vitro.2.Effect of miR-145 on TRAIL-induced apoptosis of activated LX-2 cellsLX-2 cells transfected with miR-145 inhibitor or miR-145 mimics were treated with TGF-β1（10 ng/ml）and TRAIL（10 ng/ml）,and cell apoptosis was detected by flow cytometry.The results showed that TRAIL increased the apoptosis rate of activated LX-2 cells.Silencing miR-145 significantly reduced the apoptosis rate and cleaved caspase-3 protein levels of TRAIL treated activated LX-2 cells,and increasedα-SMA and Col.I levels.However,overexpression of miR-145 significantly increased the apoptosis rate and cleaved caspase-3 protein levels of TRAIL treated activated LX-2 cells,and decreasedα-SMA and Col.I protein levels.3.Effect of miR-145-targeted ZEB2 on TRAIL induced apoptosis of activated LX-2cellsBioinformatics tools were used to predict the target genes of miR-145,and the results showed that ZEB2 was a potential target of miR-145.In order to further study the role of ZEB2 in TRAIL-induced apoptosis of activated LX-2 cells,LX-2 cells were transfected with ZEB2-siRNA and ZEB2-OE.Flow cytometry and western blot analysis showed that TRAIL could significantly increase the apoptosis rate of LX-2cells treated with TGF-β1.Overexpression of ZEB2 decreased the effect of TRAIL on apoptosis rate and cleaved caspase-3 protein leve of activated LX-2 cells,and promotedα-SMA and Col.I levels.Double luciferase reporter gene assay was used to detect ZEB2 gene 3’-UTR luciferase wild-type plasmid vector,which was co-transfected into LX-2 cells with miR-145 mimics.The results showed that the co-transfection of miR-145 mimics and ZEB2 wild-type fluorescent plasmid（ZEB2-WT）significantly reduced the relative fluorescence expression of ZEB2 in LX-2.LX-2 was transfected with miR-145 mimics or miR-145 inhibitor and corresponding negative control,and the expression of ZEB2was detected by q RT-PCR and Western blot.The results showed that miR-145 mimics inhibited the expression of ZEB2 mRNA and protein in LX-2 cells.Western blot further detected whether miR-145 enhanced TRAIL-induced apoptosis of LX-2 cells by targeting ZEB2,and the results showed that,compared with miR-145 inhibitor group,cleaved caspase-3 protein levels were significantly increased in LX-2 cells co-transfected with miR-145 inhibitor and ZEB2-siRNA.4.Effects of ZEB2 on NF-κB activity in TRAIL induced apoptosis of activated LX-2cellsActivation of transcription factor NF-κB plays a key role in HSCs apoptosis.To test whether ZEB2 plays a role in TRAIL-induced apoptosis of LX-2 cells by regulating the activity of NF-κB signaling pathway,we treated LX-2 cells with ZEB2-siRNA or ZEB2-OE.Western blot results showed that after transfection with ZEB2-siiRNA,TRAIL-induced activation of LX-2 cells significantly increased phospho-IκBαand phospho-p65 protein levels.In contrast,ZEB2 overexpression reduces phospho-IκBαand phospho-p65 protein levels.These results suggested that ZEB2 may induce apoptosis of activated LX-2 cells through TRAIL mediated NF-κB signaling pathway.5.Effects of linc-ROR/miR-145/ZEB2 on TRAIL induced apoptosis of HSCsBioinformatics analysis revealed that miR-145 was a potential target of linc-ROR.To investigate whether linc-ROR affects TRAIL-induced apoptosis of activated LX-2 cells,LX-2 cells treated with TGF-β1（10 ng/m L）and TRAIL（10ng/ml）and than transfected with linc-ROR-siRNA or linc-ROR-OE.Results of western blot showed that silencing linc-ROR significantly promoted apoptosis rate and cleaved caspase-3 protein levels of TRAIL treated LX-2 cells,and decreasedα-SMA and Col.I levels.To further explore the regulation of linc-ROR on miR-145 in TRAIL-induced apoptosis of LX-2 cells,LX-2 cells were treated with TGF-β1（10 ng/ml）and TRAIL（10 ng/ml）and then transfected with linc-ROR-siRNA or linc-ROR-OE.The expression of miR-145 was detected by q RT-PCR,and the results showed that silencing linc-ROR significantly increased the expression of miR-145.Flow cytometry showed that silencing linc-ROR significantly increased the apoptosis rate of TRAIL-treated activated LX-2 cells,while overexpression of linc-ROR significantly reduced the apoptosis rate of TRAIL-treated activated LX-2 cells.LX-2 cells co-transfected with miR-145 mimics and linc-ROR-OE were treated with TGF-β1（10 ng/ml）and TRAIL（10 ng/ml）,and cleaved caspase-3 expression was detected by Western blot.Compared with overexpression of linc-ROR,simultaneous overexpression of linc-ROR and miR-145 significantly promoted apoptosis rate and cleaved caspase-3 protein levels of TRAIL-treated activated LX-2 cells.These results suggested that linc-ROR down-regulated the sensitivity of TRAIL-induced apoptosis of activated LX-2 cells through miR-145/ZEB2 in the course of liver fibrosis. |
