【作者】 张丽娜；

【导师】 陈可欣；

【作者基本信息】 天津医科大学 ， 流行病与卫生统计学， 2011， 博士

【摘要】 乳腺癌是妇女最常见的恶性肿瘤之一。单核苷酸多态性(single nucleotide polymorphisms, SNPs)会影响癌症的发病风险和预后,但机制尚不是很清楚。微小RNA (microRNAs, miRNA)是一类高度保守的,内源的,单链非编码核苷酸序列,miRNA通过调节基因的表达参与了很多生物学过程,包括细胞增殖、细胞凋亡和肿瘤的形成。miRNA通常结合于靶基因mRNA的3’端非翻译区(3’-untranslated region,3’UTR)区域,来剪切mRNA或抑制mRNA的转录。研究者发现有越来越多与癌症和药物敏感性有关的多态位点是位于miRNA靶基因3’UTR的SNP,推测其原因是miRNA与不同基因型的3’UTR结合能力有区别。位于miRNA靶基因3’UTR结合区域的SNP代表了一类能调节miRNA与靶基因环路的遗传变异。以碱基互补原理为基础的生物信息学分析已经预测了一系列miRNA靶结合区SNPs,为进一步功能验证和病例对照研究提供了候选位点。目的近来,发现有一类单核苷酸多态位点通过影响miRNA对靶基因的调控,来影响癌症的发病风险和预后。在本项研究中,我们通过检测位于miR-367靶基因RYR3 3’UTR结合区域的多态位点rs1044129的基因型,来探究钙离子释放通道基因RYR3 3’UTR的SNP是否与乳腺癌的发病风险性、乳腺癌钙化和预后相关。从功能上验证miR-367与靶基因是否结合,rs1044129多态位点是否能影响miR-367对RYR3的调控,探究RYR3在乳腺癌细胞中发挥的重要作用和对临床医学的指导意义。方法这是一项以中国妇女为对象的病例-对照研究,对1532名乳腺癌患者和1605名健康人进行了SNP rs 1044129基因型检测。非条件logistic回归分析得出调整前后的比值比和95%可信区间。采用Kaplan-Meier曲线和Cox多因素分析评价不同基因型的生存状况。热力学模型预测和报告基因实验用来验证rs1044129对miR-367与RYR3结合的影响。在RYR3机制研究中,应用MTT实验检测细胞增殖,划痕实验评价细胞迁移,荧光染料法检测细胞内钙离子浓度。结果rs1044129 G等位基因型可以增加乳腺癌的发病风险(aOR,1.26；95% CI,1.02-1.54),并与乳腺癌钙化相关(OR,1.52;95% CI,1.21-1.90)。生存分析显示G等位基因型的乳腺癌患者预后差(P=0.036)。基于RYR3 mRNA 3’UTR和miR-367碱基互补原则和二级结构原理的热力学模型的预测结果显示miR-367与A基因型的结合能力高于G基因型。通过荧光素酶报告基因证实了rs1044129对miR-367与RYR3结合的影响。并在功能实验中发现RYR3作为钙离子通道对乳腺癌细胞生长、迁移、细胞内钙离子浓度的影响和细胞形态学的改变。结论rs1044129多态位点是一个新发现的影响miRNA调控靶基因的SNP,与响乳腺癌的发病风险、乳腺癌钙化和预后密切相关。更多还原

【Abstract】 Breast cancer is one of the most common malignant disease in women. Single nucleotide polymorphisms (SNPs) have been shown to impact both cancer risk and cancer prognosis although the underlying mechanism is largely obscure. MicroRNAs (miRNAs) are evolutionarily conserved, endogenous, single-stranded, non-coding RNA molecules that are reported to be involved in many biological processes, including cell proliferation, apoptosis, and tumorigenesis, through their regulation of gene expression. Most miRNAs bind to target sequences located within the 3’-untranslated region (3’UTR) of mRNAs by base pairing, resulting in the cleavage of target mRNAs or repression of their translation. An increasing number of 3’UTR SNPs located in miRNA binding sites have been reported to be associated with cancers and drug response, presumably due to the differential binding affinities of the SNPs for miRNA. Polymorphisms in these miRNA binding sites in the 3’UTRs of target genes represent a group of genetic variations that modulate the regulatory loop between miRNAs and their target genes. Sequence-based bioinformatic predictions have identified a number of these types of SNPs, which has provided candidates for experimental verifications and case-control studies to determine their importance.ObjectiveRecently, a new class of single nucleotide polymorphisms (SNPs) that directly affect gene regulation by microRNAs (miRs) has emerged as important for cancer risk and prognosis. In this study, we interrogated a SNP, rs 1044129, that is located in the miR-367 binding site at the 3’UTR of the calcium release channel ryanodine receptor 3 (RYR3) gene and determined whether this SNP was associated with breast cancer risk, calcification and prognosis. We then functionally validated SNP rs1044129, which is located in the miR-367 binding site in the 3’UTR of RYR3. Finally, we studied the important role of RYR3 in breast cancer cells and the significance in clinical medicine.MethodsIn a case-control study, rs 1044129 was genotyped in 1,532 breast cancer cases and 1,605 healthy Chinese women. Crude and adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were calculated with an unconditional logistic regression model. Kaplan-Meier curves and Cox regression analysis were used to evaluate the relationship between genotype and survival. A thermodynamic model and the reporter gene assay were used to functionally validate the role of rs 1044129 in miR367-RYR3 regulation in breast cancer. In RYR3 functional studies, we used MTT assay to detect cell proliferation, wound healing assay in migration experiment, Fluo-4AM to evaluate the intercellular calcium concentration.ResultsThe rs 1044129 G allele genotypes were associated with increased breast cancer risk (aOR,1.26; 95%CI,1.02 to 1.54) and increased incidence of mammographically detected microcalcifications (OR,1.52; 95%CI,1.21 to 1.90). Kaplan-Meier survival curves showed that G allele genotypes were significantly associated with poor progression-free survival in cases(P=0.036). A thermodynamic model based on base-pairing and the secondary structure of the RYR3 mRNA and miR-367 miRNA showed that miR-367 had a higher binding affinity for the A genotype than G genotype. We confirmed that miR-367 regulates the expression of a reporter gene driven by the RYR33’UTR and that the regulation was affected by the RYR3 genotype. We provided evidence that the ryanodine receptor gene (RYR3), which encodes a large protein that forms a calcium channel, is important for the growth, migration, intracellular calcium concentration and morphology of breast cancer cells.ConclusionThus, rs 1044129 is a novel SNP that resides in a miRNA-gene regulatory loop that affects breast cancer risk, calcification, and survival.更多还原