结直肠癌细胞中脾酪氨酸激酶基因甲基化状态和表达的关系

Correlation between hypermethylation and expression of Syk gene in colorectal cancer cell lines

目的:探讨结直肠癌细胞中脾酪氨酸激酶基因甲基化和表达的关系。方法:应用亚硫酸盐修饰测序、甲基化特异性聚合酶链反应和蛋白印迹技术检测结直肠癌细胞脾酪氨酸激酶的甲基化状态以及表达情况;荧光素酶报告分析法研究启动子区域CpG岛的甲基化与启动子活性的关系;甲基化转移酶抑制剂处理脾酪氨酸激酶甲基化失表达的结直肠癌细胞株,观察处理前后细胞内脾酪氨酸激酶基因甲基化状态和表达情况。结果:(1)23个结直肠癌细胞中,9个细胞启动子发生甲基化而失去蛋白质表达;其余则正常表达,甲基化发生率为39.2%;(2)9个甲基化的细胞中,7个存在微卫星不稳定;而14个未发生甲基化的细胞中,仅有4个存在微卫星不稳定。二者之间的差异显著(P<0.05);(3)脾酪氨酸激酶启动子全长和未甲基化启动子荧光素酶的活性分别是甲基化组的4.5和4.7倍;5-Aza-CdR可恢复甲基化启动子的活性;(4)5-Aza-CdR可去甲基化而使脾酪氨酸激酶基因重新表达,而且具有时间依从性。结论:结直肠癌细胞中,启动子区域的甲基化导致Syk基因丧失表达,5-Aza-CdR可以去甲基化而恢复脾酪氨酸激酶基因的表达。

AIM： To explore the relationship of methylation status and expression of spleen tyrosine kinase （Syk） gene in colorectal cancer cell lines. METHODS： Bisulfite sodium modification sequencing, methylation specific PCR and Western blotting were used to detect the methylation status and expression of Syk gene in 23 colorectal cancer （CRC） cell lines. Luciferase assay was applied to measure the activity of promoter with or without methylation in CpG islands. Meanwhile, methylation status and expression level were compared in Syk（ - ） HCT - 15 cell line before or after 5 - Aza - CdR administration. RESULTS ： （ 1 ） Among 23 cell lines, loss expression of Syk gene in 9 cell lines due to hypermethylation in promoter, and 14 expression with unmethylation status were observed. The total methylation rate was 39. 2%. （2） Microsatellite instability was found in 7 of 9 cell lines with promoter hypermethylation, 4 of 14 were wihtout hypermethylation. The difference between methylation and unmethylation group was significant （ P 〈 0. 05）. （3） 5 - Aza - CdR restored methylated - Syk gene promoter activity. Compared to methylated - promoter, luciferase activities increased to 4. 5 and 4. 7 folds with Syk full size promoter and unmethylated - promoter, respectively. （4） 5 - Aza - CdR restored methylated - Syk gene expression and the effect had time - course dependence, CONCLUSION ： Hypermethylation of CpG islands in Syk gene promoter silences Syk expression in CRC cell lines, and 5 - Aza - CdR restores Syk expression by demethylation.