SOX7基因启动子甲基化水平对乳腺癌MDA-MB-231细胞株体外迁移和侵袭的影响

Effect of SOX7 Promoter Methylation Level on Migration and Invasion of Breast Cancer MDA-MB-231 Cells Line in Vitro

目的:探讨乳腺癌MDA-MB-231细胞中,Y性别决定区基因7(SOX7)基因启动子甲基化水平对细胞的体外迁移和侵袭的影响。方法:脂质体转染pcDNA3.0-DNA甲基转移酶3a(DNMT3a)质粒至MDA-MB-231细胞中,并于24h、48h及72h后,采用蛋白质免疫印迹实验(WB)检测细胞内DNMT3a蛋白表达水平;甲基化特异性定量PCR(Q-MSP)检测DNMT3a处理组、5-aza-C处理组及对照(Control)组MDA-MB-231细胞中的SOX7基因启动子DNA甲基化水平;实时荧光定量PCR(qRT-PCR)及WB实验检测各组MDA-MB-231细胞中的SOX7 m RNA和蛋白表达水平;细胞划痕实验及细胞侵袭实验检测各组MDA-MB-231细胞的迁移和侵袭能力。结果:pcDNA3.0-DNMT3a质粒转染MDA-MB-231细胞24h时,细胞内的DNMT3a蛋白表达水平最高。DNMT3a能够显著提高SOX7基因启动子DNA甲基化水平,而5-aza-C则抑制了SOX7基因启动子DNA甲基化水平(P<0.05)。与Control组相比,DNMT3a处理组的MDA-MB-231细胞中,SOX7的m RNA及蛋白表达水平均明显下降,而5-aza-C处理组SOX7的m RNA及蛋白表达水平均明显增加(P<0.05)。与Control组相比,DNMT3a处理组的MDA-MB-231细胞的迁移和侵袭能力均显著增强(P<0.05),而5-aza-C处理组的MDA-MB-231细胞的迁移和侵袭能力变化不大(P>0.05)。结论:在恶性肿瘤中,SOX7低表达表受其基因启动子高甲基化调节,且乳腺癌MDA-MB-231细胞中低表达的SOX7能够影响细胞的外迁移和侵袭能力。

Objective: To investigate the effect of promoter methylation level of sex determining region Y-box 7(SOX7) gene on cells migration and invasion in vitro in breast cancer MDA-MB-231 cells. Methods: Liposome transfection of pcDNA3.0-DNA methylation transferase 3a(DNMT3a) plasmid into MDA-MB-231 cells, after 24 h, 48 h and 72 h, the expression of DNMT3a protein in cells was detected by Western blotting(WB). DNA methylation level of SOX7 gene promoter in MDA-MB-231 cells in DNMT3a treatment group, 5-aza-C treatment group and control groups were detected by quantitative methylation specific PCR(Q-MSP). The expression of SOX7 in MDA-MB-231 cells were detected by real-time quantitative PCR(qRT-PCR) and WB assay. Detection of migration and invasion energy of MDA-MB-231 cells by cell scratch test and cell invasion test in each groups. Results: The highest expression level of DNMT3a protein was found in MDA-MB-231 cells transfected with pcDNA3.0-DNMT3 a plasmid for 24 hours.DNMT3a significantly increased the DNA methylation level of SOX7 promoter, while 5-aza-C inhibited the DNA methylation level of SOX7 promoter(P<0.05). Compared with the control group, the expression of SOX7 in MDA-MB-231 cells treated with DNMT3a decreased significantly, while the expression of SOX7 in 5-aza-C group increased significantly(P<0.05). Compared with the control group, the migration and invasion ability of MDA-MB-231 cells treated with DNMT3a were significantly enhanced(P<0.05), while the migration and invasion ability of MDA-MB-231 cells treated with 5-aza-C did not change significantly(P>0.05). Conclusion: In malignant tumors, the low expression of SOX7 is regulated by the hypermethylation of SOX7 promoter, and the silencing of SOX7 significantly influence the migration and invasion of breast cancer MDA-MB-231 cells line in vitro.