摘要
目的:探讨肾母细胞瘤1相关蛋白(WTAP)对食管鳞状细胞癌(ESCC)细胞生物学特性的影响及分子机制。方法:收集2019年9月至2021年4月期间在川北医学院第二临床医学院手术切除的31对ESCC组织及其配对的癌旁组织,常规培养食管癌细胞KYSE30、KYSE410、KYSE150、KYSE510、TE-1和正常人食管上皮细胞HET-1A,用转染试剂将si-NC、si-WTAP#1和si-WTAP#2核酸转染至KYSE150和KYSE510细胞中,实验分为si-NC、si-WTAP#1和si-WTAP#2组,用qPCR法检测各组细胞中WTAP和MAP3K9 mRNA的表达,CCK-8法、克隆形成实验、划痕愈合实验、Transwell小室实验检测敲减WTAP表达对ESCC细胞增殖、迁移、侵袭能力和凋亡的影响,WB法检测各组ESCC细胞中WTAP、MAP3K9、EMT及MAPK通路相关蛋白的表达,免疫组化法检测ESCC组织中WTAP蛋白的表达,甲基化RNA免疫共沉淀(MeRIP)-qPCR实验检测ESCC细胞MAP3K9 m^(6)A水平,放线菌素D实验检测MAP3K9的mRNA的稳定性,用数据库数据分析WTAP的表达、靶基因、功能富集和互作RNA。结果:WTAP在ESCC组织和细胞中高表达(P<0.05或P<0.01或P<0.001)且与分化程度有关联(P<0.01);在KYSE150和KYSE510细胞中成功地敲减了WTAP mRNA和蛋白的表达(P<0.01或P<0.001);敲减WTAP可明显抑制KYSE150和KYSE510细胞的增殖、迁移和侵袭能力(P<0.05或P<0.01或P<0.001),促进KYSE150和KYSE510细胞凋亡(P<0.05或P<0.01),敲低WTAP后MAP3K9的m^(6)A水平显著下降(P<0.05),其mRNA表达水平及mRNA稳定性均显著降低(P<0.05);数据库数据分析显示,WTAP靶基因富集于MAPK信号通路;敲减WTAP后KYSE150和KYSE510细胞中MAP3K9、p-ERK、N-cadherin和MMP9表达水平显著降低(P<0.05或P<0.01),E-cadherin表达水平显著升高(P<0.05或P<0.01)。结论:WTAP在ESCC组织和细胞中呈高表达且与其分化相关,其通过m^(6)A修饰促进MAP3K9 mRNA稳定性,激活MAPK通路进而促进ESCC细胞的恶性生物学行为。
Objective:To investigate the effects and molecular mechanisms of Wilms tumor 1-associated proteins(WTAP)on the cell biological properties of esophageal squamous cell carcinoma(ESCC)cells.Methods:31 pairs of ESCC tissues and their paired paracancerous tissues that were surgically resected at the Second Clinical Medical College of Chuanbei Medical College between September 2019 and April 2021 were collected.The esophageal cancer cells KYSE30,KYSE410,KYSE150,KYSE510,TE-1,and normal human esophageal epithelial cells HET-1A were routinely cultured.Transfection reagents were used to transfect si-NC,si-WTAP#1 and si-WTAP#2 nucleic acids into KYSE150 and KYSE510 cells.The cells were divided into si-NC,si-WTAP#1 and si-WTAP#2 groups.The expressions of WTAP and MAP3K9 mRNA were detected in the cells of each group by qPCR assay.CCK-8 assay,clone formation assay,and scratch healing assay,Transwell assay were employed to detect the effects of knockdown of WTAP expression on ESCC cell proliferation,migration,invasion and apoptosis.WB assay was used to detect the expressions of WTAP,MAP3K9,EMT and MAPK pathway-related proteins in ESCC cells of each group knocked down of WTAP;immunohistochemistry to detect the expression of WTAP proteins in ESCC tissues,immunoprecipitation of methylated RNA(MeRIP)-qPCR assay to detect the level of MAP3K9 m^(6)A in ESCC cells,actinomycin D assay to detect the stability of mRNA of MAP3K9,and database data to analyze the expression,target genes,functional enrichment,and interacting RNA of WTAP.Results:WTAP was highly expressed in ESCC tissues and cells(P<0.05 or P<0.01 or P<0.001)and correlated with the degree of differentiation(P<0.01);the expression of WTAP mRNA and its protein were successfully knocked down in KYSE150 and KYSE510 cells(P<0.01 or P<0.001);the knockdown of WTAP significantly inhibited the proliferation,migration and invasion of KYSE150 and KYSE510 cells(P<0.05 or P<0.01 or P<0.001),and promoted the apoptosis of KYSE150 and KYSE510 cells(P<0.05 or P<0.01).Knockdown of WTAP resulted in a significant decrease in the m^(6)A level of MAP3K9(P<0.05),and its mRNA expression level and mRNA stability were both significantly reduced(P<0.05).Database data analysis showed that WTAP target genes clustered in the MAPK signaling pathway;the expression levels of MAP3K9,p-ERK,N-cadherin,and MMP9 were significantly reduced(P<0.05 or P<0.01),and the expression level of E-cadherin was significantly elevated(P<0.05 or P<0.01)in the KYSE150 and KYSE510 cells after knockdown of WTAP.Conclusions:WTAP is highly expressed in ESCC tissues and cells and correlates with their differentiation.It promotes the stability of MAP3K9 mRNA through m^(6)A modification,activates the MAPK pathway and thus promotes the malignant biological behaviors of ESCC cells.
作者
潘英杰
孙姗
杨航
谭劲松
陈巧玲
赵全能
杨蜜
刘康
宋桂芹
PAN Yingjie;SUN Shan;YANG Hang;TAN Jinsong;CHEN Qiaoling;ZHAO Quanneng;YANG Mi;LIU Kang;SONG Guiqin(Institute of Basic Medicine and Forensic Medicine,North Sichuan Medical College,Nanchong 637100,Sichuan,China;Institute of Tissue Engineering and Stem Cell Research,the Second Clinical College of North Sichuan Medical College&Beijing Anzhen Nanchong Hospital Affiliated to Capital Medical University&Nanchong Central Hospital,Nanchong 637000,Sichuan,China)
出处
《中国肿瘤生物治疗杂志》
北大核心
2025年第2期140-150,共11页
Chinese Journal of Cancer Biotherapy
基金
国家自然科学基金项目(No.82203851)
四川省科技计划资助项目(No.2023YFSY0045,No.2023NSFSC0731)
四川省卫健委普及应用项目(No.21PJ194)
南充市校合作项目(No.22SXQT0336)
南充市基础研究平台项目(No.23JCYJPT0027,No.22SXQT0096,No.22SXQT0087)。
关键词
食管鳞状细胞癌
肾母细胞瘤1相关蛋白
N^(6)-甲基腺苷
增殖
迁移
侵袭
凋亡
esophageal squamous cell carcinoma(ESCC)
Wilms tumor 1-associated protein(WTAP)
N^(6)-methyladenosine(m6A)
proliferation
migration
invasion
apoptosis
