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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 role of miR-204 in the regulation of mitochondrial transcription factor A (TFAM) in breast cancer cells and its relationship with cell growth and proliferation. Methods: The human breast cancer MDA-MB-231 cells were transfected with mi R-204 mimics or miR-204 inhibitors respectively. Real-time PCR and Western blot were used to detect the expression of mi R-204 and TFAM protein respectively. The enzyme reporter gene plasmid (mut-TFAM / wt-TFAM) was co-transfected into MDA-MB-231 cells with mi R-204 mimics or miR-204 inhibitor. / TFAM plasmid. The expression of TFAM protein was detected by cotransfection of MDA-MB-231 cells alone or with mi R-204 mimics. The cell growth and proliferation were detected by MTT assay and BrdU assay. Results: The expression of mi R-204 was significantly increased in MDA-MB-231 cells transfected with mi R-204 mimics, while the expression of TFAM protein was significantly decreased. The expression of miR-204 was reversed <0.05). The luciferase activity of co-transfection with wt-TFAM and mi R-204 mock was significantly decreased, and the luciferase activity was significantly increased when co-transfected with mi R-204 inhibitor (all P <0.05). After transfected with pcDNA3.1 / TFAM, the expression of TFAM m RNA and protein in MDA-MB-231 cells was significantly up-regulated and the cell growth and proliferation were significantly increased (all P <0.05). After mi R-204 mimics, MDA-MB-231 cells were significantly decreased in the expression of TFAM, while the cell growth and proliferation were significantly decreased. However, the effects of pcDNA3.1 / TFAM were partially offset (all P <0.05). Conclusion: mi R-204 can inhibit the expression of TFAM in breast cancer cells and thus inhibit the growth and proliferation of breast cancer cells.