Angâ…¡ Stimulated SMAD Pathway Byupregulating Micro RNA-21Expression In The Process Of Liver Fibrosis

BackgroundLiver fibrosis is a secondary Compensatory response in the process of liver injury and tissue repair after inflammation caused by various chronic pathogenic factors, which is pathological characteristic of extracellular matrix (extracellular matrix, EMC) excess deposits and is the common pathological changes and the only way from every kind of liver disease to liver fibrosis.Numerous studies have shown that local tissue renin-angiotensin system (RAS) is closely related to liver fibrosis, RAS significantly raised in hepatic fibrosis tissues, angiotensin Ⅱ (AngⅡ) is one of the important components of RAS, stimulating hepatic stellate cell proliferation and collagen synthesis, therefore AngⅡ is one of the important material for liver fibrosis. In recent years, another new important component of RAS:angiotensin-converting enzyme2(ACE2)-angiotensin1-7(Ang (1-7))-Mas receptor shaft aroused people’s concern, Which negatively regulate the shaft of the ACE-Ang Ⅱ-AT1R.The balance between the two axes can influence the development of liver fibrosis.Hepatic stellate cells, hepatic stellate cells(HSC) are the key cells in the liver fibrosis process. After activated by liver damage related inflammatory response, HSC make a large number of proliferation and differentiation, secrete excessive extracellular matrix, lead to liver fibrosis finally. After TGF-beta receptor2polymerization, the phosphorylation of Smad2/3start fibrosis related gene expression under the participation of Smad4,the process can be inhibited by Smad7by inhibiting phosphorylation of Smad2/3(figure1)Despite the basic pathological process of liver fibrosis and signaling pathways have been known by people, but there is no specific medicine to cure liver fibrosis. The inhibitors of TGF-beta and SMAD pathway are still in the stage of experimental research, have not been used in clinical in large-scale. therefore, Looking for new targets and new strategy for inhibiting liver fibrosis is still the main challenge at present.microRNAs (miRNAs) are a class of endogenous small RNAs,20-24nt in length, which bind to the3’UTR of target genes and thereby repress translation of target genes and/or induce degradation of target gene mRNA.miRNAs involved in the pathological process of a variety of diseases, which is an important candidate diagnostic sign of disease and potential therapeutic targets. MiRNAs have many important functions in the cells, which can regulate the target gene after transcription. Each miRNA can have multiple target genes, and a few miRNAs can also adjust the same gene. The complex regulation network either can regulate the expression of multiple genes through a micrornas, also can regulate the expression of a gene through the combination of several miRNAs (figure2). Micrornas can lead to gene silence by combing with sequence complementary mRNA molecules, even by combing with specific DNA fragments. The way of the body regulates gene expression is an important strategy. It is presumed that micrornas regulated a third of the human genes. MirRNA studies mostly made in tumors but little made in the field of organ fibrosis. In this article, we are mainly aimed at whether AngⅡ regulate critical signaling molecules of SMAD pathway attributing to liver fibrosis by promoting mir-21expression, clarifying the key role of mir-21in the process of liver fibrosis, also proving the mechanism that AngⅡ activates transcription factor AP-1SMAD3or SMAD2to induced the expression of mir-21,Discussing the mir-21as a potential targets for treatment of liver fibrosis.ObjectiveTo investigate whether AngⅡ regulate critical signaling molecules of SMAD pathway attributing to liver fibrosis by promoting mir-21expression, clarifying the key role of mir-21in the