RNAi及DNA芯片分析肝癌细胞系中受DNMT3B调控的下游基因(英文)  

Identification of Potential Genes Regulated by DNA Methyltransferase 3B in a Hepatocellular Carcinoma Cell Line by RNA Interference and Microarray Analysis

作　　者：许军[1] 樊红[1] 赵主江[1] 张建琼[1] 谢维[1] 

机构地区：[1]教育部发育与疾病相关基因研究重点实验室,江苏省基因诊断与治疗医学重点实验室,东南大学遗传学研究中心,南京210009

出　　处：《Acta Genetica Sinica》2005年第11期1115-1127,共13页

基　　金：教育部高校青年教师奖(编号:2001/182);国家自然科学基金项目(编号:30470950)资助~~

摘　　要：为揭示DNA甲基转移酶3B(DNMT3B)在肝癌中是否参与了肿瘤的发生,应用Westernblotting及细胞免疫化学方法分析DNMT3B蛋白在人的正常肝细胞株、肝癌癌旁细胞株及肝癌癌细胞株中的表达。构建了DN-MT3B的RNAi稳定表达的重组载体,并转染入肝癌细胞株SMMC-7721中。以半定量RT-PCR及Westernblot-ting分别鉴定DNMT3BRNAi表达载体对内源性DNMT3B的抑制效率。用高通量的cDNA基因芯片分析了SMMC-7721中DNMT3B抑制后有影响的下游基因谱。结果显示,DNMT3B在肝癌细胞株中的表达水平明显高于肝癌癌旁和正常肝细胞株。DNMT3B的RNAi稳定表达重组载体转染SMMC-7721细胞株2个月后,观察到DNMT3B明显受到抑制。cDNA基因芯片分析发现,DNMT3B抑制后诱导26条基因表达下调,115条基因表达上调,包括一些发育相关基因以及肿瘤相关基因,如SNCG、NOTCH1、MBD3、WNT11、MAOA、FACL4等。提示DNMT3B的高表达可能与肝癌的发生有关,并以调控其他相关基因的表达而起作用,包括与发育相关的重要基因。Whether DNA methyltransferase 3B （DNMT3B） is deregulated in hepatocellular carcinoma cell lines is still unclear, The expression levels of DNMT3B protein in normal liver cell line, pericacinoma cell line and hepatocellular carcinoma cell lines were compared by both Western blotting and immunocytochemistry, Long-term downregulated DNMT3B in a hepatocellular carcinoma cell line SMMC-7721 was achieved using a RNAi recombinant plasmid, The suppression of DNMT3B induced by RNA interference was confirmed using semi-quantitative RT-PCR and Western blotting, High throughput cDNA microarray was used to analyze the expression profi- ling of downstream genes of DNMT3B displayed in the treated cell lines and control, In the result, DNMT3B in hepatocellular carcinoma cell lines was expressed at a significantly higher level compared to those in pericacinoma cell line and normal liver cell line, A specific DNMT3B siRNA stably expressed from a plasmid vector effectively suppressed the expression of DNMT3 B in SMMC-7721 cell line. By microarray analysis,26 downregulated genes and 115 upregulated genes have been identified in the DNMT3B knockdown cell line,including some important developmental genes and tumor-related genes such as SNCG, NOTCH1, MBD3, WNT11, MAOA and FACL4. The discovery showed DNMT3B was over-expressed in most hepatocellular carcinoma cell lines examined and may be linked to the carcinogenesis of hepatocytes. An array of candidate genes that are involved in the action of DNMT3B have been identified,including those related to development.

关 键 词：甲基转移酶 DNA甲基转移酶3B RNA干扰 肝癌 

分 类 号：R735.7[医药卫生—肿瘤]