沉默XRCC2基因表达对大肠癌放射治疗敏感性的影响
发布时间：2018-09-05 09:04
【摘要】：大肠癌,包括结肠癌和直肠癌,是威胁人类生命健康的常见消化道恶性肿瘤。放射治疗是大肠癌的主要治疗手段之一,但放射治疗大肠癌的辐射耐受现象严重影响大肠癌病人的疗效,放疗抵抗性成为大肠癌放疗面临严峻且迫切需要解决的难题。电离辐射后细胞DNA损伤的修复是肿瘤放疗效果不佳的主要原因之一。X射线修复交叉互补(X-ray repair cross complementing, XRCC)基因家族(XRCC1～XRCC11)对电离辐射诱导的DNA损伤修复发挥重要作用。DNA损伤通过碱基切除修复、核苷酸切除修复、错配修复、同源重组修复(homologous recombination,HR)和非同源末端连接(non-homologous end joining, NHEJ)等多种方式进行修复,从而维持生物体基因组的完整性和抑制肿瘤的发生。XRCC2是重要的参与HR途径的基因之一,其高表达与增加辐射诱导的DNA损伤抵抗有关。XRCC2基因修复缺陷表现出对电离辐射的敏感性增高,而XRCC2蛋白过表达则对放射线耐受。提示通过抑制肿瘤细胞XRCC2的表达,有可能提高临床肿瘤放射治疗的敏感性。目前尚未见到关于大肠癌中XRCC2表达水平以及XRCC2与放射敏感性关系的研究报道。降低XRCC2的表达是否可以改变大肠癌细胞的放射敏感性,XRCC2是否可以预测大肠癌放射治疗的疗效,目前在国内外未见相关的研究报道。 目的：本实验通过大肠癌体外细胞模型和体内动物模型,探讨shRNA介导的XRCC2基因沉默是否影响大肠癌细胞的放疗敏感性及其疗效,阐明XRCC2在大肠癌放疗敏感性中的关键作用和初步相关机制。 方法：(1)体外细胞实验：将shRNA-XRCC2转染人大肠癌T84细胞以沉默XRCC2基因表达,采用蛋白免疫印迹法和实时定量PCR法检测沉默XRCC2基因的效率；采用MTT法检测T84细胞的增殖。经X-射线照射后,采用克隆形成法检测T84细胞的放射敏感性；采用碱性“彗星”电泳法测定T84细胞的DNA损伤修复；流式细胞术检测T84细胞的细胞周期；Annexin V-FITC/PI双染法检测T84细胞的细胞凋亡率。(2)体内细胞实验：同时将shRNA-XRCC2转染的大肠癌T84细胞接种于BALB/c裸鼠建立移植瘤模型,进行X-射线照射,检测肿瘤的体积和重量变化,并对肿瘤组织进行病理分析。 结果：(1)在体外细胞实验中,shRNA-XRCC2转染有效抑制了T84细胞中XRCC2蛋白和mRNA的表达。经嘌呤酶素筛选,得到了稳定的XRCC2基因沉默的大肠癌T84细胞系。细胞生长曲线表明,沉默XRCC2表达明显抑制了T84细胞的增殖。克隆形成实验显示,XRCC2基因沉默的T84细胞经X-射线照射后,克隆形成数目显著减少,表明XRCC2基因沉默提高了T84细胞的放射敏感性。彗星实验表明,沉默XRCC2表达的T84细胞DNA损伤增多,DNA损伤修复能力下降。流式细胞术检测显示,XRCC2基因沉默显著诱导了辐射导致的细胞凋亡和细胞阻滞在G2/M期。(2)在体内细胞实验中,转染shRNA-XRCC2的裸鼠种植瘤生长缓慢,肿瘤体积和重量明显减少。肿瘤病理组织学分析表明,转染shRNA-XRCC2的肿瘤组织核分裂相减少,多见大小不等的坏死区。说明沉默XRCC2表达提高了裸鼠大肠癌对辐射的敏感性,肿瘤生长受到明显的抑制作用。 结论：shRNA介导的XRCC2基因沉默有效抑制了体外大肠癌细胞和体内裸鼠大肠癌肿瘤的生长,沉默XRCC2表达对体外和体内大肠癌细胞对X射线的反应具有一致性,即均提高了大肠癌对放射的敏感性。提示XRCC2有希望在大肠癌的临床放射治疗敏感性中作为一重要的靶向基因。
[Abstract]:Colorectal cancer, including colorectal cancer and rectal cancer, is a common malignant tumor of the digestive tract that threatens human life and health. Radiotherapy is one of the main treatments for colorectal cancer. However, the radiation tolerance of colorectal cancer seriously affects the curative effect of colorectal cancer patients. Radiotherapy resistance has become a serious and urgent problem in colorectal cancer radiotherapy. X-ray repair cross complementing (XRCC) gene family (XRCC1-XRCC11) plays an important role in the repair of DNA damage induced by ionizing radiation. DNA damage is repaired by base excision and nucleotide excision. Repair, mismatch repair, homologous recombination (HR) and non-homologous end-joining (NHEJ) repair methods to maintain the integrity of the organism genome and inhibit the occurrence of tumors. XRCC2 is one of the important genes involved in the HR pathway, its high expression and increased radiation