Silencing of miR-21 regulates proliferation and invasion in trophoblastic cells and targets PTEN

Objective: MicroRNAs (miRNAs) are small non coding RNAs that post-transcriptional regulate gene expression by inducing translational inhibition or transcript degradation. MiR-21 is an oncomir and has been associated with various types of cancer. Interestingly, our group has been reported that miR-21 is the highest expressed miRNA, out of 762 analyzed, in first trimester isolated trophoblast cells. In this study, we investigated the functions of miR-21 in trophoblastic cell lines and its possible target genes.

Methods: The immortalized human trophoblast cell line HTR-8/svneo and the choriocarcinoma cell line JEG-3 were transfected with miR-21 inhibitor. Total RNA was extracted and RNA quality and quantity were determined by spectrophotometer (Nanodrop 1000). MiRNA-21 expression was quantified by qPCR using Taqman^âmiRNA single assay. Cell growth and invasion were followed after transfection with miR-21 inhibitor for up to 72 hr by BrdU assay and Matrigel invasion assay. Potential targets of miR-21 were analyzed by RT-PCR.

Results: MiR-21 expression was higher in HTR-8/svneo cells than in JEG-3 cells.

In JEG-3 cells, miR-21 inhibition significantly increased proliferation while over-expression resulted in a decreased proliferation when compared to untreated cells and negative control cells. Moreover, JEG-3 cells demonstrated significant reduction of invasion after inhibition of miR-21 expression. Knockdown of miR-21 in HTR-8/svneo cells significantly suppressed proliferation compared to untreated cells and negative control cells, but no change was observed in invasion. MiR-21 inhibitor inhibited expression of PTEN in JEG-3 cells, while increased it in HTR-8/svneo cells.

Conclusions: Both cell lines express miR-21 but in different levels. MiR-21 plays an important role on regulating cell growth and invasion in trophoblast cells possibly by a mechanism that involves control of PTEN expression.