摘要
目的:检测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。
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-24-3pincreased the expression of HOXB8,with statistically significant differences compared with the negative control group (P<0.05).Conclusion:HPV18 may down-regulate the expression of miR-24-3p through DNA methylation and histone methylation,leading to increased HOXB8 expression,and then promote the occurrence and development of cervical cancer.It provides experimental basis and intervention targets for the treatment of HPV18^+ cervical cancer.
作者
李会影
徐明鑫
徐明研
李鑫
宋超
周宇
LI Huiying;XU Mingxin;XU Mingyan;LIXin;SONG Chao;ZHOU Yu(Hongqi Hospital Affiliated of Mudanjiang Medical College,Mudanjiang 157011,China)
出处
《中国医学创新》
CAS
2020年第28期1-6,共6页
Medical Innovation of China
基金
黑龙江省卫生健康委员会项目(2018335)。
