ALKBH5对人肝癌细胞上皮间质转化的影响

Effects of ALKBH5 on epithelial-mesenchymal transition in human hepatoma cell

目的探讨ALKBH5对肝癌细胞迁移、侵袭能力及上皮间质转化(EMT)的影响。方法选择手术切除的肝癌标本50例,分析ALKBH5与临床病理学参数的关系。设计ALKBH5短发夹核糖核酸(shRNA)序列,构建pLKO.1-TRC vector载体,慢病毒感染肝癌细胞HepG2和SMMC7721,嘌呤霉素筛选并培养稳定低表达ALKBH5细胞株;检测ALKBH5 mRNA及ALKBH5蛋白质水平干扰效率;Transwell实验检测细胞的迁移及侵袭;蛋白质免疫印迹(Western blot)检测细胞EMT相关蛋白的影响。结果肝癌中ALKBH5蛋白表达与分化程度、临床分期存在相关性(P<0.05),与血清甲胎蛋白(AFP)水平、肿瘤大小、年龄、性别均无相关性(P>0.05);下调肝癌细胞ALKBH5表达,对照组和实验组HepG2细胞迁移数目分别为(123±22)、(335±21)个,侵袭数目分别为(118±29)、(282±17)个;对照组和实验组SMMC7721细胞迁移数目分别为(220±27)、(453±23)个,侵袭数目分别为(200±11)、(388±20)个,2组间差异均有统计学意义(P<0.05)。下调ALKBH5表达后,E-钙黏蛋白(E-cadherin)表达水平降低,波形蛋白(Vimentin)、锌指结构转录抑制因子(Snail)表达水平增高。结论ALKBH5扮演了抑癌基因的角色,下调ALKBH5表达能够促进肝癌细胞的迁移、侵袭及EMT。

Objective To investigate the effects of ALKBH5 on migration, invasion and epithelial-mesenchymal transformation(EMT) of hepatoma cells.Methods A total of 50 hepatocellular carcinoma specimens were selected.The relationship between ALKBH5 and clinicopathological parameters was analyzed.ALKBH5 short hairpin RNA(shRNA) sequence was designed, pLKO.1-TRC vector was constructed, then lentivirus was used to infect HepG2 and SMMC7721 cells.Puromycin screened and cultured ALKBH5 cell lines with stable low expression.Interference efficiency of ALKBH5 mRNA and ALKBH5 protein levels were detected.Transwell assay was used to detect the migration and invasion of cells.Western blot was used to detect the effects of EMT-associated proteins.Results ALKBH5 protein expression was correlated with differentiation degree and clinical stage(P<0.05), but not with serum alpha-fetoprotein(AFP) level, tumor size, age and gender(P>0.05).The expression of ALKBH5 was down-regulated, and the migration number of HepG2 cells in control group and experimental group was(123±22) and(335±21), respectively;the number of invasions was(118±29) and(282±17), respectively;the migration number of SMMC7721 cells in control group and experimental group was(220±27) and(453±23), respectively;the number of invasions was(200±11) and(388±20), respectively, and the differences among all groups were statistically significant(P<0.05).After down-regulated ALKBH5 expression, E-cadherin protein expression was decreased, and the expression level of Vimentin and Snail protein increased(P<0.05).Conclusion ALKBH5 plays the role of tumor suppressor gene, and down-regulated ALKBH5 expression can promote migration,invasion and EMT of hepatoma carcinoma cells.