摘要
目的为了揭示小分子RNA miR-630抑制肺癌细胞生长的作用,阐明其通过靶向抑制BCL2来发挥作用的分子机制。方法以CCK8检测细胞活力来评估细胞增殖的状况;以BD FACSCalibur流式细胞仪检测并分析细胞周期各个时期的变化;以mirPath和RNAhybrid两个生物信息学工具预测miR-630的下游靶点。通过一系列如PCR、琼脂糖胶回收、限制性克隆位点酶切链接以及质粒小提和大提等克隆工具,将miR-630基因构建到mi RNA表达质粒pmR-mcherry载体,使用Lipofectamine 2000转染miR-630表达质粒至细胞内以在体外表达miR-630;以Western blot检测BCL2蛋白的表达。结果成功构建表达miR-630的表达质粒,与对照组相比较,pmR-premiR630表达质粒转染48 h和72 h后miR-630的表达水平增高了(4.49±0.59)和(9.16±1.50)倍(P均<0.05)。与对照空质粒转染48 h和72 h后细胞活力为1.26±0.09和1.84±0.03相比较,miR-630表达质粒转染48 h和72 h后细胞活力降低到0.89±0.09和1.34±0.23(P均<0.05)。细胞转染miR-630表达质粒转染48 h后细胞周期中S期降低。生物信息学预测到BCL2是miR-630的一个靶点。与对照组相比较,pmR-premiR630表达质粒转染48 h和72 h后BCL2蛋白的表达分别下降了(0.6±0.03)和(0.4±0.01)倍(P均<0.05)。细胞转染miR-630表达质粒后BCL2蛋白表达降低。结论 miR-630通过抑制BCL2表达来抑制肺癌细胞的生长。
Objective To investigate the roles of miR-630 on the proliferation of lung cancer cells, and the molecular mechanisms targeting BCL2 involved. Methods The cell proliferation was examined by CCK-8 kit. The distribution data of cell cycle populations was collected and analyzed on BD FACSCalibur Flow Cytometry. Targets for miR-630 were predicted by bioinformatical tools mirPath and RNAhybrid. The miR-630 expression plasmid was constructed using a series cloning tools,such as PCR, Gel purification, restriction enzyme digestion, plasmids DNA extraction used mini-prep and midi-prep, and then transfected into lung cancer cells by Lipofectamine 2000. And BCL2 protein was detected using Western blots. Results We successfully constructed miR-630 expression plasmids, and the expression levels of miR-630 were increased by 4.49±0.59 and 9.16±1.50 fold change in cells 48 and 72 hours post-transfection of miR-630 expression plasmids, compared with those in cells transfected control plasmid（P〈0.05）. The viabilities of cells 48 and 72 hours post-transfection with miR-630 expression plasmid were decreased to 0.89±0.09 and 1.34±0.23, compared with those（1.26±0.09 and 1.84±0.03） in cell transfected with control plasmid（P〈0.05）. Cell population of S phase was significantly decreased in cells transfected with miR-630 expression plasmid（P〈0.05）. mirPath and RNAhybrid predicted that BCL2 was one of its targets, which confirmed by that the expression levels of BCL2 were decreased by（0.6±0.03）and（0.4±0.01）fold change in cells 48 and 72 hours post-transfection of miR-630 expression plasmids, compared with those in cells transfected control plasmid（P〈0.05）. BCL2 protein was significantly repressed in cells 48 hours post-transfection of miR-630 expression plasmid. Conclusion MiR-630 inhibits proliferation of human lung cancer cells through targeting BCL2.
出处
《中国热带医学》
CAS
2018年第1期22-27,共6页
China Tropical Medicine
基金
广州市医药卫生科技项目(No.20161A010007)
