miR-335通过下调Fra-1表达抑制人乳腺癌细胞增殖机制的实验研究

Research of miR-335 Inhibited Proliferation of Human Breast Cancer Cells by Down-Regulated Fra-1 Expression

目的 探讨微小核糖核酸(microRNA,miR)-335对人乳腺癌细胞增殖能力的影响,以及其可能存在的机制。方法 体外培养乳腺癌细胞及正常乳腺组织细胞,检测miR-335及Fros相关抗原1(fros related antigent-1,Fra-1)的表达量;体外转染miR-335 mimics后,通过细胞活力检测试剂盒(cell counting kit-8,CCK-8)检测细胞的增殖能力,检测Fra-1的表达变化及下游细胞增殖相关蛋白分子基质金属蛋白酶9(matrix metalloproteinase-9,MMP-9)、血管内皮生长因子C(vascular endothelial growth factor-C,VEGF-C)的表达量;生物信息学预测及双荧光霉素报告基因验证Fra-1为miR-335作用靶基因。结果 乳腺癌细胞中miR-335的表达量(0.755±0.034)低于正常乳腺细胞(1.495±0.029),Fra-1蛋白的表达量(2..347±0.120)高于正常乳腺细胞(1.319±0.038),差异均有统计学意义(t=0.075,4.191,均P<0.001)。转染mi R-335 mimic后48,72 h乳腺癌细胞增殖能力(0.881±0.062,0.887±0.082)低于对照组细胞(1.326±0.051,1.493±0.038),差异均有统计学意义(t=44.096,59.307,均P<0.001);转染miR-335 mimic后乳腺癌细胞中Fra-1的蛋白表达量(0.567±0.091)低于未转染细胞(2.347±0.204),差异有统计学意义(t=3.763,P<0.001);转染miR-335 mimic后细胞中MMP-9(0.469±0.027),VEGF-C(0.540±0.041)的蛋白表达量低于对照组(0.958±0.058,1.024±0.171),差异均有统计学意义(t=1.953,7.856,均P<0.001);荧光霉素报告基因实验结果显示miR-335下调Fra-1启动子核心转录区域(-164~-52nt)的转录活性。结论 miR-335通过抑制MMP-9和VEGF-C的表达,并下调Fra-1启动子转录活性,抑制了体外乳腺癌细胞的增殖。

Objective To explore the effect of microRNA(miR)-335 on the proliferation of human breast cancer cells and its possible mechanism.Methods Breast cancer cells and normal breast tissue cells were cultured in vitro,and the expression of miR-335 and Fros related antigent-1(Fra-1)were detected.MiR-335 mimics were transfected into breast cancer cells,the proliferation ability of breast cancer cells was detected by cell counting kit-8(CCK-8).Real-time PCR and Western blotting were used to detecte the expression of Fra-1 mRNA and protein,and detected the expression of cell proliferation-related molecules matrix metalloproteinase 9(MMP-9)and vascular endothelial growth factor-C(VEGF-C);Bioinformatics prediction and luciferase reporter gene assay were comfirmed that Fra-1 was the potential target genes of miR-335.Result The expression level of miR-335 in breast cancer cells(0.755±0.034),which was lower than that in normal breast cells(1.495±0.029),the expression level of Fra-1 protein(2.347±0.120),which was higher than that in normal breast cells(1.319±0.038),the differences were statistically significant(t=0.075,4.191,all P<0.001).MiR-335 mimic was transfected into breast cancer cells and cultured for 48 and 72 hours,the proliferation ability of breast cancer cells were 0.881±0.062 and 0.887±0.082,which were lower than that of control cells by 1.326±0.051 and 1.493±0.038,the differences were statistically significant(t=44.096,59.307,all P<0.001).The protein expression of Fra-1 in breast cancer cells which transfected with miR-335 mimic was 0.567±0.091,which was lower than that untransfected cells(2.347±0.204),the difference was statistically significant(t=3.763,P<0.001).MiR-335 mimic was transfected into breast cancer cells,the protein expressions of MMP-9 and VEGF-C in the cells were 0.469±0.027 and 0.540±0.041,which were lower than the control group by 0.958±0.058 and 1.024±0.171,the differences were statistically significant(t=1.953,7.856,all P<0.001).Luciferase reporter gene assay showed that miR-335 could down-regulate the transcription activity of the core transcription region of Fra-1 promoter(-164~-52nt).Conclusion MiR-335 inhibited the proliferation of breast cancer cells in vitro by down-regulating the expression of MMP-9,VEGF-C and the transcriptional activity of Fra-1 promoter.