摘要
目的:研究甲基转移酶样3(METTL3)介导的m6A修饰调控双同源盒A假基因8(DUXAP8)对涎腺腺样囊性癌细胞SACC-LM的增殖、迁移和侵袭能力的影响及其潜在的分子机制.方法:通过肿瘤组织和癌旁组织全转录组测序筛选出差异基因DUXAP8并进行qRT-PCR验证;采用m6A修饰位点预测网站SRAMP预测DUXAP8上的m6A修饰位点;qRT-PCR、Western blot检测m6A修饰和上皮-间质转化(EMT)相关基因mRNA及蛋白水平.干扰或过表达METTL3和DUXAP8,通过CCK-8、划痕、侵袭实验检测各组细胞的增殖、迁移和侵袭能力;结合MeRIP-qPCR检测METTL3和DUXAP8的相关性.结果:DUXAP8在SACC肿瘤组织的表达显著高于癌旁组织(P＜0.05);干扰DUXAP8可以显著抑制SACC-LM细胞的增殖、迁移和侵袭能力以及EMT相关基因表达水平(P＜0.05);DUXAP8上存在多个可信度较高的m6A修饰位点;METTL3在肿瘤组织高表达且较其他相关基因差异最为显著(P＜0.05);METTL3作为甲基转移酶调控DUXAP8的表达;下调METTL3可以显著抑制SACC-LM细胞的增殖、迁移和侵袭能力,并可部分逆转DUXAP8过表达对这些能力的促进作用(P＜0.05).结论:METTL3介导的m6A修饰上调DUXAP8的表达,从而促进了 SACC细胞的增殖、迁移和侵袭能力.
Abstract
Objective:To investigate the effects of methyltransferases like 3(METTL3)mediated m6A modification of double homology cassette A pseudogene8(DUXAP8)on the proliferation,migration and invasion of salivary adenoid cystic carcinoma SACC-LM cells and its potential molecular mechanisms.Methods:Whole-transcriptome sequencing showed that DUXAP8 was highly ex-pressed in SACC than in para-cancerous tissues(P＜0.05).The m6A modification sites on DUXAP8 were predicted using the SRAMP website,and the mRNA and protein expression of m6A-modified genes and the genes associated with the epithelial-mesen-chymal transition(EMT)was measured by qRT-PCR and Western blot,respectively.METTL3 and DUXAP8 was knocked down or overexpressed in SACC-LM cells,and the proliferation,migration,and invasion of the cells were assessed by CCK-8,scratch and Transwell assays.The correlation between METTL3 and DUXAP8 was evaluated using MeRIP-qPCR.Results:The expression of DUXAP8 in SACC tumor was higher than that in para-cancerous tissues(P＜0.05).Knockdown of DUXAP8 reduced proliferation,migration and invasion of SACC-LM cells,as well as the expression of EMT-related genes(P＜0.05).Multiple m6A modification sites of high confidence were found on DUXAP8.METTL3 was highly expressed in tumor tissues,more than other related genes(P＜0.05)and enzyme-encoding genes in SACC-LM cells(P＜0.05).METTL3 was found to function as a methyltransferase to regulate the expression of DUX-AP8,and downregulation of METTL3 inhibited prolifera-tion,migration and invasion of SACC-LM cells and partially reversed the promotion of these activities induced by DUX-AP8 overexpression(P＜0.05).Conclusion:METTL3-me-diated m6A modification upregulated DUXAP8 expression,which promotes the proliferation,migration and invasion of SACC cells.
基金项目
国家自然科学基金(82173165)
国家自然科学基金青年基金(82002867)
国家口腔疾病临床医学研究中心专项(LCB202008)
NETL
NSTL
万方数据