目的：整体分析雄激素依赖性和非依赖性前列腺癌细胞组蛋白H3甲基化差异，探讨前列腺癌从激素依赖性发展为非激素依赖性的表观遗传机制。方法：应用重甲基细胞培养条件下氨基酸稳定同位素标记（ SILAC）技术结合生物质谱分析雄激素依赖性前列腺癌细胞LNCaP和非依赖性前列腺癌细胞DU145组蛋白H3的甲基化修饰谱，寻找差异的修饰位点和模式；通过Western blotting验证所发现的差异修饰；采用real-time PCR检测2株细胞相关甲基化酶和去甲基化酶的表达差异。结果：质谱鉴定发现2株前列腺癌细胞的组蛋白H3存在5个甲基化位点，甲基化模式分别为 H3K14me2、H3R17me1、H3K36me1、H3K36me2、H3K36me3、H3R72me2、H3K79me1和H3K79me2。其中，包含甲基化位点H3K36的肽段有2种，分别为“KSAPATGGVKKPHR”和“KSAPST-GGVKKPHR”，前者鉴定到的频数高于后者，两者的区别在于第31位的氨基酸分别为A与S，前者归属于组蛋白H3变异体H31T、H31和H32，主要出现在DU145细胞中，后者归属于组蛋白H3变异体H33，在LNCaP细胞中出现次数稍多；提示2株细胞可能表达不同的组蛋白H3变异体，从而导致甲基化模式的差异。 Western blotting检测发现H3K36的一甲基化和二甲基化在2株细胞间没有差异，而其在DU145细胞中的三甲基化程度显著高于LNCaP细胞。 Real-time PCR检测显示DU145细胞的H3K36去甲基化酶mRNA表达比LNCaP细胞有所降低。结论：组蛋白H3变异体和H3K36去甲基化酶的差异表达可能导致非激素依赖性前列腺癌细胞H3K36三甲基化增加，成为前列腺癌从激素依赖性发展为非激素依赖性的一种表观遗传转变。%AIM:To study the epigenetic mechanisms involved in the evolution of prostate cancer from an an-drogen-dependent state to an androgen-independent state , and the global difference of histone H 3 methylation between an-drogen-dependent and -independent prostate cancer cells .METHODS:The methylation sites and patterns of histone H 3 in androgen-dependent prostate cancer cell line LNCaP and androgen-independent prostate cancer cell line DU 145 were ana-lyzed by heavy methyl stable isotope labeling with amino acids in cell culture ( SILAC) coupled with 2D LC-MS/MS.West-ern blotting was used to verify the results from MS .The differential expression of related methylases and demethylases was tested by real-time PCR.RESULTS:Five methylation sites on histone H3 were found in both cell lines, the patterns of which were as follows: H3K14me2, H3R17me1, H3K36me1, H3K36me2, H3K36me3, H3R72me2, H3K79me1 and H3K79me2.There were 2 different peptides both containing methylated H 3K36,“KSAPATGGVKKPHR” and“KSAPSTG-GVKKPHR”, which were different from the 31th amino acid residue “A” and “S”.The former peptide belonging to his-tone H3 variants, H31T, H31 and H32, was mainly identified in DU145 cells, the total peptide counts of which were much more than that of the latter peptide belonging to histone H 3 variant H31T, suggesting that these 2 cell lines expressed differ-ent histone H3 variants.Mono-and dimethylation of H3K36 were not different between these 2 cell lines, but the trimethyl-ation was significantly higher in DU 145 cells than that in LNCaP cells .Many H3K36 demethyltransferases were decreased in DU145 cells compared with LNCaP cells .CONCLUSION: The differential expression of histone H 3 variants and H3K36 demethyltransferases may result in up-regulation of H3K36 tri-methylation during the evolution of prostate cancer from an androgen-dependent state to an androgen-independent state .
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