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 keratinocyte differentiation in uterine cervical cancer cells. Moreover, we evaluated whether neuropilin 2 (NRP2) which are putative target molecules of miR-331-3p regulated the human papillomavirus (HPV) -related oncoproteins E6 and E7.
Cell proliferation in the human cervical cancer cell lines SKG-II and HeLa was assessed using the MTS assay. A functional assay for cell growth was performed using cell viability and cell cycle analysis. Cellular apoptosis was measured using a TUNEL assay. Quantitative RT-PCR was used to measure the mRNA expression of the E6, E7, NRP2, p63, and involucrin (IVL) genes and anti-apoptosis markers bcl2, bclXL, and BAX.
Overexpression of miR-331-3p inhibited cell proliferation, and induced G2/M phase arrest and apoptosis in SKG-II cells. The luciferase reporter assay of the NRP2 3¢-untranslated region revealed direct regulation of NRP2 by miR-331-3p. Gene expression analyses using quantitative RT-PCR in both SKG-II 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. We showed that miR-331-3p and NRP2 were key effectors in cell proliferation by regulating the cell cycle and expression of E6 and E7, and keratinocyte differentiation by down-regulating p63 and up-regulating IVL.
Our findings suggest that miR-331-3p has an important role in regulating cervical cancer cell proliferation, and overexpression of miR-331-3p through suppression of NRP2 may contribute to keratinocyte differentiation and may have anti-cancer effects. Our future studies will examine whether miR-331-3p and its target, NRP2, are useful clinical diagnostic and/or prognostic markers for histological and cytological examination using tissue specimens and liquid-based cytology in the screening and diagnosis of cervical cancer.
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Nara Medical University School of Medicine, Nara, Japan
Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (26462424)
All authors have declared no conflicts of interest.