地西他滨通过DNA甲基化调控乳腺癌细胞中miR-1301-3p的表达及机制  

Decitabine regulates the expression and mechanism of miR-1301-3p in breast cancer cells through DNA methylation

作　　者：李元 路玉祥 楚元奎[1] 杨华[1] LI Yuan;LU Yuxiang;CHU Yuankui;YANG Hua(School of Laboratory Medicine,Ningxia Medical University,Ningxia Yinchuan 750004,China)

机构地区：[1]宁夏医科大学检验学院,宁夏银川750004

出　　处：《中国医院药学杂志》2024年第24期2839-2844,共6页Chinese Journal of Hospital Pharmacy

基　　金：宁夏自然科学基金项目(编号:2022AAC03135)。

摘　　要：目的:通过分析地西他滨(decitabine,DAC)诱导miR-1301-3p的DNA甲基化状态,探讨miR-1301-3p在乳腺癌中表达降低的调控机制。方法:生物信息学软件预测miR-1301-3p启动子区甲基化岛,甲基化特异性PCR(methylation-specific PCR,MSP)检测miR-1301-3p启动子区的甲基化水平。应用MSP和实时荧光定量PCR(qRT-PCR)分别检测DAC对miR-1301-3p启动子甲基化和表达的影响。在乳腺癌细胞中转染sh-DNMT3B,qRT-PCR检测DNMT3B对miR-1301-3p表达的影响。MTT实验检测sh-DNMT3B及miR-1301-3p inhibitor对乳腺癌细胞增殖的影响。结果:miR-1301-3p启动子区存在甲基化岛,且乳腺癌细胞中miR-1301-3p启动子区的甲基化水平高于正常乳腺上皮细胞。DAC处理乳腺癌细胞MDA-MB-231和MCF748 h后,细胞中miR-1301-3p启动子区的甲基化产物均明显减少,miR-1301-3p基因表达水平显著增加;而沉默DNMT3B可促进miR-1301-3p表达,并抑制乳腺癌细胞MDA-MB-231和MCF7增殖。共转染miR-1301-3p inhibitor和shDNMT3B,可逆转sh-DNMT3B对乳腺癌细胞增殖的抑制作用。结论:miR-1301-3p位点高甲基化导致乳腺癌细胞中miR-1301-3p表达降低,DAC可能通过抑制DNMT3B的表达降低miR-1301-3p启动子区的甲基化水平,激活其表达,从而抑制乳腺癌细胞增殖。OBJECTIVE To explore the DNA methylation status of miR-1301-3p induced by decitabine(DAC)to elucidate the regulatory mechanism of lowered expression of miR-1301-3p in breast cancer.METHODS Bioinformatic software was utilized for predicting the methylation island of miR-1301-3p promoter region.And methylation-specific polymerase chain reaction(PCR)(MSP)was employed for detecting the methylation level of miR-1301-3p promoter region.The effects of DAC on the methylation and expression of miR-1301-3p promoter were detected by MSP and quantitative real-time PCR(qRT-PCR).shDNMT3B was transfected into breast cancer cells.The effect of DNMT3B on the expression of miR-1301-3p was detected by qRT-PCR.The effects of sh-DNMT3B and miR-1301-3p inhibitor on the proliferation of breast cancer cells were detected by methylthiazolyldiphenyl tetrazolium(MTT)assay.RESULTS There were methylation islands in promoter region of miR-1301-3p.The methylation level of promoter region of miR-1301-3p in breast cancer cells was higher than that in normal breast epithelial cells.After DAC treatment of MDA-MB-231 and MCF7 for 48 h,methylation level in promoter region of miR-1301-3p declined markedly in breast cancer cells and the expression of miR-1301-3p gene was significantly up-regulated.Silencing DNMT3B could promote the expression of miR-1301-3p and arrest the proliferation of breast cancer cells.Co-transfection of miR-1301-3p inhibitor and sh-DNMT3B reversed the inhibitory effect of sh-DNMT3B on the proliferation of breast cancer cells.CONCLUSION The hypermethylation of miR-1301-3p site leads to a down-regulation of miR-1301-3p in breast cancer cells.DAC may lower the methylation level of miR-1301-3p promoter region and activate its expression by blunting the expression of DNMT3B,thus suppressing the proliferation of breast cancer cells.

关 键 词：miR-1301-3p 乳腺癌 地西他滨 DNMT3B 

分 类 号：R979.1[医药卫生—药品]