Study On The Effect Of XRCC2Gene Silencing On Radiosensitivity Of Colorectal Cancer

Colorectal cancer, including colon cancer and rectal cancer, is one of the most common tumors of the digestive tract threatening the human life and health seriously. Although radiotherapy is one of methods of colorectal cancer treatment, the radioresistance in radiotherapy seriously affects the curative effect of colorectal cancer patients. DNA damage’s repair after exposure to ionizing radiation is one of reasons of tumors resistance to radiotherapy. X-ray repair cross complementing gene family (XRCC1-XRCC11) plays an important role in repairing DNA damages induced by ionizing radiation. DNA damages are repaired by various mechanisms such as base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end joining pathways to maintain the integrity of the genetic information and inhibit the formation of tumors. XRCC2is the key protein of DNA homologous recombination repair pathway, and its high expression is associated with enhanced resistance to DNA damage induced by ionizing radiation. XRCC2gene defects in some cell lines show an increased sensitivity to radiation, whereas the abnormal upregulation of XRCC2gene expression renders tumor cells resistance to radiation. Thus, we propose that the inhibiting of XRCC2expression in tumor cells may enhance their radiosensitivity. However, the studies on XRCC2expression in colorectal cancer and its association with sensitivity to radiation were not found. Until now, it is not yet known whether lowering XRCC2expression can affect the sensitivity of radiotherapy for colorectal cancer or XRCC2can predict the efficacy of colorectal cancer radiotherapy.Objective:The goal of the project is to study the effects of XRCC2gene silencing mediated by shRNA on radiosensitivity of colorectal cancer cells in vitro and in vivo and to elucidate the relationship between XRCC2role and the mechanism of colorectal cancer radiotherapy.Methods:The vector-based shRNA plasmid (shRNA-XRCC2) was transfected into colorectal cancer T84cell line to silence XRCC2gene expression. The efficiency of XRCC2silencing was determined by western blot and real-time PCR analyses. The growth curve of T84cells in vitro was examined by MTT assay. The effect of XRCC2suppression on T84cells’radiosensitivity to X-radiation was examined by colony formation assay. DNA damage’s repair of T84cells was determined by alkaline comet assay. The relationship between the sensitivity of T84cells to radiation and the cell cycle distribution or cell apoptosis was performed by flow cytometric analysis. Colorectal cancer T84cells transfected shRNA-XRCC2were transfered into BALB/c nude mice to establish a xenograft model in vivo.The curative effect and pathological analysis of xenografts were investigated after xenograft received radiotherapy.Results:(1) In vitro:XRCC2protein and mRNA expression of colorectal cancer T84cells was effectively silenced by shRNA-XRCC2transfection. Colorectal cancer T84cell line silencing XRCC2gene expression stablely was achieved successfully through selecting with purine enzyme. Knockdown of XRCC2expression by shRNA inhibited cell growth of T84cells as evaluated by MTT assay. The number of colonies formed in shRNA-XRCC2cells was significantly decreased, which showed that shRNA-mediated XRCC2suppression rendered T84tumor cells more sensitive to radiation treatment. DNA damages were increased and the capability of DNA damage’s repair was decreased in T84cells as examined by comet assay. Suppression of XRCC2expression resulted in an elevation of cell apoptosis and cell cycle arrested in G2/M phase induced by radiation through flow cytometric analysis.(2)In vivo:Tumor xenograft transfected with shRNA-XRCC2in nude mice grew slowly and the tumor volume and the tumor weight were decreased significantly. Pathological analysis showed that karyokinesis was decreased and small areas of necrosis were found in tumor xenograft treated by shRNA-XRCC2transfection. The data suggested that knockdown of XRCC2expression enhanced the tumor’s sensitivity to radiation in nude mice and tumor xenograft’s growth was retarded.Conclusion:XRCC2gene silencing mediated by shRNA inhibited the growth of colorectal tumor cells in vitro and in vivo and has radiosensitization effects on colorectal tumor cells in vitro and in vivo, ie. increased tumor’s sensitivity to radiation. These data strongly suggested that XRCC2may be further developed as a promising therapeutic target for the treatment of radioresistant human colorectal cancer.