miR-34a-5p通过调控CDK6表达和PI3K/AKT信号通路调节滋养层细胞的增殖、浸润和凋亡  

miR-34a-5p regulates viability, invasion and apoptosis of placental trophoblastic cells via modulating CDK6 and PI3K/AKT pathway

作　　者：李勤[1] 许娟秀[2] LI Qin;XU Juanxiu(Department of Obstetrics,Jiangxi Maternal and Children's Health Hospital,Nanchang 330006,China;Department of Oncology,Jiangxi Maternal and Children's Health Hospital,Nanchang 330006,China)

机构地区：[1]江西省妇幼保健院产科,江西南昌330006 [2]江西省妇幼保健院肿瘤科,江西南昌330006

出　　处：《南方医科大学学报》2020年第1期79-86,共8页Journal of Southern Medical University

摘　　要：目的探讨miR-34a-5p及CDK6对滋养层细胞增殖,浸润和凋亡的作用和下游机制以及二者的调节关系。方法培养滋养层细胞HTR-8/Svneo和人绒毛膜癌细胞系BeWo和JEG-3HTR-8/Svneo,使用RTqPCR法检测3种细胞中miR-34a-5p的表达差异。根据对HTR-8/Svneo细胞的不同处理进行如下分组:无处理的HTR-8/Svneo细胞(对照组);miR-34a-5p拟似物mimic转染细胞(mimic组),pcDNA-CDK6和mimic共转染HTR-8/Svneo细胞(pcDNA-CDK6+mimic组),miR-34a-5p抑制剂inhibitor转染HTR-8/Svneo细胞(inhibitor组)。MTT法检测细胞增殖,ELISA法测定caspase 3活性检测细胞凋亡水平,Transwell实验检测细胞浸润能力;Western blot检测细胞周期依赖性蛋白激酶CDK6,凋亡标记物cleaved-caspase 3,浸润标记物MMP-9的表达;荧光素酶报告基因实验确认miR-34a-5p对CDK-6的直接靶向关系。结果过表达miR-34a-5p抑制HTR-8/Svneo细胞的增殖活性(P=0.000)和浸润能力(P=0.049),下调细胞中MMP-9(P=0.004)和CDK6(P=0.014)的表达水平,上调caspase 3活性(P=0.018)和cleaved caspase 3的表达水平(P=0.003);CDK6是miR-34a-5p的靶基因,过表达CDK6削弱miR-34a-5p对细胞增殖(P=0.000)、凋亡(P=0.015)和浸润(P=0.046)的作用;使用IFG-1激活PI3K/AKT通路部分逆转miR-34a-5p对细胞增殖(P=0.011)、凋亡(P=0.004)和浸润(P=0.002)的作用。结论 miR-34a-5p通过调控CDK6表达和PI3K/AKT信号通路激活调节滋养层细胞的增殖,浸润和凋亡。Objective To investigate the roles of microRNA(miR)-34 a-5 p and cyclin-dependent kinase(CDK) 6 in the regulation of cell viability, apoptosis and invasion of human placental trophoblastic cells and the relationship between miR-34 a-5 p and CDK6. Methods We examined the expression of miR-34 a-5 p using RT-qPCR in cultured human trophoblast HTR-8/Svneo cells and human choriocarcinoma cell lines BeWo and JEG-3 HTR-8/Svneo. HTR-8/Svneo cells transfected with a miR-34 a-5 p-mimic,the miR-34 a-5 p-inhibitor, or pcDNA-CDK6 along with the mimic group were analyzed for changes in cell proliferation using MTT assay;the apoptosis of the cells were assessed by detecting caspase 3 activity and cleaved caspase 3 protein expression,and the cell invasion was evaluated using Transwell assay. Western blotting was used to determine the protein levels of CDK6,cleaved caspase 3, and MMP-9 in the cells. The interaction between CDK6 and miR-34 a-5 p analyzed using a luciferase reporter assay. Results Transfection with the miR-34 a-5 p mimic significantly reduced the viability(P=0.000), suppressed the invasion(P=0.049), enhanced the cell apoptosis(P=0.018), down-regulated the expressions of MMP-9(P=0.004) and CDK6(P=0.014), and up-regulated caspase 3 activity(P=0.018) and cleaved caspase 3 expression(P=0.003) in cultured HTR-8/Svneo cells. CDK6 was confirmed as one of the target gene of miR-34 a-5 p. Transfection with pcDNA-CDK6 significantly reversed the effects of miR-34 a-5 p overexpression on the cell viability(P=0.000), apoptosis(P=0.015), and invasion(P=0.046). Treatment of the cells with insulin-like growth factor 1(IGF-1), an activator of the PI3 K/AKT pathway, also significantly attenuated the effects of miR-34 a-5 p overexpression on the cell viability(P=0.011), apoptosis(P=0.004), and invasion(P=0.002). Conclusion miR-34 a-5 p promotes apoptosis and inhibits the viability and invasion of human placental trophoblastic cells by down-regulating CDK6 and inactivating the PI3 K/AKT pathway.

关 键 词：滋养层细胞 miR-34a-5p 细胞周期依赖性激酶6 增殖活性 凋亡 浸润 

分 类 号：R714.244[医药卫生—妇产科学]