摘要
目的:探究不同乳腺癌细胞(MCF-7、MDA-MB-231)中miR-21与DNA甲基化相互调节作用。方法:将荧光标记的miR-21抑制剂及阴性对照瞬时转入MCF-7、MDA-MB-231细胞中,用荧光显微镜观察其转染效率,以Real-time PCR检测miR-21的敲低水平,并以bisulfite-q MSP法检测基因组DNA甲基化水平。同时,以2.5μmol/L DNA甲基化酶抑制剂5-AZA处理细胞72 h,以单纯二甲基亚枫(DMSO)处理做为阴性对照,观察DNA甲基化改变对miR-21表达水平的影响,接着以Western blot检测miR-21下游基因人第10号染色体缺失的磷酸酶及张力蛋白同源基因(PTEN)、蛋白激酶B(AKT)蛋白的表达水平。结果:miR-21抑制剂可敲低MCF-7细胞中miR-21的表达水平(P<0.01),并引起基因组DNA甲基化水平的显著升高以及DNA甲基化转移酶Dnmt1、Dnmt3a以及Dnmt3b的普遍升高(P<0.05,P<0.01)。而在MDA-MB-231细胞中瞬转miR-21抑制剂则引起miR-21表达水平的小幅度升高(P<0.01)以及整体DNA甲基化水平的降低(P<0.05),并伴随有Dnmt3a的升高及Dnmt3b的降低。使用5-AZA处理后发现,其可显著上调MCF-7以及MDA-MB-231细胞中miR-21的表达(P<0.01),并引起其下游基因PTEN在MCF-7细胞内的表达升高,进而下调AKT的蛋白水平。结论:瞬转miR-21抑制剂对MCF-7与MDA-MB-231细胞DNA甲基化水平的调节截然相反,而DNA甲基化的降低则可使miR-21的表达一致上调。本研究可为以后不同类型乳腺癌的临床治疗提供一定的实验依据。
Objective： To determine the interaction between miR-21 and DNA methylation in different breast cancer cells. Methods： Fluorescence tagged miR-21 inhibitor and its negative control （NC） were transient transfected into MCF-7 and MDA-MB-231 cell, the transfec- tion eMciency was observed using fluorescence microscopy, and the miR-21 expression level and genome DNA methylation status before and af- ter transfection were assessed by real-time PCR and bisulfite-qMSP respectively. To investigate the regulation effect of DNA methylation on miR-21, cells were treated with 5-AZA （2.5 μmol/L） for 72 h, with dimethyl sulfoxide （DMSO） treatment as its negative control （NC）, and the expression level of phosphatase and tensin homolog deleted on chromosome ten （PTEN） and AKT（also known as Protein Kinase B）, two downstream genes of miR-21 were detected by Western blot. Results： The expression of miR-21 in MCF-7 cell was significantly knocked down （P 〈 0.01） by miR-21 inhibitor, with the genome DNA methylation level （P 〈 0.05） and all the three Dnmts： Dnmtl, Dnmt3a, and Dn- mt3b unregulated. In contrast, the miR-21 expression in MDA-MB-231 cell was elevated （ P 〈 0.01） by miR-21 ilfihitor, meanwhile, down- regulated of genome DNA methylation （P 〈 0.05） and D nmt3b expression, upregulation of Dnmt3a were also observed. In addition, treated with 5-AZA resulted in significant increases of miR-21 expression in both MCF-7 and MDA-MB-231 cells （P 〈 0.01）, with the protein level of PTEN increased in MCF-7 cell, which was further involved in the dowuregulation of AKT. Conclusion： The regulation effects of DNA methylation by transient transfection of miR-21 in MCF-7 and MDA-MB-231 cells are almost opposite, whilst the expression of miR-21 in two cell lines were all upregulated by decreased DNA methylation level, and our results may provide some experimental evidences for the future de- velopment of rational therapy for different breast cancer.
出处
《中国应用生理学杂志》
CAS
CSCD
2015年第3期220-224,共5页
Chinese Journal of Applied Physiology
基金
天津市科委项目(13JCYBJC21600)
天津市教委项目(20130601)
天津医科大学项目(2012KYM10)
