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

The primary carcinoma of liver (hepatocarcinoma) is one of the most common malignant tumors.Its fatality is at the second among the fatality of correlated malignant tumors in China. With the development of three dimensional conformal radiation therapy technology, radiotherapy has become a powerful weapon for the treatment of hepatocarcinoma. Radiosensitivity is one of the major influence factors to 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 the expression of a gene changes after irradiation and could simultaneously cause apoptosis or growth suppression in tumor cells, this gene will be very valuable to radiation treatment of cancer. IER5 gene belongs to the immediate early response gene family. In prophase times, our research found that IER5 gene showed a high expression in radiated hepatoma tissues of BALB/c-nu nude mices. This phenomenon hinted that IER5 might have some relationship with radiosensitivity of hepatocarcinomc. This gene may play an important role in the maintenance of the liver's normal physiology function, also, the genesis and development of hepatocarcinoma.RNA interference (RNAi) can close some special genes and achieve the aims of curing tumors. Small interfering RNA (siRNA) technology is an important method to study a gene's function. Using RNAi can break down the specific intracellular mRNA mediated by endogenous or exogenous double stranded RNA, and its ultimate purpose is to silence the targeted gene and result in the depletion of the homologous function. RNAi maybe cure tumor because of its distinct effect to close specified genes. It reveals the mechanism of intracellular gene silencing, moreover, RNAi is a splendid mean to analyze the gene's function in the postgenome times. One complete cell cycle is the whole process that the cell experiences from the end of the first division to generate a new cell, to the end of the second division. It is divided into interphase and mitosis phase. The interphase is divided into pre-DNA synthesis phase (G1 phase), DNA synthesis phase (S phase) and DNA synthesis post-phase (G2 phase). The mitosis phase is also called M phase, including prophase, intermediate stage, anaphase, and end stage. The end stage is also known as GO phase. In the GO phase, the cell stops dividing, and begins to carry out certain biological function. During the running of a cell cycle, from G1 phase to S phase, and from G2 phase to M phase, there are two critical "check points":one is G1/S, and the other is the G2/M. Only after the completion of the previous phase of the physiological proceeding, the cell can pass through the test points and enter into the next step. Reaction in the cell triggered by some unexpected events can influence some factors which can drive the proceeding of the cell cycle. This reaction which is called cell cycle arrest, can block the cell cycle at a certain check point.Research on IER5 is very few now, and biological function of the gene is indefinite. There was no study on the relationship between IER5 gene and hepatocarcinoma. Accordingly, we designed special RNAi fragments to transfect into HepG2 cells, selected growth curve and cell cycle as the experimental parameters, and then studied the proliferation and radiosensitivity in HepG2 cells. Furthermore, we try to explore the biological function of IER5 through the experiment.Objective:In order to research the biological function of the gene IER5, we selected the ex vivo HepG2 cells as research objects. IER5-silenced HepG2 cell model was generated by plasmid mediated siRNA technology. Growth curve and flow cytometry were used to measure the cell proliferation after silencing the gene. In addition, effect on the cell cycle has been studied after exposed to gamma-ray of cobalt 60.Methods: 1 Establishment of the the stable transfected cell lines:Designed special RNAi fragments and constructed carrying agents which could silence IER5 gene, then RNA interference technology was employed to silence the IER5 gene in HepG2 cells. At the same time, the plasmid connected with fragments which had no homology to Human gene order was also transfected into HepG2 cells as a negative control group. The expression of IER5 mRNA was confirmed by real-time PCR and the expression of IER5 protein was detected by Western blot analysis.2 Study on the HepG2 cells'proliferation and cell cycle mediated by siRNA to silence IER5:①cell proliferation:growth curve of the cells exposed to gamma-ray of cobalt 60 at the doses of 2 and 4Gy, compared with OGy.②cell cycle:using flow cytometry to detect cell cycle after being incubated for 24 hours.③effects on cell cycle exposed to gamma-ray of cobalt 60:using flow cytometry to detect cell cycle exposed to the radiation of 4Gy doses.Results:1 Establishment of the the stable transfected cell lines:Detected the transfected cells by Real-time PCR technique, HepG2 cells which were tranfected with the vector named A-3, demonstrated the lowest RNA expression level with a 79.3% degression compared to the negative control cells HepG2-NC. Western blot analysis could also certificate that IER5 gene was knocked down in this cell line. It was defined as HepG2-IER5-siRNA cell line.2 Study on the HepG2 cells'proliferation mediated by siRNA to silence IER5:Growth velocity of the HepG2-IER5-siRNA stepped up after 3 or 4 days exposed to gamma-ray of cobalt 60 with the dose of OGy,2Gy and 4Gy. Until the sixth day its cell numbers was exceeded apparently, compared to the HepG2-NC.Detected after inoculation for 24 hours:percentages of S phase in HepG2-IER5-siRNA cells was higher than that in HepG2-NC:(39.80±2.02)% vs(32.30±0.02)%, P＜0.05. 3 Study on cell cycle of the HepG2 and radiation effect mediated by siRNA to silence 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 HepG2-IER5-siRNA cells:(21.98±0.96)%vs(52.44±4.74)%, P＜0.05; percentages of G2/M phase in HepG2-NC cells were (23.36±2.20)% vs(49.00±4.58)%, P＜0.05. At the end of zeroth hour and 24th hour, percentages of G2/M phase in HepG2-NC cells were (23.36±2.20)%vs (31.85±4.586)%, P＜0.05; the percentages in HepG2-IER5-siRNA cells at the end of zeroth hour and 24th hour were (21.98±0.96)%vs(20.73±1.04)%, P＞0.05.It suggested that at the time of 24th, the G2/M phase arrest in HepG2-IER5-siRNA was removed, while in HepG2-NC cells G2/M phase arrest was still maintained partially. At the end of 24th hour, percentages of S phase in HepG2-IER5-siRNA and HepG2-NC cells were (14.71±1.04)% vs(6.14±0.44)%, P＜0.05.Conclusions:1 A kind of monoclonal cell line with IER5 silenced was established, named HepG2-IER5-siRNA.2 Inhibiting the expression of IER5 gene could speed up the growth rapid in HepG2 cells, at the same time, quantity of the cells entered into S phase was increased.3 Radiosensitivity was weakened after silencing IER5 gene, and ability to reparation was strengthened in the cells exposed to gamma-ray of cobalt 60.This study carried out beneficial investigations about the function of IER5 gene. We also inquired into the therapy to tumors using RNAi technology. Point of innovation in the experiment was the effect on proliferation and cell cycle in HepG2 cells when IER5 gene was silenced. In addition, radiosensitivity was weakened in HepG2-IER5-siRNA cells, and ability to reparation of irradiation was intensified in the HepG2 cells.It suggested that IER5 gene may play an important role in regulating the cell cycle. This gene will be very valuable to radiation treatment of hepatocarcinoma.