摘要
microRNAs(miRNA)是一种内源性的长22nt的单链小分子RNA,是一组不编码蛋白质的短序列RNA。miRNA通过与靶基因mRNA 3'非翻译区(3'Untranslated regions,3'UTR)的结合,在转录后水平降解mRNA或抑制翻译,调节基因的表达。miRNA与靶基因结合的关键部位称为“种子区域”(seed regionmiRNA 5'末端开始2—7个核甘酸),与miRNA3'UTR通过碱基配对结合。单核甘酸多态性是(SNPs)人类可遗传的变异中最常见的一种,前期研究已经证实miRNA靶位点序列中,存在有功能的SNPS。那么位于miRNA靶标位点的SNP会不会影响miRNA的功能及靶基因的表达呢?从2007年开始,部分学者开始从事这方面的研究,最近的研究已经证实了某些miRNA靶标位点的SNP与乳腺癌、结肠癌、肺癌、胃肠癌发病有关。
然而,miRNA靶位点的单核酸多态性对鼻咽癌的发病是否有影响,与疾病发生、发展、预后的关系目前仍知之甚少,因此本研究的目的是通过生物信息学预测、筛选鼻咽癌易感基因3'UTR潜在的microRNA靶位点,并发掘靶标区域SNP位点,结合病例一对照研究,探讨miRNA靶标位点基因多态性与鼻咽癌发病的关系。
首先通过文献回顾,我们总结了鼻咽癌的七大特征所涉及到的82个易感基因:持续生成新生血管、逃避凋亡、生长信号的自足性、抗生长信号的不敏感性、无限增殖潜能、组织侵袭与转移、肿瘤相关的炎症等。在NCBI数据库中下载了82个候选基因3'UTR的SNPs,利用miRanda,PicTar,TargetScans,和RNAfold webserve网站预测SNP位点的microRNA靶标和自由能,以与“种子”序列严格碱基互补配对的位点作为候选microRNA靶位点。最终在82个基因里找到220个SNPs存在于潜在的miRNA靶位点中,挑选在鼻咽癌中高表达,3'UTRSNP等位基因频率(MAF＞0.05)且miRNA结合自由能相对较小的9个基因(EGFR,COX2,CCNE1,hTERT,MMP2,MMP9,NF-κB VEGF and WNT3)进行实验验证。
实验首先随机选取12例患者和12例对照DNA,通过直接测序法筛选9个候选基因3'UTR的SNPs,结果筛选出EGFR rs884225,CCNE1 rs3218073 andMMP2 rs7201 3个SNPs用于下一步实验。然后,采用病例-对照的研究方法,选取广东地区167例鼻咽癌患者和171例正常对照为研究对象,针对已经筛选出来的SNPs进行批量PCR扩增和测序分型。
研究结果表明:在鼻咽癌总体样本中CCNE1基因多态位点(rs3218073)中,携带T等位基因的基因型TC+TT(OR＝1.585;95%CI＝1.023～2.458 P＝0.046),及等位基因T(OR＝1.464,95%CI＝1.012～2.118;P＝0.042)显著增加了鼻咽癌患病风险。进一步分层分析表明,杂合子变异型TC(OR＝1.959,P＝0.043)和等位基因T(OR＝2.123,P＝0.006)在原发肿瘤T3-T4分期中分布明显,此外,基因型TC(OR＝1.959,P＝0.043)和等位基因T(OR＝2.123,P＝0.006)与较晚临床分期(Ⅲ-Ⅳ期)有关。MMP2 rs7201位点基因型分析,选取了116例鼻咽癌患者和143例正常对照,MMP2 rs7201多态性与鼻咽癌易感性不存在关联,(杂合子变异型AC OR＝0.69.95%CI＝0.404～1.124,p＝0.209;等位基因C OR＝0.769,95%CI＝0.509～1.16,p＝0.135)。以167例鼻咽癌患者和171例为研究对象分析EGFR多态位点(rs884225)基因型分布,EGFR rs884225多态性与鼻咽癌发病风险无统计学差异(杂合子AG OR＝1.375,95%CI＝0.850～2.224 p＝0.194;纯合子变异型GG OR＝0.844,95%CI＝0.460～1.550 p＝0.584:等位基因G OR＝0.969,95%CI＝0.714～1.315,p＝0.839)。
上述结果提示,生物信息学预测miRNA-151靶基因CCNE1 3'UTR区miRNA结合位点的(rs3218073)多态性,携带T等位基因与鼻咽癌易感关联程度较高,并且与肿瘤分期有关,miRNA-151功能与rs3218073多态性的关系,还需进一步实验证实。
MicroRNAs(miRNAs) are endogenous 22nt small non-coding single stranded RNAs that bind with target mRNAs and function as posttranscriptional regulators of gene expression by either promoting mRNA degradation or translational silencing. The critical region for miRNA binding to mRNA is the 'seed region'(nucleotides 2-7 from the 5'end of the miRNA),which most often binds to a target site in the 3'UTR of the genes by perfect Watson-Crick complementarity.Single nucleotide polymorphisms(SNPs) build the essence of human genetic diversity.
