| Objective: PTEN is closely related to the development of diabetic nephropathy renal interstitial fibrosis,but the specific mechanism is not clear,this study aims to explore whether miR-22 regulates autophagy and renal interstitial fibrosis through targeting at PTEN/AKT/mTOR signaling pathway,in order to further clarify the pathogenesis of diabetic nephropathy renal interstitial fibrosis,to explore a new effective target for the prevention and treatment of diabetic nephropathy.Methods:（1）The models of diabetic nephropathy rats and renal tubular epithelial cells（NRK-52E）that cultured in high glucose were established,and the expression of Autophagy related molecules was detected by western blot.（2）Autophagy promoter rapamycin（100nm）or autophagy inhibitor chloroquine（40μM）were put in NRK-52 E cells respectively,to collect cell protein samples after cultured in high glucose for48 h,and detect the protein expression of LC3、P62、Col-IV by western blot.（3）To detect the expression of PTEN by immunohistochemistry,western blot and Real-time PCR in diabetic nephropathy rat models.（4）To collect 24 h and 48 h protein and RNA samples of NRK-52 E cells cultured in high glucose,and detect the expression of PTEN by Western blot and Real-time PCR.（5）The expression of Akt、p-AKT(Thr308)mTOR、p-mTOR(Ser2448)were detected by Western blot in diabetic nephropathy rat models.（6）To detect the expression of miR-22 by Real-time PCR in diabetic nephropathy rat models and renal tubular epithelial cells.（7）The binding site of miR-22 and PTENmRNA 3’UTR were predicted by bioinformatics software targetscan and miranda,and the PTENmRNA 3’UTR which contains the binding sites was constructed to the vector pmiR-reporter,and the targeted regulation of miR-22 toPTEN was detected by the experiment of double luciferase reporter gene.（8）To overexpress mi R-22 in NRK-52 E cells,collect protein and RNA samples after culturing cells in medium of low or high glucose for 48 hours,to detect transfection efficiency by Real-time PCR.The protein expression of PTEN,LC3,p62,and Col-IV were detected by western blot after transfected miR-22 mimics for 48 h.Results:（1）The expression of LC3 decreased significantly in diabetic nephrapathy renal tissues and NRK-52 E cells cultured in high glucose（P<0.05）,and the expression of p62 increased significantly.（2）Rapamycin could obviously promote the conversion of LC3 I to LC3 II,promote the degradation of p62 and reduce the expression of Col-IV in NRK-52 E cells that cultured in high glucose.（3）PTEN mainly located in the cytoplasm of renal tissues,and the protein and mRNA expression of PTEN in renal tissues of diabetic nephropathy rats were significantly decreased（P<0.05）.（4）The protein and mRNA expression of PTEN were obviously lower in NRK-52 E cells cultured in high glucose for 24 h and 48h（P<0.05）.（5）The expression of p-AKT(Thr308)and p-mTOR(Ser2448)in renal tissues of diabetic nephropathy rats were increased significantly（P<0.01）.（6）The expression of miR-22 in the model of diabetic nephropathy rats and NRK-52 E cells cultured in high glucose were significantly increased（P<0.05）.（7）miR-22 can target at the PTENmRNA 3’UTR.（8）Overexpression of miR-22 can significantly reduce the expression of PTEN and LC3,and promote the expression of p62 and Col-IV.Conclusion:（1）To promote autophagy could significantly inhibit the synthesis of Col-IV in renal tubular epithelial cells cultured in high glucose.（2）The low expression of PTEN may be regulated by miR-22 in renal tubular epithelial cells cultured with high glucose.（3）miR-22 could inhibit the level of autophagy of renal tubler and promote the development of renal insteinal fibrosis by targeting at PTEN/AKT/m TOR signaling pathway. |
