Research On Mechanism Of MiR-294 Regulating Aging Renal Interstitial Fibrosis Through TGF-?1 Pathway

Objective: Aging kidney was a pathological basic of aging chronic renal diseases, and renal interstitial fibrosis was the major manif'estation. Renal interstitial fibrosis, characterized by deposition of ECM, resulted in loss of renal structure and function. Preventing and curing renal interstitial fibrosis was the major way to prevent the process of aging CKD.We discovered that less Effects of aging microvesicles released from mesenchymal stem cells (MSC-MV) depressing TGF-?1-mediated EMT may be relative with the downregulation of miR-294, and miR-294 weakened epithelial-mesenchymal transition ( EMT) induced by TGF-?1 .The purpose was to find out the direct target gene and analyze the mechanism of miR-294 regulating renal fibrosis through TGF-?1 pathway, and discuss the therapeutic effect that miR-294 treating aging renal interstitial fibrosis.Methods: (1)we overexpressed miR-294 in HK-2 cells and established TGF-?1-induced HK-2 EMT model, Western Blot?reverse transcription polymerase chain reaction(RT-PCR) analyses and Immunofluorescence were used to evaluate the makers of EMT in HK-2 cells. We tried to find possible targets of miR-294 in TGF-?1 pathway by bioinformatics and verify the direct target by dual luciferase reporter assay system.(2)We established renal injury of unilateral ureteral obstruction (UUO) model in aging mice, injected transfection reagents with miR-294 through caudal vein and evaluated therapeutic effect miR-294 treating UUO renal injury in aging mice by pathological examination of renal tissue and Western Blot.Results: (1) In vitro experiment :?after inducing EMT with TGF-?1 (10ng/ul) for 48h and transfecting HK-2 cells with miR-294 mimic/NC- miR (20pm/ul) for 24h,PCR showed that mir-294 was overexpressed in group miR-294 + TGF-?1.?TGF-?1-induced HK-2 cells lost the original epithelial form but showed fibroblast form;after transfecting NC-miR, the form of cells did not change; after transfecting miR-294,part of the form of cells recovered to normal. ? Western Blot showed that the markers of epithelium - ?-SMA?fibronectin?COL3A1 was observably downregulated ,and marker of mesenchyme-E-cadherin was upregulated compared with group TGF-?1; immunofluorescence showed the markers of EMT- ?-SMA was observably upregulated in group TGF-?1 compared with the control, and observably downregulated after transfecting miR-294. ? bioinformatics analysis showed RBL1 may be the target gene of miR-294; dual luciferase reporter assay system showed that after cotransfecting miR-294 mimic , the luciferase activity of pGL3-WT-RBL1 was remarkably downregulated (p<0.05),but the luciferase activity of pGL3-Mut-RBL1 changed little; the luciferase activity of pGL3-WT-RBL1 in group NC did not change.Western Blot showed RBL1 was remarkably upregulated after TGF-?1-induced EMT.(2)In vivo experiment: ? we built the UUO model in aging mice successfully. ?RT-PCR showed that the expression of miR-294 was remarkably higher in 7th day?14th day after injecting miR-294 compared with group sham and group UUO.?The expression of RBL1 was remarkably downregulated in group miR-294 compared with group UUO.?after curing for 14 days,the serum creatinine in group miR-294 was lowered than serum creatinine in group UUO.Conclusion: (1)RBL1 was the direct target gene of miR-294;miR-294 can inhibit TGF-?1-induced EMT in renal tubular epithelial cells through regulating target gene RBL1;(2) miR-294 can depress renal interstitial fibrosis of UUO renal injury in aging mice and improve renal function.