Recently,genome-wide analysis(GWA) studies of human SNPs have revealed that nocoding variation in the regulatory sites are more like to be a associated with disease than the coding region variations.
Previous studies have described several putative functional SNPs in miRNA target sites.Whether SNPs located at miRNA binding sites can affect the expression of the miRNA target? Several researchers have conducted on these projects since 2007and recent studies have demonstrated that several putative functional SNPs in miRNA target sites may contribute to the susceptibility of humans to common diseases,For example,breast cancer,colorectal cancer,lung cancer and gastrointestinal cancers.However,Little is known about whether variability of miRNA target sites could have impact on the nasopharyngeal carcinoma risk.Thouse,The purpose of this study was to screen SNPs of NPC candidate genes,which might modify miRNA-binding predicted by bioinformatics analysis and to assess the association with NPC.
Based on the literatures related to genetic susceptibility of nasopharyngeal carcinoma,by analyzing seven hallmarks,including self-sufficiency in growth signals, evasion of apoptosis,insensitivity to growth inhibitory signals,limitless replicative potential,sustained angiogenesis,tissue invasion and metastasis,and cancer related inflammation that are involved in NPC,We summarized 82 genes were the most characterized and studied in NPC.We downloaded human SNPs in the 3' UTRs of 82 candidate genes from NCBI database and identified putative miRNA-binding sites by specialized algorithms(PicTar,miRanda,TargetScan,and RNAfold webserve).220 SNPs were identified in miRNA-binding sites.Thus,We focused on the 9 genes,including EGFR,COX2,CCNE1,hTERT,MMP2,MMP9,NF-κB VEGF and WNT3 for further investigation due to significant overexpression in NPC.We evaluated the miRNA-binding sites SNPs by assessing the MAF(MAF＞0.05) and free energy.
Therefore,a sequence analysis of targeted region was carried out to identify the potential variants of the candidate genes in 3'UTR.SNPS in 3'UTRs were identified by directly sequencing of genomic DNAs derived from 12 randomly selected patients and 12 controls,three SNPs,such as EGFR rs884225,CCNE1 rs3218073 and MMP2 rs7201 were found for next stage work.The study was carried out on a series of167 NPC cases and 171 controls from Guangdong province,a population with the highest worldwide incidence of NPC to research the distribution frequencies of the genotype. Here,we report results from the study.
We found statistically significant associations between risk of NPC and variant genotypes of CCNE1 rs3218073,for TC+TT(OR＝1.585;95%CI＝1.023～2.458 P＝0.046),for allele T(OR＝1.464,95%CI＝1.012～2.118;P＝0.042).A significant association between rs3218073 genotype TC(OR＝1.959,P＝0.043),T allele (OR＝2.123,P＝0.006) and later primary tumor(T3-T4) was retrieved.Moreover,the genotype TC(OR＝1.959,P＝0.043),T allele(OR＝2.123,P＝0.006) of rs3218073 also showed an increased risk of higher stage(Ⅲ-Ⅳ).
Analysis of the MMP2 rs7201and EGFR rs884225 genotype frequencies were performed in 116 NPC cases vs 143 controls and 167 NPC cases vs 171 controls, respectively.Allele case-control,and genotype case-control test statistics did not identify statistically significant association involving the two polymorphisms in NPC (p＞0.05),according to MMP2 rs7201(the variant heterozygous AC OR＝0.69,95% CI 0.404～1.124;allele C OR＝0.769,95%CI＝0.509～1.161 p＝0.135) and to EGFR rs884225(the variant heterozygous AG OR＝1.375,95%CI＝0.850～2.224,p＝0.194; the homozygotes variant GG OR＝0.844,95%CI＝0.460～1.550 p＝0.584;allele G OR＝0.969,95%CI＝0.714～1.315,p＝0.839).
In conclusion,CCNE1 rs3218073 polymorphisms located at miRNA-151 binding sites through bioinformation prediction,T allele was associated with susceptibility to NPC and correlated with staging.Experimental assays will determine the functional effects of miRNA- 151 and rs3218073 polymorphisms.
引文
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