过表达miR-153非小细胞肺癌细胞稳转株的构建及鉴定

目的:构建过表达miR-153的非小细胞肺癌的稳转细胞株,为进一步探讨miR-153在肺癌发生发展中的作用奠定基础。方法:构建具有嘌呤霉素抗性的miR-153慢病毒载体;体外培养非小细胞肺癌细胞系SPC-A-1和A549,加入浓度梯度的嘌呤霉素溶液,筛选出最佳浓度;使用慢病毒转染细胞株,并在转染72 h后用荧光显微镜检测绿色荧光蛋白(EGFP)的表达,评价转染效率;体外实验使用PCR检测miR-153在稳转株的表达;稳转株移植裸鼠体内成瘤,取出瘤体后检测瘤体内EGFP和miR-153的表达。结果:完成过表达miR-153慢病毒载体及阴性对照载体的构建;确定嘌呤霉素最佳筛选浓度:在SPC-A-1细胞中的浓度为2.0 g/mL,A549细胞中的浓度为3.0 g/mL;目的基因miR-153被慢病毒成功导入SPC-A-1细胞和A549细胞中,荧光显微镜下能直接观察到EGFP。转染miR-153组和阴性对照组的PCR实验结果显示:在SPC-A-1细胞中,miR-153的表达量为92.9±20.6,明显高于阴性对照组(P=0.016);在A549细胞中,miR-153的表达量为2624.6±153.4,显著高于阴性对照组(P0.001)。稳转株能在裸鼠体内形成移植瘤,瘤体内明显有EGFP的表达;与阴性对照组的瘤体相比,移植瘤组miR-153的表达量为显著上调(P=0.048)。结论:成功构建过表达miR-153的非小细胞肺癌的稳转细胞株。

Construction and Identification of miR-153 Overexpression in the Non-small Cell Lung Cancer Cell Stable Lines

ABSTRACT Objective: To construct stable cell lines of non-small-cell lung cancer overexpressed miR-153 and further explore its role in the development of non-small-cell lung cancer. Methods: Mir-153 lentivirus vector with purinomycin resistance was constructed. Non-small cell lung cancer cell lines SPC-A-1 and A549 were cultured in vitro and the concentration gradient of purinomycin solution was added to determine the optimal concentration. Lentivirus was used to transfect cell lines, and the expression of green fluorescent protein (EGFP) was detected with a fluorescence microscope 72 h after transfection to evaluate the transfection efficiency. In vitro, PCR was used to detect the expression level of mir-153 in stable cell lines. Tumor formation in nude mice was obtained by transplanting the stable cell lins, and the expression of EGFP and miR-153 in the tumor was detected. Results: The construction of mir-153 lentivirus vector and negative control vector was completed. The optimal concentration of purinomycin was determined: 2.0 g/mL in SPC-A-1cells and 3.0 g/mL in A549 cells. Target gene miR-153 was successfully introduced into SPC-A -1 and A549 cells by lentivirus, and EGFP expression could be directly observed under fluorescence microscope. PCR results of transfection of miR-153 group and negative control group showed that the expression of miR-153 in SPC-A-1 cells was 92.9±20.6, which was significantly higher than that in the negative control group (t=7.79 P=0.016). The expression of miR-153 in A549 cells was 2624.6±153.4, significantly higher than that in the negative control group (t=29.63 P<0.001). Stable transplanting cell lines can form transplanted tumor in nude mice. EGFP was obviously expressed in the tumor. Compared with the tumor in the negative control group, the expression of miR-153 in the miR-153 group was 559±220.6, which was significantly up-regulated (t=4.38 P=0.048). Conclusion: A stable cell line of non-small cell lung cancer overexpressed miR-153 was successfully constructed.