The Effect Of SIK2 In DNA Damage Response

Objective: By means of experiments to explore the effect of SIK2 on radiation-induced DNA damage repair and mitotic catastrophe. Known that SIK2 interacts with DNA-PKcs, to investigate whether the interaction regulating mitotic catastrophe of radiation damage cell,then further reveal the regulation mechanism,so as to provide experimental basis for further study of biological function of SIK2.Methods: Using pulsed field gel electrophoresis to detect relative content of remaining fracture DNA at the indicatead time points,to infer the repair capacity of SIK2 in DNA DSBs damage. Then,using immunofluorescence assays at different time points after 4Gy 60 Co γ-ray irradiation to statistics the abnormal spindle ratio and multinucleated cells ratio during mitosis. In order to determine whether the interaction between SIK2 and DNA-PKcs regulate radiation damage mitotic catastrophe. He La cells irradiated by 4 Gy 60 Co γ-ray, co-immunoprecipitation showed the strength of the interaction between and SIK2 and DNA-PKcs,suggesting the effect of γ-ray on the interaction. Co-immunoprecipitation experiments, testing whether there is interaction between DNA-PKcs and Cdc20, SIK2 and Cdc20 in He La cells. After the knockdown of DNA-PKcs in He La cells, Western blot detected the expression of Cdc20 and Cdc20 protein degradation rate. To explore the regulation of DNA-PKcs on the stability of Cdc20 in these above experiments. After the knockdown or the increase of Cdc20,we used Western blot to detect the expression of SIK2, exploring the regulation of Cdc20 on SIK2. To determine whether the DNA-PKcs achieve regulation of SIK2 by Cdc20, and suggest the possible mechanism of SIK2 interact with DNA-PKcs regulating mitotic catastrophe in radiation damage cells.Results: 1.Pulse field gel electrophoresis results showed that 0.5-4h after 20 Gy 60Co γ ray irradiation, relative content of remaining fracture DNA in He La-sh SIK2 cells higher than in He La-NC cells. This indicated that the knockdown of SIK2 cause the decrease of DNA DSBs repair ability. 2.Immunofluorescence experiments showed that He La-sh SIK2 cells and He La-sh DNA-PKcs cells compared with the control cells(in 48 h, for example), the proportion of abnormal spindle respectively by 25% or 28% increase to 45% or 55%; and percentage of multinucleated cells respectively by 6% or 8% increased to 13% or 18%.Which shows that SIK2 or DNA-PKcs knockdown induced mitotic catastrophe in radiation damage cells. After overexpression of SIK2 in the DNA-PKcs knockdown cells, abnormal spindle ratio compared to the control cells(in 48 h, for example), was reduced from 50% to 30%; and multinuclear cell proportion by 17% down to 8%. That is to say, the overexpression of SIK2 can save mitotic catastrophe. After overexpression of SIK2Δ400-700 in the DNA-PKcs knockdown cells, abnormal spindle and multinucleated cells ratio is similar to the ratio in control cells,but have a more significant increase compared to cells overexpressed full-length SIK2.In 48 h, as an example,abnormal spindle ratio increased from 27% to 51%, proportion of multinucleated cells increases from 7% to 15%, which shows that destruct the interaction between SIK2 and DNA-PKcs can prevent SIK2 from saving mitotic catastrophe in radiation damage cells. 3.Using co-immunoprecipitation, after 4Gy and 0Gy 60 Co γ ray irradiation, detecting the interaction strength of SIK2 and DNA-PKcs had little difference, suggesting that γ ray can not enhance the interaction between SIK2 and DNA-PKcs. 4.We found that DNA-PKcs interact Cdc20 and SIK2 also interact Cdc20 by co-immunoprecipitation assays.Western blot results showed that after DNA-PKcs knockdown, the expression level of Cdc20 protein increased; the degradation rate was slow, indicating that DNA-PKcs can adjust stability of Cdc20. In DNA-PKcs knockdown cell lines, knockdown of Cdc20 caused the increase of SIK2 expression levels and after overexpression of Cdc20 lead to downregulation of SIK2 protein. It shows that DNA-PKcs may promote the degradation of SIK2 protein by enhancing the stability of Cdc20.Conclusions: 1.SIK2 involved in radiation-induced DNA damage repair. 2.SIK2 knockdown induced mitotic catastrophe,and SIK2 interacts with DNA-PKcs can regulate mitotic catastrophe in radiation damage cells. 3.DNA-PKcs may regulate mitotic catastrophe in radiation damage cells through the regulation of SIK2 via APC / C Cdc20 pathway.