Study On The Regulation Of Ionizing Radiation On SIK2Gene Expression

Objective：This study was designed to investigate the effect and related mechanism ofionizing radiaiton (⁶⁰Coγ rays) on SIK2gene expression on the levels of mRNA,protein and promoter activity. A eukaryotic expression recombinant vector of thepCMV-Tag-2B-SIK2has been successfully constructed for future functional studiesof SIK2involved in radiation damage response.Methods：HepG2cells were irradiated with⁶⁰Co γ-rays. Western blot and Real-time PCRwere used to detect the expression levels of the protein and mRNA, respectively. TdRdouble-blocking method was used to synchronize cells at G1/S boundary. The cellcycle distribution was analyzed by flow cytometry. Promoter activity was analyzed bythe Dual-Luciferase Reporter Assay System.Results：1. The mRNA expression of SIK2gene in HepG2cells significantly increasedat10h after irradiation（P<0.05）, but this radiaiton-inducible expression didn’t exhibitdose dependency （P>0.05）.2. The protein level of SIK2was significantly increased at4,6, and10hpost-2Gy γ-ray irradiation. The same increasing tendency of SIK2protein level wasalso observed in the cells irradiated by10Gy irradiation, but the increased extent wasless than that induced by2Gy. The alteration of SIK2protein level exhibited a doesdependency.3. The G2/M arrest was shown to reach the peak at10h after irradiation of2Gyand10Gy（P<0.05）. The result of mRNA level analysis indicated that there is no obvious change among different phases of the synchronized cells （P>0.05）.4. The recombinant plasmids containing the different length of promoterregion of SIK2gene were constructed. The data of transactivity detections suggestedthe SIK2promoter contained the region-2000to+119bp and the its upstream region,the transcriptional activity of the promoter significantly increased at10hpost-irradiation （P <0.05）.5. The pCMV-Tag-2B-SIK2recombinant plasmid was constructed, whichexpression was further verified by western blotting analysis in293T cells.Conclusions：1. SIK2mRNA expression is inducible by ionizing radiation, but this is noobvious dose dependency.2. The level of SIK2protein can be significantly induced by both2Gymedium dose and10Gy high dose of irradiation, a part of reason of the increasedprotein level at10h post-irradiation is attributed to the induction of mRNAtranscription. The increased SIK2mRNA expression is not the consequence of theG2/M arrest by irradiation but it is a result of the activation of its promotertransactivity by irradiation at the same time.