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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。
To reveal whether DNMT3B is involved in the development of hepatocellular carcinoma, Western blotting and immunocytochemistry were used to analyze the expression of DNMT3B protein in human normal liver cell line, hepatocarcinoma cell line and hepatoma cell line expression. The recombinant vector of RNAi stable expression of DN-MT3B was constructed and transfected into hepatocellular carcinoma cell line SMMC-7721. Semi-quantitative RT-PCR and Western blotting were used to identify the inhibitory efficiency of DNMT3B RNAi expression vector on endogenous DNMT3B. High-throughput cDNA microarray was used to analyze the downstream gene profiles influencing DNMT3B inhibition in SMMC-7721. The results showed that the expression level of DNMT3B in hepatocellular carcinoma cell lines was significantly higher than that in paracancer and normal liver cell lines. DNMT3B RNAi stably expressing recombinant vector transfected SMMC-7721 cell line after 2 months, was observed DNMT3B was significantly inhibited. cDNA microarray analysis showed that DNMT3B down-regulated 26 genes and up-regulated 115 genes, including some development-related genes and tumor-related genes such as SNCG, NOTCH1, MBD3, WNT11, MAOA and FACL4. It is suggested that the high expression of DNMT3B may be related to the occurrence of hepatocellular carcinoma and play a role in regulating the expression of other related genes, including the important gene related to development.