Effects Of MiR-552-5p On DNA Repair-Related Proteins In Esophageal Squamous Carcinoma Cell Line EC9706 After X-ray Irradiation

BackgroundRadiotherapy is the main treatment for advanced esophageal cancer,but the recurrence of local advanced esophageal cancer and the failure of radiotherapy due to the presence of DNA repair.At present,many reports have confirmed that DNA-PKcs(DNA-dependent protein kinase catalytic subunit),PARP1(poly(ADP-ribose)polymerase-1)and RAD51(recombination protein A)are involved in DNA damage repair induced X-ray irradiation.However,there is a lack of research on the effect of miR-552-5p on DNA repair-related proteins(DNA-PKcs,PARP1 and RAD51)in esophageal squamous carcinoma cell line EC9706 after X-ray irradiation.In this study,the biological role of miR-552-5p in esophageal squamous cell carcinoma cell EC9706 was first explored,the binding relationships between miR-552-5p and DNA-PKcs,PARP1 and RAD51 were predicted through online websites,and the regulatory relationships of miR-552-5p and DNA-PKcs,PARP1 and RAD51 in EC9706 were verified.To observe the effects of miR-552-5p on other DNA repair pathway-related proteins in EC9706 cells and the cell clone formation and cell cycle after X-ray irradiation.Objective(1)To investigate the biological roles of miR-552-5p in EC9706 cells.(2)To predict the regulatory relationships between miR-552-5p and DNA-PKcs,RAD51 and PARP1 through online website.(3)To observe the effects of miR-552-5p on the expression of DNA-PKcs,RAD51 and PARP1 before and after EC9706 cells X-ray irradiation(8Gy)in EC9706 cells.(4)To observe the effects of miR-552-5p on clone formation and cell cycle distribution before and after X-ray irradiation in EC9706 cells.Methods(1)The effects of EC9706 cells transfection with miR-552-5p mimic/inhibitor on cell proliferation,migration,and invasion were examined using CCK-8,scratch,and Transwell experiments,respectively.(2)The online database TargetScan,RNA22 and starBase were used to predict the relationship between miR-552-5p and DNA-PKcs.(3)qRT-PCR and Western blot were used to detect the relative mRNA and protein expression levels of of DNA repair pathway related proteins(DNA-PKcs,PARP1 and RAD51)before and after cell transfection with miR-552-5p mimic/inhibitor combined with X-ray irradiation.At the same time,DNA-PKcs inhibitor NU7441 was used as a control.(4)The effects of EC9706 cells transfection with miRNA-552-5p mimic/inhibitor combined X-ray irradiation on the number of cell clones and cycle distribution were examined using clone formation experiments and flow cytometry,respectively.(5)After transfection,the cells were irradiated with linear accelerator line 6MV X-ray,the irradiation dose was 8 Gy,and total RNA and protein were extracted 24 hours after irradiation for subsequent experiments.Results(1)Compared with mimic NC,the cell viability,migration and invasion capacity of EC9706 cells transfected with miR-552-5p mimic were increased(P<0.05);compared with inhibitor NC,cell viability,migration and invasion ability of EC9706 cells transfected with miR-552-5p inhibitor were reduce(P<0.05).(2)It was predicted online that miR-552-5p has binding sites with DNA-PKcs,RAD51 and PARP1.(3)The results of qRT-PCR showed that compared with the blank group,the relative mRNA expression level of DNA-PKcs in NU7441 group was reduced(P<0.05);compared with mimic NC,the relative mRNA expression levels of DNA-PKcs,RAD51 and PARP1 in the miR-552-5p mimic group were decreased(P<0.01,P<0.05,P<0.05,respectively);compared with inhibitor NC,the relative mRNA expression levels of DNA-PKcs,RAD51,and PARP1 in miR-552-5p inhibitor group were increased(P<0.05,P<0.05,P<0.01,respectively).After X-ray irradiation,the relative expression mRNA level of DNA-PKcs in NU7441 group was decreased compared with blank group(P<0.01);the relative mRNA expression level of DNA-PKcs in miR-552-5p mimic group was decreased compared with NU7441 group(P<0.01);compared with mimic NC,the relative mRNA expression levels of of DNA-PKcs,RAD51 and PARP1 in miR-552-5p mimic group were