摘要
目的:探讨乳腺癌细胞中miR-204对线粒体转录因子A(TFAM)的靶向调控作用及其与细胞生长、增殖的关系。方法:将人乳腺癌MDA-MB-231细胞分别转染mi R-204模拟物或miR-204抑制物,用real-time PCR和Western blot分别检测mi R-204与TFAM蛋白的表达;构建荧光酶报告基因质粒(mut-TFAM/wt-TFAM),将其与mi R-204模拟物或miR-204抑制物共转染MDA-MB-231细胞后检测荧光酶活性变化;构建pc DNA3.1/TFAM质粒,将其单独或与mi R-204模拟物共转染MDA-MB-231细胞后检测TFAM蛋白表达,并用MTT法和Brd U法检测细胞生长与增殖情况。结果:MDA-MB-231细胞转染mi R-204模拟物后mi R-204的表达明显升高,而TFAM蛋白表达明显降低,转染miR-204抑制物后则呈反向变化(均P<0.05)。wt-TFAM与mi R-204模拟物共转染时荧光酶活性明显下降,与mi R-204抑制物共转染时荧光酶活性明显升高(均P<0.05)。转染pc DNA3.1/TFAM后,MDA-MB-231细胞的TFAM m RNA及蛋白表达量明显上调,细胞生长与增殖能力明显升高(均P<0.05);mi R-204模拟物后,MDA-MB-231细胞在TFAM表达降低的同时,细胞生长与增殖能力明显降低,而与pc DNA3.1/TFAM共转染后其上述作用均被部分抵消(均P<0.05)。结论:mi R-204能靶向抑制乳腺癌细胞TFAM的表达,从而抑制乳腺癌细胞的生长与增殖。
Objective： To investigate the targeted regulation of miR-204 on mitochondrial transcription factor A （TFAM） in breast cancer cells and their relations with cell growth and proliferation.Methods： Human breast cancer MDA-MB-231 cells were transfected with miR-204 mimics or inhibitors, and then, the miR-204 and TFAM protein expressions were determined by real-time PCR and Western blot, respectively. The luciferase reporter plasmids （mut-TFAM/wt-TFAM） were constructed and co-transfected with miR-204 mimics or inhibitors into MDA-MB-231 cells, and then, changes in luciferase activities were detected. The pcDNA3.1/TFAM plasmids were constructed and transfected alone or co-transfected with miR-204 mimics into MDA-MB-231 cells, and then, the TFAM protein expressions were measured, and cell growth and proliferation were analyzed by TTC and BrdU assay.Results： The miR-204 mRNA expression was significantly increased, and TFAM protein expression was significantly decreased in MDA-MB-231 cells after transfection with miR-204 mimics, while, opposite directional changes were found after transfection with miR-204 inhibitors （all P〈0.05）. The luciferase activity was significantly decreased after transfection with miR-204 mimics, but was significantly increased after transfection with miR-204 inhibitors （both P〈0.05）. In MDA-MB-231 cells, both expressions of TFAM mRNA and protein were significantly up-regulated, and the growth and proliferation were significantly enhanced after transfection of pcDNA3.1/TFAM （all P〈0.05）, and the growth and proliferation were significantly impaired along with significant down-regulation of TFAM protein expression after transfection of miR-204 mimics, which were all partially abolished by co-transfection with pcDNA3.1/TFAM （all P〈0.05）.Conclusion： MiR-204 exerts targeted inhibition on TFAM expression in breast cancer cells, and thereby suppresses the growth and proliferation of breast cancer cells.
出处
《中国普通外科杂志》
CAS
CSCD
北大核心
2016年第11期1615-1621,共7页
China Journal of General Surgery
