慢病毒载体对MDA-MB-231 细胞中 microRNA-18a-3P 表达的影响

Effect of lentiviral vector on the expression of microRNA-18a-3P in MDA-MB-231 cells

摘要:

目的 构建人microRNA-18a-3P（miR-18a-3P）过表达及干扰慢病毒载体，研究病毒感染人乳 腺癌细胞系MDA-MB-231 的步骤和方法。方法 PCR 技术扩增相应目的基因片段并进行酶切，回收后与目 的基因连接，产物转化细菌感受态细胞，对阳性克隆测序行对比分析，构建miR-18a-3P 过表达及干扰慢病毒 载体，荧光法测定病毒滴定；倒置荧光显微镜观察人乳腺癌细胞MDA-MB-231 转染及筛选过程的转染效率， 筛选最佳MOI 值；qRT-PCR 检测慢病毒转染后MDA-MB-231 细胞内miR-18a-3P 的表达。结果 测序 分析证实重组慢病毒构建正确；过表达及干扰慢病毒滴度分别为2×109 和1×109 TU/ml ；加Polybrene 较未 加Polybrene 转染效率升高，当感染复数为10 TU/ml，Polybrene 浓度为1μg/ml 时，转染效率最佳；miR- 18a-3P 转染组过表达miR-18a-3P 的相对表达量较空白对照组、阴性对照组高（P <0.05），miR-18a-3P 转染组干扰表达miR-18a-3P 的相对表达量较空白对照组、阴性对照组低（P <0.05）。结论 成功构建了 miR-18a-3P 过表达及干扰慢病毒表达载体，对人乳腺癌细胞MDA-MB-231 进行转染和筛选后，可快速高 效率获得所需目的细胞。

Abstract:

Objective To construct a lentivirus vector expressing miRNA-18a-3P, and to discuss the optimal steps and methods in infecting human breast cancer cell line MDA-MB-231. Methods Target gene which was digestied to be amplified by PCR, and then those target genes were connected with carriers. The products were transformed into bacterial competent cells. The positive clones were sequenced and analyzed to construct miR-18a- 3P overexpression or interference lentiviral vectors. The virus titer was determined by fluorescence method. In the process of transfection and screening of human breast cancer cell line MDA-MB-231, the transfection efficiency was observed by inverted microscope, and the optimal MOI value was screened. Real-time PCR was used to detect the expression of miR-18a-3P in MDA-MB-231 cells after lentivirus transfection. Results Sequencing proved that recombinant lentiviral expression vector was constructed correctly. The titer of obtained overexpression and suppression expression recombinant lentivirus was 2×109 TU/ml and 1×109 TU/ml. The transfection efficiency was higher in the polybrene group than in the non-polybrene group, and the transfection efficiency was the best when the polybrene concentration was 1 μg/ml. The expression of mir-18a-3p in mir-18a-3p transfection group was higher than that in blank control group and negative control group (P < 0.05). The expression of mir-18a-3p in mir-18a-3p interference group was lower than that in blank control group and negative control group. Conclusions The lentiviral expression or interference vector for miR-18a-3P is successfully constructed, and target cells can be obtained after transfection and selection of human breast cancer MDA-MB-231 cells.