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Int J Mol Sci. 2016 Aug 18;17(8). pii: E1351. doi: 10.3390/ijms17081351.

MicroRNA-331-3p Suppresses Cervical Cancer Cell Proliferation and E6/E7 Expression by Targeting NRP2.

Author information

1
Department of Pathology, Nara Medical University School of Medicine, Nara 634-8521, Japan. fujiit@naramed-u.ac.jp.
2
Department of Diagnostic Pathology, Nara City Hospital, Nara 630-8305, Japan. k-shimada@nara-jadecom.jp.
3
Department of Pathology, Nara Medical University School of Medicine, Nara 634-8521, Japan. ayaasano1018@yahoo.co.jp.
4
Department of Pathology, Nara Medical University School of Medicine, Nara 634-8521, Japan. tatsumi3@naramed-u.ac.jp.
5
Department of Central Clinical Laboratory, Nara Medical University Hospital, Nara 634-8521, Japan. nyama@naramed-u.ac.jp.
6
Department of Central Clinical Laboratory, Nara Medical University Hospital, Nara 634-8521, Japan. masayama@naramed-u.ac.jp.
7
Department of Pathology, Nara Medical University School of Medicine, Nara 634-8521, Japan. nkonishi@naramed-u.ac.jp.

Abstract

Aberrant expression of microRNAs (miRNAs) is involved in the development and progression of various types of cancers. In this study, we investigated the role of miR-331-3p in cell proliferation and the expression of keratinocyte differentiation markers of uterine cervical cancer cells. Moreover, we evaluated whether neuropilin 2 (NRP2) are putative target molecules that regulate the human papillomavirus (HPV) related oncoproteins E6 and E7. Cell proliferation in the human cervical cancer cell lines SKG-II, HCS-2, and HeLa was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Cellular apoptosis was measured using the TdT-mediated dUTP nick end labeling (TUNEL) and Annexin V assays. Quantitative RT-PCR was used to measure the messenger RNA (mRNA) expression of the NRP2, E6, E7, p63, and involucrin (IVL) genes. A functional assay for cell growth was performed using cell cycle analyses. Overexpression of miR-331-3p inhibited cell proliferation, and induced G2/M phase arrest and apoptosis in SKG-II, HCS-2 and HeLa cells. The luciferase reporter assay of the NRP2 3'-untranslated region revealed the direct regulation of NRP2 by miR-331-3p. Gene expression analyses using quantitative RT-PCR in SKG-II, HCS-2, and HeLa cells overexpressing miR-331-3p or suppressing NRP2 revealed down-regulation of E6, E7, and p63 mRNA and up-regulation of IVL mRNA. Moreover, miR-331-3p overexpression was suppressed NRP2 expression in protein level. We showed that miR-331-3p and NRP2 were key effectors of cell proliferation by regulating the cell cycle, apoptosis. NRP-2 also regulates the expression of E6/E7 and keratinocyte differentiation markers. Our findings suggest that miR-331-3p has an important role in regulating cervical cancer cell proliferation, and that miR-331-3p may contribute to keratinocyte differentiation through NRP2 suppression. miR-331-3p and NRP2 may contribute to anti-cancer effects.

KEYWORDS:

E6/E7 mRNA; cervical cancer; human papillomavirus; keratinocyte differentiation; miR-331-3p; neuropilin 2

PMID:
27548144
PMCID:
PMC5000747
DOI:
10.3390/ijms17081351
[Indexed for MEDLINE]
Free PMC Article

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