miR-34s增强结直肠癌细胞对放射敏感性的作用研究

The study of miR-34s enhancing the radiosensitivity of colorectal cancer cells

目的:探讨miR-34s对结直肠癌细胞放射敏感性的影响以及在电离辐射致DNA损伤中的作用。方法:利用实时荧光定量PCR(real-time quantitative fluorescence PCR,qRT-PCR)检测miR-34a/b/c-5p在结直肠癌细胞中的表达水平,以及电离辐射后miR-34a/b/c-5p表达水平变化趋势。基于克隆形成法和单击多靶模型建立细胞存活曲线,分析miR-34a/b/c-5p对结直肠癌细胞放射敏感性的影响。过表达miR-34a/b/c-5p并进行照射,利用免疫荧光法检测照射后γH2AX焦点形成情况,进而分析miR-34a/b/c-5p对电离辐射致DNA损伤的作用。结果:miR-34a/b/c-5p在HCT116细胞中的表达水平明显高于HT29细胞(P<0.05)。HCT116细胞经4 Gy照射后24 h内,miR-34a/b/c-5p表达水平呈双峰变化趋势。与miR-NC组相比,过表达miR-34a/b/c-5p可显著增加结直肠癌细胞的放射敏感性,miR-34a/b/c-5p组细胞平均致死剂量(D_(0))和准阈值剂量(D_(q))均明显降低,且辐射增敏比(SER)明显增加。过表达miR-34a/b/c-5p可显著增加电离辐射诱导的DNA双链断裂(double strand breaks,DSBs)水平,照射后1 h和8 hγH2AX焦点数明显高于miR-NC组(P<0.05)。结论:miR-34s为放射响应miRNA分子,过表达miR-34s可增加电离辐射诱导的DNA损伤水平并增强结直肠癌细胞的放射敏感性。

Objective:To investigate the effects of miR-34s on the radiosensitivity of colorectal cancer(CRC)cells and the role of miR-34s in ionizing radiation-induced DNA damage.Methods:Real-time quantitative fluorescence PCR(qRT-PCR)was used to detect the expression of miR-34a/b/c-5p in CRC cells and the expression trend within 24 h after ionizing radiation.Cell survival curves were established based on clonogenic assay and multitarget/single-hit model to analyze the effects of miR-34a/b/c-5p on the radiosensitivity of CRC cells.Cells overexpressing miR-34a/b/c-5p were exposed to ionizing radiation,andγH2AX foci were detected using immunofluorescence assay after irradiation to analyze the effects of miR-34a/b/c-5p on ionizing radiation-induced DNA damage.Results:The expression levels of miR-34a/b/c-5p in HCT116 cells were significantly higher than that in HT29 cells(P<0.05).Within 24 h after 4 Gy irradiation,miR-34a/b/c-5p showed a bimodal expression pattern in HCT116 cells.Compared with the miR-NC group,overexpression of miR-34a/b/c-5p could significantly enhance the radiosensitivity of CRC cells.The mean lethal dose(D_(0))and quasi-threshold dose(D_(q))were significantly decreased in the miR-34a/b/c-5p group,while the sensitizing enhancement ratio(SER)was significantly increased.Overexpression of miR-34a/b/c-5p significantly increased the level of ionizing radiation-induced DNA double strand breaks(DSBs),and the number ofγH2AX foci in miR-34a/b/c-5p group were significantly higher than that in miR-NC group at 1 h and 8 h after irradiation(P<0.05).Conclusion:miR-34s are radioresponsive miRNA molecules,and overexpression of miR-34s can increase the level of ionizing radiation-induced DNA damage and enhance the radiosensitivity of CRC cells.