Establishment Of The Cell Line By Up-regulating IER5 Gene In HepG2 Cells And Its Study On Radiation Effect

The primary carcinoma of liver (hepatocarcinoma) is a common malignant tumor in China and emerging liver cancer in China accounts for 55% of the world. Diagnosis and treatment of primary liver cancer is very difficult. Clinical research confirmed that the three-dimensional conformal radiation treatment of liver cancer was valuable. Radiation sensitivity is an important factor in cancer radiotherapy effects, and radiation sensitive is related to the radiation-sensitive genes. If a certain gene's expression changes after radiation, and which can promote tumor cell apoptosis or inhibit the growth of tumor cells, then the gene may be the radiation-sensitive gene for the tumor. Our previous studies have shown that radiation can cause the expression of early response gene IER5 mRNA upregulate. IER5 gene on human hepatocellular carcinoma cell was very sensitive to radiation. The level of mRNA expression in hepatoma cells after 4Gy radiation was 11.54 times than the non-radiation cells. Foreign literature revealed that IER5 gene was related for the cell division and apoptosis.This study aimed to explore the radiation sensitivity of biological function of IER5 gene in the development of liver cancer. In order to explore the radiation sensitivity of biological function of IER5, we study the relation between IER5 gene high expression and gene silencing with the cell radiation effects. Stably and highy expression is that to put the foreign gene into the target cell technology and establishing the cells which can express the protein stably and highly.Common gene transfer technology is following. The plasmid of bacteriophague expression vector, which contains the objective gene,was ex-tracted,and identified with agarose gel electrophoresis after cut by restricted endonuclease.The plasmids and empty vector were transduced into a cell line,which express protein at a lower level,with LipofectAMINE Lipid gene transfection method. The positive cell clones,which had been transduced with objective gene,were obtained with hygromycin or neomycin screening. We selected the ex vivo HepG2 cells as research objects. In order to research the radiation sensitivity and biological function of the IER5 gene, we establish the HepG2 cells which can express IER5 protein stably and highly. At the same time, other person designed special RNAi fragments and constructed carrying agents which could silence IER5 gene, then transfected HepG2 cells using siRNA technology. Comparing the tow kinds of results to discover the radiosensitive and biological functions of IER5 gene in the liver cancer. We tried to confirm whether IER5 gene is the radiation sensitive genes in the liver cancer, and whether it can become a radiotherapy sensitization target of liver cancer, for future drug development of molecular targeted therapy to provide theoretical guidance.We hope that this study could provide theoretical guidance for the future development of molecular targeted therapy.Object:In order to research the radiosensitive and biological function of the IER5 gene, we selected the ex vivo HepG2 cells as research objects to establish the HepG2 cell line which can express IER5 protein stably and highly, named IER5-overexpression-HepG2. Growth curve and flow cytometry were used to measure the cell proliferation. In addition, effects on the cell cycle and apoptosis have been studied exposed to gamma-ray of cobalt 60.Methods:1 Establishment of the the stable transfected cell lines:we used cells stably transfected technology to form the vector and to transfecting HepG2 cells by plasmid mediated, then to screen by G418 and to culture molocllonal cells. We establish the HepG2 cell line which can express IER5 protein stably and highly, named IER5-overexpression- HepG2. The plasmids and empty vector were transduced into a cell line,which express protein at a lower level as a negative control group,with LipofectAMINE Lipid gene transfection method. The expression changes of protein were detected by western blotting analysis.