摘要
目的探讨微小RNA 203-3p(miR-203)靶向调控枯灵素2(CUL2)对人乳头瘤病毒16型(HPV16)阳性宫颈癌细胞生物学特性的影响。方法收集2018年9月-2019年9月于山西医科大学第二医院妇产科行宫颈癌筛查,HPV检测为单一HPV16阳性,病理检查为宫颈鳞癌(SCC)的样本及相应癌旁组织(对照组)各10份。采用实时定量反转录聚合酶链反应(qRT-PCR)检测SCC与相应癌旁组织,以及人SCC细胞(SiHa)与人永生化表皮细胞(HaCaT)中miR-203的表达。采用GO和KEGG富集法分析miR-203参与的功能和通路,TargetScan网站及双荧光素酶报告基因实验验证miR-203与CUL2的靶向调控作用。将miR-203模拟物或抑制剂转染至SiHa细胞,建立过表达或低表达miR-203的细胞模型,采用qRT-PCR和Western blotting检测CUL2 mRNA及蛋白的表达;采用CCK-8法、划痕实验、AnnexinⅤ-APC/PI双染色法评估SiHa细胞的增殖、迁移和凋亡能力。结果qRTPCR检测分析结果显示,与相应癌旁组织和HaCaT细胞相比,miR-203在SCC组织和SiHa细胞中的表达水平均明显降低(P<0.01)。GO和KEGG富集结果显示,miR-203广泛参与泛素化过程并与恶性肿瘤参与的信号通路有关。TargetScan与双荧光素酶报告基因实验表明,miR-203与CUL2存在靶向调控关系(P<0.01)。qRT-PCR及Western blotting结果表明,过表达miR-203时,CUL2 mRNA及蛋白表达水平均明显降低(P<0.05或P<0.01),而低表达miR-203则可上调CUL2 mRNA及蛋白的表达(P<0.01)。CCK-8、划痕实验和AnnexinⅤ-APC/PI双染结果显示,过表达miR-203可降低SiHa细胞的增殖率和迁移率(P<0.01),升高细胞凋亡率(P<0.05);相反,低表达miR-203可升高SiHa细胞的增殖率和迁移率(P<0.01),降低细胞凋亡率(P<0.01)。结论miR-203可能通过靶向CUL2抑制HPV16阳性宫颈癌细胞SiHa的生物学特性,有望成为宫颈癌诊断和治疗的一个新的候选基因。
Objective To explore the effect of hsa-microRNA-203-3p(miR-203)targeted-regulating Cullin2(CUL2)on the biological characteristics of human papillomavirus 16(HPV16)positive cervical cancer cells.Methods A total of 10 patients underwent cervical cancer screening from September 2018 to September 2019 in the Department of Obstetrics and Gynecology,the Second Hospital of Shanxi Medical University.HPV genotyping was performed as single HPV16 positive,and pathological examination showed cervical squamous cell carcinoma(SCC).Ten corresponding paracancer normal tissue samples were collected as control group.Real-time quantitative reverse transcription PCR(qRT-PCR)was performed to detect the expression of miR-203 in cervical squamous cell carcinoma and corresponding adjacent tissues,cervical cancer cell line(SiHa)and human immortal keratinocyte line(HaCaT)cells.GO and KEGG enrichment were applied to analyze the functions and pathways miR-203 involved.The regulatory relationship between miR-203 and CUL2 were verified via TargetScan website and dual luciferase reporter assay.The miR-203 mimics or inhibitor were transfected into SiHa cells to establish cell models of high and low expression of miR-203,and the expressions of CUL2 mRNA and protein were detected by qRT-PCR and Western blotting.The proliferation,migration and apoptosis of SiHa cells were assessed by CCK-8,scratch assay and Annexin V-APC/PI double staining,respectively.Results Results of qRT-PCR indicated that,compared with the corresponding adjacent tissues and HaCaT cells,the relative expression level of miR-203 decreased obviously in both cervical SCC tissues and SiHa cells(P<0.01).The results of GO and KEGG enrichment methods showed that miR-203 was widely involved in the ubiquitination process and the signaling pathways involved in the malignancies.TargetScan website and dual-luciferase reporter assay showed that the targeting regulatory relationship existed between miR-203 and CUL2(P<0.01).qRT-PCR and Western blotting indicated that overexpression of miR-203
作者
李亚芹
刘亚涛
张宏蕾
赵卫红
Li Ya-Qin;Liu Ya-Tao;Zhang Hong-Lei;Zhao Wei-Hong(Department of Obstetrics and Gynecology,the Second Clinical Medical College,Shanxi Medical University,Taiyuan,Shanxi 030001,China;Department of Obstetrics and Gynecology,the Second Hospital of Shanxi Medical University,Taiyuan,Shanxi 030001,China;Department of Pathology and Pathophysiology,Basic Medical College,Shanxi Medical University,Taiyuan,Shanxi 030001,China)
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2022年第10期968-975,共8页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金(81702583)
山西省高等学校科技创新项目(201701D31111191)
山西省优秀青年基金项目(201901D211506)
山西省回国留学人员科研教研资助项目(2022-195)。
