摘要
Beclin 1是哺乳动物中首个被发现的具有调节细胞自我吞噬作用的抑癌基因,在细胞自噬作用的过程中能够调节吞噬泡的形成,并通过结合Bcl-2参与细胞凋亡的调控,实现对肿瘤的抑制功能。本工作通过研究beclin 1在乳腺癌、胃癌和直结肠癌中的表达情况及调控机制,为进一步研究beclin 1基因与肿瘤发生、发展的关系奠定基础。
我们首先对60例乳腺癌、胃癌和直结肠癌的组织标本beclin1 mRNA及蛋白表达水平进行了检测,发现beclin 1基因的表达在乳腺癌和胃癌中与癌旁组织表达呈现显著差异,并筛选到19例表达显著下调的标本。同时检测了P53、BRCA1和BRCA2三种蛋白的表达,结果表明beclin 1的mRNA表达水平与BRCA1阳性着色的乳腺癌标本之间存在显著关联。为了阐明影响beclin 1表达可能的调控机制,我们从杂合子缺失、点突变和DNA甲基化修饰三个方面进行了研究。结果表明:①在乳腺癌和胃癌标本中分别存在至少45%和20%的杂和子缺失,且缺失标本与beclin 1的mRNA表达水平呈现显著关联,而在直结肠癌中未发现任何缺失,说明杂合子缺失是乳腺癌和胃癌中beclin 1表达的一种调控机制;②对20例来自乳腺癌组织标本的基因组DNA中beclin 1外显子区分析未发现任何突变,说明beclin 1在乳腺癌中突变发生较为罕见;③生物信息学分析结果提示在beclin 1基因5‘端存在一个CpG岛,横跨启动子至第二个内含子区域,对显著下调的肿瘤标本进行检测发现乳腺癌、胃癌和直结肠癌组织中均存在启动子区域和第二个内含子区域的异常甲基化,且甲基化与belcin 1基因表达相关,用5-aza-dC处理卵巢癌细胞SKOV3,beclin 1基因的表达得到恢复,DNA甲基化对其表达有沉默作用。
总之,我们在乳腺癌和胃癌中发现了调控beclin 1表达的两种重要机制:杂合子缺失和DNA甲基化。直结肠癌中除DNA甲基化外可能还存在未知beclin 1表达的调控机制。由于beclin 1在抑制肿瘤发生中起到重要作用,其调控方式的研究有助于为肿瘤的治疗提供新的靶标。
Beclin 1, was the first autophagic gene identified in mammarlian cells with roles in mediating autophagy and apoptosis. Earlier studies showed that beclin 1 expression was down-regulated with mono-allelic deletions in human breast cancer cells and the decreased expression of beclin 1 could contribute to tumorigenesis. But the status of its expression and regulatory mechanisms in other cancers were remained to be determined. In the present work, we investigated the effects of possible mechanisms on beclin 1 expression in breast, gastric and colorectal cancers for estabilishing the basis between autophagic genes and tumorigenesis of beclin 1 in these cancers.
In our present work, we detected the expression status of beclin 1 in the tumor tissues and corresponding adjacent normal tissues from 60 pairs of breast, gastric and colorectal tumors. We found that beclin 1 expression was associated with breast and gastric cancers, in which 19 samples were obtained with significantly decreased expression of beclin 1. Association between beclin 1 mRNA expression and clinocopathologic parameters was found in none of the three different kinds of cancer. Furthermore, in breast tumors, we examined the expression of p53, BRCA1 and BRCA2, which play important roles in the development of breast cancer and found that beclin 1 mRNA level was significantly greater in the BRCA1 positive tumors than in the negative ones, suggesting beclin 1 expression may be parallel to cell growth in breast cancer.
Then we explored the possible regulatory mechanisms of beclin 1 expression in three aspects, loss of heterozygosity (LOH), point mutation and DNA methylation. The results showed a LOH rate of 45% and 20% at the beclin 1 locus respectively in breast and gastric tumors. But LOH was not found in colorectal tumors. We also examined mutations throughout 12 exons of beclin 1 and no mutation was found, which showed that mutation might not be a common mechanism affecting beclin 1 expression. Inspection of beclin 1 genomic location, we found a large and dense CpG island ranging from the promoter to the intron 2 of the beclin 1 gene. Therefore, we detected the methylation status of the CpG island in breast, gastric and colotectal tumors. In the promoter from -528 to -65 (from transcription start site) and the intron 2 from 733 to 977, methylation was detected in parts of tumors with significant decreased beclin 1 expression and hardly detected in the corresponding normal tissues. Furthermore, beclin 1 mRNA expression was restored in an ovarian cancer cell line, SKOV3, with 5-aza-dC treatment. The results indicated that aberrant DNA methylation of beclin 1 was responsible for its decreased expression in these tumors.
In summary, our work indicated that decreased expression of beclin 1 is frequently observed in breast, gastric and colorectal cancer. Aberrant DNA methylation and LOH were demonstrated to be responsible for the decrease of beclin 1 expression in breast and gastric cancer, and DNA methylation was also found to play a role in colorectal tumors. Aberrant DNA methylation and LOH of beclin 1 associated with decreased expression suggest that epigenetic and genetic alterations of this gene may be interesting targets for cancer therapy.
引文
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