The Regulation Of PIG3 Gene Expression By Ionizing Radiation And Its Mechanism

Objective: 1) to investigate the induction of mRNA level of PIG3 gene by ⁶⁰Coγradiation at the dose range from 0.05 to10Gy, and provide the informative data for the purpose of developing novel molecular bio-dosimeter of radiation injury detection; 2) to identify the critical element of PIG3 promoter, which involves in the regulation of ionizing radiation on PIG3 gene expression; 3) to investigate the status of PIG3 expression in different tumor tissues.Methods: Quantitative real-time PCR was performed to detect the mRNA expression level in human lymphoblastoid AHH-1 cells at 4 h after exposure with ⁶⁰Coγ-ray at doses of 0.05, 0.5, 2, 4, 6, 8and 10Gy, further Northern Blot was used to confirm the expression alterations; Dual luciferase assay was used to analyze the activity of PIG3 gene promoter driving the reporter gene expression. Cancer profiling array II was used to observe the difference of PIG3 gene expression between tumor tissues and their compatible normal tissues.Results: 1) The induction of PIG3 mRNA expression by ionizing radiaiton was unveiled in this study. The result given by quantitative real-time PCR has shown that the radiation-induced expression level of PIG3 was increased in a clear dose-dependent manner from 0.5 to 10 Gy. After exposure of 4 Gy, the induced mRNA level of PIG3 remained almost no decrease at least to 48 h post-irradiation.2) Northern blot analysis further indicated that PIG3 gene expression was induced by radiation and H₂O₂ in A549 cells, but not by cisplatin. In comparison with the parental human cervical carcinoma C33A-C ( 627) cells, the HPV E6 transfected C33A-E6 (673) cells displayed a deficiency of p53 protein level as well as a much lower level of PIG3 mRNA.3) A series of truncated promoters of PIG3 were constructed, which diriving the expression of GFP reporter gene. By using these vectors, we have identified that the radiation-induced PIG3 expression was mainly mediated by the microsatellite sequences.4) Recombinant vector of pGEX-5T-PIG3 was constructed, and fusion protein GST-PIG3 was expressed in E.Coli and purified by glutathione agrose affinity chromatography, which is for the purpose of functional study of PIG3 product.5) The level of PIG3 mRNA in kidney carcinoma was found to be only half of that in its compatible normal tissue.Conclusion: Our study, in the first time, revealed that PIG3 gene was inducible by ionizing radiation, and the induced expression was shown a good dose-effect relationship with radiation. These data make PIG3 very appealing for future development as a novel radiological bio-dosimeter. Our results also provided the information for the further study of the biological function of PIG3.