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目的:构建miRNA-126的重组真核表达载体,研究其对小鼠乳腺癌4T1细胞增殖和迁移的影响。方法:设计合成miRNA-126的正义和反义寡核苷酸,构建真核表达载体pcDNA6.2-miR-126,体外瞬时转染至4T1细胞,荧光显微镜下观测转染效率。Real-time PCR检测4T1细胞中miRNA-126的表达,MTT法和克隆形成实验检测4T1细胞的增殖和克隆形成能力,划痕法观察4T1细胞的体外迁移。结果:成功构建pcDNA6.2-miR-126真核表达载体,其可在4T1细胞中有效表达miR-126。与转染空质粒组(pcDNA6.2-Ctrl)相比,瞬时转染72 h后,pcDNA6.2-miR-126转染组4T1细胞的体外增殖能力受到明显抑制[(0.30±0.03)vs(0.51±0.04),P<0.05];瞬时转染48 h后,pcDNA6.2-miR-126组4T1细胞迁移能力也受到明显抑制[(8.17±2.30)vs(28.33±2.16)个,P<0.05]。结论:miRNA-126过表达可抑制乳腺癌4T1细胞的增殖及迁移。
OBJECTIVE: To construct recombinant eukaryotic expression vector of miRNA-126 and study its effect on the proliferation and migration of mouse breast cancer 4T1 cells. Methods: The sense and antisense oligonucleotides of miRNA-126 were designed and synthesized. The eukaryotic expression vector pcDNA6.2-miR-126 was constructed and transiently transfected into 4T1 cells in vitro. The transfection efficiency was observed under a fluorescence microscope. The expression of miRNA-126 in 4T1 cells was detected by Real-time PCR. The proliferation and colony formation ability of 4T1 cells were detected by MTT assay and colony formation assay. The in vitro migration of 4T1 cells was observed by scratch assay. Results: The pcDNA6.2-miR-126 eukaryotic expression vector was successfully constructed, which can effectively express miR-126 in 4T1 cells. Compared with the pcDNA6.2-Ctrl group, the proliferation of 4T1 cells was significantly inhibited in 72h after transfection ([(0.30 ± 0.03) vs ( 0.51 ± 0.04), P <0.05]. After transient transfection for 48 h, the migration ability of 4T1 cells in pcDNA6.2-miR-126 group was also significantly inhibited (8.17 ± 2.30 vs 28.33 ± 2.16, P <0.05 ]. Conclusion: Overexpression of miRNA-126 can inhibit the proliferation and migration of 4T1 breast cancer cells.