Ct on Snail, mRNA and protein expression of Snail was examined
Ct on Snail, mRNA and protein expression of Snail was examined in ES-2 and SKOV-3 cells following transfection of miRNA mimics or inhibitors, respectively. As expected, Snail mRNA and protein were down-regulated in ES-2 cells transfected with GW 4064 site miR-137 or miR-34a mimic (Fig. 2d and f). In contrast, transfection with miR-137 or miR-34a inhibitor into SKOV-3 cells (which has relatively higher levels of miR-34a and miR-137) up-regulated Snail expression at mRNA and protein levels (Fig. 2e and f). Then we examined the expression of epithelial marker Ecadherin, mesenchymal marker N-cadherin and Vimentin in OC cells after the overexpression or knockdown of miR-137 and miR-34a, using qPCR analysis. ES-2 cells transfected with miR-137 or miR-34a mimic showed increased levels of E-cadherin and decreased expression of N-cadherin and Vimentin (Fig. 2g). However, transfection of SKOV-3 cells with miR-137 or miR-34a inhibitor reversed these effects (Fig. 2h). Next, we examined the correlation between the expression of miR-137, miR-34a and Snail mRNA expression. We found that Snail mRNA level was significantly increased in OC samples compared with their nontumor counterparts (Fig. 2i). Next, we chose to compare our data with existing published gene expressiondatabase on OC. Importantly, our analysis of the TCGA OC samples revealed significantly higher Snail transcript levels in OC samples, relative to normal ovaries (Fig. 2j), supporting a negative correlation between miR137 or miR-34a and Snail expression in human OCs. Collectively, these results suggest that both miR-137 and miR-34a suppress Snail PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27597769 by targeting specific sites within the 3-UTR of Snail.MiR-137 and miR-34a inhibit EMT, invasion and sphere-forming ability of OC cellsWe also examined the potential effects of miR-137 and miR-34a on EMT features, invasive and sphere-forming abilities of OC cells. For this, we transiently reduced or overexpressed miR-137 and miR-34a in SKOV-3 and ES-2 cells, which express relatively higher or lower expression of miR-137 and miR-34a, respectively (Fig. 2a). The down-regulation of miR-137 or miR-34a in SKOV-3 cells induced a spindle-like mesenchymal morphology (Fig. 3a), and the overexpression of miR-137 or miR-34a in ES-2 cells resulted in an epithelial morphology change (Fig. 3b). We further investigated the effects of knockdown and overexpression of these two miRNAs on EMT, OC cell invasion and sphere formation ability. Matrigel invasion assay and sphere formation assayFig. 3 MiR-137 and miR-34a inhibit EMT, invasion and sphere-forming ability of OC cells. a, b Cellular morphology of OC cells after transient knockdown (a) or overexpression (b) of miR-137 and miR-34a. Scale bar = 200 m (a) and 100 m (b). c, d Cell invasion (c) and sphere formation (d) of OC cells after transient knockdown or overexpression of miR-137 and miR-34a. e, f qPCR analysis of indicated mRNAs in SKOV-3 (e) and ES-2 (f) cells after transient knockdown or overexpression of miR-137 and miR-34a. g Representative images of invaded SKOV-3 cells after transient transfection with miR-137 inhibitor or miR-34a inhibitor or negative control inhibitor (Neg inhibitor). h Representative images of spheres formed from ES-2 cells transfected with miR-137 or miR-34a mimic or Neg mimic. Scale bar =100 m. **P < 0.Dong et al. Journal of Experimental Clinical Cancer Research (2016) 35:Page 6 ofdemonstrated that knockdown of miR-137 or miR-34a using antisense oligonucleotides in SKOV-3 cells significantl.
