| It is one of the most common malignant tumors for primary carcinoma of liver (hepatocarcinoma). And 55% of the new hepatocarcinoma cases occur in China. The mortality rate of hepatocarcinoma is 3thamong malignant tumors. Therefore, it is very important to diagnose and treat hepatocarcinoma. The commonly applied non-operative treatments of hepatocarcinom 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. In recent years, with the development of three dimensional conformal radiation therapy technology, radiotherapy has become a powerful weapon for the treatments of hepatocarcinoma. Radiosensitivity is one of the major factors on the radiation effect of the tumor. Radiosensitivity is induced by gene regulation. So they are a great significance for improving the effect of tumor radiotherapy and prolonging survival of cancer patients, which are radiation sensitive genes of searching for the tumor and clarifying the relationship between the induction of radiation on gene expression mechanism and tumor radiosensitivity.IER5 gene (Immidiate Early Response 5) belongs to the immediate early response gene family and the activation of early response genes is first and most important step of cells and chromosomes to external stimuli respond. Research shows that IER5 exists in tumor and normal tissues and seems to have to do with mitotic cycle and cell apoptosis. In 2005, we and Kis almost simultaneously used gene chip technology to a number of genes which were screened radiation effects and the results showed that expression increased after radiation IER5 gene. Therefore, We started to pay attention to the genes, then utilized gene silencing and overexpression to research radiation effects and biological functions on IER5 gene. In addition, we also confirmed the promoter region of radiation response on IER5 gene. It was found that radiation could induce the upregulation of mRNA levels on IER5 gene; that RNAi could promote cell growth and showed antagonistic for the radiation; o- verexpression could inhibit cell growth and promoted radiation-induced apopt- osis. Bioinformatic analysis predicted that IER5 gene promoter most likely ranged -408bp to-238bp. In the case of radiation and its biological function similar to the tumor suppressor gene, these experiments showed that IER5 gene was a gene related to cancer radiotherapy. Because of this, We decided to do in-depth exploration of IER5 gene, so we will intend to use bioinformatics, molecular and cell biology methods and use HepG2 cell to research the transcriptional regulation mechanism of IER5 gene in order to provide a theoretical basis for clinical liver cancer radiosensitization and drug development . These results suggest that IER5 gene may be radiation sensitive genes for liver cancer, therefore we should make it more research.Objective: IER5 gene belongs to early response gene, and its role is similar to tumor suppressor gene, and it can not only upregulated after radiation but also promote cell apoptosis. At present, the researchs on IER5 gene are few at home and abroad. We retrieved some literatures which elaborate structural information and biological functions on IER5 gene. Therefore the purpose of this study is to analyze the transcription factors on transcription activity of IER5 gene promoter in order to reveal IER5 gene transcriptional regulation mechanism in liver cancer radiotherapy, and to provide a theoretical basis for future clinical liver cancer radiosensitization and drug development.Methods: First, according to Web site"http://thr.cit.nih.gov/molbio/Pro- scan",we analyz the results of IER5 gene promoter containing potential cis-acting element, and we constructe the luciferase reporter vector of the IER5 gene, and we ultlize promoter mutation experiments to determine the types and distribution of IER5 gene promoter containing cis-acting element. Second, in the HepG2 cells,we ultilze co-transfection methods to analyze the transcription factors on the effect of the transctriptional activity of IER5 gene promoter before and after radiation; we ultilze EMSA to anlyze the transcription factor GCF whether it bind with the corresponding binding site of the IER5 gene promoter in vitro, and whether the transcription factor change with the corresponding binding site of the IER5 gene promoter before and after radiation; and we ultilze CHIP to anlyze the transcription factor GCF whether it bind with the corresponding binding site of the IER5 gene promoter in vivo, and whether the transcription factor change with the corresponding binding site of the IER5 gene promoter before and after radiation.Results:1The mutations of GCF promoter binding site on IER5 gene promoter -388~-382 region,-274~-270 region can cause a significant increase in transcriptional activity (P<0.05), and transcriptional activity change significantly before and after radiation (P<0.05); but the mutations of NFI promoter binding site on the -362~-357 region can cause a significant decrease in transcriptional activity (P<0.05), and transcriptional activity change significantly before and after radiation (P<0.05); however the mutations of TTR-inverted-repeat promoter binding site on the -351~-345 region can cause little effect on transcriptional activity (P>0.05), and transcriptional activity has no change before and after radiation (P>0.05).2 GCF transcription factor combines with GCF binding site on IER5 gene promoter -388~-382 region, -274~-270 region in vitro, and with the increase in radiation dose (0Gy, 2Gy, 4Gy), GCF transcription factor binding with corresponding region of the promoter on binding capacity diminish.3 GCF transcription factor combines with GCF binding site on IER5 gene promoter -388~-382 region, -274~-270 region in HepG2 cells, and with the increase in radiation dose (0Gy, 2Gy, 4Gy), GCF transcription factor binding with corresponding region of the promoter on binding capacity diminish.Consultation:1 IER5 gene promoter -388~-382 region, -274~-270 region exist transcription factor GCF cis-acting element of the negative regulatory role, and with the increase in radiation dose, the inhibition of transcription factor GCF decreases; -362~-357 region exist transcription factor NFI cis-acting element of the positive regulatory role, and with the increase in radiation dose, the promotion of transcription factor NFI increases.2 Transcription factor GCF interact with cis-acting element on IER5 gene promoter, then it inhibit the expression of IER5 gene, further transcriptional regulation on IER5 gene plays a negative regulatory role, and with the increase in radiation dose, transcription factor GCF plays a negative regulatory role to following to decrease. |
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