The Regulatory Effect And Mechanism Of MiR-34s In DNA Damage Repair

Purpose:As tumor suppressor genes,miR-34s have been widely studied and reported.These studies focused mainly on cell cycle,proliferation,senescence,apoptosis and other processes,which based on the effects of miR-34s through inhibiting associated target molecules.In this study,we layed emphasis on the precipitating factor for these events and analyzed whether miR-34s are involved in DNA damage repair,the molecular mechanisms that cause DNA damage and the effect events of DNA damage,such as cell cycle arrest,proliferation inhibition and apoptosis.Methods:?1?HCT116,HT29 and U2OS cells were transfected with miR-34s or negative control miR-NC mimic respectively.As a DNA double-strand break marker,?H2AX protein level was detected by western blot analysis.Then the same treatment was given to HCT116 cells,and?H2AX foci were detected by immunofluorescent staining of?H2AX and the percentage of tail DNA was determined via neutral comet assays.?2?HCT116 cells were transfected with miR-34s or miR-NC mimic.Cell cycle distributions were analyzed by flow cytometry?n=3?.?3?HCT116 cells were transfected with miR-34s or miR-NC mimic.Cell growth was closly observed under an inverted microscope.CCK-8 assays and clonogenic assays were performed to detect cell proliferation and survival.?4?HCT116 cells were transfected with miR-34s or miR-NC mimic.The proportions of apoptosis were determined by flow cytometric analysis of Annexin V-FITC/PI-staining?n=3?.?5?HCT116 cells were transfected with miR-34c-5p or miR-NC mimic.The expression of miR-34c-5p was detected using quantitative real-time PCR and the expression of proteins associated with the HR or NHEJ pathways was analyzed by western blot analysis.?6?HCT116 cells were co-transfected with DR-GFP-and I-Sce I-expressing plasmids and miR-34a/b/c-5p mimic,miR-NC mimic or si-RAD51,and the percentage of GFP⁺cells was detected by flow cytometry?n=3?.?7?HCT116 cells were co-transfected with miR-34a/b/c-5p or miR-NC mimic and pcDNA3.1-MYC-RAD51 or pcDNA3.1-MYC plasmid.The expression of RAD51 and?H2AX was determined by western blot analysis.?8?Using qPCR to detect RAD51 mRNA level after miR-34a/b/c-5p overexpression.Using bioinformatics to analyze the potential binding sites of miR-34a/b/c-5p and RAD51 mRNA.Dual-luciferase reporter assays were performed to verify the binding sites?n=3?.?9?HCT116p53^wt and HCT116 p53^-/-cells were transfected with miR-34a/b/c-5p or miR-NC mimic respectively.The protein levels of p53,RAD51 and?H2AX were detected by western blot.At the same time,we detected the mRNA levels of p53 and RAD51 using qPCR.Results:?1?At 48 h post-transfection,the levels of?H2AX protein in miR-34s group significantly increased in HCT116,HT29 and U2OS cells,although the results were somewhat different anmong the three type of cells.In immunofluorescence assays and comet assays,miR-34s except miR-34b-3p could significantly enhanced the number of?H2AX foci positive cells??H2AX foci>5?and the percentage of tail DNA.?2?At 48 h and 72 h post-transfection,miR34s except miR34b-3p and miR34c-3p resulted in significant G1 arrest with a reduction of cells in the S and G2/M phases to different extents.?3?After up-regulation of miR-34s in HCT116 cells,the morphology of cells was significantly changed.The cell shapes change from long spindle or polygon to round or oval with rough cell surfaces.There were many adhered cells and the cell density was significantly reduced.At 24 h post-transfection,differences were apparent between the miR-NC group and miR-34s groups,and these differences were more distinct at 48 h and 72 h.Both cell proliferation assays and colonogenic assays showed significant inhibition of cell proliferation and viability.However,the effect of miR-34b-3p was weak in the results above.?4?After the up-regulation of miR-34s levels in HCT116 cells,the proportions of apoptosis in the miR-34s except miR-34a-3p and miR-34b-3p groups were higher than that in the miR-NC group at 48 h post-transfection.At 72 h after transfection,apoptosis was induced to different degrees in all the experimental groups,and the difference was statistically significant,among which miR-34a-3p and miR-34b-3p showed weak apoptosis induction ability.?5?HCT116 cells were transfected with miR-34c-5p or miR-NC mimic.At 24 h and 48 h post-transfection,compared with the miR-NC group,the expression level of miR-34c-5p in the miR-34c-5p group was up-regulated by about 20 times.What's more,RAD51 and PLK1 protein levels were significantly down-regulated.The expression of other HR or NHEJ pathway-associated proteins was also found to be upregulated(RAD50 and DNA-PKcs Ser²⁰⁵⁶)or downregulated?BCL2,and Lig4?to some extents.?6?Overexpression of miR-34a/b/c-5p or transfection of si-RAD51 significantly inhibited the RAD51 protein level and reduced the percentage of GFP+cells.The percentage of GFP+cells represents HR repair efficiency.?7?Overexpression of RAD51 partially reversed the high level of?H2AX induced by miR-34a/b/c-5p.?8?The qPCR results showed that miR-34a/b/c-5p inhibited RAD51 mRNA level.Dual-luciferase reporter assays confirmed that miR-34a/b/c-5p could directly bind to 137-158 nt,137-159 nt and 136-158 nt sites of RAD51 mRNA 3'UTR respectively.?9?miR-34a/b/c-5p upregulated the expression of p53 at the mRNA and protein levels.The degree of RAD51 inhibition in HCT116 p53^wt cells was more significant than that in HCT116p53^-/-cells and the level of?H2AX protein in HCT116 p53^wt was significantly higher than that in HCT116 p53^-/-cells.Conclusion:?1?The miR-34s family can induce DSBs in different degrees and the effect inducing DSBs varies in different cells.The effect of miR-34b-3p is weak in HCT116 cells.?2?MiR-34s can significantly arrested cells at the G1 phase of the cell cycle,inhibited cancer cell proliferation and promoted apoptosis to different extents,the consequences of which were highly consistent with the results of the DSB phenotype.These findings indicate that miR-34s overexpression induces G1 arrest,inhibits proliferation and promotes apoptosis through increasing endogenous DSBs.?3?We show that miR34a/b/c-5p overexpression downregulates RAD51 expression at the mRNA and protein levels.RAD51 restoration partially rescue the DSB phenotype induced by miR-34a/b/c-5p.These results confirm that miR-34s inhibit the HR pathway by downregulating RAD51,which ultimately increases the accumulation of DSBs.?4?miR-34a/b/c-5p has direct and indirect inhibitory effect on RAD51.We finally confirmed the participation of p53 in the inhibition of RAD51.