Role of miR-146a-5p on proliferation and apoptosis of normal human oral keratinocytes and oral squamous cell carcinoma cells

Abstract

Objectives: miR-146a-5p is implicated in cell cycle regulation and apoptosis by suppressing the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) and Smad4, which are signal transducers of the transforming growth factor-β (TGF-β) signaling pathway. The purpose of this study was to verify the association of miR-146a-5p in oral squamous cell carcinoma (OSCC). Furthermore, experiments were designed to assess the expression pattern and role of miR-146a-5p in replicative senescence of normal human oral keratinocytes (NHOKs). Lastly, the study was intended to determine the role of miR-146a-5p on proliferation and apoptosis of OSCC.

Materials and Methods: To identify potential miRNAs involved in transformation of normal oral keratinocytes, we compared miRNA expression profile between NHOKs and HPV-16 immortalized human oral keratinocyte (HOK-16B) by miRNA microarray. Subsequently, miR-146a-5p was quantified in NHOKs and HOK-16B, CTHOK-16B-BaP, CTHOK-16B-DMB, SCC-4, SCC-9, KOSCC-11, KOSCC-25A, KOSCC-25B, KOSCC-25C, KOSCC-25D, KOSCC-25E, KOSCC-33A, KOSCC-33B, Spt-HOK80, HEp-2, FaDu, HeLa, and SiHa cell lines and in whole blood and saliva of patients with OSCC by quantitative real-time PCR. To investigate the roles of miR-146a-5p in regulating proliferation and apoptosis of NHOKs and OSCC cells, cells were transfected with miR-146a-5p mimic, miR-146a-5p inhibitor, and siRNAs against human TRAF6. Cell proliferation and apoptosis were analyzed by water soluble tetrazolium reduction assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, respectively, and protein levels of miR-146a-5p target genes were assayed by immunoblotting.

Results: miRNAs including miR-146a-5p were overexpressed in HOK-16B compared with NHOKs. miR-146a-5p expression was significantly upregulated in all the OSCC cell lines examined compared with NHOKs. miR-146a-5p was also overexpressed in blood and saliva of OSCC patients. miR-146a-5p expression was gradually increased during replicative senescence of NHOKs, which was in line with increased NF-κB expression by TGF-β1 signaling. In addition, exogenous miR-146a-5p expression suppressed TRAF6, which was a mediator of TGF-β1-induced apoptosis and suppression of proliferation. Exogenous miR-146a-5p expression enhanced proliferation of NHOKs and SCC-9, but did not so in HEp-2 and HeLa cells. Exogenous miR-146a-5p also inhibited apoptosis of NHOKs but enhanced apoptosis in SCC-9. These pleiotropic responses were partially based on the contextual responses of c-Jun N-terminal kinase and p38 mitogen activated kinase which are downstream of TRAF6. In support of the proliferation promoting effect of miR-146a-5p, inhibition of endogenous miR-146a-5p significantly reduced proliferation of SCC-9 cells and sensitized it to cisplatin.

Conclusion: The present study confirmed overexpression of miR-146a-5p in OSCC. miR-146a-5p forms a negative regulatory circuit within TGF-β signaling during replicative senescence of NHOKs. miR-146a-5p enhances proliferation of OSCC cells and confers resistance against cisplatin. These findings demonstrate that miR-146a-5p affects cell proliferation and apoptosis in a cellular context dependent manner and potentiates proliferation of OSCC cells.