Effect of RNAi against 53BP1 gene on radiosensitivity of human esophageal carcinoma cells

Background and purpose:Cell cycle checkpoint kinases p53-binding protein 1(53BP1) plays an important role in regulating cell cycle distribution.53BP1 has no effect on the growth of somatical cells.When irradiation induces DNA damage,53BP1 is activated immediately,and arrest of cell cycle is induced to enhance repair for DNA lesions.The study explored the influence of 53BP1 gene expression by RNAi on the radiosensitivity of human esophageal carcinoma cell line ECA109.Methods:Three pairs of siRNA based on the sequences of the 53BP1 mRNA were synthesized(siRNA1,siRNA2,and siRNA3),and a negative one was synthesized to be used as control.53BP1-siRNA positive recombinant plasmids(pSIH1-H1-siRNAl,pSIH1-H1-siRNA2,pSIH1-H1-siRNA3) were thus constructed and then transfected into the cultured ECA109 cells.Inhibitory effects of siRNA on 53BP1 mRNA and protein expression were detected by real-time PCR and Western blot respectively in instantaneously transfected cells.The siRNA with the best inhibitory effect or empty vector was co-transfected along with pPACKH1-Lentivector Packaging system into 293T to package lentivirus particles,48 hours after transfection,stable integrants with specific or control lentiviral vectors,selected by using copGFP reporter gene,were defined as ECA109/B(pSIH1-H1-siRNAl) and ECA109/N(pSIH1-H1-negtive),respectively.The effect of siRNA was identified by real-time RT-PCR and Western blot.The effect of knockdown of 53BP1 on proliferation ability of Eca109 cell was observed by MTT.The sensitivity of ECA109 cells to radiotherapy was detected by flow cytometry(FCM) and clone formation assay,so as to elucidate whether knockdown of 53BP1 can increase the sensitivity to radiotherapy.Results:Eukaryotic expression plasmid expressing siRNA targeting 53BP1 gene was constructed successfully.The results of real-time PCR and Western blot showed in instantaneously transfected cells,53BP1 mRNA and protein expression were inhibited.The mRNA and protein expression level of 53BP1 in group transfected with pSIH1-H1-siRNA1 were both significantly lower than the other groups.The results of FCM showed the degree of retardance of G2/M stage was more serious in ECA109/B cells than that in ECA109 and ECA109/N cells.Percent of S stage and apoptosis were not influenced in this report.D0 value and SF2 value were 3.06 Gy and 0.91 in ECA109 cells.D0 values were 2.90,2.07 Gy and SF2 values were 0.89,0.79 respectively with clonegenetic assay in ECA109/N,ECA109/B cells.Conclusion:RNAi could inhibit 53BP1 gene expression and enhance radiosensitivity of ECA109 cell.