The Mechanism Of Long Non Coding RNA MALAT1 Promotes Renal Interstitial Fibrosis In Obstructive Nephropathy By Targeting MiR-124-3p/ITGB1 Axis

Background:Obstructive nephropathy(ON)is one of commom urinary system diseases that cause renal interstitial fibrosis.Given its high incidence,ON seriously threaten human health.Timely relief of obstruction to save renal function is the common treatment means.However,there is still a lack of targeted drug therapy for renal interstitial fibrosis caused by ON.Recently,long non-codingRNAs(lncRNAs)have attracted much attention.And accumulating evidence suggests that lncRNA/miRNA/mRNA axis is one of the most well-established regulatory mechanisms in the pathogenesis of different diseases.Furthermore,lncRNA metastasis-associated lung adenocarcinoma transcript-1(LncRNA MALAT1)has been reported to play a pro-fibrotic role in the progression of liver fibrosis,pulmonary fibrosis and myocardial fibrosis by regulating the expression of multiple miRNA/mRNA axes.Studies have also shown that miR-124-3p is an important fibrosis related miRNA,which plays an important regulatory role in wound healing,scar formation and liver fibrosis,and it could participate in renal ischemia-reperfusion injury by regulating the expression of integrin β1(ITGB1).However,it remains unclear whether lncRNA MALAT1 can play a role in ON induced renal interstitial fibrosis by targeting miR-124-3p /ITGB1 axis.Objective: To investigate the effect of lncRNA MALAT1 on ON induced renal interstitial fibrosis in vitro,in vivo and through analyzing clinical specimens,and to further explored the mechanism of lncRNA MALAT1 targeting miR-124-3p / ITGB1 axis in ON induced renal interstitial fibrosis.Methods:1、In vitro cell experiments:(1)HK2 and normal rat kidney fibroblast(NRK-49F)cells were treated with different concentrations of transforming growth factor β1(TGF β1).Then,the expression of fibrosis related proteins in HK2 and NRK-49 F cells were detected by Western blotting(WB).(2)Quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the relative expression of lncRNA MALAT1 and ITGB1 in HK2 and NRK-49 F cells induced by different concentrations of TGF β1.The protein expression of ITGB1 was assessed by WB.(3)Firstly,knockdown of lncRNA MALAT1 expression by lentivirus transfection in HK2 and NRK-49 F cells.Meanwhile,the cells were interfered with or without TGF β1,then the expression of fibrosis related proteins in the two cell lines were detected by WB.(4)Fluorescence in situ hybridization(FISH)method were used to predict the subcellular localization of lncRNA MALAT1 in HK2 and NRK-49 F cells.Then bioinformatics software analysis,q RT-PCR,dual luciferase reporter assay,andRNA immunoprecipitation(RIP)assay were used to determine the relationship between lncRNA MALAT1 and miR-124-3p.(5)q RT-PCR was used to detect the relative expression of miR-124-3p in HK2 and NRK-49 F cells induced by different concentrations of TGF β1.Bioinformatics software analysis predicted that ITGB1 might be a direct target of miR-124-3p and verified by dual luciferase reporter assay.In addition,ITGB1 mRNA level were detected by q RT-PCR after miR-124-3p was overexpressed or silenced in HK2 and NRK-49 F cells.After that,the expression of ITGB1 and fibrosis related proteins in the TGF β1 induced HK2 and NRK-49 F cell were detected by WB after inhibiting the expression of miR-124-3p.(6)After knockdown of lncRNA MALAT1,the expression levels of ITGB1 mRNA and protein in HK2 and NRK-49 F cells were evaluated by q RT-PCR and WB,respectively.In addition,lncRNA MALAT1 and miR-124-3p were knockdown in TGF β1 treated HK2 or NRK-49 F cells,the protein level of ITGB1 and fibrosis related proteins were detected by WB.2、In vivo animal experiments:(1)C57BL/6J mice were divided into two groups: sham group and unilateral ureteral obstruction(UUO)group(n=7 mice/group).After sampling,whether UUO model is established successfully were assessed by visual observation,hematoxylin-eosin staining(HE),Masson staining,immunohistochemistry(IHC)and WB method.(2)q RT-PCR was used to detect the relative expression of lncRNA MALAT1,miR-124-3p,and ITGB1.Moreover,IHC and WB were used to detect the protein expression of ITGB1 in renal of the two groups.(3)UUO+ Len-sh-control group and UUO+ Len-sh-MALAT1 group(n=7 mice/group)were successfully constructed by tail vein injection of lentivirus in mice.Then,q RT-PCR was used to detect the relative expression of lncRNA MALAT1/miR-124-3p/ ITGB1 axis.Meanwhile,renal pathology and fibrosis related markers were detected by HE,Masson staining,IHC and WB.3、Clinical specimen validation:(1)The renal interstitial fibrosis tissues of 20 ON patients and 20 normal controls were collected.HE,Masson staining,WB and IHC were used to detect the pathological changes and the expressions of fibrosis markers in the renal of the two group.(2)The relative expression of lncRNA MALAT1/miR-124-3p/ITGB1 axis were detect by q RT-PCR,IHC and WB.(3)The correlation between the expression of lncRNA MALAT1/miR-124-3p /ITGB1 axis and fibrosis related markers in renal tissues of ON patients.Results:1、In vitro cell experiments:(1)TGF β1 can successfully induce the fibrosis process of HK2 and NRK-49 F cells;(2)LncRNA MALAT1 and ITGB1 were highly expressed in TGF β1treated HK2 and NRK-49 F cells;(3)LncRNA MALAT1 may promote the expression of fibrosis related markers in TGF β1 induced HK2 and NRK-49 F cells by targeting miR-124-3p /ITGB1 axis.2、In vivo animal experiments:(1)UUO surgery could successfully induce renal interstitial fibrosis;(2)The expression of lncRNA MALAT1 and ITGB1 were higher and miR-124-3p expression was lower in renal interstitial tissues of UUO group than that in the sham group;(3)Knockdown of lncRNA MALAT1 expression in vivo partially reversed the degree of renal interstitial fibrosis induced by UUO.3、Clinical specimen validation:(1)The expression of lncRNA MALAT1,ITGB1,and fibrosis related markers were higher and miR-124-3p expression was lower in renal interstitial tissues of ON patients than that in the control group.Conclusion:In this study,lncRNA MALAT1 may promote the progression of ON induced renal interstitial fibrosis in vivo and in vitro by regulating the expression of miR-124-3p/ITGB1 axis,which is expected to become a specific therapeutic target.