CAJ | 학술논문

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。
목적：정체분석웅격소의뢰성화비의뢰성전렬선암세포조단백H3갑기화차이，탐토전렬선암종격소의뢰성발전위비격소의뢰성적표관유전궤제。방법：응용중갑기세포배양조건하안기산은정동위소표기（ SILAC）기술결합생물질보분석웅격소의뢰성전렬선암세포LNCaP화비의뢰성전렬선암세포DU145조단백H3적갑기화수식보，심조차이적수식위점화모식；통과Western blotting험증소발현적차이수식；채용real-time PCR검측2주세포상관갑기화매화거갑기화매적표체차이。결과：질보감정발현2주전렬선암세포적조단백H3존재5개갑기화위점，갑기화모식분별위 H3K14me2、H3R17me1、H3K36me1、H3K36me2、H3K36me3、H3R72me2、H3K79me1화H3K79me2。기중，포함갑기화위점H3K36적태단유2충，분별위“KSAPATGGVKKPHR”화“KSAPST-GGVKKPHR”，전자감정도적빈수고우후자，량자적구별재우제31위적안기산분별위A여S，전자귀속우조단백H3변이체H31T、H31화H32，주요출현재DU145세포중，후자귀속우조단백H3변이체H33，재LNCaP세포중출현차수초다；제시2주세포가능표체불동적조단백H3변이체，종이도치갑기화모식적차이。 Western blotting검측발현H3K36적일갑기화화이갑기화재2주세포간몰유차이，이기재DU145세포중적삼갑기화정도현저고우LNCaP세포。 Real-time PCR검측현시DU145세포적H3K36거갑기화매mRNA표체비LNCaP세포유소강저。결론：조단백H3변이체화H3K36거갑기화매적차이표체가능도치비격소의뢰성전렬선암세포H3K36삼갑기화증가，성위전렬선암종격소의뢰성발전위비격소의뢰성적일충표관유전전변。
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 .