The Anti-renal Fibrosis Effect Of Mouse Bone Marrow Mesenchymal Stem Cells Through Promoting MiR-146a-5p Expression In Mouse Renal Tubular Epithelial Cells

[Objective]To explore the biological function and molecular mechanism of miRNA in renal tubular epithelial cells regulated by bone marrow mesenchymal stem cells in the progression of renal fibrosis,and to provide new clinical therapeutic molecular targets and new prognosis biomarkers of renal fibrosis.[Method]Part I:Verified the effect of BM-MSC on renal fibrosis in vivo and vitro:1.Cisplatin was used to stimulate C57BL/6 mouse kidney fibrosis models,and mouse bone marrow mesenchymal stem cell(BM-MSCs)suspension was injected through tail vein.Collected mouse kidney tissues,and used Hematoxylin-Eosin(HE)staining,Masson staining,Sirius red staining to observe the degree of fibrosis in kidney tissues;2.Real-time fluorescence quantitative PCR and western blot were used to detect?-SMA,Coll?1 mRNA and protein expression levels in mouse kidney tissues which influenced by BM-MSCs.3.Collected the serum of mouse renal fibrosis models and detected the serum creatinine(Cre)and urea nitrogen(BUN)expression levels;4.Exogenous cytokine TGF-?1 was used to stimulate mouse renal tubular epithelial cells(mRTEC)and then mRTECs were co-cultured with BM-MSCs,detected the expression levels of ?-SMA,Collal mRNA and protein in mRTECs by real-time fluorescence quantitative PCR and western blot.Part ?:Verified target miRNA in mRTECs regulated by BM-SMC:1.mRTECs were co-cultured with BM-MSCs after stimulated by the exogenous cytokine TGF-?1,collected mRTECs and used miRNA high-throughput sequencing to detect differentially expressed miRNAs,and then selected the top five high expression miRNAs compared to the control group.The five miRNAs with the most obvious change were verified and screened by real-time fluorescence quantitative PCR.The most highest changed miRNA in mRTECs after co-cultured with BM-MSCs was miR-146a-5p;2.Real-time fluorescence quantitative PCR and western blot were used to detect the expression levels of ?-SMA,Collal mRNA and protein in mRTECs which were transfeced with miR-146a-5p mimics after stimulated by exogenous cytokine TGF-?1.3.Real-time fluorescence quantitative PCR and western blot were used to detect the expression levels of ?-SMA,Collal mRNA and protein in mRTECs which transfected miR-146a-5p inhibitor after stimulated by exogenous cytokine TGF-?1.Part III:Verification of miR-146a-5p target genes:1.Real-time fluorescence quantitative PCR was used to screen candidate target genes with elevated expression levels in mRTECs which stimulated by exogenous TGF-?1 cytokines;2.Detected the mRNA and protein levels of the candidate target gene Tfdp2 in mRTECs with real-time quantitative PCR and western blot after transfected the miR-146a-5p mimic.3.Real-time fluorescence quantitative PCR was used to detect Tfdp2 mRNA expression level in mRTECs after the transfection with different concentration gradients of miR-146a-5p mimics.Part ?:Verification of anti-renal fibrosis effect of Tfdp2:1.Real-time fluorescence quantitative PCR was used to detect the inhibitory efficiency of Tfdp2 siRNA on the expression level of Tfdp2 in mRTECs.2.The effects of Tfdp2 siRNA on ?-SMA,Collal mRNA and protein expression levels in mRTECs were detected by real-time fluorescence quantitative PCR and western blot.3.Immunohistochemical staining was used to observe the expression level of Tfdp2 in renal tubular epithelial cells in kidney tissue of mice.[Result]Part I:1.In the cisplatin-induced C57BL/6 mouse renal fibrosis models,mice received tail vein injection of BM-MSCs had a less fibrosis degree than mice which receivied tail vein injection of saline;2.The expression of ?-SMA and Coll?1 in kidney tissues of fibrotic mice with tail vein injection of BM-MSCs decreased;3.The expression of Cre and BUN in serum of renal fibrotic mice with tail vein injection of BM-MSCs decreased;4,The expression of ?-SMA,Collal mRNA and protein was decreased in mRTECs co-cultured with BM-MSCs.Part ?:1.The high-throughput sequencing of miRNA showed the top five increased expression of miRNAs in mRTECs which were stimulated by exogenous cytokine TGF-?1 and co-cultured with BM-MSCs compared with controls were miR-146a-5p,miR-12194-3p,let-7i-3p,miR-210-5p,and miR-210-3p;2.The most significant change in the expression levels of the five miRNAs verified by real-time fluorescent quantitative PCR is miR-146a-5p;3,After transfection of miR-146a-5p mimic,the expression of ?-SMA and Col1?1 in mRTECs which stimulated by exogenous cytokine TGF-?1 were significantly reduced;4.After transfection of miR-146a-5p inhibitors,The expression of ?-SMA and Collal in mRTECs stimulated by exogenous cytokine TGF-?1 were significantly increased.Part ?:1.In mRTECs,only the expression level of Tfdp2 mRNA was significantly increased under the stimulation of exogenous cytokine TGF-?1;2.After the transfection of miR-146a-5p mimic,the mRNA and the expression level of Tfdp2 was significantly reduced;3.As the concentration of miR-146a-5p mimic transfection increased in mRTECs,the expression level of the candidate target gene Tfdp2 gradually decreased.Part ?:1.Tfdp2 siRNA can effectively inhibit the expression level of Tfdp2 in mRTECs;2.Tfdp2 siRNA can reduce the expression of ?-SMA,Coll?l in mRTECs stimulated by exogenous cytokine TGF-?1.3.The ratio of Tfdp2 immunohistochemically stained nuclei in renal tubular epithelial cells of renal fibrosis mice injected with BM-MSCs via tail vein decreased.[Conclusions]In the process of renal fibrosis,the increase in the concentration of TGF-?1 can inhibit the expression level of miR-146a-5p.This inhibitory effect makes miR-146a-5p suppress the regulatory effect of its downstream target gene Tfdp2 weakened,so that the expression level of Tfdp2 increased,and the increase in the expression level of the target gene Tfdp2 will increase the expression of ?-SMA,Collal in mRTECs,thereby promoting the renal fibrosis process.BM-MSC can increase the expression of miR-146a-5p in renal tubular epithelial cells,thereby enhancing the inhibitory effect of miR-146a-5p on the downstream target gene Tfdp2,and reducing the expression level of the target gene Tfdp2 reduces the expression of?-SMA and Col1?1,So as to achieve the effect of treating renal fibrosis.