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

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.

Figure 1

Cell proliferation and apoptosis assay in the cervical cancer cells. (A) MTS assay in SKG-II, HCS-2 and HeLa cells. Cell proliferation was suppressed by transient transfection of the miR-331-3p precursor. (pre: precursor, * p < 0.05 (24 h), + p < 0.05 (48 h), # p < 0.05 (72 h)); (B) TdT-mediated dUTP nick end labeling (TUNEL) assay for SKG-II, HCS-2 and HeLa cells. The Y-axis shows the number of positive cell counts per 10 or 20 high-power fields (10 or 20 HPFs). Positive cells were induced by transfection of the miR-331-3p precursor (* p < 0.05); (C) Expression of miR-331-3p was up-regulated in SKG-II, HCS-2 and HeLa cells by miR-331-3p precursor transfection compared to control cells (* p < 0.05).

Figure 2

Annexin V assay for cervical cancer cells. Early and total apoptotic cells were significantly increased by miR-331-3p overexpression in SKG-II, HCS-2 and HeLa cells (* p < 0.05).

Figure 3

mRNA expression of HPV-related oncogenes, E6/E7 and keratinocyte related genes, p63 and IVL. E6/E7 (A) p63 and IVL (B) mRNA expression in SKG-II, HCS-2 and HeLa cells. The Y-axis (fold) shows relative expression compared with control (normalized with actin mRNA expression). E6/E7 and p63 mRNA expression was significantly decreased in SKG-II, HCS-2 and HeLa cells transiently transfected with miR-331-3p precursor, whereas IVL mRNA was significantly increased in SKG-II, HCS-2 and HeLa cells transiently transfected with the miR-331-3p precursor (* p < 0.05).

Figure 4

mRNA expression of neuropilin 2 (NRP2), which are the putative target molecules of miR-331-3p. (A) mRNA expression of NRP2 in cervical cancer cell lines. NRP2 expression was higher in SKG-II than other two cervical cancer cell lines; (B,C) NRP2 expression under overexpression of miR-331-3p in cervical cancer cell lines. Images were shown from quantitative RT-PCR (B) and western blot (C). miR-331-3p down-regulates NRP2 mRNA (B) and protein (C) expression in SKG-II, HCS-2 and HeLa cells; (D) Luciferase reporter activity for NRP-2 3′-UTR. NRP2 3′-UTR reporter activity was reduced by miR-331-3p overexpression (* p < 0.05).

Figure 5

The effect of NRP2 on cell proliferation in SKG-II cells. (A) MTS assay in SKG-II, HCS-2 and HeLa cells. Cell proliferation was suppressed by transient transfection with NRP2 siRNA. (* p < 0.05 (24 h), + p < 0.05 (48 h), # p < 0.05 (72 h)); (B) TUNEL assay for SKG-II, HCS-2 and HeLa cells. The Y-axis shows the number of positive cell counts per 10 or 20 high-power fields (10 or 20 HPFs). Positive cells were induced by transfection of NRP2 siRNA (* p < 0.05).

Figure 6

Annexin V assay for cervical cancer cells. Early and total apoptotic cells were significantly increased by NRP2 suppression in SKG-II, HCS-2 and HeLa cells (* p < 0.05).

Figure 7

mRNA expression of HPV-related oncogenes, E6/E7 and keratinocyte related genes, p63 and IVL. E6/E7 and p63 mRNAs were significantly decreased by transient transfection with NRP2 siRNA. whereas IVL mRNA was significantly increased in SKG-II, HCS-2 cells transiently transfected with NRP2 siRNA (* p < 0.05).

Figure 8

Cell cycle analysis in the SKG-II cells. Cell cycle analysis using the Muse™ Cell Analyzer (Millipore; Hayward, CA, USA). (A) The upper panels show the DNA content profile by transient transfection of with the miR-331-3p precursor (miR-331-3p pre) or NRP2 siRNA. The DNA histogram results show the distribution of the cell cycle phases; G0/G1 (blue), S (red) and G2/M (green). The lower panel shows the percentage of G1, S, or G2/M phase; (B) Western blotting (upper panel) and immunocytochemistory (lower panel) for p16INK4a (p16) protein expression in SKG-II cells.

Figure 9

(Upper panel) miR-331-3p down-regulates NRP2, a putative direct target of miR-331-3p, and inhibits cell proliferation by regulating the expression of E6/E7, keratinocyte differentiation, cell cycle, and apoptosis in cervical cancer cells (thin black arrows); (Lower panel) HPV infection promotes cell proliferation through up-regulation of E6/E7, p16 and p63, and progression to cervical cancer (thin blue arrows). miR-331-3p acts as an anticancer factor through suppression of NRP2 (the thick blue arrow).