microRNA-331-3p inhibits cell proliferation and E7 expression by targeting NRP2 in cervical cancer

Background

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.

Methods

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.

Results

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.

Conclusions

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.

Clinical trial identification

Legal entity responsible for the study

Nara Medical University School of Medicine, Nara, Japan

Funding

Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (26462424)

Disclosure

All authors have declared no conflicts of interest.