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.
Nan Fang Yi Ke Da Xue Xue Bao. 2020 Jan 30;40(1):79-86. doi: 10.12122/j.issn.1673-4254.2020.01.13.
To investigate the roles of microRNA (miR)-34a-5p 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-34a-5p and CDK6.
We examined the expression of miR-34a-5p using RT-qPCR in cultured human trophoblast HTR-8/Svneo cells and human choriocarcinoma cell lines BeWo and JEG-3HTR-8/Svneo. HTR-8/Svneo cells transfected with a miR-34a-5p-mimic, the miR-34a-5p-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-34a-5p analyzed using a luciferase reporter assay.
Transfection with the miR-34a-5p mimic significantly reduced the viability (=0.000), suppressed the invasion (=0.049), enhanced the cell apoptosis (=0.018), down-regulated the expressions of MMP-9 (=0.004) and CDK6 (=0.014), and up-regulated caspase 3 activity (=0.018) and cleaved caspase 3 expression (=0.003) in cultured HTR-8/Svneo cells. CDK6 was confirmed as one of the target gene of miR-34a-5p. Transfection with pcDNA-CDK6 significantly reversed the effects of miR- 34a-5p overexpression on the cell viability (=0.000), apoptosis (=0.015), and invasion (=0.046). Treatment of the cells with insulin-like growth factor 1 (IGF-1), an activator of the PI3K/AKT pathway, also significantly attenuated the effects of miR-34a- 5p overexpression on the cell viability (=0.011), apoptosis (=0.004), and invasion (=0.002).
miR-34a-5p promotes apoptosis and inhibits the viability and invasion of human placental trophoblastic cells by down-regulating CDK6 and inactivating the PI3K/AKT pathway.
探讨微小RNA（miR）-34a-5p和细胞周期蛋白依赖性激酶（CDK）6在调控人胎盘滋养层细胞活力、凋亡和侵袭中的作用以及miR-34a-5p与CDK6之间的关系。
我们采用逆转录定量聚合酶链反应（RT-qPCR）检测培养的人滋养层HTR-8/Svneo细胞、人绒毛膜癌细胞系BeWo和JEG-3HTR-8/Svneo中miR-34a-5p的表达。对转染了miR-34a-5p模拟物、miR-34a-5p抑制剂或pcDNA-CDK6的HTR-8/Svneo细胞以及模拟物组，使用MTT法分析细胞增殖的变化；通过检测半胱天冬酶3活性和裂解的半胱天冬酶3蛋白表达评估细胞凋亡，使用Transwell法评估细胞侵袭。采用蛋白质免疫印迹法测定细胞中CDK6、裂解的半胱天冬酶3和基质金属蛋白酶9（MMP-9）的蛋白水平。使用荧光素酶报告基因检测法分析CDK6与miR-34a-5p之间的相互作用。
转染miR-34a-5p模拟物显著降低了培养的HTR-8/Svneo细胞的活力（P = 0.000），抑制了侵袭（P = 0.049），增强了细胞凋亡（P = 0.018），下调了MMP-9（P = 0.004）和CDK6（P = 0.014）的表达，并上调了半胱天冬酶3活性（P = 0.018）和裂解的半胱天冬酶3表达（P = 0.003）。CDK6被确认为miR-34a-5p的靶基因之一。转染pcDNA-CDK6显著逆转了miR-34a-5p过表达对细胞活力（P = 0.000）、凋亡（P = 0.015）和侵袭（P = 0.046）的影响。用胰岛素样生长因子1（IGF-1）（一种PI3K/AKT途径激活剂）处理细胞，也显著减弱了miR-34a-5p过表达对细胞活力（P = 0.011）、凋亡（P = 0.004）和侵袭（P = 0.002）的影响。
miR-34a-5p通过下调CDK6并使PI3K/AKT途径失活，促进人胎盘滋养层细胞凋亡并抑制其活力和侵袭。
