miR-145过表达增强非小细胞肺癌细胞的放疗敏感性

Overexpression of miR-145 enhancing the radiosensitivity of non-small cell lung cancer cells

目的探讨miR-145过表达对非小细胞肺癌细胞放疗敏感性的影响。方法 RT-PCR检测非小细胞肺癌细胞株A549、Calu-3、H460、SPC-A-1及支气管黏膜上皮细胞16HBE中miR-145的表达水平。细胞转染miR-145模拟物(miR-145 mimics)、阴性对照(mimics control),RT-PCR检测转染效果。噻唑蓝(MTT)、流式细胞术分别检测miR-145过表达、放疗和miR-145过表达联合放疗后细胞的增殖、凋亡能力,细胞克隆实验检测放疗敏感性。Western印迹检测细胞中活化的含半胱氨酸的天冬氨酸蛋白水解酶3(酶切Caspase-3)、Wnt信号通路下游靶基因(c-myc)、β-连环蛋白(β-catenin)表达水平。结果非小细胞肺癌细胞株A549、Calu-3、H460、SPC-A-1中miR-145的表达水平均明显低于支气管黏膜上皮细胞16HBE(P<0. 05),且Calu-3细胞中miR-145水平最低,后续选用Calu-3细胞为研究对象。miR-145 mimics有效促进Calu-3细胞中miR-145的表达,而mimics control没有明显作用。miR-145过表达或者放疗均能够抑制Calu-3细胞增殖,促进Calu-3细胞凋亡,促进细胞中酶切Caspase-3的表达,抑制细胞中c-myc、β-catenin的表达,且miR-145过表达联合放疗后细胞存活率最低,凋亡率最高,酶切Caspase-3表达水平也最高,c-myc、β-catenin表达水平最低。miR-145能够增加细胞放疗敏感性,增敏比为1. 363。结论 miR-145过表达能够抑制非小细胞肺癌细胞增殖,促进非小细胞肺癌细胞凋亡,增加非小细胞肺癌细胞放疗敏感性,作用机制可能与Wnt信号通路有关。

Objective To observed effects of miR-145 overexpression on radiosensitivity of non-small cell lung cancer ceUs. Methods Expression of miR-145 in non-small cell lung cancer cell line A549, Calu-3, HZ460, SPC-A-1 and bronchial epithelial cells 16HBE were detected by RT-PCR. Cells were transfected with miR-145 mimics and mimics control, RT-PCR was used to detect transfection effect. MTT and flow cytometry were used to detect the proliferation, apoptosis in ceils treated with miR-145 overexpression, radiotherapy and miR- 145 combined with radiotherapy, radiosensitivity was detected by cell cloning. Western blot was used to detect the expression levels of cleaved Caspase-3, c-myc, and β-Catenin in ceils. Results The expression of miR-145 in non-small cell lung cancer cell line A549, Calu-3, H460, SPC-A-1 was significantly lower than that of 16HBE （P〈0.05）. Level of miR-145 in Calu-3 cells was the lowest. Calu-3 cells were selected as the research object, miR-145 mimics promoted the expression of miR-145 in Calu-3 cells, mimics control had no obvi- ous effect, miR-145 overexpression or radiotherapy could inhibit the proliferation of Calu-3 cells, promote apoptosis of Calu-3 cells, promote the expression of cleaved Caspase-3 in cells, inhibit the expression of c-myc and β-catenin in cells, and after treated with the miR-145 over- expression combined with radiotherapy, survival rate was the lowest, apoptosis rate was the highest, the expression of cleaved Caspase-3 was the highest, expressions of c-myc and beta -eatenin were the lowest, miR-145 could increase cell radiosensitivity, the sensitization ratio was 1. 363. Conclusions Overexpression of miR-145 could inhibit the proliferation of non-small cell lung cancer cells, promote apoptosis of non-small cell lung cancer, increase radiosensitivity of non-small cell lung cancer cells, the mechanism may be related to Wnt signaling pathway.