Research On The Radiosensitivity Of HepG-2 Cancer Cells By RNAi Targeting RAD51 Gene

Radiotherapy and chemotherapy are two major methods for cancer treat, but the radiosensitivity of the majority of cancers is similar to that of normal tissues. The results of radiotherapy are not satisfying. The radiosensitizer with good performance and low toxicity has not been found yet. It has become a hot spot and difficult issue of cancer research. Recently it has been found that radiosensitivity of cancers is related to repair efficiency of DSB damages, cell cycle arrest, apoptosis and so on. Moreover, repair efficiency of DSB damage is the most important factor among the above factors. Ionizing radiation and DNA damaging agents(such as MMC)give rise to DSBs of DNA. DSB are one of the most detrimental DNA lesions. To combat these detrimental lesions, two major pathways have evolved for the repair of DSB: HR and NHEJ. The fundamental difference in these pathways is the requirement for a homologous DNA sequence. HR repairs DSB by retrieving genetic information from an undamaged homologue (sister-chromatid or homologous chromosome). In contrast to HR, NHEJ rejoins DSB via direct ligations of the DNA ends without any requirement for sequence homology, which is an important mechanism of DNA damage response.HR is a complex process. A lot of proteins are involved. Rad51, the homolog of the E. coli RecA protein, is critical to HR-mediated DSB repair. Rad51 forms nucleo-protein filaments on single-stranded DNA and mediates homologous pairing and strand exchange between DNA duplexes. RAD51 is a kind of ATPase, which is activated when binding to ssDNA. It also binds to dsDNA or ssDNA, and plays a role in DNA repair and recombination. Moreover, RAD51 involves in mitotic and meiotic recombination. Defect of RNA51 leads to increased chromosome breaks owning to reduced DSB repair. If RAD51 protein is overexpressed, the cells are resistant to IR and the apoptosis of the cellsis reduced by IR. These imply that if RAD51 expression is inhibited by RNAi, the radiosensitivity of cancers may increase.RNA interference (RNAi) represents a phenomenon of double-stranded RNA-mediated post-transcriptional gene silencing (PTGS). During this process, exogenous cellular dsRNA is cleaved by an ribonuclease, Dicer, to generate 19²3 nt small RNA fragments, referred to as short interfering RNA (siRNA). By joining an effector complex termed RISC, siRNA binds to the cellular mRNA with homologous sequences and leads to the degradation of the corresponding mRNA. At present, RNAi has been widely applied in the research of gene function and gene therapy as an efficient tool for specific gene silencing. In gene therapy of cancers, RNAi can specificly inhibit target gene expression through deliverying siRNA or siRNA expression vector to cancer cells.This research aims to reveal whether inhibited RAD51 gene by RNAi could enhance radiosensitivity of HepG-2 cell line. According to the design results of Biocomputer and Oligoengine software, siRNAs were designed to target 449⁴67 nU 876⁸94 nt and 374—392 nt of the mRNA sequence of RAD51 gene(NM₀02875). The siRNA-encoding complementary single-stranded oligonucleotides hybridize to give BamH I and Hindffl— compatible overhang. After annealing and 5' phosphorylation by T4 polynucleotide kinase, oligonucleotides were ligated into plasmid pSilencer2.1. Then transform the plasmids in DH5a. Name designation of the resulting plasmids was pSilencer-1, pSilencer-2, pSilencer-3 or pSilencer-C. PCR and sequencing identified these plasmids.To reduce the amount of carbohydrate and increase transfection efficiency, Qiagen plasmid purification kit was used. The pcDNA3.0-GFP vector, which encodes GFP protein, was used to assess transfection efficiency. The efficiency will be best if cell confluence is 90% during transfection and the ratio of DNA: lipofectamine 2000 is 0.8 ug: 2 ul. The experiment included five groups: pSilencer-1, pSilencer-2, pSilencer-3, pSilencer-C and untransfection. After transfection each group was cultured in DMEM medium with 200 ug/ml hygromycin B. About two weeks later, cell clones wereobtained. Three clones of each group were selected and transfered to a fresh 96 well culture plate. After amplification culture, these cells were transfered to culture dishes. pSilencer vector sequences in each clone were demonstrated by PCR experiment.