The Regulatory Relationship between E2F4 and MiRNA-363, and it’s Relevance to Oral Cancer

Abstract

BACKGROUND Oral Squamous Cell Carcinoma (OSCC) is the most common type of head and neck cancer and persists a leading cause of cancer associated mortality and morbidity universally. Survival rate is still poor at less than 50% urging the need for biomarkers to allow better diagnosis, prognosis and therapeutic strategies. In this study we focus on E2F4, a repressor of cell cycle, and further propose its regulatory relationship with MiRNA-363. METHODS Western blot (WB) was used to measure E2F4 protein expression and RT-qPCR was used to measure relative gene expression of E2F4, in UT-SSC-24A against UT-SCC-24B. Immunohistochemistry (IHC) of human tongue tissues was also applied to detect location and expression pattern of E2F4. Furthermore, transient transfection of the cell lines with miRNA-363 was applied to detect changes in E2F4 protein and gene expression. RESULTS E2F4 gene expression did not show major differences in the two cell lines. However, the protein levels did show difference both in the whole cell lysates and in the cytoplasmic fractions of cell lines. The IHC study revealed a relatively higher expression in cytoplasm of the cells belonging to the invasive front and areas with budding. Nearly 50% cell number decrease was observed in UT-SCC-24B, as a result of transfection with miRNA-363. This effect was not observed for UT-SCC-24A. A decrease of E2F4 protein levels in transfected UT-SCC-24B cells was further demonstrated by WB. Similar effect was observed in a immunoblot of transfected cytoplasmic fraction, but not observed for the nuclear fraction. Lastly, downregulation of E2F4 gene expression was exhibited by the transfected UT-SCC-24B cells with miRNA-363. CONCLUSION Our data indicate the involvement of E2F4 and miRNA-363 in OSCC and show a regulatory relationship between them. Furthermore, due to the findings of E2F4’s dynamic expression pattern, this study also proposes E2F4 as a potential biomarker for OSCC.