title: 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.
authors.affiliation: Department of Pathology, Nara Medical University School of Medicine, Nara 634-8521, Japan. fujiit@naramed-u.ac.jp.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Fujii
authors.affiliation: Department of Diagnostic Pathology, Nara City Hospital, Nara 630-8305, Japan. k-shimada@nara-jadecom.jp.
authors.forename: Keiji
authors.initials: K
authors.lastname: Shimada
authors.affiliation: Department of Pathology, Nara Medical University School of Medicine, Nara 634-8521, Japan. ayaasano1018@yahoo.co.jp.
authors.forename: Aya
authors.initials: A
authors.lastname: Asano
authors.affiliation: Department of Pathology, Nara Medical University School of Medicine, Nara 634-8521, Japan. tatsumi3@naramed-u.ac.jp.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Tatsumi
authors.affiliation: Department of Central Clinical Laboratory, Nara Medical University Hospital, Nara 634-8521, Japan. nyama@naramed-u.ac.jp.
authors.forename: Naoko
authors.initials: N
authors.lastname: Yamaguchi
authors.affiliation: Department of Central Clinical Laboratory, Nara Medical University Hospital, Nara 634-8521, Japan. masayama@naramed-u.ac.jp.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Yamazaki
authors.affiliation: Department of Pathology, Nara Medical University School of Medicine, Nara 634-8521, Japan. nkonishi@naramed-u.ac.jp.
authors.forename: Noboru
authors.initials: N
authors.lastname: Konishi
mesh_terms: D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics / Q000502:physiology; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D005260:Female; D006367:HeLa Cells; D006801:Humans; D020287:In Situ Nick-End Labeling; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D039943:Neuropilin-2 / Q000235:genetics / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism*; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000378:metabolism*
chemical_list: C544460:MIRN331 microRNA, human; D035683:MicroRNAs; D039943:Neuropilin-2; D009856:Oncogene Proteins, Viral
keywords: E6/E7 mRNA; cervical cancer; human papillomavirus; keratinocyte differentiation; miR-331-3p; neuropilin 2