Snail 基因转录启始区组蛋白H3K4修饰的变化在EMT中的作用及对前列腺癌转移的影响

Alteration of whole genome histone modification occurred in EMT progress, however, relationship of EMT- associated transcription factors expression with histone modification remains elusive. Our results demonstrated H3K4Me3 enrichment changes notable around Snail Transcription Start Site, with the same tendency to Snail expression, during TGFb induced prostate cancer cell EMT. Knocking-down of WDR5 & RBBP5 - two crucial components of COMPASS/-like complex which catalyze H3K4Me3 formation, inhibited Snail expression and abrogated EMT progress. Previous studies show that phosphorylation of SAMD2/3 - a downstream effector of TGFb pathway regulated Snail transcription level, while is there possibility that SMAD2/3 promote H3K4Me3 at Snail TSS by recruiting COMPASS/-like complex? Does WDR5 & RBBP5 sensitize the susceptibility of prostate cancer cell to TGFb? Does WDR5/RBBP5 and EMT associated molecules expression positive correlated with tumor metastasis ？ If above hypothesis is correct, it will pave a way in solving of metastatic prostate cancer detection and treatment.

有研究显示,EMT过程存在组蛋白修饰的变化,但该变化与EMT相关转录因子表达的关系尚不明确,我们前期结果显示:EMT重要转录因子Snail转录起始区(TSS)附近H3K4Me3的富集在TGFb诱导前列腺癌细胞EMT中发生显著变化,且与Snail表达相关; 进一步用RNAi 抑制COMPASS/-like复合体(催化H3K4Me3生成)中重要组份WDR5&RBBP5也能抑制Snail表达上调及EMT过程.另有研究报道,TGFb通路下游组份pSMAD2/3能调控Snail表达,SMAD2/3能否通过募集COMPASS/-like复合体促进H3K4Me3在Snail TSS富集,进而促进Snail表达和EMT？WDR5,RBBP5能否调控细胞对TGFb的敏感性? WDR5/RBBP5及EMT相关分子的表达与PC转移是否相关?我们将回答并阐明这些问题。以上假设如能证实,将为PC治疗提供新的靶点。

本研究采用前列腺癌细胞为研究对象，研究内源性TGF-Beta的表达、自分泌及其相应受体的表达模式及其变化对前列腺癌细胞EMT过程的调控；TGF-Beta诱导的EMT中，SNAI1 转录起始区组蛋白H3K4、H3K9甲基化对SNAI1转录水平的影响，去甲基化酶KDM4D在其中的作用，继而研究SNAI1 转录起始区组蛋白甲基化改变对体内外前列腺癌侵袭的影响及可能的作用机制。研究结果显示，DU145细胞自分泌产生的TGF-β1作用于自身受体进而促进细胞发生EMT。PC3细胞能高度表达TGF-β1，TβR-Ⅱ，但不能发生明显的EMT。后续实验利用TGF-Beta1诱导DU145细胞，其形态学，转录，及蛋白质水平，细胞均发生了显著的EMT现象, 其中EMT相关转录因子SNAI1上调最为明显；TGF-Beta1诱导DU145细胞中，SNAI1 转录起始区H3K4me3、H3K9me3及RbBP5/WDR5 (COMPASS复合体组分) 的富集程度显著上升。干扰KDM4D后，SNAI1 转录起始区H3K9Me3的富集程度增加，SNAI1表达下调。RNAi干扰RbBP5后，TGF-Beta1诱导的细胞迁移增加被显著抑制；SNAI1上调及CDH1下调均被抑制，同时H3K4me3、RbBP5在SNAI1转录起始区域富集情况显著下调。在293T细胞中的模拟SNAI1报告基因实验表明，RbBP5和WDR5均能够显著促进报告基因表达，SMAD2/3及CBP在SNAI1转录起始区域富集程度显著升高；在TGF-Beta1诱导后，SMAD2/3通过CBP使得RbBP5募集于SNAIL TSS，促进H3K4me3富集继而诱导SNAIL1转录，从而促进EMT的发生。RbBP5控制着DU145细胞对TGF-Beta1的敏感性。通过敲减RbBP5 的DU145细胞建立的裸鼠皮下荷瘤模型及前列腺癌肺转移模型，皮下肿瘤中可以观察到 SNAIL1及vimentin表达水平显著降低，同时降低前列腺癌的肺转移能力。