miR-99b-5p通过靶向TRIB1基因调控乳腺癌MCF-7细胞增殖和凋亡的研究

miR-99b-5p regulates the proliferation and apoptosis of breast cancer MCF-7 cells by targeting TRIB1 gene

目的:探讨miR-99b-5p靶向Tribbles同源蛋白1(Tribbles pseudokinase 1,TRIB1)基因调控人乳腺癌MCF-7细胞增殖和凋亡。方法:采用RT-PCR检测人乳腺上皮细胞HBL-100和乳腺癌MCF-7细胞中miR-99b-5p的表达水平,采用Western blotting检测人乳腺上皮细胞HBL-100和乳腺癌MCF-7细胞中TRIB1蛋白表达量。在MCF-7细胞中转染miR-99b-5p mimics或mimics对照,分别记为miR-99b-5p组和miR-NC组,RT-PCR检测细胞中miR-99b-5p表达变化,Western blotting检测TRIB1蛋白表达量,MTT法检测细胞增殖能力,AnnexinV-FITC/PI双染色法检测细胞凋亡水平,双荧光素酶报告基因实验检测miR-99b-5p和TRIB1的靶向关系。结果:与人乳腺上皮细胞HBL-100比较,乳腺癌MCF-7细胞中miR-99b-5p的表达量明显降低(P<0.01),而TRIB1蛋白的表达明显升高(P<0.01)。与miR-NC组比较,miR-99b-5p组MCF-7细胞中miR-99b-5p的表达量升高(P<0.05),TRIB1蛋白表达水平降低(P<0.05),细胞增殖能力减弱(P<0.05),细胞凋亡率升高(P<0.05)。双荧光素酶报告基因检测实验结果显示,miR-99b-5p能够明显降低TRIB1-Wt MCF-7细胞相对荧光素酶活性(P<0.01),而对TRIB1-Mut MCF-7细胞相对荧光素酶活性无明显影响(P>0.05)。结论:miR-99b-5p在乳腺癌MCF-7细胞中呈低表达,TRIB1蛋白呈高表达,过表达miR-99b-5p能够靶向抑制TRIB1阻碍MCF-7细胞增殖并促进细胞凋亡。

Objective:To investigate the regulation of proliferation and apoptosis of human breast cancer MCF-7 cells by miR-99 b-5 p targeting Tribbles pseudokinase 1(TRIB1) gene.Methods:The expression of miR-99 b-5 p in human breast epithelial cells HBL-100 and breast cancer MCF-7 cells was detected by RT-PCR,and the expression of TRIB1 protein in human breast epithelial cells HBL-100 and breast cancer MCF-7 cells was detected by Western blotting.After transfection with miR-99 b-5 p mimics or mimics control in MCF-7 cells,which was named as miR-99 b-5 p group and miR-NC group,respectively,the expression of miR-99 b-5 p in the cells was detected by RT-PCR,the expression of TRIB1 protein was detected by Western blotting,the cell proliferation was detected by MTT assay,the apoptosis level was detected by AnnexinV-FITC/PI double staining,and the targeting relationship between miR-99 b-5 p and TRIB1 was detected by dual-luciferase reporter gene assay.Results:Compared with human breast epithelial cells HBL-100,the expression of miR-99 b-5 p in breast cancer MCF-7 cells was significantly decreased(P0.05).Conclusions:MiR-99 b-5 p is down-regulated in breast cancer MCF-7 cells,and TRIB1 protein is up-regulated.Overexpression of miR-99 b-5 p can inhibit the proliferation and promote apoptosis in MCF-7 cells by targeted inhibiting TRIB1.