Fujii Tomomi, Shimada Keiji, Asano Aya, Tatsumi Yoshihiro, Yamaguchi Naoko, Yamazaki Masaharu, Konishi Noboru
Department of Pathology, Nara Medical University School of Medicine, Nara 634-8521, Japan.
Department of Diagnostic Pathology, Nara City Hospital, Nara 630-8305, Japan.
Int J Mol Sci. 2016 Aug 18;17(8):1351. doi: 10.3390/ijms17081351.
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.
微小RNA（miRNA）的异常表达与多种癌症的发生和发展有关。在本研究中，我们调查了miR-331-3p在子宫颈癌细胞增殖及角质形成细胞分化标志物表达中的作用。此外，我们评估了神经纤毛蛋白2（NRP2）是否为调控人乳头瘤病毒（HPV）相关癌蛋白E6和E7的假定靶分子。使用3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲氧基苯基)-2-(4-磺基苯基)-2H-四唑鎓内盐（MTS）试验评估人宫颈癌细胞系SKG-II、HCS-2和HeLa中的细胞增殖。使用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）和膜联蛋白V试验检测细胞凋亡。采用定量逆转录聚合酶链反应（qRT-PCR）检测NRP2、E6、E7、p63和内披蛋白（IVL）基因的信使核糖核酸（mRNA）表达。利用细胞周期分析进行细胞生长功能试验。miR-331-3p的过表达抑制了SKG-II、HCS-2和HeLa细胞的增殖，并诱导G2/M期阻滞和凋亡。NRP2 3'-非翻译区的荧光素酶报告基因试验揭示了miR-331-3p对NRP2的直接调控。在过表达miR-331-3p或抑制NRP2的SKG-II、HCS-2和HeLa细胞中，使用qRT-PCR进行基因表达分析，结果显示E6、E7和p63 mRNA表达下调，IVL mRNA表达上调。此外，miR-331-3p的过表达在蛋白水平上抑制了NRP2的表达。我们发现miR-331-3p和NRP2是通过调控细胞周期、凋亡来影响细胞增殖的关键效应分子。NRP-2还调控E6/E7的表达和角质形成细胞分化标志物。我们的研究结果表明，miR-331-3p在调控宫颈癌细胞增殖中起重要作用，并且miR-331-3p可能通过抑制NRP2促进角质形成细胞分化。miR-331-3p和NRP2可能具有抗癌作用。
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