inducement. XRCC2 gene repair deficiency shows increased sensitivity to ionizing radiation, while XRCC2 protein overexpression is radioresistant. It suggests that inhibiting the expression of XRCC2 in tumor cells may enhance the sensitivity of clinical tumor radiotherapy. Whether reducing the expression of XRCC2 can change the radiosensitivity of colorectal cancer cells and whether XRCC2 can predict the efficacy of radiotherapy for colorectal cancer have not been reported at home and abroad.
Objective: To investigate whether shRNA-mediated XRCC2 gene silencing affects the radiosensitivity of colorectal cancer cells in vitro and in vivo, and to elucidate the key role of XRCC2 in the radiosensitivity of colorectal cancer cells.
Methods: (1) In vitro cell experiment: shRNA-XRCC2 was transfected into human colorectal cancer T84 cells to silence XRCC2 gene expression, and the efficiency of XRCC2 gene silencing was detected by Western blotting and real-time quantitative PCR, and the proliferation of T84 cells was detected by MTT method. DNA damage and repair of T84 cells were detected by alkaline comet electrophoresis, cell cycle of T84 cells was detected by flow cytometry, and apoptosis rate of T84 cells was detected by Annexin V-FITC/PI double staining. (2) In vivo cell experiment: T84 cells transfected with shRNA-XRCC2 were inoculated into BALB/c nude mice to establish a transplanted tumor model. X-ray irradiation was used to detect tumor volume and weight changes and pathological analysis of tumor tissues.
Results: (1) In vitro, shRNA-XRCC2 transfection effectively inhibited the expression of XRCC2 protein and mRNA in T84 cells. A stable colon cancer T84 cell line with XRCC2 gene silencing was obtained by purinase screening. After X-ray irradiation, the number of cloned T84 cells with gene silencing decreased significantly, suggesting that XRCC2 gene silencing increased the radiosensitivity of T84 cells. Comet assay showed that the DNA damage of T84 cells expressing XRCC2 increased and DNA damage repair ability decreased. Flow cytometry showed that XRCC2 gene silencing significantly induced radiation-induced damage. Cell apoptosis and cell block were observed in G2/M phase. (2) In vivo, shRNA-XRCC2-transfected nude mice showed slow growth, significantly reduced tumor volume and weight. Tumor histopathological analysis showed that shRNA-XRCC2-transfected tumors showed less mitosis and more necrotic areas of different sizes. The sensitivity of colorectal cancer to radiation in nude mice was significantly inhibited by tumor growth.
