Methylation-associated silencing of miR-193a-3p promotes ovarian cancer aggressiveness via targeting GRB7

Abstract

The human growth factor receptor-bound protein-7 (GRB7) is a pivotal mediator involved in receptor tyrosine kinase signaling and governing diverse cellular processes. We and others have reported that GRB7 is frequently upregulated and associated with the progression of human cancers. Clinicopathological analysis has shown that the overexpressed GRB7 is correlated with high grade and metastatic ovarian cancers. However, the molecular mechanisms leading to the upregulation of GRB7 remain largely unknown. In this study, we propose that the epigenetic modification of GRB7 at the post-transcriptional level may be a crucial factor leading to GRB7 upregulation in ovarian cancers. We found that the protein level of GRB7 was much higher than its mRNA levels in a subset of ovarian cancer cell lines, indicating aberrant post-transcriptional alterations in GRB7. Using an in silico study, we identified miR-193a-3p and its isoform, miR-193b-3p, by specifically targeting the conserved site (GGCCAGT) at position 332–338 in the 3’ UTR (length: 387) of the human GRB7 gene. QPCR and western blot analyses revealed that only the expression levels of miR-193a-3p but not miR-193b-3p were inversely correlated in ovarian cancer cell lines accompanied with high levels of GRB7 protein levels. Further biochemical analyses using luciferase reporter assays in combination with mutational analyses identified miR-193a-3p specifically targeting GRB7 3’UTR, confirming miR-193a-3p, instead of its isoform miR-193b-3p, has a more important role in GRB7 post-transcriptional regulation in ovarian cancer cells. Constitutive expression or knockdown of miR-193a-3p could significantly alter GRB7 expression, as well as oncogenic capacities of ovarian cancer cells such as cell growth, cell migration and cell invasion. Importantly, we observed that there was a significant stepwise decrease of miR-193a-3p expression from low to high grade ovarian cancers, and was inversely correlated with GRB7 expressions. Intriguingly, treatment with the DNA methyl transferase inhibitor (5’-Aza-dc) could restore the expression of miR-193a-3p, suggesting the epigenetic inhibition of miR-193a-3p is clinically relevance. Importantly, such epigenetic alteration may be the clue leading to overexpression of GRB7 in ovarian cancer cells. Taken together, our findings suggest that epigenetically silencing of miR-193a-3p upregulates GRB7 and contribute to ovarian cancer aggressiveness in tumor progression.