HPV16编码蛋白与宫颈癌细胞内CALCA和TFPI-2表达的关系

doi:10.3969/j.issn.1004-616x.2017.06.006

癌变·畸变·突变 ›› 2017, Vol. 29 ›› Issue (6): 431-436.doi: 10.3969/j.issn.1004-616x.2017.06.006

HPV16编码蛋白与宫颈癌细胞内CALCA和TFPI-2表达的关系

迪拉热·力迪甫¹, 木塔力甫·艾买提², 盛磊², 古扎力努尔买提沙², 艾尔肯·肉孜比拉力³, ABULIZI Abudula^2,3

1. 新疆医科大学基础医学院形态中心, 新疆乌鲁木齐 830011;
2. 新疆医科大学中心实验室, 新疆乌鲁木齐 830011;
3. 新疆医科大学生物化学教研室, 新疆乌鲁木齐 830011

收稿日期:2017-06-09 修回日期:2017-10-24 出版日期:2017-11-30 发布日期:2017-11-30
通讯作者: ABULIZI Abudula,E-mail:abulizi_a@126.com E-mail:abulizi_a@126.com
作者简介:迪拉热·力迪甫,E-mail:dilara17@163.com。
基金资助:
新疆维吾尔自治区研究生教育创新计划科研创新项目（XJGRI2015071）

Expression of HPV16 coding proteins and regulation of CALCA and TFPI-2 expression in cervical carcinoma cells

DILARE Lidifu¹, MUTALIFU Aimaiti², SHENG Lei², GUZHALINUER Maitisha², AIERKEN Rozibilali³, ABULIZI Abudula^2,3

1. Morphological Center, College of Basic Medicine, Xinjiang Medical University, Urumqi 830011;
2. Research Center, Xinjiang Medical University, Urumqi 830011, Xinjiang;
3. Department of Biochemistry, Xinjiang Medical University, Urumqi 830011, China

Received:2017-06-09 Revised:2017-10-24 Online:2017-11-30 Published:2017-11-30

摘要/Abstract

摘要： 目的：研究人乳头瘤病毒16（HPV16）编码蛋白的表达与宫颈癌细胞内CALCA和TFPI-2基因表达水平的关系，探讨依赖于HPV感染的宫颈癌发病机制。方法：设计与合成HPV16编码基因E6和E7序列特异性短发夹RNA（shRNA）寡聚核苷酸片段，构建表达shRNA的pRNAi载体，其中携带绿色荧光蛋白（GFP）报告基因，以此感染HPV16阳性的SiHa宫颈癌细胞，采用荧光成像和实时荧光定量PCR（qPCR）分析等方法，评价载体转染效率、抑制效率及候选基因转录表达水平的变化。结果：在激光共聚焦显微镜下，观察到释放绿色荧光的细胞群，转染成功；根据定量qPCR分析，HPV16编码基因E6和E7的mRNA表达水平明显下降，shRNA表达载体转染产生抑制效率；CDK4和BCL-2 mRNA表达水平明显下降，推测抑制HPV基因转录后，可能抑制宫颈癌细胞生长，引起细胞周期停滞；抑制E7基因表达后，CALCA和TFPI-2 mRNA表达水平明显上升，而抑制E6基因表达后，对上述基因表达水平的影响不明显。结论：HPV16编码蛋白质E7可能引起宫颈癌细胞内CALCA和TFPI-2基因表达下调，此为揭示依赖于HPV感染的宫颈癌发病机制提供了依据。

关键词: HPV16, 编码蛋白, SiHa细胞, 宫颈癌, CALCA, TFPI-2

Abstract: OBJECTIVE:We investigated the expression of CALCA and TFPI-2,and their association with expression of human papillomaviurs (HPV) 16 coding proteins in cervical carcinoma cells. METHODS:We designed and synthesized small-hairpin RNAs (shRNA) which were specific to the sequences of E6 or E7 genes of HPV16. In addition,we prepared a series of shRNA expression constructs using pRNAi plasmid that could simultaneously express shRNA and green fluorescent protein (GFP) reporter gene. After transient expression of shRNA vectors in HPV16-positive SiHa cervical carcinoma cells by lipid transfection,we analyzed transfection efficiency,inhibition of HPV-coding gene expression and its effect on candidate gene expression using fluorescent imaging and quantitative RT-PCR. RESULTS:Relatively high transfection efficiency was confirmed by using confocal microscopy on cell population based on their release of green fluorescence. According to quantitative RT-PCR analysis,E6 or E7 expressions were,to a very high extent,inhibited after transfection of shRNA vectors. Expressions of CDK4 and BCL-2 were significantly decreased after selective inhibition of E6 or E7 gene expression. These observations suggest that the inhibition of HPV16 coding gene expression might have inhibited cell proliferation and induced cell-cycle arrest. Expressions of CALCA and TFPI-2 were significantly upregulated after inhibition of E7 gene expression but not remarkable after inhibition of E6 gene expression. CONCLUSION:Results of this study together with previous reports suggest that HPV16 E7 protein may promote the downregulation of CALCA and TFPI-2 in cervical carcinoma cells. The mechanism may be involved with HPV-driven cervical carcinogenesis.

Key words: HPV16, coding proteins, SiHa cells, cervical carcinoma, CALCA, TFPI-2

中图分类号:

R349.7

迪拉热·力迪甫, 木塔力甫·艾买提, 盛磊, 古扎力努尔买提沙, 艾尔肯·肉孜比拉力, ABULIZI Abudula. HPV16编码蛋白与宫颈癌细胞内CALCA和TFPI-2表达的关系[J]. 癌变·畸变·突变, 2017, 29(6): 431-436.

DILARE Lidifu, MUTALIFU Aimaiti, SHENG Lei, GUZHALINUER Maitisha, AIERKEN Rozibilali, ABULIZI Abudula. Expression of HPV16 coding proteins and regulation of CALCA and TFPI-2 expression in cervical carcinoma cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2017, 29(6): 431-436.

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HPV16编码蛋白与宫颈癌细胞内CALCA和TFPI-2表达的关系

Expression of HPV16 coding proteins and regulation of CALCA and TFPI-2 expression in cervical carcinoma cells

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