微小RNA 124-5p对宫颈癌细胞增殖和肿瘤恶化程度的影响

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摘要目的: 建立稳定表达微小RNA 124-5p（miR-124-5p）的宫颈癌细胞系，探讨miR-124-5p对宫颈癌细胞增殖和肿瘤恶化程度的影响。方法: 采用聚合酶链式反应（PCR）扩增miR-124-5p的前体序列，将其克隆至mpCDH-CMV-MCS-EF1-tRFP（简称为mpCDH）载体中，构建重组质粒mpCDH-miR-124-5p。通过慢病毒包装系统将目的质粒mpCDH-miR-124-5p或空载体mpCDH与包装质粒psPAX2以及包膜质粒pMD2.G共转染入人胚肾上皮细胞（293 T细胞），收集病毒液分别感染宫颈癌HeLa和SiHa细胞系。观察48 h后红色荧光蛋白（RFP）阳性细胞的比例。进一步采用实时荧光定量PCR（qRT-PCR）检测感染后宫颈癌细胞中miR-124-5p的表达水平，运用虫荧光素酶报告实验验证miR-124-5p的活性。通过平板克隆实验和软琼脂集落形成实验观察miR-124-5p对宫颈癌细胞增殖能力和肿瘤恶化程度的影响。结果：成功构建重组质粒mpCDH-miR-124-5p。重组miR-124-5p慢病毒感染宫颈癌细胞系后细胞状态良好，且荧光率高达90%。建立了稳定表达miR-124-5p的宫颈癌细胞系。过表达miR-124-5p后，宫颈癌细胞的增殖能力以及软琼脂集落形成能力明显减弱。结论： miR1245p能够抑制宫颈癌细胞的增殖和肿瘤恶化程度。

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关键词 ： miR-124-5p, 宫颈癌, 慢病毒, 增殖, 落形成

Abstract：Objective: To establish cervical cancer cell lines stably expressing miR1245p and to investigate the effect of miR1245p on the proliferation and transformation of cervical cancer cells. Methods: The precursor sequence of miR1245p was amplified by polymerase chain reaction (PCR) and then cloned into the mpCDHCMVMCSEF1tRFP (abbreviated as mpCDH). The target plasmid miR1245p, or the control empty vector, the packaging plasmid psPAX2 and the envelope plasmid pMD2.G were cotransfected into human embryonic kidney epithelial cells(293 T cells), and the lentiviruses were respectively collected to infect cervical cancer cells, and the red fluorescent protein (RFP) was observed with fluorescent microscope after 48 hours. The expression levels of miR1245p in cervical cancer cells infected with control or target plasmids were detected with realtime quantitative PCR (qRTPCR) and luciferase reporter assay. Meanwhile, the effect of miR1245p on the proliferation and tumor progression of cervical cancer cells was measured with colony formation assay and soft agar colony formation assay. Results: The recombinant plasmid mpCDHmiR1245p was successfully constructed and packaged into lentivirus. The results showed that the infected cells grew well and the rate of cells expressing RFP was up to 90%. qRTPCR and dual luciferase activity assays confirmed that the cervical cancer cell line stably expressing miR1245p was established. The colony formation assay and soft agar colony formation assay showed that the ability of proliferation and colonyforming of cervical cancer cells was significantly decreased after overexpression of miR1245p. Conclusion: miR1245p could significantly inhibit the proliferation and tumor progression of cervical cancer cells.

收稿日期: 2016-11-07

通讯作者: 卢春（通讯作者），教授，博士生导师，E-mail：clu@njmu.edu.cn

作者简介: 徐静云（1992—），女，硕士研究生

引用本文:

徐静云，丁祥亚，卢春. 微小RNA 124-5p对宫颈癌细胞增殖和肿瘤恶化程度的影响[J]. 江苏大学学报:医学版, 2017, 27(01): 53-57,60. XU Jing-yun, DING Xiang-ya, LU Chun. Effects of miR-124-5p on the proliferation of cervical cancer cells and progression of cervical cancer. Journal of Jiangsu University(Medicine Edition), 2017, 27(01): 53-57,60.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2017/V27/I01/53

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