摘要
目的研究微小核糖核酸(miR)-324-3p对体外培养的乳腺癌MCF-7细胞放射敏感性的影响并探讨其作用机制。方法采用qRT-PCR法和Western blot法分别检测miR-324-3p、丝氨酸/苏氨酸蛋白激酶(AKT)1蛋白的表达量;采用LipofectamineTM2000介导,将过表达miR-324-3p模拟物、沉默和过表达AKT1质粒转染乳腺癌MCF-7细胞并联合4 Gy放射照射,应用克隆形成实验、CCK-8试剂盒、流式细胞术法检测细胞存活率、细胞活性、细胞凋亡率,并绘制单击多靶模型拟合曲线;利用TargetScan在线预测、荧光素酶基因报告试验及Western blot法验证miR-324-3p的靶向关系。结果与正常乳腺上皮细胞MCF10A相比,乳腺癌MDA-MB-435S、MCF-7细胞中miR-324-3p的表达下调,AKT1蛋白表达上调(P均<0.05)。过表达miR-324-3p联合放射照射,MCF-7细胞放射敏感性增加,存活率呈剂量依赖性降低,细胞活性降低、凋亡率升高(P均<0.05)。miR-324-3p靶向调控AKT1的表达,当沉默AKT1联合放射照射时,MCF-7细胞放射敏感性增加,存活率下降并呈剂量依赖性,细胞活性降低、凋亡率升高(P均<0.05)。过表达AKT1逆转了miR-324-3p对乳腺癌放射敏感性、细胞增殖及凋亡的作用。结论过表达miR-324-3p可以靶向调控AKT1的表达,增强乳腺癌MCF-7细胞的放射敏感性。
Objective To study the effects of microRNA(miR)-324-3 p on the radiosensitivity of breast cancer MCF-7 cells cultured in vitro and its mechanism.Methods The expression levels of miR-324-3 p and serine/threonine protein kinase(AKT)1 were respectively detected by qRT PCR and Western blot.The overexpressed mir-324-3 p mimics,silenced and overexpressed AKT1 plasmids were transfected into breast cancer MCF-7 cells by lipofectamine2000 and irradiated with 4 Gy.The cell survival fraction,cell viability,apoptosis rate were determined by colony formation assay,cell counting kit 8 assay(CCK-8 kit)and flow cytometry,respectively,and the fitting curve of click multi-target model was drawn.TargetScan online prediction,luciferase reporter gene assay and Western blot were used to verify the target orientation of miR-324-3 p.Results Compared with normal mammary epithelial cells line MCF10 A,the expression of miR-324-3 p were down-regulated,and the expression of AKT1 protein were up-regulated in MDA-MB-435 S and MCF-7 breast cancer cells(all P<0.05).Overexpression of miR-324-3 p combined with radiation increased radiosensitivity of MCF-7 cells,decreased survival rate in a dose-dependent manner and cell activity,and increased the apoptosis rate(all P<0.05).MiR-324-3 p targeting AKT1 expression,when silencing AKT1 combined with radiation,the radiosensitivity of MCF-7 cells increased,survival fraction decreased in a dose-dependent manner cell activity decreased,and the apoptosis rate of MCF-7 cells increased(all P<0.05).Overexpression of AKT1 reversed the effect of miR-324-3 p on radiosensitivity,cell proliferation and apoptosis of breast cancer.Conclusion Overexpression of miR-324-3 p can target the regulation of AKT1 expression and enhance the radiosensitivity of breast cancer MCF-7 cells.
作者
张亚珍
宋杰峰
吴煌福
黄光钺
ZHANG Ya-zhen;SONG Jie-feng;WU Huang-fu;HUANG Guang-yue(Department of Oncology Surgery,the Second Affiliated Hospital of Hainan Medical College,Haikou,Hainan 570311,China;不详)
出处
《中国临床研究》
CAS
2020年第7期879-883,889,共6页
Chinese Journal of Clinical Research
基金
海南省重点研发计划项目(ZDYF2017087)。
