Wang Nan, Xu Meilan, Liao Shuting
Laboratory of Cell and Molecular Biology, College of Life Sciences, Meizhou 514015, China.
Clinical Microbiology and Immunology Laboratory, Medical College, Jiaying University, Meizhou 514015, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2018 Jul 30;38(7):861-866. doi: 10.3969/j.issn.1673-4254.2018.07.15.
To investigate the role of AGGF1 in DNA damage repair and modulating chemotherapy resistance in human colon cancer cells.
Cisplatin-induced human colon cancer HCT116 cells transfected with AGGF1 siRNA and siNC Lipofectamine 2000 were examined for AGGF1, γH2AX and pNBS1 expressions using Western blotting. Immunofluorescence analysis was used to detect the recruitment of phosphorylated γH2AX and AGGF1 at the site of cisplatin-induced double-strand DNA breaks, and MTS method was used to investigate the proliferation of the damaged cells. Immunohistochemical method was used to detect the expression level of AGGF1 in human colon cancer and adjacent normal tissues.
Western blotting showed that AGGF1 expression was significantly down-regulated in HCT116 cells after cisplatin exposure, and transfection withAGGF1 siRNAobviously inhibited the expression of phosphorylated γH2AX and NBS1. Immunofluorescence assay showed the co-localization of AGGF1 and γH2AX. Down-regulation of AGGF1 mediated by siRNA obviously increased the chemosensitivity of the cells ( < 0.01). In the clinical specimens, AGGF1 was found to be overexpressed in colon cancer tissues as compared with the adjacent normal tissues ( < 0.01), suggesting its association with the malignant phenotype of the tumor.
Down-regulation of AGGF1 inhibits DNA damage repair and increases the chemosensitivity in colon cancer cells possibly in relation with the suppressed phosphorylation of NBS1.
研究血管生成素样因子1（AGGF1）在人结肠癌细胞DNA损伤修复及调节化疗耐药中的作用。
用脂质体2000将AGGF1小干扰RNA（siRNA）和阴性对照siRNA（siNC）转染至顺铂诱导的人结肠癌HCT116细胞，采用蛋白质免疫印迹法检测AGGF1、γH2AX和磷酸化NBS1（pNBS1）的表达。用免疫荧光分析法检测顺铂诱导的双链DNA断裂部位磷酸化γH2AX和AGGF1的募集情况，并用MTS法研究受损细胞的增殖情况。采用免疫组织化学方法检测AGGF1在人结肠癌组织及癌旁正常组织中的表达水平。
蛋白质免疫印迹结果显示，顺铂处理后的HCT116细胞中AGGF1表达明显下调，转染AGGF1 siRNA可显著抑制磷酸化γH2AX和NBS1的表达。免疫荧光分析显示AGGF1与γH2AX共定位。siRNA介导的AGGF1下调明显增加了细胞的化学敏感性（P<0.01）。在临床标本中，发现结肠癌组织中AGGF1的表达高于癌旁正常组织（P<0.01），提示其与肿瘤的恶性表型有关。
AGGF1下调可抑制结肠癌细胞的DNA损伤修复并增加其化学敏感性，这可能与NBS1磷酸化受抑制有关。
