miR-31慢病毒载体质粒构建及其对宫颈癌HeLa细胞增殖和迁移能力的影响

Construction of miR-31 lentiviral vector and its effect on proliferation and migration of cervical cancer He La cells

目的构建微小核糖核酸31(miR-31)慢病毒载体质粒,并观察其对宫颈癌HeLa细胞增殖和迁移能力的影响。方法以包含miR-31基因的序列为目的片段,设计末端含有相应酶切位点的引物,PCR扩增目的片段,产物双酶切后插入线性化慢病毒载体中,构建miR-31慢病毒载体质粒并鉴定。将miR-31慢病毒载体质粒与包装质粒pPACKH1-GAG、pPACKH1-REV、pVSV-G共转染宫颈癌HeLa细胞,并进行慢病毒包装,检测miR-31慢病毒载体质粒滴度。取对数生长期HeLa细胞,随机分为空白对照组、miR-31组、空载体组,空白对照组不做任何处理,miR-31组予miR-31慢病毒载体质粒处理,空载体组予空载体慢病毒质粒处理。采用实时荧光定量PCR法检测各组miR-31 mRNA表达;MTT法和细胞克隆实验检测各组细胞增殖能力,Transwell小室法检测各组细胞迁移能力。结果成功构建重组慢病毒表达质粒,其病毒滴度为3×107TU/m L。miR-31组miR-31 mRNA相对表达量明显高于空白对照组、空载体组(P均<0.05),而空白对照组与空载体组比较P>0.05。miR-31组细胞增殖和迁移能力均高于空白对照组、空载体组(P均<0.05),而空白对照组与空载体组比较P均>0.05。结论成功构建了miR-31慢病毒载体质粒,建立了稳定过表达miR-31的宫颈癌HeLa细胞。稳定过表达miR-31的宫颈癌HeLa细胞增殖和迁移能力明显增强。

Objective To construct the miR-31 lentiviral vector and to investigate its effect on proliferation and migration of cervical cancer He La cells. Methods PCR was used to amplify the target gene which included gene miR-31. The target gene was designed with appropriate enzyme sites at the end of the primer pairs. We inserted the PCR products into linearization of lentiviral vector after the double digestion. Restriction endonuclease analysis and DNA sequencing had confirmed the sequence of the recombinant plasmid p CDH-CMV-MCS-EF1-cop GFP-miR-31. He La cells were co-transfected with the lentiviral vectors p CDH-CMV-MCS-EF1-cop GFP-miR-31,p PACKH1-GAG,p PACKH1-REV and p VSV-G,the titer of the lentivirus was detected. He La cells in the logarithmic phase were randomly divided into the control group without any treatment,the miR-31 group treated with lentivirus miR-31,and the negative control group given empty lentivirus. The q PCR was used to detect the expression of miR-31 in He La cells of each group. The cell proliferation was detected by clone formation assay and MTT assay,and the cell migration ability was detected by Transwell assay. Results The recombinant lentivirus expression plasmid was constructed successfully,with lentivirus titer of 3 × 10^7TU/m L. The expression of miR-31 in He La cells of the miR-31 group was significantly higher than that of the other two groups（ all P〈0. 05）; no significant difference was found between the control group and the negative control group（ P〈0. 05）. Compared with the other two groups,the proliferation and migration abilities of He La cells were increased in the miR-31 group（ all P〈0. 05）; no significant difference was found between the control group and the negative control group（ P〈0. 05）. Conclusion The miR-31 lentivirus expression plasmid is constructed successfully,and the proliferation and migration abilities of He La cells with stale expression of miR-31 are significantly enhanced.