Elucidating the Role of miR-617 in Oral Squamous Cell Carcinoma Pathogenesis

Abstract

Oral Squamous Cell Carcinoma (OSCC) or oral cancer is the malignancy of the epithelial lining of the oral cavity. It is the leading cause of cancer-related mortality in Indian males and despite technological advancements, the 5-year survival rates of OSCC patients have not improved in the past few decades. Therefore, there is a compelling need to find novel ways to diagnose and treat OSCC. Incidentally, microRNAs, a group of small non-coding RNAs that regulate gene expression, are often dysregulated in cancer. Thus, studying microRNAs and their downstream effectors has gained importance in discovering novel diagnostic signatures and therapeutic modalities in cancer, in general. In cancer, microRNAs can be categorized into tumour suppressor miRs, oncomiRs or metastamiRs. Therefore, targeting the silencing mechanisms behind tumour suppressor microRNAs in OSCC can serve as a novel approach to discover a cure for OSCC. In cancer, tumour suppressor genes are predominantly silenced by extensive focal DNA hypermethylation at the promoter CGIs (CpG islands). Thus, by harnessing the reversible nature of this epigenetic modification, use of passive DNA demethylating agents like 5-Azacytidine emerged in cancer therapeutics to reprogram cancer cells to a more normal-like state by reactivating tumour suppressor genes. Thus, we hypothesized that the tumour suppressor microRNAs can be similarly silenced by promoter DNA hypermethylation and by treating OSCC cells with 5-Azacytidine, those tumour suppressor microRNAs can be reactivated and detected by a microRNA microarray analysis. In the present study, we validated that miR-617 expression is reactivated upon 5-Azacytidine treatment of OSCC cells in comparison to vehicle-control-treated cells. It is one of the 50 upregulated miRNAs, which were previously identified by a microRNA microarray analysis following 5- Azacytidine treatment of cells from an OSCC cell line SCC131 in our laboratory. Next, we characterised the independent promoter of MIR617 and deciphered that its demethylation is one of the causes responsible for the reactivation of miR617 expression upon 5-Azacytidine treatment of SCC131 cells. As per our speculation, next we tested the anti-proliferative potential of miR-617 in OSCC and found that miR-617 negatively regulates cell proliferation. MiRNAs bring about their biological functions primarily by downregulating their gene targets. We shortlisted eight protein-coding gene targets (DDX27, ZFX, FAM179B, RFXAP, MAP2K4, SORBS1, MEX3C, and JPH1) by employing five miRNA target prediction tools. Since miR-617 shows an anti- proliferative function, we expected its gene target to have pro-proliferative role in OSCC cells. Thus, we decided to further study DDX27 as a gene target for miR-617 as it has an established oncogenic role in breast, hepatocellular, gastric, and colorectal cancers. Contrary to our expectation, DDX27 transcript and protein levels are upregulated by miR-617 instead of downregulating it. To unveil the reason behind DDX27 upregulation by miR-617, we looked for alternative modes of miRNA action. One of the non- canonical ways of miRNA action entails the binding of a miRNA to the promoter region of its gene target to induce gene expression. Thus, we tested for the binding preference of miR-617 for the promoter and/ or 3’UTR of DDX27 and found that miR-617 interacts with the promoter of DDX27. Then, we tested the biological relevance of this interaction in cancer cell lines and OSCC tissues and obtained a positive correlation between the expression of miR-617 and DDX27, in general. Eventually, we found that miR-617 induces apoptosis and negatively regulates cell proliferation and anchorage-independent growth of OSCC cells by modulating DDX27 levels. Additionally, the tumour suppressive function of miR-617 is found to be associated with the reduced activation of phospho-S6K1 in the pro-survival PI3K/AKT/MTOR pathway. Finally, we tested the therapeutic potential of miR-617 by OSCC xenograft studies in nude mice. The results indicated that miR-617 acts as a tumour suppressor in OSCC, in part, by activating DDX27 levels. In conclusion, we have shown that miR-617 is a tumour suppressor microRNA whose expression is reactivated upon 5-Azacytidine treatment of SCC131 cells in comparison to vehicle- control-treated cells. We have established the anti-tumorigenic activity of miR-617 via targeting the promoter of DDX27. The downregulation of miR-617 in OSCC is due to hypermethylation of MIR617promoter. miR-617 regulates cell proliferation, anchorage-independent growth, and apoptosis of OSCC cells by modulating DDX27 levels. Further, our pre-clinical in vivo nude mice study indicates that synthetic miR-617 mimics can be potential candidates for miRNA replacement therapy in OSCC and perhaps in other cancers