背景:异常隐窝灶(ACF)是人类结肠中最早的肿瘤前病变,在染色体内镜筛查中可识别。我们的目标是评估APC的甲基化百分比,CDKN2A,MLH1,RASSF1,MGMT,ACF中的WIF1抑癌基因(TSG),相应的结直肠癌(CRC),和正常结肠粘膜对照。
方法:在本研究中,从302份新鲜结肠切除术标本中,在距肿瘤块5-10cm处取样宏观上外观正常的黏膜瓣,以鉴定ACF样病变.选择35例具有多个ACF的患者(n35)作为主要研究组,以CRC(n35)的相应切片作为疾病对照,和20个结肠切除术标本(正常对照)的粘膜组织块,手术治疗非肿瘤性病变。提取基因组DNA,甲基化特异性聚合酶链反应(PCR)在定制的甲基化阵列模型上进行.%甲基化数据在组间进行比较,并与临床病理参数进行比较。进行选择的靶mRNA和蛋白质表达研究。
结果:ACF中TSGs的%甲基化介于正常结肠和CRC之间,尽管仅在WIF1基因中观察到统计学上的显着差异(P<0.01)。此外,与对照组相比,ACF和CRCs中核β-catenin表达增加,CD44阳性癌干细胞上调.右侧ACFs和发育不良ACFs的CDKN2A甲基化百分比较高(P<0.01),而增生性ACFs的RASSF1甲基化百分比较高(P0.04)。ACF的拓扑特征与TSG%甲基化无关。
结论:WIF1基因的早期表观遗传甲基化是人结肠ACF发生的机制之一。
BACKGROUND: Aberrant crypt foci (ACF) are the earliest preneoplastic lesions in human colon, identifiable on chromoendoscopic screening. Our objective was to evaluate the %methylation of APC, CDKN2A, MLH1, RASSF1, MGMT, and WIF1 tumor suppressor genes (TSG) in ACF, corresponding colorectal carcinomas (CRC), and normal colonic mucosal controls.
METHODS: In this study, macroscopically normal-appearing mucosal flaps were sampled 5-10 cm away from the tumor mass from 302 fresh colectomy specimens to identify ACF-like lesions. Thirty-five cases with multiple ACFs were selected (n 35) as the main study group, with corresponding sections from CRC (n 35) as disease controls, and mucosal tissue blocks from 20 colectomy specimens (normal controls), operated for non-neoplastic pathologies. Genomic DNA was extracted, and methylation-specific polymerase chain reaction (PCR) was performed on a customized methylation array model. %Methylation data were compared among the groups and with clinicopathological parameters. Selected target mRNA and protein expression studies were performed.
RESULTS: %Methylation of TSGs in ACF was intermediate between normal colon and CRC, although a statistically significant difference was observed only for the WIF1 gene (P < 0.01). Also, there was increased nuclear β-catenin expression and upregulation of CD44-positive cancer-stem cells in ACF and CRCs than in controls. Right-sided ACFs and dysplastic ACFs had a higher %methylation of CDKN2A (P < 0.01), whereas hyperplastic ACFs had a higher %methylation of RASSF1 (P 0.04). The topographic characteristics of ACFs did not correlate with TSG %methylation.
CONCLUSIONS: Early epigenetic methylation of WIF1 gene is one of the mechanisms for ACF development in human colon.