PRMT5调控m6A修饰介导三阴性乳腺癌细胞对多柔比星敏感性的机制研究

Mechanism research of PRMT5 regulating m6A modification to mediate sensitivity of triple-negative breast cancer cells to doxorubicin

目的探讨蛋白精氨酸甲基转移酶5(PRMT5)通过调控RNA m6A修饰降低三阴性乳腺癌细胞对多柔比星(DOX)敏感性的分子机制。方法利用TCGA数据库分析PRMT5在乳腺癌组织中的表达水平及其与患者预后的相关性;构建稳定过表达和敲降PRMT5的人乳腺癌MDA-MB-231细胞株;利用CCK-8和克隆形成实验,分析PRMT5对乳腺癌细胞DOX敏感性的影响;通过qRT-PCR和Western blotting等方法,检测在DOX作用下PRMT5对MDA-MB-231细胞m6A相关分子表达水平的影响;利用MeRIP-seq测序分析PRMT5调控DOX作用下m6A修饰显著差异的靶基因,并通过qRT-PCR检测靶基因的表达水平以及RNA稳定性。结果TCGA数据库分析结果显示,PRMT5在包括乳腺癌在内的多种肿瘤中高表达(P<0.05),并且高表达PRMT5预示不良预后;过表达PRMT5能够降低MDA-MB-231细胞对DOX的敏感性(P<0.01),敲降或者采用PRMT5抑制剂JNJ能显著增加MDA-MB-231细胞对DOX的敏感性(P<0.01,P<0.05)。PRMT5过表达可以显著抑制DOX所致的细胞m6A水平增加(P<0.01),特别是可以降低DNA损伤修复相关分子ERCC4、REV3L的RNA甲基化水平,提高这些分子的RNA稳定性。结论PRMT5通过降低MDA-MB-231细胞DNA损伤修复相关分子的RNA甲基化水平,提高其稳定性,进而导致细胞对DOX敏感性降低。

Objective To explore the molecular mechanism of protein arginine methyltransferases 5(PRMT5)reducing the sensitivity of triple-negative breast cancer cells to doxorubicin(DOX)by regulating RNA m6A modification.Methods The TCGA database was used to analyze the expression level of PRMT5 in breast cancer tissues and its correlation with prognosis of patients.Human breast cancer MDA-MB-231 cell lines with stable overexpression and knockdown of PRMT5 were constructed.The effect of PRMT5 on DOX sensitivity of breast cancer cells was analyzed by CCK-8 and colony formation assay.The effect of PRMT5 on the expression level of m6A-related molecules in MDA-MB-231 cells under DOX was detected by qRT-PCR and Western blotting.MeRIP-seq was used to analyze the target genes with significant differences in m6A modification under the regulation of DOX by PRMT5,and the expression level and RNA stability of the target genes were detected through qRT-PCR.Results The results of TCGA database analysis showed that PRMT5 was highly expressed in breast cancer and other various tumors(P<0.05),and high expression of PRMT5 predicted poor prognosis.Overexpression of PRMT5 reduced the sensitivity of MDA-MB-231 cells to DOX(P<0.01),while knockdown of PRMT5 or using PRMT5 inhibitor JNJ significantly increased it(P<0.01,P<0.05).Overexpression of PRMT5 notably inhibited the increase of m6A levels in cells induced by DOX(P<0.01),particularly reduced the RNA methylation levels of molecules associated with DNA damage,such as ERCC4 and REV3L,and promoted RNA stability of these molecules.Conclusion PRMT5 reduces RNA methylation level of molecules related to DNA damage repair in MDA-MB-231 cells to increase its stability,thereby reducing the cell sensitivity to DOX.