微小RNA-29b对乳腺癌细胞增殖和迁移的影响及其分子生物学机制

目的：研究微小RNA（miRNA）-29b对乳腺癌细胞增殖和迁移的影响及其分子生物学机制。方法：构建miRNA-29b重组慢病毒表达载体lenti-miRNA-29b转染293T细胞获得重组慢病毒，提纯慢病毒颗粒感染乳腺癌细胞MCF-7。通过绿色荧光蛋白肿瘤细胞比例来验证转染情况；实时定量PCR检测miRNA-29b的表达水平；CCK-8实验和Transwell实验检测miRNA-29b过表达后乳腺癌细胞生物学行为的变化。采用生物信息学软件对miRNA-29b调控的下游靶基因进行预测和筛选，使用双荧光素酶检测、实时定量PCR和蛋白质印迹法进行验证。对筛选出的miRNA-29b调控的下游靶基因RTKN通过siRNA-RTKN验证其对MCF-7细胞的增殖和迁移的影响。结果：成功构建了miRNA-29b的重组慢病毒表达载体。感染乳腺癌细胞MCF-7后，lenti-miRNA-29b组和空载体转染组分别有90%和60%左右的细胞出现绿色荧光；lenti-miRNA-29b组miRNA-29b表达量较空载体转染组和空白对照组显著增加（均P<0.05），MCF-7细胞的增殖和迁移能力较空载体转染组显著减弱（均P<0.05）。RTKN的基因编码3'UTR区中具有miRNA-29b的结合位点；野生型RTKN-WT-3'UTR报告基因载体与lenti-miRNA-29b共转染MCF-7细胞后可使荧光素酶的活性显著下降（P<0.05）。转染siRNA-RTKN后，MCF-7细胞中RTKN蛋白的水平显著下降，细胞增殖和迁移能力显著减弱（均P<0.05）。结论：miRNA-29b能够通过抑制RTKN的表达来抑制乳腺癌细胞的增殖和迁移能力。
Abstract: Objective:To investigate the effects of microRNA(miRNA)-29b on the proliferation and migration of breast cancer cells and its molecular mechanism. Methods:The recombinant lentiviral expression vector (lenti-miRNA-29b) was constructed and transfected into 293T cells to obtain lentivirus particles that were used to infect breast cancer MCF-7 cells. Transfection efficiency of lenti-miRNA-29b in MCF-7 cells was identified by the expression of green fluorescent protein (GFP). The expression of miRNA-29b was detected by real-time PCR. The cell proliferation and migration were detected by CCK8 assay and Transwell assay, respectively. The bioinformatics softwares were used to predict and screen the downstream target genes regulated by miRNA-29b, which were verified by double luciferase reporter gene assay, RT-PCR and Western blot. The effects of screened target gene RTKN on the growth and migration of MCF-7 cells were verified by RTKN siRNA. Results:Recombinant lentiviral expression vector of miRNA-29b were successfully constructed. About 90% and 60% of the breast cancer cells showed green fluorescence in lenti-miRNA-29b and lenti-miRNA-NC groups, respectively. The expression of miRNA-29b in lenti-miRNA-29b group increased significantly compared with the lenti-miRNA-NC group and blank control group (all P<0.05); the proliferation and migration ability of MCF-7 cells significantly reduced compared with the control group (all P<0.05). The screening with bioinformatics softwares found that the 3'UTR coding region RTKN had the binding site to miRNA-29b; the dual luciferase reporter gene assay showed that the luciferase activity decreased significantly after the MCF-7 cells were co-transfected with wild type RTKN-WT-3'UTR and miRNA-29b mimics report gene vector (P<0.05). The RTKN proteins in MCF-7 cells were