| Objective:DNA damage is closely related to the occurrence and development of diabetic nephropathy,but the specific mechanism is still unclear[1].This study aims to explore the specific mechanism of CREB/miR-22/DDIT4pathway in promoting DNA damage of renal tubular epithelial cells in diabetic nephropathy,so as to further clarify the pathogenesis of diabetic nephropathy and find new effective targets for the prevention and treatment of DN.Methods:（1）Diabetic nephropathy model was established in C57BL mice and sacrificed 16 weeks later.Biochemical indexes were analyzed and the renal tissues were observed by HE staining.The ATM phosphorylation level,Chk2 phosphorylation level and protein expression levels of DDIT4 and CREB were detected by western blot.（2）NRK-52E cells were cultured in high-glucose medium and normal glucose medium respectively.After that comet experiment was performed to detect the extent of DNA double-strand breakage,the influence of cell cycle was detected by cell flow technique,ATM phosphorylation level and Chk2 phosphorylation level were detected by western blot so as protein expression levels of DDIT4 and CREB in cells.（3）DDIT4overexpressed plasmids were constructed and transfected in NRK-52E cells,then the DNA double-strand breakage in cells was detected by comet experiment.The ATM phosphorylation level,Chk2 phosphorylation level and DDIT4 protein expression level in cells were detected by western blot.（4）MiR-22 targeted regulation effect on DDIT4 was predicted by miRanda and TargetScan.（5）MiR-22 mimics was constructed and transfected in NRK-52E cells.Then The impact of overexpression of mir-22 on DNA double-strand breakage was detected by comet assay,and the impact of overexpression of mir-22 on DDIT4 protein expression and ATM and Chk2phosphorylation was detected by western blot.（6）The promoter binding sites of CREB and miR-22 were predicted by clustalX2 software,luciferase reporter plasmid and CREB overexpression plasmid were constructed,and the regulatory effect of CREB on miR-22 was verified by luciferase experiment and q-pcr experiment after the overexpression of CREB in NRK-52E cells.（7）After the overexpression of CREB in NRK-52E cells,the effect on DDIT4 protein expression was detected by western blot,and the effect on cell cycle was detected by cell flow.Results:（1）In the kidney of diabetic nephropathy mice,ATM phosphorylation level and Chk2 phosphorylation level were increased,DDIT4 protein expression level was decreased,and CREB expression level was increased than normal mice.（2）the high-glucose cultured NRK-52E cells had longer tails,and the levels of ATM and Chk2 phosphorylation were increased.The expression level of DDIT4 protein was decreased,and the expression level of CREB was increased.（3）After DDIT4 overexpressed in high glucose treated cells,the comet tail became shorter,and after the overexpression of DDIT4,the expression level of DDIT4 protein in NRK-52E cells increased,and the level of ATM phosphorylation and Chk2 phosphorylation decreased.（4）Software prediction showed that miR-22 could regulate DDIT4 in a targeted manner.（5）After miR-22 overexpressed,the comet tail of cells was longer,the phosphorylation level of ATM and Chk2 increased,and the expression level of DDIT4 protein decreased than control group cells.（6）After the over-expression of CREB,the luciferase fluorescence activity was increased,miR-22 expression increased.（7）the protein expression of DDIT4 decreased after overexpression of CREB in cells.Conclusion:（1）In vitro and vivo,DNA damage may be enhanced in high glucose.（2）The expression of DDIT4in NRK-52E cells was decreased under the environment of high glucose,and DNA damage can be reduced by overexpressing DDIT4 in NRK-52E cells.（3）The increased expression of CREB in rat renal tubular epithelial cells in the high-glucose environment may down-regulate the expression of DDIT4 by targeting mir-22,thereby inducing DNA damage in renal tubular epithelial cells and promoting the occurrence and development of diabetic nephropathy. |
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