HPV-16 URR突变对病毒早期启动子活性影响的研究

doi:10.19401/j.cnki.1007-3639.2017.02.005

中国癌症杂志 ›› 2017, Vol. 27 ›› Issue (2): 109-114.doi: 10.19401/j.cnki.1007-3639.2017.02.005

HPV-16 URR突变对病毒早期启动子活性影响的研究

宋　丹¹，史　茜¹，侯向前¹，玛依努尔·尼亚孜²，马正海¹

1. 新疆大学生命科学与技术学院，新疆乌鲁木齐 830046 ；
2. 新疆维吾尔自治区人民医院妇产科，新疆乌鲁木齐 830001

出版日期:2017-02-28 发布日期:2017-03-22
通信作者: 马正海　E-mail：mzhxju@126.com
基金资助:
国家自然科学基金项目(31060025)。

The effect of mutations in the upstream regulatory region of HPV-16 on the activity of virus early promoter

SONG Dan¹, SHI Qian¹, HOU Xiangqian¹, MAYINEUR·Niyazi², MA Zhenghai¹

1. College of Life Science and Technology, Xinjiang University, Urumqi 830046, Xinjiang Uyghur Autonomous Region, China; 2. Gynecology and obstetrics Xinjiang Uyghur Autonomous Region People’s Hospital, Urumqi 830001, Xinjiang Uygur Autonomous Region, China

Published:2017-02-28 Online:2017-03-22
Contact: MA Zhenghai E-mail: mzhxju@126.com

摘要/Abstract

摘要： 背景与目的：新疆是宫颈癌高发区，该地区宫颈癌高发与人乳头瘤病毒16型(human papillomavirus type 16，HPV-16)感染密切相关。该研究旨在分析新疆地区妇女宫颈病样组织中HPV-16上游调控区(upstream regulatory region，URR)的突变及其功能。方法：以新疆妇女子宫颈上皮非典型增生(cervical intraepithelial neoplasia，CIN)和宫颈癌病样组织标本DNA为模板，PCR扩增HPV-16 URR片段，PCR产物经测序比对，筛选代表性的URR突变体构建至pGL3-Basic载体，将其转染Vero细胞，48 h后检测荧光素酶活性，分析URR突变体启动子活性。结果：采用聚合酶链反应(polymerase chain reaction，PCR)获得了55个HPV-16 URR DNA片段，测序及序列分析发现44个突变位点，其中nt7192(G→T)、nt7433(-→T)、nt7435(C→G)和nt7863(A→-)4个位点的突变为所有序列共有，nt7520(G→A)位点的突变存在于54个样品中，剩余39个位点的突变存在于不同样品中。根据突变的位置、频率和程度，筛选出9个URR突变体分别克隆至pGL3-Basic中荧光素酶基因前并转染Vero细胞。荧光素酶活性分析表明，不同URR突变体的启动子活性差异较大，来源于宫颈癌的URR突变体启动子活性显著高于来源于CIN的URR突变体(P<0.01)，部分宫颈癌URR突变体的启动子活性显著高于SiHa和Caski细胞来源的URR参照序列的启动子活性。结论：新疆地区分离的HPV-16URR发生多位点突变，其中部分突变增强了URR内部启动子的活性，导致HPV-16致癌活性增强。

关键词: 人乳头瘤病毒16型, 上游调控区, 突变, 启动子

Abstract: Background and purpose: The incidence of cervical cancer is rather high in Xinjiang, which is closely associated with the infection of human papilloma virus type 16 (HPV-16). The purpose of this study was to analyze the variants and function of HPV-16 upstream regulatory region (URR) in the tissues of cervical cancer biopsies from Xinjiang. Methods: The DNAs were extracted from the tissues of cervical epithelial atypical hyperplasia (CIN) and cervical cancer biopsies. HPV-16 URR segments were amplified by PCR. Based on the sequence analysis of the URR, the representative URR variants were selected and cloned into pGL3-Basic. The recombinant plasmids were transfected into Vero cell lines respetively. Luciferase activity of transfected cells was detected 48 h after transfection. Results: Fifty-five HPV-16 URR DNA fragments were obtained through PCR, and 44 mutations were found from the URR fragments. 4 of these mutations, including nt7192(G→T) , nt7433(- →T), nt7435 (C→G) and nt7863 (A→-) occurred in all sequences. The mutation at nt7520 (G→A) occurred in 54 URR sequences, and the 39 other mutations were present in different samples. Based on the location and frequency of the mutations in the URR fragments, 9 URR variants were selected and cloned into pGL3-Basic. Then the luciferase activity of the cells transfected with pGL3- URR plasmids was detected respectively. Promoter activity of URR mutants from cervical cancer are significantly higher than that of URR mutants from CIN (P<0.01). Promoter activity of URR fragments from some cervical cancer was significantly higher than that of the URR fragments from SiHa and Caski cells. Conclusion: Multiple mutations occurred in HPV-16 URR of cervical cancer patients from Xinjiang. The promoter activity and carcinogenicity of some URR mutants have been enhanced.

Key words: Human papillomavirus type 16, Upstream regulatory region, Mutation, Promoter

宋　丹,史　茜,侯向前,等. HPV-16 URR突变对病毒早期启动子活性影响的研究[J]. 中国癌症杂志, 2017, 27(2): 109-114.

SONG Dan, SHI Qian, HOU Xiangqian, et al. The effect of mutations in the upstream regulatory region of HPV-16 on the activity of virus early promoter[J]. China Oncology, 2017, 27(2): 109-114.

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HPV-16 URR突变对病毒早期启动子活性影响的研究

The effect of mutations in the upstream regulatory region of HPV-16 on the activity of virus early promoter

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