Motiwala Tasneem, Ghoshal Kalpana, Das Anindita, Majumder Sarmila, Weichenhan Dieter, Wu Yue-Zhong, Holman Kristen, James S Jill, Jacob Samson T, Plass Christoph
Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
Oncogene. 2003 Sep 25;22(41):6319-31. doi: 10.1038/sj.onc.1206750.
A diet lacking folic acid and choline and low in methionine (folate/methyl deficient diet, FMD diet) fed to rats is known to produce preneoplastic nodules (PNNs) after 36 weeks and hepatocellular carcinomas (tumors) after 54 weeks. FMD diet-induced tumors exhibit global hypomethylation and regional hypermethylation. Restriction landmark genome scanning analysis with methylation-sensitive enzyme NotI (RLGS-M) of genomic DNA isolated from control livers, PNNs and tumor tissues was performed to identify the genes that are differentially methylated or amplified during multistage hepatocarcinogenesis. Out of the 1250 genes analysed, 2 to 5 genes were methylated in the PNNs, whereas 5 to 45 genes were partially or completely methylated in the tumors. This analysis also showed amplification of 3 to 12 genes in the primary tumors. As a first step towards identifying the genes methylated in the PNNs and primary hepatomas, we generated a rat NotI-EcoRV genomic library in the pBluescriptKS vector. Here, we describe identification of one methylated and downregulated gene as the rat protein tyrosine phosphatase receptor type O (PTPRO) and one amplified gene as rat C-MYC. Methylation of PTPRO at the NotI site located immediate upstream of the trancription start site in the PNNs and tumors, and amplification of C-MYC gene in the tumors were confirmed by Southern blot analyses. Bisulfite genomic sequencing of the CpG island encompassing exon 1 of the PTPRO gene revealed dense methylation in the PNNs and tumors, whereas it was methylation free in the livers of animals on normal diet. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed significant decrease in the expression of PTPRO in the tumors and in a transplanted rat hepatoma. The expression of PTPRO mRNA in the transplanted hepatoma after demethylation with 5-azacytidine, a potent inhibitor of DNA methyltransferases, further confirmed the role of methylation in PTPRO gene expression. These results demonstrate alteration in methylation profile and expression of specific genes during tumor progression in the livers of rats in response to folate/methyl deficiency, and further implicate the potential role of PTPRO as a novel growth regulatory gene at least in the hepatocellular carcinomas.
已知给大鼠喂食缺乏叶酸和胆碱且蛋氨酸含量低的饮食（叶酸/甲基缺乏饮食，FMD饮食），36周后会产生癌前结节（PNN），54周后会产生肝细胞癌（肿瘤）。FMD饮食诱导的肿瘤表现出全基因组低甲基化和区域高甲基化。对从对照肝脏、PNN和肿瘤组织中分离的基因组DNA进行了用甲基化敏感酶NotI的限制性地标基因组扫描分析（RLGS-M），以鉴定在多阶段肝癌发生过程中差异甲基化或扩增的基因。在分析的1250个基因中，有2至5个基因在PNN中发生甲基化，而在肿瘤中有5至45个基因部分或完全甲基化。该分析还显示原发性肿瘤中有3至12个基因扩增。作为鉴定在PNN和原发性肝癌中甲基化基因的第一步，我们在pBluescriptKS载体中构建了大鼠NotI-EcoRV基因组文库。在此，我们描述了鉴定一个甲基化且下调的基因，即大鼠蛋白酪氨酸磷酸酶受体O型（PTPRO），以及一个扩增基因，即大鼠C-MYC。通过Southern印迹分析证实了PNN和肿瘤中转录起始位点紧邻上游的NotI位点处PTPRO的甲基化，以及肿瘤中C-MYC基因的扩增。对包含PTPRO基因外显子1的CpG岛进行亚硫酸氢盐基因组测序显示，PNN和肿瘤中存在密集甲基化，而正常饮食动物的肝脏中无甲基化。逆转录-聚合酶链反应（RT-PCR）分析显示，肿瘤和移植的大鼠肝癌中PTPRO的表达显著降低。用DNA甲基转移酶的有效抑制剂5-氮杂胞苷去甲基化后，移植肝癌中PTPRO mRNA的表达进一步证实了甲基化在PTPRO基因表达中的作用。这些结果表明，在大鼠肝脏中，响应叶酸/甲基缺乏，肿瘤进展过程中甲基化谱和特定基因的表达发生改变，并且进一步暗示了PTPRO至少在肝细胞癌中作为一种新型生长调节基因的潜在作用。
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