Investigating the role of miR-198 in oral squamous cell carcinoma pathogenesis

Abstract

Evidence supports the critical role of microRNA (miRNA) dysregulation in several diseases including cancers. The aberrant expression of numerous tumor suppressor and oncogenic miRNAs is a well-established driving factor in tumor progression. Though several mechanisms are reported to cause downregulation of tumor suppressor miRNAs, epigenetic silencing seems to play a major role. Pharmacological drugs are mostly employed for the reactivation and identification of epigenetically silenced tumor suppressor miRNAs. To this end in our laboratory, the miRNA microarray analysis of miRNA enriched RNA samples from OSCC SCC131 cells treated separately with DMSO (vehicle control) and 5-Azacytidine (a DNMT inhibitor) had previously revealed the upregulation and downregulation of 50 and 28 miRNAs respectively. In the present study, we have elucidated the role of miR-198 which is one of the miRNAs found to be upregulated following the 5-Azacytidine treatment. We confirmed the upregulation of miR-198 induced by 5-Azacytidine treatment using qRT-PCR and established its tumor suppressive function by checking its cell proliferating ability using trypan blue dye exclusion assay in OSCC cells from SCC084 and SCC131 cell lines. Since miRNAs bind to their target genes to bring about changes in gene expression, we wanted to identify a novel target gene for miR-198. Using bioinformatics and molecular approaches, we showed that miR-198 regulates the expression of the oncogene TOPORS at both the transcript and protein levels in OSCC cells in a dose-dependent manner. Using the dual-luciferase reporter assay, we also established the direct interaction of miR-198 with the 3’UTR of TOPORS in a sequence-specific manner. We hypothesised that miR-198 downregulation might be the cause of increased expression of TOPORS in OSCC and miR-198 might play a critical role in tumorigenesis. Thus, we analysed the expression of miR-198 and TOPORS in six different cancer cell lines and 39 matched normal oral tissue and OSCC patient samples and established the biological relevance of this interaction. We also showed that miR-198 suppressed the cell proliferation and anchorage-independent growth, and enhanced apoptosis of OSCC cells, in part, by targeting the 3’UTR of TOPORS. To elucidate the downstream effectors of miR-198 and TOPORS interaction, we analysed the p53/p21 signaling, and our results showed that miR-198 enhances this signaling pathway, in part, through the miR-198-TOPORS-p53-p21 axis. We also investigated the expression of TP53 (p53) and CDKN1A (p21) transcripts in our OSCC patient cohort and showed the clinical relevance of the p53-p21 pathway in these patients. To uncover the mechanism for upregulation of miR-198 following 5-Azacytidine, we identified an independent promoter for the MIR198 gene using the dual-luciferase reporter assay. We then analysed the methylation status of the MIR198 promoter in DMSO and 5-Azacytidine treated SCC131 cells, using bisulphite sequencing PCR. The results showed that the promoter methylation of MIR198 is responsible for its downregulation/silencing in SCC131 cells. We also showed that the promoter methylation of MIR198 causes differential miR-198 expression in MCF7 and MDA-MB-231 cells (breast cancer) and speculate the involvement of the same mechanism in other cancers. We also showed that the differential methylation pattern of the MIR198 promoter is inversely correlated with p21 levels in MCF7 and MDA-MB-231 cells, indicating the relevance of miR-198-TOPORS-p53-p21 axis in these cell lines. Taken together, our study suggests that miR-198 is epigenetically silenced by hypermethylation of the MIR198 promoter in OSCC, and exerts its tumor suppressive effect, in part, by regulating TOPORS, and the interaction between miR-198 and TOPORS is biologically relevant.