摘要
目的:
筛选肝细胞肝癌(hepatocellular carcinoma, HCC)相关基因中miRNA结合靶点内的基因多态并验证其与HCC发生的关联性进而探讨其中的具体分子机制。
方法:
(1)利用生物信息学方法结合文献报道筛选HCC相关基因,并在HCC相关基因3′UTRs或5′UTRs中预测目前人类已知miRNA的结合靶点,然后在预测靶点内筛出候选多态。(2)基于第一步预测结果,采用病例对照研究方法,PCR扩增结合聚丙烯胺凝胶电泳技术对部分候选多态进行基因分型,Logistic统计分析基因位点与HCC的关联性。(3)对发现的阳性关联基因多态位点,利用生物信息学的方法预测该位点可能结合的miRNA;采用定点突变和分子克隆技术,构建不同基因型荧光素酶表达载体,与特定miRNA共转染人肝癌细胞系(HepG2和SMCC7721),采用双荧光素酶报告系统对荧光素酶表达量进行检测。(4)采用Real-time RT-PCR方法,分析阳性关联位点不同基因型的肝癌组织样本目的基因的表达水平。
结果:
(1)利用生物信息学方法结合文献报道筛选出HCC相关基因105个,并在HCC相关基因3′UTRs或5′UTRs中已知miRNA结合靶点内筛出16个候选多态(含SNPs和插入缺失多态)。(2)我们从16个候选多态中选取了两个插入缺失多态进行病例对照研究,分别是位于IL1A-3′UTR的rs3783553(“TTCA”4bp插入缺失)和位于VEGF-5′UTR的rs35569394(“TCCCACTCTTCCCACAGG”18bp插入缺失)。基因分型结果显示,rs3783553和rs35569394均具有较好多态性,其在对照样本中最低等位基因频率分别为0.385和0.255。两位点在对照样本中的等位基因频率分布均符合Hardy-weinberg平衡。卡方检验显示rs3783553的基因型和等位基因频率在HCC组和对照组均存在显著性差异。用Logistic回归分析校正年龄、性别、吸烟、饮酒及HBV感染因素后发现,与4N Ins/4N Del和4N Del/4N Del相比,携带有4N Ins/4N Ins的个体HCC易感性明显降低(OR=0.30,95%CI:0.17-0.54),趋势检验P值<0.001。考虑到HBV感染与HCC发生的密切关系,我们对HBV进行了分层分析,结果显示,分层后插入型等位基因的保护作用仍能体现,但在HBV阳性的患者中更为突出。该结果提示,rs3783553多态可能在HBV感染的免疫调节过程中发挥作用。而rs35569394经上述分析后发现与HCC易感性不存在显著性关联,趋势检验P值=0.87。(3)根据rs3783553病例对照研究结果,我们分别构建了rs3783553两种不同基因型荧光素酶载体,基于生物信息学预测结果,将Pre-mir-122或Pre-mir-378与重组载体共转染HepG2和SMCC7721两种肝癌细胞系。实验结果显示,在加入不同浓度的Pre-mir-122后,与含有“TTCA”插入型等位基因的载体比较,含有“TTCA”缺失型等位基因载体的荧光强度显著下降,并呈现梯度依赖趋势。就miR-378而言,当Pre-mir-378浓度加大到10pmol以上时,我们观察到了同样的变化趋势,但rs3783553对miR-378与靶序列的结合的影响较miR-122小。(4)Taqman荧光定量PCR方法对不同rs3783553基因型肝癌组织样本的IL-1αmRNA表达水平进行比较后发现,4N Ins/4N Ins纯合子组的样本IL-1αmRNA表达水平最高,依次高于杂合子组和4N Del/4N Del纯合子组。26个4N Del/4N Del纯合子样本中有3个IL-1αmRNA表达水平未达到检测阈值,Ct按最大循环数40计算。非参数Mann–Whitney U检验显示三组间IL-1αmRNA表达水平存在显著性差异(P=0.042),其中4N Ins/4N Ins组和杂合子组IL-1αmRNA的表达水平分别是4N Del/4N Del组的5.57和3.76倍。
结论:
(1)病例对照研究数据分析结果显示,位于IL1A-3′UTR的rs3783553与HCC易感性存在显著性关联,HBV分层后分析结果提示rs3783553多态可能在HBV感染的免疫调节过程中发挥作用,值得进一步深入研究;位于VEGF-5′UTR的rs35569394与HCC易感性未发现显著性关联;(2)根据体外及体内试验结果,我们初步阐明了rs3783553参与HCC发生的具体分子机制,即rs3783553位点“TTCA”四碱基插入破坏了miR-122与IL1A-3′UTR的紧密结合,使得miR-122对IL-1α转录后调控水平的抑制作用减弱,因此携带有“TTCA”插入型个体IL-1α的抗肿瘤免疫调控作用增强,进而使HCC易感性降低。(3)考虑到IL-1α不仅可以影响肿瘤发生、生长和侵润等病理过程,而且可以影响肿瘤与机体之间的相互作用,我们的实验结果提示IL-1α有望成为研究HCC发生机制、早期诊断以及免疫治疗的新靶点。
Objective: To screen polymorhisms residing in the MicroRNAs binding sites of HCC-related genes and investigate their associations with HCC susceptibility and the underlined molecular mechanism.
