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 ~60% reduction of telomerase reverse transcriptase (TERT) transcription followed by ~35% decrease of telomerase activity. Furthermore, miR-375-mediated regulation of 14-3-3ζ contributes to decrease telomerase activity by altering nuclear translocation of TERT.


CONCLUSIONS
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.
Hyun Min
HM
Jung
Brittany L
BL
Phillips
Department of Oral Biology, University of Florida, 1395 Center Drive, Gainesville, FL 32610, USA. echan@ufl.edu.
Edward Kl
EK
Chan
D048948:14-3-3 Proteins / Q000096:biosynthesis*; D001324:Autoantigens / Q000096:biosynthesis; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D015967:Gene Expression Regulation, Viral; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000096:biosynthesis; D035683:MicroRNAs / Q000378:metabolism*; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000378:metabolism*; D050725:Papillomavirus E7 Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000096:biosynthesis; D016160:Retinoblastoma Protein / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis; D020741:rho GTP-Binding Proteins / Q000096:biosynthesis
D048948:14-3-3 Proteins; D001324:Autoantigens; C500266:CIP2A protein, human; D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; C052603:E6 protein, Human papillomavirus type 18; D047908:Intracellular Signaling Peptides and Proteins; C540933:MIRN375 microRNA, human; D008565:Membrane Proteins; D035683:MicroRNAs; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; C059731:oncogene protein E7, Human papillomavirus type 16; C471639:UBE3A protein, human; D044767:Ubiquitin-Protein Ligases; D020741:rho GTP-Binding Proteins