ALKBH5对食管鳞状细胞癌恶性生物学行为的影响及相关机制研究  

Effects of ALKBH5 on the malignant biological behavior of esophageal squamous cell carcinoma and the related mechanism

作　　者：马培晗 张灵敏[2] 李茜 路宁 温华 张明鑫 Ma Peihan;Zhang Lingmin;Li Qian;Lu Ning;Wen Hua;Zhang Mingxin(Department of Anesthesiology,First Affiliated Hospital of Xi'an Medical University,Xi'an 710077,China;Department of Anesthesiology,First Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710061,China;Department of Gastroenterology,First Affiliated Hospital of Xi'an Medical University,Xi'an 710077,China)

机构地区：[1]西安医学院第一附属医院麻醉科,西安710077 [2]西安交通大学第一附属医院麻醉科,西安710061 [3]西安医学院第一附属医院消化内科,西安710077

出　　处：《国际肿瘤学杂志》2025年第2期79-88,共10页Journal of International Oncology

基　　金：陕西省重点研发计划(2022JM-502);浙江省消化系肿瘤微创诊治与快速康复研究重点实验室开放课题资助项目(21SZDSYS16);西安医学院第一附属医院配套基金(XYFYPT-2023-04);西安医学院创新团队(2021TD15)。

摘　　要：目的研究m^(6)A去甲基化酶ALKBH5在食管鳞状细胞癌(ESCC)细胞中的作用及潜在机制。方法通过实时荧光定量PCR及蛋白质印迹法检测ALKBH5在正常食管黏膜上皮细胞(Het-1A)和ESCC细胞株(Eca109、KYSE30、KYSE150、KYSE410)中的表达;构建ALKBH5过表达/沉默的瞬转细胞株(siRNA转染后分为si-ALKBH5-1组、si-ALKBH5-2组)及对照细胞株;分别采用MTT法、细胞划痕实验、细胞凋亡实验研究ALKBH5对ESCC细胞增殖、迁移和凋亡的影响;通过转录组测序(RNA-seq)及甲基化RNA免疫共沉淀测序(MeRIP-seq)技术测序交集筛选出差异表达基因并采用RT-qPCR检测ALKBH5对该基因表达的影响。结果实时荧光定量PCR结果显示,ALKBH5 RNA在Het-1A、Eca109、KYSE30、KYSE150及KYSE410细胞株中的相对表达量分别为1.03±0.28、0.46±0.02、0.23±0.10、0.04±0.02、0.05±0.00,差异有统计学意义(F=444.60,P<0.001);蛋白质印迹法显示,ALKBH5蛋白在Het-1A、Eca109、KYSE30、KYSE150及KYSE410细胞株中的相对表达量分别为1.14±0.03、0.88±0.04、0.66±0.01、0.69±0.01、0.95±0.01,差异有统计学意义(F=139.90,P<0.001);MTT实验显示,KYSE30细胞对照组与ALKBH5过表达组72 h的吸光度(A)值分别为0.86±0.01、1.25±0.01,差异有统计学意义(t=46.93,P<0.001);KYSE150细胞对照组与ALKBH5过表达组72 h的A值分别为1.00±0.03、1.43±0.02,差异有统计学意义(t=16.80,P<0.001);KYSE30细胞对照组与si-ALKBH5-1、si-ALKBH5-2组96 h的A值分别为0.98±0.01、0.85±0.02、0.80±0.09,差异有统计学意义(F=72.97,P<0.001),KYSE30细胞对照组的A值均大于si-ALKBH5-1、si-ALKBH5-2组(均P<0.001);KYSE410细胞对照组与si-ALKBH5-1、si-ALKBH5-2组72 h的A值分别为1.28±0.02、1.15±0.02、1.08±0.05,差异有统计学意义(F=16.97,P=0.003),KYSE410细胞对照组A值均大于si-ALKBH5-1、si-ALKBH5-2组(P=0.020;P=0.003)。细胞划痕实验显示,划痕后48 h,KYSE30细胞对照组与过表达ALKBH5组的迁移率分别为(27.39±0.54)%、(48.89±5.12)%,Objective To investigate the role and potential mechanism of m^(6)A demethylase ALKBH5 in esophageal squamous cell carcinoma(ESCC).Methods Real time fluorogenic quantitative PCR and Western blotting were used to detect ALKBH5 expression in normal esophageal epithelial cells(Het-1A)and ESCC cell lines(Eca109,KYSE30,KYSE150,KYSE410).Transient cell lines with overexpression/knockdown of ALKBH5(siRNA transfection was divided into si-ALKBH5-1 group and si-ALKBH5-2 group)and control cell lines were constructed.The effects of ALKBH5 on ESCC cell proliferation,migration and apoptosis were studied by MTT assay,cell scratch assay and cell apoptosis assay respectively.The differentially expressed gene was screened by the intersection of RNA sequencing(RNA-seq)and methylated RNA immunoprecipitation sequencing(MeRIP-seq)techniques,and the effect of ALKBH5 on the gene expression was detected by RT-qPCR.Results Real time fluorogenic quantitative PCR results showed that,the relative expression levels of ALKBH5 RNA in Het-1A,Eca109,KYSE30,KYSE150 and KYSE410 were 1.03±0.28,0.46±0.02,0.23±0.10,0.04±0.02,0.05±0.00,respectively,with a statistically significant difference(F=444.60,P<0.001).Western blotting showed that,the relative expression levels of ALKBH5 protein in Het-1A,Eca109,KYSE30,KYSE150 and KYSE410 were 1.14±0.03,0.88±0.04,0.66±0.01,0.69±0.01,0.95±0.01,respectively,with a statistically significant difference(F=139.90,P<0.001).MTT test showed that the absorbance(A)values of KYSE30 control group and ALKBH5 overexpression group were 0.86±0.01 and 1.25±0.01 after 72 hours,respectively,with a statistically significant difference(t=46.93,P<0.001).The A values of KYSE150 control group and ALKBH5 overexpression group were 1.00±0.03 and 1.43±0.02 after 72 hours,respectively,with a statistically significant difference(t=16.80,P<0.001).The A values of KYSE30 control group,si-ALKBH5-1 group and si-ALKBH5-2 group were 0.98±0.01,0.85±0.02 and 0.80±0.09 after 96 hours,respectively,with a statistically significant differe

关 键 词：ALKBH5 食道鳞癌 IGF2BP3 

分 类 号：R73[医药卫生—肿瘤]