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

Objective: 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.

Methods: 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.

Results: Transfection with the miR-34a-5p 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-34a-5p. Transfection with pcDNA-CDK6 significantly reversed the effects of miR- 34a-5p 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 PI3K/AKT pathway, also significantly attenuated the effects of miR-34a- 5p overexpression on the cell viability (P=0.011), apoptosis (P=0.004), and invasion (P=0.002).

Conclusions: 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.