DNMT3a通过提升基因内部甲基化介导紫杉醇诱导的LINE-1异常表达

DNMT3a mediates paclitaxel-induced abnormal expression of LINE-1 by increasing the intragenic methylation

药物诱导的长散在重复序列LINE-1异常激活可促进细胞基因组不稳定,而基因组不稳定是促进肿瘤发生发展和耐药表型形成的重要因素。因此,探索LINE-1异常激活的分子机制具有重要的理论和临床意义。DNA甲基化是调控基因表达的重要方式,已知DNA甲基转移酶家族成员DNMT3a不仅能通过促进基因启动子甲基化抑制基因表达,还可通过增强基因内部甲基化上调基因表达。本实验室前期研究发现,将乳腺癌细胞暴露于化疗药物可诱导LINE-1异常高表达,但LINE-1启动子甲基化水平并无显著改变。本研究进一步探讨了在化疗药物压力下DNMT3a是否可通过增强LINE-1基因内部甲基化水平促进LINE-1在乳腺癌细胞中的异常高表达。ChIP实验和甲基分析结果显示,用化疗药物紫杉醇(PTX)处理乳腺癌细胞,不仅可以诱导DNMT3a表达,而且可以促进DNMT3a与LINE-1基因内部区域的结合,提升其基因内部甲基化水平,进而上调LINE-1的表达水平。利用表达载体增加细胞内DNMT3a的表达水平,可显著上调LINE-1基因内部的甲基化及基因的表达水平,而下调DNMT3a的表达可有效抑制LINE-1表达。上述研究结果表明,DNMT3a介导的基因非启动子区甲基化在药物诱导的LINE-1异常激活中发挥重要作用,为认识LINE-1在乳腺癌化疗耐药性形成过程中异常激活的机制提供了新思路。

The activation of long interspersed nuclear element-1(LINE-1)leads to genomic instability,which promotes carcinogenesis and drug resistant.Therefore,exploring the mechanism underlying LINE-1 abnormal activation has the theoretical and clinical significance.DNA methylation is an important way to regulate gene expression.DNMT3a,one member of the DNA methyltransferase family,not only inhibits gene expression by inducing promoter hypermethylation,but also activates gene expression by increasing the intragenic DNA methylation.Our previous studies found that the expression of LINE-1 did not increase significantly in the promoter methylation in breast cancer cells treated with paclitaxel(PTX),a first-line chemotherapeutic drug for breast cancer.Here we explored whether DMNMT3a could directly mediate the drug-induced activation of LINE-1 in breast cancer cells through increasing the LINE-1 intragenic methylation.Our ChIP experiments and methyl analysis showed that treatment of breast cancer cells with PTX not only induced DNMT3a expression,but also promoted the binding of DNMT3a to the inner region of the LINE-1 gene to increase its methylation,resulting in upregulation of LINE-1 expression.Using expression vectors or RNA interference to alter the DNMT3a expression levels in the cells significantly changed the intragenic methylation degree and LINE-1 expression.Moreover,down-regulation of DNMT3a expression effectively inhibited the expression of LINE-1.These results indicate that DNMT3a-mediated intragenic methylation plays an important role in drug-induced abnormal activation of LINE-1,which provides a new idea for understanding the mechanism of abnormal activation of Line-1 induced by chemotherapy drug stress in breast cancer cells.