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目的构建miR-145高效表达载体,研究其对宫颈癌HeLa细胞增殖的影响。方法人工合成miR-145基因序列,构建真核重组质粒pcDNATM6.2-GW-miR-145,并转染HeLa细胞,将HeLa细胞分为转染miR-145组、空载体组和未经处理的HeLa细胞组。采用实时荧光定量PCR(qRT-PCR)和Western blot法分别检测miR-145及其下游靶基因细胞周期蛋白依赖性激酶6(CDK6)在各组中的表达,采用MTT法检测miR-145对HeLa细胞增殖活性的影响。结果经测序和qRT-PCR分析鉴定证明重组质粒pcDNATM6.2-GW-miR-145构建成功,qRT-PCR和Western blot结果显示转染miR-145组的HeLa细胞中CDK6的表达水平明显降低,MTT试验结果显示转染miR-145的HeLa细胞其增殖活性明显受到抑制(P<0.05)。结论成功构建miR-145真核表达载体pcDNATM6.2-GW-miR-145,miR-145能够抑制宫颈癌HeLa细胞中CDK6的表达。
Objective To construct miR-145 high expression vector and study its effect on the proliferation of cervical cancer HeLa cells. Methods The recombinant plasmid pcDNATM6.2-GW-miR-145 was constructed by artificial synthesis of miR-145 gene and transfected into HeLa cells. HeLa cells were divided into miR-145 group, empty vector group and untreated HeLa cell group. The expression of miR-145 and its downstream target gene cyclin-dependent kinase 6 (CDK6) in each group were detected by real-time quantitative PCR (qRT-PCR) and Western blot. MTT assay was used to detect the effect of miR- Effect of Cell Proliferation Activity. The results of sequencing and qRT-PCR analysis showed that the recombinant plasmid pcDNATM6.2-GW-miR-145 was successfully constructed. The results of qRT-PCR and Western blot showed that the expression of CDK6 in HeLa cells transfected with miR- The results showed that the proliferation of HeLa cells transfected with miR-145 was significantly inhibited (P <0.05). Conclusion The miR-145 eukaryotic expression vector pcDNATM6.2-GW-miR-145 was successfully constructed and miR-145 could inhibit the expression of CDK6 in HeLa cells.