Study On The Radiation Effect Of IER5 Gene In HepG2 Cells In Vivo And Its Promoter Region

The incidence of primary carcinoma of liver (hepato- -carcinoma ) is 6th among malignant tumors all over the world. And 55% of the new hepatocarcinoma cases occur in China. It is one of the most common malignant neoplasm of digestive system in our country. The commonly applied non-operative treatments of hepatocarcinoma include transcatheter hepatic arterial chemoembolization, absolute alcohol injection, radiofrequency ablation, etc. But the treatment effects of them are not ideal. Radiotherapy is one of the three effective treatments for cancer. With the development of three dimensional conformal radiation therapy technology, radiotherapy in the treatment of hepatocarcinoma is put forward again. Recent clinical researches have shown that three dimensional conformal radiotherapy can significantly improve the patients'median survival time and quality of life. At present, radiotherapy has become a powerful weapon for the treatments of hepatocarcinoma.Radiosensitivity is one of the major influence factors of the radiotherapeutic effect. In order to increase the radiosensitivity and improve the radiotherapeutic effect, people try their best to identify the radiosensitivity related genes. If a gene expression changes after irradiation and could simultaneously cause apoptosis or growth suppression in tumor cells, this gene will be very useful to radiation treatment of cancer. Based on the above-mentioned reasons, we applied microarray technology to filter the radiosensitive genes. The result had shown that about 30 genes, including IER5 gene (Immidiate Early Response 5), were up-regulated after irradiation. IER5 gene belongs to the immediate early response gene family, which exists in tumor and normal tissues and seems to have to do with mitotic cycle and cell apoptosis. At present, the research on IER5 gene is few, the mechanism of IER5 gene expression induced by radiation has not yet entirely clear, and there was no study on the radiation effect of IER5 gene in HepG2 cells in vivo.Based on these, we selected mRNA, protein expression and apoptosis as the experimental parameters to study the radiation effect of IER5 gene in HepG2 cells in vivo and the IER5 promoter region.Objective: In the present study we selected the HepG2 cells in vivo as research objects to investigate the radiation effects of IER5 gene. Secondly, we analysed the IER5 gene promoter region that regulate gene expression after irradiation in HepG2 cells in vitro.Methods:1 BALB/c-nu nude mice were inoculated human HepG2 cells which were cultured in vitro. When the hepatic tumors grew to be 1.0cm×1.0cm size, the mice were exposed to gamma-ray of cobalt 60 at the doses of 2 and 4Gy. At the end of 4h and 12h after irradiation we cut the tumor tissues as required to detect the mRNA level of IER5 using real-time PCR. At the end of 12h after irradiation we detect the level of IER5 protein using western-blot and analysed the tumor cells'apoptosis using TUNEL method.2 In order to study the regulatory mechanism of the radiation-induced IER5 gene expression, we performed bioinformatics analysis of IER5 promoter sequence and made different mutations on promoter region, recombinanted the reporter gene plasmid vectors in IER5 gene sequence upstream of 2KB, and finally transfected HepG2 cells in vitro. We analyzed the activity of IER5 gene promoter driving the reporter gene expression before and after irradiation by Dual Luciferase Reporter gene assays.Results:1 Radiation-induced changes in the mRNA levels of IER5 by real-time PCR analysis in HepG2 cells in vivo: A radiation induced increase in the IER5 mRNA were evident 4h after irradiation with 2Gy and 4Gy. And 4Gy radiation could more significantly increase the IER5 mRNA than 2Gy radiation. The mRNA levels of 2Gy and 4Gy radiation groups respectively increased by 1.344 times and 11.542 times compared with non-radioactive group. But the radiation-induced expression of IER5 gene was down-regulated 12h after irradiation. Both of 2Gy and 4Gy radiation groups the IER5 mRNA were lower than that of the non-radiation group. The mRNA levels of IER5 expression after 4Gy radiation were lower than after 2Gy radiation. The mRNA levels of them were respective 0.327 and 0.349 times of non-radioactive group.2 Radiation-induced changes in the protein levels of IER5 by western-blot analysis in HepG2 cells in vivo:A radiation induced increase in the IER5 protein was evident 12h after irradiation with 2 Gy and 4Gy. And the protein levels of IER5 after 4Gy radiation increased more than after 2Gy.3 Radiation-induced apoptosis in HepG2 cells in vivo by TUNEL method: Radiation could accelerate hepatomacells apoptosis on 12h after irradiation. Its change trend was similar to the change of IER5 mRNA and protein after irratiation, suggesting that radiation might increase IER5 gene and protein expression to accelerate hapatomacell apoptosis. It needs to be researched more deeply.4 The IER5 promoter region of the radiation-induced expression by Dual luciferase assay: The IER5 promoter region might be in the range of -408bp ~-238bp. In the range, there were four regulatory elements: TGGCGC, GGCCGT, GCGCACA and CGCGG. The deletants of the IER5 promoter region (IER5-1, IER5-2, IER5-3) were exposed to 2Gy radiation. The activity of IER5-3 after irradiation increased more significantly than that of the control group (P=0.01). The result indicated that the 2Gy radiation could induce IER5 gene promoter transcription activity.Consultation: Radiation can increase the expression levels of IER5 mRNA and protein in HepG2 cells in vivo. The radiation-induced expression of IER5 protein with 4Gy was significantly higher than that with 2Gy radiation. With the expression of IER5 mRNA and protein increase, the apoptosis of tumor cells increased. These results indicate that IER5 gene may be a sensitive gene of radiation. The promoter region of IER5 gene is in the range of -408bp ~-238bp. In the range, there have four regulatory elements: TGGCGC, GGCCGT, GCGCACA, CGCGG, building the basis for the further stdudy on the mechanism of radiation-induced expression of IER5 gene. This study amplifies people's awareness of the biological function and regulatory mechanism of IER5 gene and it is worth studying more deeply on this gene in the future.Taken together, this study made constructive exploration of the radiation effects and regulatory mechanism of IER5 gene. This study, for the first time, investigated the radiation effects of IER5 gene in HepG2 cells in vivo, narrowed down the IER5 promoter region to -408bp ~-238bp and found four potential regulatory elements. These are the main innovations of this study. This study has formed the basis for the further research on regulatory mechanism of the IER5 expression.