摘要： 目的 通过DNA甲基转移酶抑制剂5-氮杂-2’-脱氧胞苷（5-Aza-CdR）与siRNA干扰改变乳腺癌细胞MCF-7中DNA甲基化水平，探讨DNA甲基转移酶3b（Dnmt3b）表达对乳腺癌细胞相关mRNA、微小RNA（miRNA）的影响。方法 通过5-Aza-CdR抑制乳腺癌细胞整体甲基化水平，免疫荧光染色检测其抑制效果，高通量测序检测乳腺癌细胞miRNA的表达，荧光定量PCR（qRT-PCR）检测乳腺癌细胞miRNA及乳腺癌相关基因的表达；通过siRNA干扰抑制乳腺癌细胞中Dnmt3b的表达，qRT-PCR和Western blotting检测siRNA干扰效率，细胞生长曲线反映细胞生长情况。结果 5-Aza-CdR降低细胞整体甲基化水平，siRNA干扰后Dnmt3b的表达下降，细胞生长显著抑制；高通量测序显示5-Aza-CdR作用后67个miRNA表达有统计学差异（均P<0.05）；qRT-PCR结果显示5-Aza-CdR作用后乳腺癌细胞中miR-200a、miR-411、miR-382、miR-409、miR-493、miR-127、miR-520a、miR-654表达显著上调（P<0.05）；siRNA干扰Dnmt3b后乳腺癌细胞中miR-29b、miR-506、miR-200a、miR-203b、miR-409、miR-520a、miR-654的表达显著上调（P<0.05），而miR-200b、miR-127的表达显著下调（均P<0.05）；5-Aza-CdR作用后乳腺癌细胞WWOX、MMP7的表达下调，TCF21、TIMP1的表达上调（均P<0.05）；siRNA干扰Dnmt3b后乳腺癌细胞抑癌基因BRCA1、BRCA2及癌基因MMP7、survivin的表达均下调（均P<0.05）。结论 Dnmt3b可影响乳腺癌细胞相关基因WWOX、TCF21、BRCA1、BRCA2、MMP7、TIMP1、PKM2、survivin及miR-29b、miR-506、miR-200a、miR-200b、miR-203b、miR-409、miR-127、miR-520a、miR-654的表达。

DNA甲基转移酶3b对乳腺癌细胞微小RNA表达的影响

王奕丹, 王一涵, 黄钰雯, 阴迪, 于运, 刘丽梅

北华大学医学技术学院实验中心, 吉林 吉林 132013

收稿日期:2020-04-30出版日期:2021-02-28发布日期:2021-01-21
通讯作者:刘丽梅E-mail:liulm74@163.com
作者简介:王奕丹(1997-),女,硕士研究生.
基金资助:国家自然科学基金（81201354）；吉林省教育厅科研项目（2017-196）


关键词: DNA甲基转移酶3b, 乳腺癌, 微小RNA
Abstract: Objective 5-Aza-2'-deoxycytidine (5-Aza-CdR),a methyltransferase inhibitor,and siRNA interference were used to explore the effect of DNA methyltransferase 3b (Dnmt3b) expression to study mRNA and microRNA (miRNA) expression landscape in breast cancer. Methods 5-Aza-CdR was used to inhibit methylation of MCF-7 breast cancer cells,and its effects were evaluated using immunofluorescence staining. High-throughput sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to detect and assess the expression of miRNA and breast cancer-related genes,respectively. siRNA interference was used to inhibit Dnmt3b expression in breast cancer cells,and its impact on cell growth and miRNA and breast cancer-related gene expression were evaluated. Results Addition of 5-Aza-CdR reduced the methylation levels of the cells,as well as the expression of Dnmt3b. Cell growth was significantly inhibited by siRNA interference. High-throughput sequencing revealed 67 miRNAs whose expression levels were significantly altered after 5-Aza-CdR treatment (P<0.05). Particularly,expression levels of miR-200a,miR-411,miR-382,miR-409,miR-493,miR-127,miR-520a,and miR-654 were significantly upregulated in breast cancer cells (all P<0.05). Moreover,expression levels of miR-29b,miR-506,miR-200a,miR-203b,miR-409,miR-520a,and miR-654 were significantly upregulated upon Dnmt3b knockdown,whereas those of miR-200b and miR-127 were significantly downregulated (all P<0.05). WWOX and MMP7 levels were downregulated in breast cancer cells in the presence of 5-Aza-CdR,whereas those of TCF21 and TIMP1 were upregulated (all P<0.05). Furthermore,Dnmt3b knockdown led to the expression of the BRCA1 and BRCA2 tumor suppressors,and the downregulation of MMP7 and surviving oncogenes. Conclusion Dnmt3b affects the expression of breast cancer cell-related genes WWOX,TCF21,BRCA1,BRCA2,MMP7,TIMP1,PKM2,survivin,and miR-29b,miR-506,miR-200a,miR-200b,miR-203b,miR-409,miR-127,miR-520a,and miR-654.
Key words: DNA methyltransferase 3b, breast cancer, microRNA
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