Effect And Mechanisms Of XRCC3 On Radiosensiti-vity Of Esophageal Squamous Cell Carcinoma

Objective:The present study aimed to investigate the effect of X-ray repair cross-complementing groups 3(XRCC3) on the radiotherapy response of esophageal squamous cell carcinoma(ESCC) and the underlying mechanism of the roles of XRCC3 in ESCC radiosensitivity.Materials and Methods:1. The expression levels of XRCC3 in normal esophageal cell line and five ESCC cell lines were tested by Reverse transcriptase-PCR(RT-PCR) and Western Blot.Immunohistochemistry(IHC) was used to test the XRCC3 expression in both epithelial mucosa specimens and ESCC tissues. The expression of XRCC3 was assessed by IHC evaluation method. The relationships among high XRCC3 expression and most patient characteristics, including age, gender, TNM stage et al,and chemoradiotherapy(CRT) were detected by univariate analysis. Then the correlation between the 60 ESCC patients' disease-specific survival(DSS) and high XRCC3 expression was also tested by univariate analysis. Further multivariate analysis was used to detect the relativity among XRCC3 expression,CRT response, N stage, M stage and DSS.2. A sh RNA targeting XRCC3 m RNA(sh XRCC3) was introduced into two ESCC cell lines(TE-1 and Kyse30) for stable knockdown of XRCC3 through recombinant lentiviral infection. Then, p CDH-XRCC3 lentiviral particles were transduced into the above XRCC3-silenced ESCC cells(sh XRCC3 + XRCC3) to replenish the expression of XRCC3. The levels of XRCC3 were examined by Western blot.3. Inspecting the colony formation capacity among the sh XRCC3, sh XRCC3 +XRCC3 and control group in Kyse30 and TE-1 cells. The responses of ESCC cells to ionizing radiation(IR) were examined by clonogenic survival assays. After IR the volume of the TE-1 cell xenografts was measured with a caliper. Then,observing the variation of the xenografts volume between sh XRCC3 group and control group.4. Flow cytometry was used to determine the percentage of cells undergoing apoptosis. Cells were stained with DAPI and examined for nuclear morphology.Western blot was used to detect the expression levels of cleaved PARP and cleaved caspase-3 in both TE-1 and Kyse30 cells exposed to 6 Gy IR,and then detect the protein expression of XRCC2 and Rad51.3.5. Immunofluorescence was used to analyze the ?-H2 AX and the telomere dysfunction induced foci(TIF) among the sh XRCC3, sh XRCC3 + XRCC3 and control group in Kyse30 and TE-1 cells.Results:1. High expression of XRCC3 in esophageal squamous cell carcinoma(ESCC) is associated with chemoradiotherapy resistance and predicts poor patient survival.2. Compared with normal esophageal cell line and epithelial mucosa specimens,ESCC cell lines and tissues were higher expressing XRCC3. And then TE-1 and Kyse30 cell with XRCC3 silencing or XRCC3 replenishing expression was successfully constructed.3. Silencing of XRCC3 enhances esophageal squamous cell carcinoma cell radiosensitivity in vitro and in vivo. The results showed that inhibition of XRCC3 had no significant effect on the colony formation capacity of untreated cells.However, after ionizing radiation(IR) treatment, clonogenic survival was obviously reduced in XRCC3 silencing cell lines compared with control cells. We also found that silencing of XRCC3 did not affect tumor growth under normal conditions but xenograft tumor volumes were decreased significantly compared with the control group after the same IR treatment.4. Inhibition of XRCC3 promotes ionizing radiation-induced apoptosis and mitotic catastrophe in esophageal squamous cell carcinoma cells. Compared with control cells, inhibition of XRCC3 has a significant increase in the proportion of apoptosis and mitotic catastrophe(MC) after IR. Moreover, Western blot analysis indicated that significant downregulation of XRCC2 and RAD51.3 in both KYSE30-sh XRCC3 and TE-1-sh XRCC3 cells.5. Inhibition of XRCC3 enhances ionizing radiation-induced DNA damage and telomere dysfunction. After 48 h of IR treatment, the unrepaired DNA damage detected by ?-H2 AX and telomere dysfunction as judged by telomere dysfunction induced foci(TIF) were significantly increased in XRCC3-silenced ESCC cells compared with the control cells.Conclusion:XRCC3 protects ESCC cells from ionizing radiation-induced death by promoting DNA damage repair and ? or enhancing telomere stability. XRCC3 may be a novel radiosensitivity predictor and promising therapeutic target for ESCC.