Conclusion: XRCC2 gene silencing mediated by shRNA can effectively inhibit the growth of colorectal cancer cells in vitro and nude mice colorectal cancer cells in vivo. The silencing of XRCC2 expression is consistent with the response of colorectal cancer cells to X-ray in vitro and in vivo, that is to say, it increases the sensitivity of colorectal cancer to radiation. As an important target gene in therapeutic sensitivity.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R735.34;R730.55
[Abstract]:Colorectal cancer, including colorectal cancer and rectal cancer, is a common malignant tumor of the digestive tract that threatens human life and health. Radiotherapy is one of the main treatments for colorectal cancer. However, the radiation tolerance of colorectal cancer seriously affects the curative effect of colorectal cancer patients. Radiotherapy resistance has become a serious and urgent problem in colorectal cancer radiotherapy. X-ray repair cross complementing (XRCC) gene family (XRCC1-XRCC11) plays an important role in the repair of DNA damage induced by ionizing radiation. DNA damage is repaired by base excision and nucleotide excision. Repair, mismatch repair, homologous recombination (HR) and non-homologous end-joining (NHEJ) repair methods to maintain the integrity of the organism genome and inhibit the occurrence of tumors. XRCC2 is one of the important genes involved in the HR pathway, its high expression and increased radiation inducement. XRCC2 gene repair deficiency shows increased sensitivity to ionizing radiation, while XRCC2 protein overexpression is radioresistant. It suggests that inhibiting the expression of XRCC2 in tumor cells may enhance the sensitivity of clinical tumor radiotherapy. Whether reducing the expression of XRCC2 can change the radiosensitivity of colorectal cancer cells and whether XRCC2 can predict the efficacy of radiotherapy for colorectal cancer have not been reported at home and abroad.
Objective: To investigate whether shRNA-mediated XRCC2 gene silencing affects the radiosensitivity of colorectal cancer cells in vitro and in vivo, and to elucidate the key role of XRCC2 in the radiosensitivity of colorectal cancer cells.
Methods: (1) In vitro cell experiment: shRNA-XRCC2 was transfected into human colorectal cancer T84 cells to silence XRCC2 gene expression, and the efficiency of XRCC2 gene silencing was detected by Western blotting and real-time quantitative PCR, and the proliferation of T84 cells was detected by MTT method. DNA damage and repair of T84 cells were detected by alkaline comet electrophoresis, cell cycle of T84 cells was detected by flow cytometry, and apoptosis rate of T84 cells was detected by Annexin V-FITC/PI double staining. (2) In vivo cell experiment: T84 cells transfected with shRNA-XRCC2 were inoculated into BALB/c nude mice to establish a transplanted tumor model. X-ray irradiation was used to detect tumor volume and weight changes and pathological analysis of tumor tissues.
Results: (1) In vitro, shRNA-XRCC2 transfection effectively inhibited the expression of XRCC2 protein and mRNA in T84 cells. A stable colon cancer T84 cell line with XRCC2 gene silencing was obtained by purinase screening. After X-ray irradiation, the number of cloned T84 cells with gene silencing decreased significantly, suggesting that XRCC2 gene silencing increased the radiosensitivity of T84 cells. Comet assay showed that the DNA damage of T84 cells expressing XRCC2 increased and DNA damage repair ability decreased. Flow cytometry showed that XRCC2 gene silencing significantly induced radiation-induced damage. Cell apoptosis and cell block were observed in G2/M phase. (2) In vivo, shRNA-XRCC2-transfected nude mice showed slow growth, significantly reduced tumor volume and weight. Tumor histopathological analysis showed that shRNA-XRCC2-transfected tumors showed less mitosis and more necrotic areas of different sizes. The sensitivity of colorectal cancer to radiation in nude mice was significantly inhibited by tumor growth.
Conclusion: XRCC2 gene silencing mediated by shRNA can effectively inhibit the growth of colorectal cancer cells in vitro and nude mice colorectal cancer cells in vivo. The silencing of XRCC2 expression is consistent with the response of colorectal cancer cells to X-ray in vitro and in vivo, that is to say, it increases the sensitivity of colorectal cancer to radiation. As an important target gene in therapeutic sensitivity.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R735.34;R730.55
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