decreased(P<0.001,P<0.01,P<0.01,respectively);compared with inhibitor NC,the relative mRNA expression levels of DNA-PKcs,RAD51 and PARP1 in miR-552-5p inhibitor group were increased(P<0.001,P<0.01,P<0.01,respectively).Compared with NU7441 alone,the mRNA expression level of DNA-PKcs was increased after NU7441 combined with X-ray irradiation(P<0.01);compared with transfection of miR-552-5p mimic alone,mRNA expression levels of DNA-PKcs,RAD51 and PARP1 were decreased after transfection of miR-552-5p mimic combined with X-ray irradiation(P<0.01,P<0.05,P<0.05,respectively);compared with the transfection of miR-552-5p inhibitor alone,the mRNA expression levels of DNA-PKcs,RAD51 and PARP1 were decreased after transfection of miR-552-5p inhibitor combined with X-ray irradiation(P<0.01,P<0.05,P<0.05,respectively).(4)The results of Western blot showed compared with the blank group,the relative protein expression level of DNA-PKcs in NU7441 group was decreased(P<0.05);ompared with mimic NC,the relative protein expression levels of DNA-PKcs,RAD51 and PARP1 in miR-552-5p mimic group were reduced(all P<0.05);compared with inhibitor NC,the relative protein expression levels of DNA-PKcs,RAD51 and PARP1 in miR-552-5p inhibitor group were increased(P<0.05).After X-ray irradiation,compared with the blank group,the relative protein expression level of DNA-PKcs in the NU7441 group was decreased(P<0.05);compared with NU7441 group,the relative protein expression level of DNA-PKcs in miR-552-5p mimic group was reduced(P<0.05);compared with mimic NC,the relative protein expression levels of DNA-PKcs,RAD51 and PARP1 in miR-552-5p mimic group were decreased(P<0.01,P<0.05,P<0.05,respectively);compared with inhibitor NC,the relative protein expression levels of DNA-PKcs,RAD51 and PARP1 in miR-552-5p inhibitor were increased(all P<0.05).Compared with NU7441 alone,the protein expression level of DNA-PKcs was increased after NU7441 combined with X-ray irradiation(P<0.001);compared with transfection of miR-552-5p mimic alone,protein expression levels of DNA-PKcs,RAD51 and PARP1 were increased after transfection of miR-552-5p mimic combined with X-ray irradiation(P<0.001,P<0.01,P<0.01,respectively);compared with the transfection of miR-552-5p inhibitor alone,the protein expression levels of DNA-PKcs,RAD51 and PARP1 were increased after transfection of miR-552-5p inhibitor combined with X-ray irradiation(P<0.01,P<0.05,P<0.05,respectively).(5)After X-ray irradiation,the results of colony formation experiments showed that compared with mimic NC,the number of clones in miR-552-5p mimic group was significantly reduced(P<0.001);compared with inhibitor NC,the number of clones in miR-552-5p inhibitor group was increased(P<0.05);compared with transfection of miR-552-5p mimic alone,the number of cell clones after transfection of miR-552-5p mimic combined with X-ray irradiation was significantly reduced(P<0.001).(6)The results of cell cycle showed that after X-ray treatment,compared with mimic NC,the number of G2/M phase cells was increased(P<0.05)and the number of S phase cells was significantly reduced in miR-552-5p mimic group(P<0.01);compared with inhibitor NC,there was no significant difference in cell cycle distribution of miR-552-5p inhibitor group(P>0.05).Compared with the transfection of miR-552-5p mimic alone,the number of cells in S phase after transfection of miR-552-5p mimic combined with X-ray irradiation was significantly reduced(P<0.001)and the number of cells in G2/M phase was significantly increased(P<0.001);compared with the transfection of miR-552-5p inhibitor alone,the number of S phase cells after transfection of miR-552-5p inhibitor combined with X-ray irradiation was significantly reduced(P<0.01)and the number of G2/M phase cells was significantly increased(P<0.001).Conclusions(1)miR-552-5p promoted the proliferation,migration and invasion of EC9706 cells.(2)miR-552-5p inhibited the mRNA and protein expression leves of DNA-PKcs,RAD51 and PARP1 in EC9706 cells,inhibited the formation of EC9706 cell clones after X-ray irradiation and promoted G2/M phase arrest.