【作者】 陈翱翔；

【导师】 邵志敏；

【作者基本信息】 复旦大学 ， 肿瘤学， 2011， 硕士

【摘要】 既往研究证实let-7可以转录后抑制LIN28基因的表达,而LIN28又能够阻断let-7成熟通路。let-7和LIN28以及二者组成的双重负反馈调控通路的异常与恶性肿瘤的发生存在关联。本课题主要研究let-7/LIN28通路相关基因胚系变异对不同个体生理状态下该通路水平的影响,以及这种影响是否与乳腺癌发生风险存在相关。通过计算机分析以及荧光报告实验,我们发现一个位于let-7在LIN28 mRNA上结合序列附近的单核苷酸多态性位点(SNP)rs3811463能够改变LIN28 mRNA的局部二级结构并影响let-7对LIN28的调控作用,导致let-7/LIN28通路水平的异常。通过对正常乳腺组织内let-7、LIN28mRNA、LIN28蛋白水平的分析,我们发现let-7在携带rs3811463少见等位基因(rs3811463-C)的个体正常乳腺组织内的水平显著高于携带野生型rs3811463等位基因(rs3811463-T)的个体正常乳腺组织内的水平而LIN28蛋白的表达则呈相反的趋势。基于此,我们推测le-7/LIN28通路相关的SNP可能会影响该通路的水平且潜在地改变不同个体罹患肿瘤的风险。接下来,通过病例对照研究,我们着重研究了let-7及LIN28基因常见SNP与乳腺癌的关系。在第一个病例对照研究(n=2,300)中,我们发现LIN28基因上的两个SNP位点与乳腺癌的发生显著相关。这两个位点分别为rs3811463与rs6697410,均位于LIN28基因的3’非编码区(3’UTR).rs3811463的C等位基因能增加乳腺癌的发病风险(OR为1.25,P=0.0091),而rs6697410的少见等位基因G则能降低乳腺癌的发病风险(OR为0.76,P=0.0086)。二期于独立人群中进行的病例对照研究(n=1,156)证实了最初的结果。两个研究(n=3,456)的合并P值为8.0×10-5(rs3811463)和7.0×10-4(rs6697410)。通过软琼脂实验,我们进一步揭示了高水平的LIN28蛋白能促进正常乳腺上皮细胞(MCF 10A)的转化。总体上,本课题发现let-7/LIN28通路相关基因的胚系变异能引起不同个体体内该通路水平的变化,这种变化在汉族女性中可导致乳腺癌发生易感性的差异。更多还原

【Abstract】 Previous studies have shown that let-7 post-transcriptionally represses the expression of LIN28, and LIN28, in turn, blocks the maturation of let-7, forming a double-negative feedback loop. In this study, we investigated the effect of germline genetic variants on regulating the homeostatic status of the let-7/LIN28 loop and the influence of these variations on breast cancer risk. We demonstrated that the single nucleotide polymorphism (SNP), rs3811463, located near the binding site of let-7 in LIN28 may lead to a differential regulation of let-7 binding to LIN28 by altering the local mRNA secondary structure, thus disturbing the feedback loop between these two factors. Moreover, normal breast tissue harboring the rs3811463-C allele had significantly lower let-7 levels and higher LIN28 protein levels relative to the normal breast tissue harboring the wild-type T allele. Because the in vitro and ex vivo experiments consistently suggested that LIN28 modified breast cancer susceptibility, we subsequently evaluated the relationship between common SNPs found in LIN28 and breast cancer in a stepwise manner. The first hospital-based association study (n= 2,300) demonstrated that two SNPs (including rs3811463) significantly associated with breast cancer risk. The C allele of the rs3811463 SNP corresponded to an increased risk with an odds ratio (OR) of 1.25 (P=0.0091), which was successfully replicated in another independent study (n=1,156) with community-based controls. The combined P-value of the two studies was 8.0×10-5. Furthermore, a soft agar assay indicated that LIN28 was able to transform MCF-10A cells, which is a normal breast epithelial cell line. Taken together, our study demonstrated that host genetic variation disturbing the regulation of the let-7/LIN28 double-negative feedback loop can alter breast cancer risk.更多还原