process of liver fibrosis, also proving the mechanism that AngⅡ activates transcription factor AP-1、 SMAD3or SMAD2to induced the expression of mir-21.MethodsT6cells were divided into different groups under different experimental purposes:1.HSC were cultured with AngⅡ in the concentration of0mol/L、10-5mol/L、10-7mol/L、10-9mol/L,RT-QPCR were done to investigate the gene levels of micro-21.2.Cells were stimulated by AngII(10-7mol/L) for0h、6h and24h, RT-QPCR were done to investigate the gene levels of micro-21.3.HSC were cultured with Ang(1-7) in the concentration of0mol/L、10-5mol/L、10-7mol/L、10-9mol/L,RT-QPCR were done to investigate the gene levels of micro-21.4.Cells were stimulated by Ang(1-7)(10"7mol/L) for0h、6h and24h, RT-QPCR were done to investigate the gene levels of micro-21.5.T6cells were divided into control group、AngⅡ group、Ang (1-7) group、 AngⅡ+Ang (1-7) group. RT-QPCR was done to investigate the gene levels of micro-21.6.T6cells were divided into NC group and pre-microRNA-21group or anti-microRNA-21group, these groups were transfected with NC and pre-microRNA-21or anti-microRNA-21,72h later, Western blot was used to test the protein levels of a-SMA、CTGF、SNAD7、type Ⅰ collagen.7.T6cells were divided into control+AngII stimulating0min group、 pre-microRNA-21+AngII stimulating0min group、control+AngⅡ stimulating5min group、pre-microRNA-21+AngⅡ stimulating5min group or control+AngⅡ stimulating0min group、anti-microRNA-21+AngII stimulating0min group、 control+AngⅡ stimulating5min group、anti-microRNA-21+AngⅡ stimulating5min group、Western blot was used to test the protein levels of p-SMA2、 p-SMA3.8.T6cells were divided into control group、SMAD2-siRNA group、control+AngⅡ group、SMAD2-siRNA+AngⅡ group, or control group、SMAD3-siRNA group、control+AngⅡ group、SMAD3-siRNA+Angll group,6h later, RT-QPCR was done to investigate the gene levels of micro-21.9.T6cells were divided into NC+microRNA-21mimics group、Smad73’UTR wild type+micro RN A-21mimics group, or NC+microRN A-21mimics group、 Smad73’UTR mutant type+microRNA-21mimics group,48h later, the fluorescence ratio of sea rennin to fireflies glow was detected.lO.Wistar rats were divided into SHAM group、BDL group、and BDL+Ang (1-7) group, after4weeks. Fibrosis score, masson staining, hydroxyproline assay were used to evaluate the level of liver fibrosis. Immunohistochemistry was used to detect expression of a-SMA, ISH was done to test the gene levels and positions of microRNA-21. 11.Wistar rats were divided into SHAM group、BDL group、and BDL+Ang (1-7) group, after4weeks, RT-QPCR was done to investigate the gene levels of micro-21.12.Wistar rats were divided into SHAM group%Angll group、Ang (1-7) group、Angll+Ang (1-7) group, after4weeks. Fibrosis score, masson staining, hydroxyproline assay were used to evaluate the level of liver fibrosis. Immunohistochemistry was used to detect expression of a-SMA.13.Wistar rats were divided into SHAM group、AngⅡ group、Ang (1-7) group、Angll+Ang (1-7) group, RT-QPCR was performed to investigate the gene levels of microRNA-21.Result1. Compared with control group, the gene level of microRNA-21was increased in AngⅡ10-5mol/L stimulation group、10-7mol/L stimulation group、10-9mol/L stimulation group (P<0.05, P<0.01, P<0.01), the gene level of microRNA-21was increased mostly in10-7mol/L stimulation group.2.After cultured in AngⅡ(10-7mol/L) for6h, the gene levels of microRNA-21were increased (P<0.05) compared with control group. however, compared with AngⅡ(10-7mol/L) stimulating24h group, the gene level of microRNA-21were up-regulated (P<0.05) in AngⅡ(10-7mol/L) stimulating6h group.3.Compared with control group, the gene level of microRNA-21was decreased (P<0.05) in Ang(1-7)10-7mol/L stimulation group, while the gene levels of microRNA-21were not changed (P=0.224, P=0.286) in the Ang(1-7)10"5mol/L stimulation group、10-9mol/L stimulation group.4.After