The Molecular Mechanism Research Of Mir-145 In The Rugulation Of Human Aortic Smooth Muscle Cells Proliferation By IRE1?-XBP1 Signaling Pathway Based On MiRWalk2.0

Part ? Prediction and screening of the target genes of miR-145Objective:In order to predict and screen potential target genes of miR-145 by miRWalk 2.0 prediction software.Methods:We used the miRWalk2.0 software to find predicted miR-145 targets.The selection of possible target genes containing target site for miR-145 in the 3'-untranslated region(UTR)was based on prediction overlap between at least 2 of 4 miRNA target prediction databases including miRWalk,RNA22,miRanda,Targetscan7.1.Based on the literature report results,the potential target genes related to smooth muscle cells proliferation were selected as the research object.Then,we applied Targetscan7.1 to analysis their binding sites with miR-145.Results:IRE1? and XBP1 in the 3'-untranslated region(UTR)contains target sites for miR-145.Conclusion:By using miRWalk2.0 software and considering literature report results,ERN1 and XBP1 that we had screened out may be the target genes of miR-145.Part ? The Effect of Silencing IREla-XBP1 signaling pathway onproliferation and the Expression of CyclinDl in HASMCObjective:To investigate the effect of silencing IREl?-XBP1 signaling pathway on the proliferation and the expression of CyclinDl in human aortic smooth muscle cells(HASMC).Methods:We transfected siERN1(siERN1-a,siERNl-b,siERN1-c),siXBP1 into HASMC with Lipofectamine-TM2000 as interference group,the cells without any treatment as the blank control group,with being transefected a scramble sequence as negative control group.Cell transfection was observed under fluorescence microscope at 24 hours.The mRNA and protein expression levels of ERN1,XBP1 were detected by RT-qPCR and Western blotting.The effective siRNA(siERNl-c,siXBP1)was further transfected into HASMC with LipofectamineTM2000.The changes of cell cycle and CyclinDl expression of HASMC cells were were assessed using flow cytometry methods,RT-qPCR and Western blotting,respectively.The proliferation of HASMC were assessed using MTS assay before transfection Oh and after transfecting siERNl-c,siXBP1 at 24h,48h,72h.Results:(1)The expression level of ERN1,XBP1 in HASMC transfected with siERN1-c,siXBP1 were decreased(P<0.05);(2)MTS revealed that the number of cell growth in siERN1-c group and siXBP1 group were significantly lower than BC group and NC group in 24h,48h,72h(P<0.05);(3)the percentages of S phases in the cell cycle of siERN1-c group and siXBPl group were decreased and G1 phases were increased for 48h(P<0.05).In addition,the expression of cyclinDl was decreased in siERN1-c group and siXBP1 group(P<0.05).Conclusion:Silencing IRE 1 ?-XBP1 signaling pathway can inhibit the proliferation of HAVSMC,and its mechanism may be related to decreasing the expression of cyclinD1.Part ? miR-145 regulating IREla-XBP1 Signaling Pathway and its effect on proliferation of HASMCObjective:The aim of this study was to test if microRNA-145(miR-145)are endogenous regulators of IRE1? and XBP1 expression and whether overexpression of miR-145 can inhibit IRE1?-XBP1-dependent HASMC proliferation.Methods:We transfected miR-145mimics and negative control(NC)into HASMC with Lipofectamine-TM2000.HASMC were divided three groups:miR-145mimics group,blank control group,negative control group.Cell transfection was observed under fluorescence microscope,then transfection efficiency was detected by flow cytometry at 24 hours.The expression of miR-145 in HASMC was detected by real time fluorescence quantitative PCR.The proliferation of HASMC was assessed using MTS assay before transfecting Oh and after transfecting at 24 h,48 h,72 h.The mRNA and protein expression levels of ERN1,XBP1 were detected by RT-qPCR and Western blotting.Differential expression of the target gene ERN1,XBP1 of miR-145 were detected by real time fluorescence quantitative PCR and western blot in HASMC,the relationship between miR-145 and ERN1,XBP1 was analyzed.Results:(1)The level of miR-145 expression in HASMC transfected with miR-145mimics is dramatically increased,the difference is significant(P<0.05);(2)MTS revealed that the number of cell growth in miR-145mimics group was significantly lower than those of the blank control group and negative control group at 24h,48h,72h(P<0.05);(3)There was no significant difference of ERN1 and XBP1mRNA in the three groups.(4)Pearson relativity analysis shows that there is no correlation between the level of miR-145 and ERN1,XBP1mRNA in HASMC.Conclusion:we conclude that miR-145 has not an role in regulating HASMC proliferation by inhibiting the IRE1?-XBP1 pathway,which implys the cause of issue occurred the applications of the miRWalk2 need to be discussed and to be put forward some countermeasures concerned.