摘要
目的探索人食管癌细胞系Hedgehog信号通路相关基因的表达状况、HHIP基因启动子区甲基化状态与HHIP表达的关系。方法采用RT-PCR方法检测5个食管癌细胞系Hedgehog信号通路SHH、PTCH1、SMO、HHIP和Gli的表达情况。用基于甲基化敏感酶和甲基化依赖酶酶切、并结合荧光定量PCR方法分析食管癌细胞系HHIP基因启动子区甲基化状态,并检测DNA甲基转移酶抑制剂5-氮杂-2-脱氧胞苷(5-Aza-CdR)处理前后食管癌细胞系HHIP mRNA表达的变化。结果 SHH、PTCH1、SMO和Gli mRNA在5个食管癌细胞系中都有较高表达。HHIP在Kyse450细胞系的表达较高,约为其余4个细胞系表达量的3～5倍。与之相应,Kyse450细胞HHIP基因CpG岛为非甲基化状态,其余4个细胞系CpG岛都发生了高甲基化。采用5-Aza-CdR去除甲基化后,HHIP基因表达较处理前增加了10～20倍。结论 Hedge-hog信号通路的异常激活可能参与了食管癌的发生、发展过程,食管癌细胞系HHIP基因的表达抑制与其基因启动子区CpG岛高甲基化相关。
Objective To study gene expression involved in hedgehog signaling pathway in human esophageal cancer cell lines,and to investigate the relationship between human hedgehog interacting protein（HHIP） expression and CpG island methylation of HHIP gene promoter.Methods In esophageal squamous cell carcinoma cell lines Kyse450,Kyse510 and Yes2 and esophageal adenocarcinoma cell lines Skgt4 and Te7,the expression of hedgehog pathway components SHH,PTCH1,SMO,HHIP and Gli was studied via RT-PCR.HHIP gene promoter methylation in esophageal cancer cell lines was analyzed through digestion with methylation-sensitive restriction enzyme HhaⅠ and methylation-dependent restriction enzyme McrBC as well as real-time PCR.The esophageal cancer cell lines were treated with 5-aza-2′-deoxycitydine（5-Aza-CdR）,and the changes of HHIP mRNA expression before and after 5-Aza-CdR treatment were tested.Results SHH,PTCH1,SMO and Gli had relatively high mRNA expression levels in all of the five cell lines,while HHIP only showed higher mRNA expression in Kyse450 cell line,with 3-5 times that in the other 4 cell lines.The CpG islands of HHIP gene were in an unmethylated state in Kyse450 cell line,but in a highly methylated state in the other 4 cell lines.In Yes2 cell line that had highly methylated CpG islands,HHIP gene expression level was increased by 10-20 times after 5-Aza-CdR demethylation.Conclusion Abnormal activation of hedgehog signaling pathway may be involved in carcinogenesis and development of esophageal cancer.The inhibition of HHIP gene expression is correlated to the high methylation level of the CpG islands in its gene promoter.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2011年第5期477-480,共4页
Journal of Third Military Medical University
基金
国家自然科学基金(30771099)~~
关键词
Hedgehog信号
HHIP基因
食管癌
细胞系
hedgehog esophageal carcinoma cell line human hedgehog interacting protein methylation
