微小RNA 124-5p对宫颈癌细胞增殖和肿瘤恶化程度的影响

Effects of miR-124-5p on the proliferation of cervical cancer cells and progression of cervical cancer

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mp CDH-CMVMCS-EF1-tRFP(简称为mp CDH)载体中,构建重组质粒mp CDH-miR-124-5p。通过慢病毒包装系统将目的质粒mp CDH-miR-124-5p或空载体mp CDH与包装质粒ps PAX2以及包膜质粒p MD2.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He La和Si Ha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mp CDH-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。

Objective： To establish cervical cancer cell lines stably expressing miR-124-Sp 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-Sp was amplified by polymerase chain reaction （PCR） and then cloned into the mpCDH-CMV-MCS-EFI-tRFP （abbreviated as mpCDH）. The target plasmid miR-124-Sp, 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. Mean- while, the effect of miR-124-Sp on the proliferation and tumor progression of cervical cancer cells was meas- ured 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-Sp.Conclusion： miR-124-5p could significantly inhibit the proliferation and tumor progression of cervical cancer cells.