MicroRNAs (miRNAs) are small regulatory non-coding RNAs that have been reported to play an important role in the tumorigenesis of many cancers. 3-UTR and its mutants into luciferase reporter plasmids, and co-transfected SKOV3 cell lines with the constructed plasmids and miR-212-3p mimics. As shown in Figure 1C, luciferase activity was decreased in cells co-transfected with miR-212-3p, as compared to that in cells expressing miR-NC, and this was statistically significant ( 0.01). In addition, mutations in the putative binding site abolished the repression of luciferase activity mediated by miR-212-3p. These findings suggest that miR-212-3p regulates MAP3K3 expression through direct interactions with its 3-UTR. To further verify whether is the direct target of miR-212-3p, we performed RNA immunoprecipitation. The result demonstrated that endogenous Dabrafenib kinase activity assay MAP3K3 pull-down by AGO2 was significantly enriched in miR-212-3p-overexpressing cells (Figure 1D; 0.05). Open in a separate window Figure 1 The MAP3K3 gene is a direct target of miR-212-3p in high-grade serous ovarian cancer (HGSOC) cells. A. The target genes of miR-212-3p were predicted using bioinformatics tools (TargetScan, miRTarBase, miRDB, and StarBase databases). B. The binding site mixed up in interaction between your MAP3K3 miR-212-3p and 3-UTR was predicted using these directories. C. Dual-luciferase reporter gene assays demonstrated that miR-212-3p binds the 3-UTR of MAP3K3 and inhibits its manifestation. D. RNA immunoprecipitation assays proven that endogenous MAP3K3 pull-down by AGO2 was considerably enriched in miR-212-3p-overexpressing Dabrafenib kinase activity assay cells; IgG was utilized as the control. MiR-212-3p suppresses MAP3K3 manifestation To verify the regulatory aftereffect of miR-212-3p on MAP3K3 manifestation, we first established the manifestation degrees of MAP3K3 and miR-212-3p in a variety of HGSOC cell lines (Shape 2A). The effect demonstrated that MAP3K3 manifestation was saturated in A2780 and SKOV3 cell lines fairly, whereas it had been lower in OV2008 cell lines relatively. Consequently, qRT-PCR and traditional western blot assays had been performed to check MAP3K3 manifestation in A2780 and SKOV3 cell lines after transfecting them with miR-212-3p mimics and in OV2008 cell lines after transfecting them with miR-212-3p inhibitors. The outcomes revealed that MAP3K3 expression was remarkably decreased after transfection with miR-212-3p mimics Dabrafenib kinase activity assay compared to that in negative controls in A2780 (Figure 2B) and SKOV3 (Figure 2C) cell lines. However, MAP3K3 expression was significantly increased after transfection with a miR-212-3p inhibitor compared to levels with the negative control inhibitor in OV2008 cell lines (Figure 2D). These results indicated that miR-212-3p deregulates the expression level of MAP3K3. Open in a separate window Figure 2 miR-212-3p can downregulate MAP3K3 expression in high-grade serous ovarian cancer (HGSOC) cells. (A) mRNA and protein expression of miR-212-3p and MAP3K3 in six ovarian cancer cell lines and 293T cells. MAP3K3 expression was remarkably decreased after transfection with miR-212-3p mimics in A2780 (B) and SKOV3 (C) cells at both the mRNA and protein levels. MAP3K3 expression was significantly increased after transfection with a miR-212-3p inhibitor in OV2008 (D) cell lines. MiR-212-3p suppresses HGSOC cell proliferation, migration and invasion To investigate the function of miR-212-3p with respect to HGSOC biological behaviors, we performed cell proliferation, invasion and migration assays using cell lines transfected with miR-212-3p mimics or inhibitors; specifically, A2780 and Rabbit polyclonal to DUSP3 SKOV3 cell lines with high endogenous expression of MAP3K3 were transfected with miR-212-3p mimics, whereas OV2008 cells with low endogenous MAP3K3 expression were transfected with a miR-212-3p inhibitor. Based on CCK8 assays, we observed that miR-212-3p markedly reduced cell proliferation at 24, 48, and 72 h, compared to that in negative controls (Figure 3A, ?,3B).3B). In contrast, the proliferation of cells transfected with the miR-212-3p inhibitor was significantly increased (Figure 3C). Colony forming assays demonstrated that miR-212-3p mimics could promote the formation of A2780 (Figure 3D) and SKOV3 (Figure 3E) cell clones; however clone formation in OV2008 cells was diminished upon inhibiting miR-212-3p.