二氢杨梅素通过抑制甲基转移酶诱导人乳腺癌MCF-7细胞PTEN基因去甲基化  

Dihydromyricetin induces PTEN demethylation by down-regulating DNA methyltransferases in human MCF-7 breast cancer cells

作　　者：白倩[1] 谢琦[1] 彭晓莉[1] 常徽[1] 朱俊东[1] 糜漫天[1] 

机构地区：[1]第三军医大学军事预防医学院营养与食品安全研究中心,重庆市营养与食品安全重点实验室,重庆400038

出　　处：《第三军医大学学报》2014年第1期20-24,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金(81372974)~~

摘　　要：目的观察二氢杨梅素对人乳腺癌MCF-7细胞中第10号染色体缺失的磷酸酶和张力蛋白同源基因(phosphatase and tensin homology deleted on chromosome ten,PTEN)甲基化及其表达的影响,并探讨相关作用机制。方法以CCK-8法检测细胞活力,qRT-PCR检测PTEN和DNA甲基转移酶(DNA methyltransferase,DNMT)DNMT1、DNMT3a、DNMT3b的mRNA表达,Western blot法检测PTEN的蛋白表达,荧光法检测细胞DNA甲基转移酶总活性,甲基化特异性PCR法(methylation-specific PCR,MSP)检测PTEN基因甲基化水平。结果细胞活力检测结果表明,二氢杨梅素可剂量依赖性降低乳腺癌MCF-7细胞活力(P<0.05);qRT-PCR和Western blot检测结果表明,二氢杨梅素可显著上调PTEN的表达(P<0.05),并呈现剂量-效应关系;MSP检测结果显示,二氢杨梅素可显著诱导MCF-7细胞PTEN基因去甲基化;qRT-PCR和荧光法检测结果显示,二氢杨梅素可显著降低MCF-7细胞DNMT1表达和DNMT活性(P<0.05)。结论二氢杨梅素显著诱导乳腺癌细胞PTEN基因去甲基化,促进其表达,其机制可能主要涉及对DNMT1表达和活性的抑制。Objective To determine the effects of dihydromyricetin on methylation status and expression of phosphatase and tensin homology deleted on chromosome ten（ PTEN） gene in human MCF-7 breast cancer cells and explore the relevant mechanisms. Methods Cells viability was assessed by CCK-8 assay in MCF-7 cells after the treatment of dihydromyricetin at 10,20,40 and 80 μmol / L for 48 h. The expression of DNA methyltransferases（ DNMT,including DNMT1,DNMT3a,and DNMT3b） and PTEN was measured by quantitative real-time PCR and Western blotting. DNMT activity was detected by DNMT activity / inhibition assay kit（ fluorometrics）. Modified methylation specific PCR（ MSP） was used to screen the methyla-tion level of PTEN. Results Dihydromyricetin significantly decreased the viability of MCF-7 cells in a dose-dependent manner. The qRT-PCR analysis and Western blotting showed that dihydromyricetin dose-dependently increased the expression of PTEN in MCF-7 cells. MSP showed that dihydromyricetin markedly induced PTEN demethylation in MCF-7 cells. Further studies indicated that dihydromyricetin decreased the expression of DNMT1 and inhibited the activity of DNMT. Conclusion Dihydromyricetin significantly induces PTEN demethylation and promotes its expression in breast cancer MCF-7 cells,and this effect may be mainly correlated with inhibition of DNMT1 expression and activity.

关 键 词：二氢杨梅素 乳腺癌细胞 PTEN 去甲基化 DNA甲基转移酶 

分 类 号：R282.71[医药卫生—中药学] R73-361[医药卫生—中医学]