Xia Yu-Fang, Pei Gui-Hua, Wang Ning, Che Yan-Ci, Yu Feng-Sheng, Yin Fu-Fen, Liu Hai-Xia, Luo Bing, Wang Yan-Kui
Department of Obstetrics and Gynecology, The Affiliated Hospital of Qingdao University, Qingdao, China.
Department of Obstetrics and Gynecology, The Third Hospital of Qingdao, Qingdao, China.
Virol J. 2017 Feb 4;14(1):20. doi: 10.1186/s12985-017-0695-7.
Cervical cancer (CC) is the second most common cancer in females in developing countries. The two viral oncoproteins E6 and E7 mediate the oncogenic activities of high-risk human papillomavirus (HR-HPV), and HR-HPV, especially HPV16 or/and HPV18 (HPV16/18) play critical roles in CC through different pathways. microRNAs (miRNAs) may be associated with CC pathogenesis. Researches have indicated that human papillomavirus (HPV) may regulate cellular miRNA expression through viral E6 and E7. Herein, the purposes of this study were to identify the relationship between HPV infection and aberrantly expressed miRNAs and to investigate their pathogenic roles in CC.
miRNA expression was assessed using a microRNAs microarray in HPV16 E6- and E7-integrated HPV-negative HT-3 cell lines and mock vector-transfected HT-3 cells. The microarray results were validated, and the expression of miR-3156-3p was identified in HPV-positive and -negative CC cell lines as well as primary CC and normal cervical epithelium tissues using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Cell Counting Kit-8 (CCK8), flow cytometry, transwell analysis, tube formation, and Western blotting were used to identify the functional role of miR-3156-3p in CaSki, SiHa, and HeLa cell lines.
Six underexpressed microRNAs (miR-3156-3p, 6779-3p, 4779-3p, 6841-3p, 454-5p and 656-5p) were consistently identified in HPV16 E6- and E7-integrated HT-3 cells. Further investigation confirmed a significant decrease of miR-3156-3p in HPV16/18 positive CC lesions. CCK8, flow cytometry, transwell analysis, tube formation assays, and Western blotting of the CC cell lines with miR-3156-3p over/under-expression in vitro showed that miR-3156-3p was involved in cell proliferation, apoptosis, migration, neovascularization, and SLC6A6 regulation.
Our findings indicate that miR-3156-3p plays a suppressor-miRNA role in CC and that its expression is associated with HR-HPV infection.
宫颈癌（CC）是发展中国家女性中第二常见的癌症。两种病毒癌蛋白E6和E7介导高危型人乳头瘤病毒（HR-HPV）的致癌活性，并且HR-HPV，尤其是HPV16或/和HPV18（HPV16/18）通过不同途径在宫颈癌中起关键作用。微小RNA（miRNA）可能与宫颈癌的发病机制有关。研究表明，人乳头瘤病毒（HPV）可能通过病毒E6和E7调节细胞miRNA的表达。在此，本研究的目的是确定HPV感染与异常表达的miRNA之间的关系，并研究它们在宫颈癌中的致病作用。
使用微小RNA微阵列评估HPV16 E6和E7整合的HPV阴性HT-3细胞系以及空载体转染的HT-3细胞中的miRNA表达。对微阵列结果进行验证，并使用定量逆转录聚合酶链反应（qRT-PCR）在HPV阳性和阴性的宫颈癌细胞系以及原发性宫颈癌和正常宫颈上皮组织中鉴定miR-3156-3p的表达。使用细胞计数试剂盒8（CCK8）、流式细胞术、Transwell分析、管形成实验和蛋白质印迹法来鉴定miR-3156-3p在CaSki、SiHa和HeLa细胞系中的功能作用。
在HPV16 E6和E7整合的HT-3细胞中一致鉴定出6种低表达的微小RNA（miR-3156-3p, miR-6779-3p, miR-4779-3p, miR-6841-3p, miR-454-5p和miR-656-5p）。进一步研究证实，在HPV16/18阳性的宫颈癌病变中miR-３１５６-３ｐ显著降低。对miR-3156-3p过表达/低表达的宫颈癌细胞系进行体外CCK8、流式细胞术、Transwell分析、管形成实验和蛋白质印迹检测，结果表明miR-3156-3p参与细胞增殖、凋亡、迁移、新生血管形成以及SLC6A6调节。
我们的研究结果表明，miR-3156-3p在宫颈癌中起抑制性微小RNA的作用，其表达与HR-HPV感染有关。
