Role Of MiR-342-5p In Cellular Senescence Of Zmpste24-deficient Mouse Embryonic Fibroblasts And DNA Damage Response

Part Ⅰ Role of miR-342-5p in cellular senescence of Zmpste24-deficient mouse embryonic fibroblastsObjective:To investigate the role of miR-342-5p in cellular senescence of mouse embryonic fibroblasts in Zmpste24-deficient progeria mouse model.Methods:（1）The differential expression of miR-342-5p in Zmpste24^-/-progeria MEFs,replicative senescent MEFs,H₂O₂ induced senescent MEFs and tissues from physiological aging mice was measured by quantitative real-time PCR（qRT-PCR）.（2）Effects of miR-342-5p overexpression on the cell phenotypes of Zmpste24^-/-MEFs transiently transfected with miR-342-5p Mimics（342M）were investigated as follow:a.Cellular senescence was detected by senescence-associatedβ-galactosidase（SA-β-Gal）staining assay;b.Cell growth curve was monitored by MTT assay and cell proliferation was measured by Ed U incorporation assay;c.Cell cycle was analyzed by flow cytometer and the protein level of cell cycle regulating proteins was detected by Western blotting.Meanwhile,the parallel experiments were performed in wild-type（WT）MEFs transfected miR-342-5p Inhibitor（342I）.（3）Target gene identification:The potential target genes of miR-342-5p were picked out using microRNA（miRNA）target gene prediction software,then the 3′-Untranslated Regions（3′-UTR）fragments（at least 500bp）of potential target genes containing the putative miR-342-5p binding sites were cloned into the pGL3-MCS vector,dual luciferase assay was used to verified whether miR-342-5p could bind to the3′-UTR of potential target genes,Western blotting was used to detect whether the protein level of these potential target genes was regulated by miR-342-5p.Results:（1）miR-342-5p was down-regulated in Zmpste24^-/-progeria MEFs,replicative senescent MEFs and H₂O₂ induced senescent MEFs.（2）miR-342-5p overexpression was sufficient to reduce the SA-β-Gal positive cells,increase the cell viability and Ed U positive cells,increase the G2+M cell cycle phase and up-regulate Cdk1 expression in Zmpste24^-/-MEFs.However,the opposite phenotypes were hardly observed in WT MEFs while suppressing miR-342-5p compared with which in Zmpste24^-/-MEFs.（3）The3′-UTR fragments of potential target genes were successfully cloned into the pGL3-MCS vector,results of dual luciferase assay showed that miR-342-5p could bind to GAS2 3′-UTR in vitro,results of Western blotting showed that Gas2 protein level was negatively regulated by miR-342-5p in Zmpste24^-/-MEFs.In addition,GAS2 was up-regulated in replicative senescent MEFs and in kidney from aging mice.Conclusions:miR-342-5p targets GAS2 and promotes cell proliferation and inhibits the senescence phenotypes in Zmpste24^-/-MEFs.Part Ⅱ Role of miR-342-5p in DNA damage response induced by doxorubicin in NIH/3T3 cells Objective: To investigate the role of miR-342-5p in DNA damage response induced by doxorubicin in NIH-3T3 cells.Methods:（1）NIH/3T3 cells were treated with doxorubicin and the expression of miR-342-5p and DNA damage response relative genes were detected by q RT-PCR and Western blotting.（2）Effects of miR-342-5p on cell cycle of NIH/3T3 cells treated with doxorubicin were evaluated using flow cytometer.（3）The potential target genes of miR-342-5p were picked out using target gene prediction software,then the 3′-UTR fragments of potential target genes were cloned into the p GL3-MCS vector and potential target genes were verified by dual luciferase assay and Western blotting.（4）Effects of miR-342-5p on DNA damage responsive proteins and cell cycle regulating proteins were evaluated using Western blotting.Results:（1）miR-342-5p,p53 and p21 were up-regulated in DNA damage response induced by doxorubicin in NIH/3T3 cells.（2）miR-342-5p overexpression promoted the G1/S transition in cell cycle and up-regulated Cyclin A2 in NIH/3T3 cells,while miR-342-5p suppression could hardly affect the cell cycle.（3）Cdkn1a was predicted to be a potential target gene of miR-342-5p,the results of dual luciferase assay showed that miR-342-5p could bind to the 3′-UTR of Cdkn1 a,the results of Western blotting showed that miR-342-5p overexpression down-regulated p21 and up-regulated p Rb and Cdk1.（4）In miR-342-5p overexpressed cells,γ-H2 AX was up-regulated at 6h⁴8h and then down-regulated at 72 h,p21 was down-regulated at 24 h,while Cyclin A2 and Cdk1 were up-regulated at 48 h after doxorubicin treatment.Conclusions: miR-342-5p targets p21 and participates in the regulation of G1/S DNA damage checkpoint in NIH/3T3 cells.