HPV18阳性和HPV18阴性子宫颈癌组织中miR-24-3p和HOXB8蛋白的表达差异及其机制研究

来源:用户上传      作者:李会影 徐明鑫 徐明研 李鑫 宋超 周宇

　　 【摘要】 目的：檢测HPV18阳性和HPV18阴性子宫颈癌患者癌组织中miR-24-3p和HOXB8的表达差异并探讨其变化机制。方法：选取2015年1月-2019年1月于本院就诊的HPV18阳性子宫颈癌患者40例（HPV18+组）和HPV18阴性子宫颈癌患者40例（HPV18-组）的手术标本。实时荧光定量PCR（RT-qPCR）检测两组miR-24-3p和HOXB8 mRNA的表达水平;免疫印迹法检测两组HOXB8蛋白的表达水平;巢式降落式甲基化特异性PCR（nMS-PCR）检测两组miR-24-3p启动子区DNA甲基化水平;染色质免疫共沉淀-定量PCR（ChIP-qPCR）检测两组miR-24-3p启动子区H3K27me3水平;培养人子宫颈癌细胞系HeLa细胞，分别转染miR-24-3p模拟物和miR-24-3p抑制物后检测HOXB8的表达水平。结果：HPV18-组miR-24-3p的相对表达水平高于HPV18+组（P<0.01）。HPV18-组HOXB8 mRNA和蛋白表达水平均低于HPV18+组（P<0.01）。miR-24-3p启动子区富含CpG位点和CpG岛，HPV18-组miR-24-3p启动子区DNA甲基化水平低于HPV18+组，（P<0.01）。HPV18-组miR-24-3p启动子区H3K27me3水平低于HPV18+组，（P<0.01）。在Hela细胞过表达miR-24-3p可抑制HOXB8的表达，而敲减miR-24-3p的表达可提高HOXB8的表达，与阴性对照比较，差异均有统计学意义（P<0.05）。结论：HPV18可能通过DNA甲基化和组蛋白甲基化下调miR-24-3p的表达导致HOXB8表达升高，进而促进子宫颈癌的发生发展，为HPV18+子宫颈癌的治疗研究提供了实验基础和干预靶点。
　　 【关键词】 HPV18 子宫颈癌 miR-24-3p HOXB8 DNA甲基化 H3K27me3
　　 Study on the Expression Differences of miR-24-3p and HOXB8 Protein in HPV18 Positive and HPV18 Negative Cervical Cancer Tissues and Its Mechanism/LI Huiying， XU Mingxin， XU Mingyan， LI Xin， SONG Chao， ZHOU Yu. //Medical Innovation of China， 2020， 17（28）： 00-006
　　 [Abstract] Objective： To detect the differential expression of miR-24-3p and HOXB8 in cancer tissues of HPV18 positive and HPV18 negative cervical cancer patients and exploring the mechanisms. Method： From January 2015 to January 2019， surgical specimens were selected from 40 patients with HPV18 positive cervical cancer （HPV18+ group） and 40 patients with HPV18 negative cervical cancer （HPV18- group） visited our hospital. miR-24-3p and HOXB8 were detected by real-time quantitative PCR （RT-qPCR）. The expression of HOXB8 protein was detected by Western blotting. The DNA methylation level of miR-24-3p promoter was detected by nMS-PCR. The H3K27me3 level in miR-24-3p promoter region was detected by chromatin immunoprecipitation quantitative PCR （ChIP-qPCR）. Hela cells were cultured and expression levels of HOXB8 were detected after transfection with miR-24-3p mimics and miR-24-3p inhibitors， respectively. Result： The relative expression level of miR-24-3p in the HPV18- group was higher than that in the HPV18+ group （P<0.01）. Both mRNA and protein expression levels of HOXB8 in the HPV18- group were lower than those in the HPV18+ group （P<0.01）. The promoter region of miR-24-3p was rich in CpG sites and CpG islands， and the DNA methylation level of the promoter region of miR-24-3p in the HPV18- group was lower than that in the HPV18+ group （P<0.01）. The promoter H3K27me3 level of miR-24-3p in the HPV18- group was lower than that in the HPV18+ group （P<0.01）. Over expression of miR-24-3p in HeLa cells inhibited the expression of HOXB8， while knockdown of miR-24-3p
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