微小RNA-452-5p对乳腺癌细胞增殖和凋亡的影响及可能机制

MicroRNA-452-5p on proliferation and apoptosis of breast cancer cells and the possible mechanism

目的探讨微小RNA-452-5p(miR-452-5p)对乳腺癌细胞增殖和凋亡的影响及可能的机制。方法采用实时荧光定量PCR(QPCR)检测miR-452-5p在乳腺癌细胞株MDA-MB-231、MCF-7、MDA-MB-361、HCC70和正常乳腺细胞株MCF-10A中的表达水平,选取表达水平最低的细胞株进行后续实验。脂质体转染法将miR-452-5p mimic及阴性对照转染至人乳腺癌MDA-MB-231细胞,并设空白对照组。QPCR检测转染效果,EdU增殖实验和克隆形成实验检测细胞增殖,流式细胞术检测细胞凋亡,Western blotting检测凋亡相关蛋白procaspase-3和procaspase-9的表达水平,生物信息学软件TargetScan预测miR-452-5p与血管内皮生长因子A(VEGF-A)的靶向结合位点,Western blotting和双荧光素酶报告基因实验验证二者靶向关系。结果miR-452-5p在乳腺癌细胞株MDA-MB-231(0.26±0.02)、MCF-7(0.35±0.04)、MDA-MB-361(0.49±0.05)和HCC70(0.67±0.07)中的表达量均明显低于正常乳腺细胞株MCF-10A(1.00±0.11),差异有统计学意义(P<0.05),选取表达量最低的MDA-MB-231细胞进行后续实验。QPCR检测显示,与阴性对照组比较,miR-452-5p组MDA-MB-231细胞中miR-452-5p的表达量上调(0.95±0.09 vs.4.28±0.44,P<0.05),而空白对照组与阴性对照组的差异无统计学意义(P>0.05)。EdU检测显示,与阴性对照组比较,miR-452-5p组细胞增殖率降低(P<0.05),而空白对照组与阴性对照组的差异无统计学意义(P>0.05)。克隆形成实验显示,与阴性对照组比较,miR-452-5p组细胞克隆形成数目均减少[(77.65±7.84)个vs.(53.22±6.16)个,P<0.05)],空白对照组与阴性对照组的差异无统计学意义(P>0.05)。流式细胞术检测显示,与阴性对照组比较,miR-452-5p组细胞凋亡率升高[(2.21±0.46)%vs.(20.61±3.09)%,P<0.05)],而空白对照组与阴性对照组的差异无统计学意义(P>0.05)。Western blotting检测显示,与阴性对照组比较,miR-452-5p组procaspase-3(1.20±0.13 vs.0.49±0.05)和procaspase-9(1.49±0.20 vs.0.38±0.04)蛋白表达下调(P<0.05),空白对照组与阴性对照组的差异无统计学意义(P>0.05)。Western blotting检测显示,与阴性对照组比较,miR-452-5p组细胞中VEGF-A蛋白表达下降(P<0.05)。双荧光素酶报告基因显示,VEGF-A-Wt组与阴性对照比较,miR-452-5p相对荧光素酶活性降低(P<0.05),而VEGF-A-Mut组和阴性对照miR-452-5p相对荧光素酶活性无显著改变(P>0.05)。结论miR-452-5p在乳腺癌细胞株中呈低表达,miR-452-5p通过调控VEGF-A的表达影响乳腺癌细胞的增殖和凋亡。

Objective To investigate the effect of microRNA-452-5p(miR-452-5p)on proliferation and apoptosis in breast cancer cell lines and the possible mechanism.Methods The expression level of miR-452-5p in breast cancer cells MDA-MB-231,MCF-7,MDA-MB-361 and HCC70,and normal breast cell line MCF-10A was detected by the realtime-PCR(QPCR),and the cell line with the lowest expression was chosen for the following experiment.The miR-452-5p mimic and negative control were transfected into human breast cancer cell line with the lowest expression of miR-452-5p by lipofection,and blank control was set.Cell proliferation was examined by EdU proliferation assay and colony formation assay.Flow cytometry was employed to detect cell apoptosis.The expression levels of apoptosis-related proteins procaspase-3 and procaspase-9 were detected by Western blotting.Bioinformatics the software TargetScan predicted the binding site of miR-452-5p and vascular endothelial growth factor A(VEGF-A),and Western blotting and dual luciferase reporter gene experiment were used to verify the targeting relationship.Results The expression of miR-452-5p in breast cancer cells MDA-MB-231(0.26±0.02),MCF7(0.35±0.04),MDA-MB-361(0.49±0.05)and HCC70(0.67±0.07)cells were significant lower than that of normal breast cell MCF-10A(1.00±0.11)with significance(P<0.05).The expression level was the lowest in breast cancer cell line MDA-MB-231(P<0.05).QPCR detection showed that the expression of miR-452-5p in MDA-MB-231 cells was up-regulated in the miR-452-5p group compared with negative control group(0.95±0.09 vs.4.28±0.44,P<0.05),and the difference had no significance between negative control group and blank control group(P>0.05).EdU test showed that the cell proliferative rate in miR-452-5p group was decreased compared with negative control group(P<0.05),and the difference had no significance between negative control group and blank control group(P>0.05).The clone formation assay showed that the number of cell clones in miR-452-5p group decreased(77.65±7.84 vs.53.22±6.16,P<0.05)compared with negative control group,and the difference had no significance between negative control group and blank control group(P>0.05).Flow cytometry showed that the apoptotic rate of miR-452-5p group was higher than that of negative control group[(2.21±0.46)%vs.(20.61±3.09)%,P<0.05)],and the difference had no significance between negative control group and blank control group(P>0.05).Western blotting analysis showed that the expression of apoptosis-related proteins procaspase-3(1.20±0.13 vs.0.49±0.05)and procaspase-9(1.49±0.20 vs.0.38±0.04)in miR-452-5p group were down-regulated compared with negative control group(P<0.05),and the difference had no significance between negative control group and blank control group(P>0.05).Western blotting analysis showed that the expression of VEGF-A protein in miR-452-5p group was decreased compared with negative group(P<0.05).The dual luciferase reporter gene showed that the relative luciferase activity of miR-452-5p was decreased in the VEGF-A-Wt group compared with negative control group(P<0.05),while luciferase activity had no significant change in VEGF-A-Mut group(P>0.05).Conclusion MiR-452-5p is down-regulated in breast cancer cell lines.MiR-452-5p affects the proliferation and apoptosis of breast cancer cells by regulating the expression of VEGF-A.