(According to the monoclonal cell's backup number the cell serial number is HepG2-10, HepG2-11, HepG2-13, HepG2-14, HepG2-15, HepG2-20, HepG2-24, HepG2-21.)2 Investigated the effect of IER5 in the cellular level:The experimental parameters were cell proliferation (growth curve of the cells exposed to gamma-ray of cobalt 60 at the doses of 2 and 4Gy), cell cycle (using flow cytometry to detect cell cycle exposed to the radiation of 4Gy doses ).Results: 1 Establishment of the the stable transfected cell lines:The NO.21certificate clone cell is the effective cell oppositing in the control group HepG2 by Western Blotting technology testing.Its IER5 gene was the most highest expression, named IER5-over-expression-HepG2.The volume of the IER5-overexpressio-HepG2 cell is smaller than the HepG2 cell .2 Study on the HepG2 cells's proliferation mediated by Up-regulating IER5 gene:Growth velocity of the IER5-over-expression-HepG2 stepped up after 3 or 4 days exposed to gamma-ray of cobalt 60 with the dose of 0Gy, 2Gy and 4Gy. Until the sixth day the cell number was lesser apparently, compared to the HepG2-Control (P<0.05).Detected after inoculation for 24 hours: percentages of S phase in IER5-overexpression-HepG2 cells was lower than that in the HepG2-Control: (21.80±0.42) % vs (29.36±2.08) %, P<0.05. Corresponding with it, the G0-G1 time above two kind of cell percentage respectively is (46.96±2.26) % and (47.08±1.26) %, P>0.05. It explained that the IER5 gene has expressed the HepG2 cell's multiplication ability to weaken, is consistent with the growth curve result.3 Study on the HepG2's cell cycle and radiation effect mediated by Up-regulating IER5 gene: Exposed to gamma-ray of cobalt 60 with the dose of 4Gy, there happened a G2-M phase arrest in two kinds of the cells at the end of the 12th hour. At the zeroth hour and 12th hour, percentages of G2-M phase in IER5-over-expression-HepG2 cells :(18.98±2.96)%vs(46.64±3.78) P<0.05, percentages of G2-M phase in HepG2-Control cells were (20.36±2.26) %vs (51.00±4.58) %, P<0.05. At the end of 24th hour, percentages of S phase IER5-overexpression-HepG2 and HepG2-Control cells were (4.68±2.34) %vs (8.04±1.46)%, P<0.05. Showing shines the latter 12 hours, IER5-overexperssion-HepG2 and the HepG2-Control cell present the G2-M time to hinder.At the end of 24th hour, percentages of G2-M phase in HepG2-control cell and IER5-overexperssion-HepG2 were (27.85±4.56)%vs(20.36±2.26)%, P<0.05; (40.64±3.28)%vs(18.98±2.86)%, P<0.05. And IER5-overexperssion -HepG2 cell percentage of G2-M phase is higher than the HepG2-control group obviously (40.64±3.28)%vs(27.85±4.56)%, P<0.05. The results explained that up-regulating the IER5 gene expression has increased the HepG2 cell to have the cell ratio which the G2-M time hinders, reduced the ratio of HepG2 cell to enter the synthesis time of DNA S time. And the cell's anti-emissivity was weakened.4 Study on the HepG2's cell apoptosis by up-regulating IER5 gene: Be radiated in 2Gy and 4Gy Co60γradiation, cell's apoptosis percentages in IER5-overexperssion-HepG2,HepG2-control and IER5-SiRNA-HepG2 (this cell line originated from this experimental group cooperation member) had the significance difference (P<0.05).The ratio of IER5-overexpression-HepG2's cell apoptosis compared to IER5-SiRNA-HepG2 and HepG2-Control markup obviously. The ratio of IER5-SiRNA-HepG2's cell apoptosis was lowest. The result explained that up-regulating the IER5 gene was able to promote the HepG2 cell to apoptosis, to weaken its anti-emissivity. The IER5 gene silence can reduce the HepG2 cell to occurs apoptosis, increases its radiation resistance, prompt the IER5 gene to be able to increase the HepG2 cell radio sensitivity, indicated that this gene possibly is HepG2 cell's radiation sensitive gene.Consultation:1 We established the monoclonal cell line which can express the IER5 protein stably and highly, named IER5-overexperssion- HepG2 cell. And obtains the state-level patent of invention, the patent number: 200910082178.4。2 Up-regulating the IER5 gene has been possible to suppress the HepG2 cell's growth, the increment and the fission, and promotion cell apoptosis. 3 Up-regulating the IER5 gene has weakened the repair ability of the HepG2 cell to radiation damage and cell's anti-emissivity. The radio sensitivity was strengthened. It indicated that this gene possibly for liver cancer's radiation sensitive gene.This study carried out beneficial investigations about the function of IER5 gene. The main innovation was the effect on proliferation or cell cycle and apoptosis. In addition,the radio sensitivity of HepG2 cell was weakened by expressing the IER5 gene highly . Therefore, we could forecast that the IER5 gene will be possible to be advantageous to liver cancer's radiotherapy. Similarly, we were used of radiotherapy to enhance tumor cell death or stop the growth of tumor cells and cell division in clinical. The IER5 gene will be very valuable to radiotherapy of hepatocarcinoma.