Methods: (1) HCC-related genes were selected using bioinformatic methods and literature search, then 3′UTRs or 5′UTRs of these genes were identified. We analized putative miRNA-binding sites by means of specialized algorithms. Then we identified polymorphism within the putative binding sites for their ability to affect the binding with miRNA. (2) Based on the polymorphisms obtained from the first step, we performed case-control association studies using PCR-PAGE method. Logistic regression model was used for evaluating the association between polymorphisms and HCC susceptibility. (3) For the polymorphisms discovered in the second step affecting the HCC sucseptibility, putative miRNAs which would bind within polymorphisms were predicted using specialized algorithms. Meanwhile, Renilla luciferase reporter gene containing alternative alleles of specific polymorphisms were constructed using site-directed mutagenesis and molecular cloning methods. Alternative Renilla luciferase reporter genes were cotransfected with specific miRNAs into HepG2 and SMCC7721 cells and Renilla luciferase activities were measured with the Dual Luciferase assay system. (4) Lastly, Taqman? gene expression method was also used to detect target gene mRNA expression levels in different genotypic HCC tissue samples.
Results: (1) 105 HCC-related genes were recruited using bioinformatic methods and literature search and 16 candidate polymorphisms (including single nucleotide polymorphism and insertion/deletion polymorphism) were indentified within 3′UTRs or 5′UTRs of these genes. (2) Two insertion/deletion (Indel) polymorphisms (rs3783553 and rs35569394) were chosen from 16 candidate polymorphisms for the following case-control association studies. rs3783553 (“TTCA”Indel) and rs35569394 (“TCCCACTCTTCCCACAGG”Indel) were located within 3′UTR of IL1A and 5′UTR of VEGF, respectively. The genotyping results revealed that the two polymorphisms we studied were highly polymorphic; the minimal allele frequencies for rs3783553 and rs35569394 were 0.385 and 0.255, respectively in our control samples. No polymorphisms genotyped showed significant evidence for deviation from Hardy-Weinberg equilibrium in controls. Significant diffrence was observed for allelic and genotypic frequencies between HCC and control groups after chi-square testing. Using unconditional logistic regression model adjusted for sex, age, smoking status, drinking status and HBV infection, we found that the homozygote 4N Ins/4N Ins of rs3783553 was associated with a significantly reduced risk of HCC compared with its heterozygote 4N Ins/4N Del and homozygote 4N Del/4N Del (odds ratio=0.30, 95% confidence interval: 0.17-0.54, Ptrend<0.001). As HBV infection was one of the major risk factors, a stratified analysis by HBVinfection status was performed using binary logistic regression model. Significant differences were seen between cases and controls after stratification. The overall trend is that the differences between cases and controls were more obvious in HBV positive than the HBV negative population, suggesting a possible role of this polymorphism in the immune regulation during HBV infection. However, we found that rs35569394 was not associated with HCC after analyzing using the above method, at both the allele and genotype levels (Ptrend=0.87). (3) Based on the positive association results, Renilla luciferase reporter gene containing“TTCA”insertion or deletion alleles of rs3783553 were constructed using site-directed mutagenesis. According to the bioinformatic prediction results, two different Renilla luciferase reporter genes were separately cotransfected with Pre-mir-122 or Pre-mir-378 into HepG2 and SMCC7721 cells. Dual Luciferase assay results showed that compared with the constructs containing“TTCA”insertion alleles, the translation of Renilla luciferase from constructs containing the“TTCA”deletion allele was significantly reduced in the presence of Pre-mir-122 in a concentration dependent manner. For miR-378, we observed same change pattern only when >10 pmol pre-miR-378 were added, suggesting miR-378 binding to IL-1αtranscript may be less affected by rs3783553 genotypes. (4) Taqman gene expression analysis showed that“TTCA”insertion homozygous group had the highest IL-1αmRNA level, followed by heterozygous and“TTCA”deletion group. There were 3 of 26 samples in“TTCA” deletion group in which their IL-1αmRNA is under the detection threshold for this method and their Ct value was conservative determined by the maximum cycle number 40. Non-parametric Mann-Whitney U-test ofΔCts of three genotypic groups showed that the P value was 0.042. Compared with TTCA deletion homozygous group, the average IL-1αexpression levels of heterozygous and TTCA insertion homozygous group were 3.76 and 5.57-fold higher, respectively.
Conclusion: (1) rs3783553 polymorphism which located within IL1A-3′UTR was significantly associated with HCC susceptibility, stratification analysis by HBV infection status suggested a possible role of this polymorphism in the immune regulation during HBV infection. However, no significant association was observed for rs35569394 which located within VEGF-5′UTR; (2) Our in vitro and in vivo experiments demonstrated the molecular mechanism between rs3783553 and HCC susceptibility: miR-122 binds and negatively regulates the transcription of IL-1αwhich promote anti-tumor immunity and this regulation is negatively influenced by the presence of the“TTCA”insertion allele, presumably affecting miR-122 binding to the IL-1αtranscript. (3) Considering IL-1αaffects not only various phases of the malignant process, such as carcinogenesis, tumor growth and invasiveness, but also patterns of interactions between malignant cells and the host's immune system. Our results indicated that IL-1αmay be a promising target for immunotherapy, early diagnosis and intervention of HCC.
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