cultured in Ang(1-7)10-7mol/L for6h, the gene levels of microRNA-21were decreased (P<0.001) compared with control group, however, the gene level of microRNA-21in Ang(1-7)10-7mol/L stimulating24h group was not changed (P=0.230) compared with control group. compared with AngII(10-7mol/L) stimulating24h group, the gene level of microRNA-21were down-regulated (P<0.01) inAngⅡ(10-7mol/L) stimulating6h group.5.Compared with control group, the gene level of microRNA-21was increased (P<0.001) in AngⅡ stimulation group, however, compared with AngⅡ stimulation group, the gene level of microRNA-21was decreased (P<0.001) in AngⅡ+Ang (1-7) stimulation group.6.Compared with NC group, the protein level of a-SMA、CTGF、type I collagen were up-regulated (P<0.001, P<0.001, P<0.01) and the protein level of SMAD7was down-regulated (P<0.01) in the pre-microRNA-21group;while the protein level of α-SMA、CTGF、type I collagen were down-regulated (P<0.001, P<0.001, P<0.001) while the protein level of SMAD7was up-regulated (P<0.001) in the anti-microRNA-21group.7.Compared with control+AngII stimulating0min group, the protein levels of p-SMAD2、p-SMA3in pre-microRNA-21+AngⅡ stimulating0min group were up-regulated (P<0.001, P<0.001) and the protein levels of p-SMAD2^p-SMA3in control+AngII stimulating5min group were up-regulated(P<0.001, P<0.001), Compared with control+AngⅡ stimulating5min group, the protein levels of p-SMAD2、p-SMA3in pre-microRNA-21+AngII stimulating5min group were up-regulated (P<0.001, P<0.001). Compared with control+AngII stimulating0min group, the protein levels of p-SMAD2、p-SMA3in anti-micro RNA-21+AngII stimulating0min group were down-regulated (P<0.05, P<0.01) while the protein levels of p-SMAD2, p-SMA3in control+AngⅡ stimulating5min group were up-regulated (P<0.001, P<0.001), Compared with control+AngⅡ stimulating5min group, the protein levels of p-SMAD2、p-SMA3in anti-microRNA-21+AngⅡ stimulating5min group were down-regulated (P<0.001, P<0.001)8.Compared with control group, the gene levels of microRNA-21were obviously down-regulated in SMAD2-siRNA group while up-regulated in the control+AngⅡ stimulating group (P<0.05, P<0.05). Compared with SMAD2-siRNA group, the gene levels of microRNA-21were up-regulated in the SMAD2-siRNA+AngⅡ stimulating group (P<0.05).Compared with control group, the gene levels of micro RNA-21were obviously down-regulated in SMAD3-siRNA group (P<0.05)9.Compared with NC+microRNA-21mimics group,the fluorescence ratio of sea rennin to fireflies glow in Smad73’UTR wild type+microRNA-21mimics group was down-regulated(P<0.05),while the fluorescence ratio of sea rennin to fireflies glow in Smad73’UTR mutant type+micro RNA-21mimics group was not changed(P=0.605).lO.Compared with SHAM group, the fibrosis score, hydroxyproline and a-SMA expression were obviously up-regulated(P<0.05, P<0.05, P<0.05)in the BDL group, the gene level of microRNA-21was obviously up-regulated and the micro RNA-21was found in portal area or around the central vein in the BDL group.11.Compared with SHAM group, the gene levels of micro RNA-21in BDL group was obviously up-regulated (p<0.001). However, Compared with BDL group, the gene levels of microRNA-21in BDL+Ang(1-7) group was obviously down-regulated (P<0.001)12.Compared with SHAM group, the fibrosis score, hydroxyproline and a-SMA expression were obviously up-regulated(P<0.05, P<0.05, P<0.05) in the AngⅡ group; Compared with AngⅡ group, the fibrosis score, hydroxyproline and a-SMA expression were obviously down-regulated. 13.Compared with SHAM group, the gene level of microRNA-21was obviously up-regulated (P<0.001) in the AngⅡ group; Compared with AngⅡ group, the gene level of microRNA-21was obviously down-regulated (P<0.001) in the AngⅡ+Ang (1-7) group.ConclusionAngⅡ can up-regulate microRNA-21expression to stimulate SMAD pathway, which can lead to liver fibrosis.