miR-375 activates p21 and suppresses telomerase activity by coordinately regulating HPV E6/E7, E6AP, CIP2A, and 14-3-3ζ

Background: While microRNAs (miRNAs) are extensively studied in post-transcriptional regulation of gene expressions in many biological processes, cellular miRNA-mediated regulation of viral genes remains unclear. In particular, the interplay between human papillomavirus (HPV) genes and miRNAs and how these interactions contribute to HPV-associated cancers remain elusive. Methods: Transient transfection of miR-375-mimic was used to compensate the loss-of-function of miR-375 in HPV-positive cancer. Regulation of oncogenic molecules and their downstream molecules via miR-375 in HPV-positive cancer was investigated using qRT-PCR, western blot, dual luciferase assay, indirect immunofluorescence analysis. All experiments were conducted at least three times to achieve statistical significance determined by Student t-test. Results: In this study, we demonstrated how miR-375 negatively regulates HPV16 and 18 transcripts. We also found a cellular protein, E6-associated protein (E6AP), directly regulated by miR-375. miR-375-mediated repression of HPV transcripts and E6AP elevated major tumor suppressors p53, p21, and retinoblastoma protein 1 (RB). Cooperative regulation of miR-375 targets along with the increase of tumor suppressors led to similar to 60% reduction of telomerase reverse transcriptase (TERT) transcription followed by similar to 35% decrease of telomerase activity. Furthermore, miR-375-mediated regulation of 14-3-3 zeta contributes to decrease telomerase activity by altering nuclear translocation of TERT. Conclusion: Taken together, miR-375-mediated suppression of multiple oncogenic components in HPV-associated carcinogenesis generates a cumulative biological response to rescue key tumor suppressors and diminish telomerase activity, which results in cell cycle arrest and cell proliferation inhibition.