喉癌N6-甲基腺嘌呤甲基化修饰差异基因的初步筛查与验证  

The initial screening and verifying of differential genes related to m^(6)A methylation modification in laryngeal carcinoma

作　　者：赵学辉 李秋影[1] 阚轩[1] 王婧婷[1] 孙亚男[1] ZHAO Xuehui;LI Qiuying;KAN Xuan;WANG Jingting;SUN Ya'nan(Department of Otolaryngology Head and Neck Surgery,the Second Affiliated Hospital of Harbin Medical University,Harbin,Heilongjiang,150080,China)

机构地区：[1]哈尔滨医科大学附属第二医院耳鼻咽喉头颈外科,黑龙江哈尔滨150080

出　　处：《中国耳鼻咽喉头颈外科》2022年第4期239-242,共4页Chinese Archives of Otolaryngology-Head and Neck Surgery

基　　金：国家自然科学基金面上项目(81772874),国家自然科学基金面上项(81272965);黑龙江省自然科学基金(LH2020H055);黑龙江省博士后基金(LBH-Z18182);哈尔滨医科大学附属第二医院中青年创新科学研究基金(KYCX2018-06)。

摘　　要：目的 分析喉癌中N^(6)-甲基腺嘌呤(N^(6)-methyladenosine,m^(6)A)甲基化修饰差异基因表达水平,探讨其在喉癌进展中的作用。方法 采用m^(6)A-mRNA表观转录组芯片检测3对喉癌组织及相应癌旁组织中m^(6)A甲基化修饰差异表达基因,并选择显著差异表达的含有6个跨膜BAX抑制子基序(transmembrane BAX inhibitor motif containing 6,TMBIM6)基因行逆转录实时定量PCR(RT-qPCR)、甲基化RNA免疫沉淀定量PCR(methylated RNA immunoprecipitation-qPCR,MeRIP-qPCR)及免疫组织化验证,最后检测其甲基化位点。结果 在差异表达基因和m^(6)A甲基化基因的联合分析中,共获得135个高甲基化高表达基因,2958个高甲基化低表达基因,129个低甲基化高表达基因,682个低甲基化低表达基因。对显著高表达的TMBIM6基因行RT-qPCR、MeRIP-qPCR和免疫组化验证结果与基因芯片一致,鉴定出TMBIM6基因甲基化位点位于3’非编码区的2222碱基位。结论 TMBIM6基因通过m^(6)A修饰机制在喉癌中发挥癌基因的作用,可望成为喉癌的分子标记物及潜在的治疗靶点。OBJECTIVE To analyze the expression level of N^(6)-methyladenosine(m^(6)A) methylated differential genes in laryngeal cancer and explore their roles in the progression of laryngeal cancer.METHODS Three pairs of m^(6)A methylated differentially expressed genes in laryngeal cancer tissues and corresponding adjacent tissues were detected by m^(6)A-mRNA epigenome microarray.With significantly differentially expressed genes were detected,the transmembrane BAX inhibitor motif containing 6(TMBIM6) gene were selected for the reverse-transcription quantitative real-time PCR(RT-qPCR),the methylated RNA immunoprecipitation-qPCR(MeRIP-qPCR) and the immunohistochemistry(IHC) verification.RESULTSIn the conjoint analysis of differentially expressed genes and m6A methylated genes,135 high methylation-high expression genes(Hyper-Up),2958 high methylation-low expression genes(Hyper-Down),129 low methylation-high expression genes(Hypo-Up) and 682 low methylation-low expression genes(Hypo-Down) were obtained.RT-qPCR,MeRIP-qPCR and IHC verification results of TMBIM6 gene with significantly high expression were consistent with the gene chip,and the methylation site of TMBIM6 gene was identified at the 2222 base of 3’Untranslated regions(3’UTR).CONCLUSION The TMBIM6 gene plays an oncogene role in laryngeal cancer through m6A modification mechanism,which is expected to be a molecular marker and a potential therapeutic target for laryngeal cancer.

关 键 词：喉肿瘤 逆转录聚合酶链反应 免疫组织化学 m^(6)A甲基化修饰 差异基因 

分 类 号：R739.65[医药卫生—肿瘤]