hsa-mir-147通过5'－UTR功能性SNP介导的等位基因不平衡表达的作用机制研究及其应用

miRNAs are 21-nt RNAs that, in traditonal, bind through imperfect base pairing to the 3'- UTR of their target mRNAs and interfere with translational output. Even though, Lytle found that target mRNAs are repressed as efficiently by microRNA-binding sites in the 5'- UTR as in the 3'- UTR.The allelic expression imbalance (AEI) can be measured in individuals heterozygous for a transcribed polymorphism. And it has been suggested that variation in regulatory regions may be of particular interest for common diseases with a complex genetic background. We have found that a haplotype of the Catalase was associated with essential hypertension, and more interesting rs1049982 could serve as a marker for AEI. The expression levels of CATH1 and will be shifted in different oxidative stress conditions. miRNA target prediction shown that MIR147B can regulate CAT AEI through the SNP in the 5'- UTR. And, the dual-luciferase assay gives us the similar results. Although current computational predictions of miRNA-UTR interactions provide important guidance for experimental analysis of miRNAs, little functional data exists on which to train prediction algorithms. So we want to quantitative detect the MIR147B and CAT expression levels and the AEI in essentioanl hypertension, colorectal cancer and breast cancer. And then, detecting the MIR147B and CAT in cell lines with different culture contions and cells in different phases of the cell cycle. Furthermore, detecting the mechanism of MIR147B regulation the CAT AEI using a dual luciferase reporter system and normalized to the control. Finally, to make a molecular model of miRNA regulation the AEI, and extrapolating its mechanism.

传统认为miRNA与靶基因3'-UTR结合，在转录后水平降解mRNA或抑制翻译，而Lytle发现miRNA与5'-UTR结合同样可以有效降低mRNA表达；同时有研究表明相当数量的基因在个体水平上存在等位基因的不平衡表达（AEI），并且可能与某些疾病密切相关。本课题组前期发现过氧化氢酶基因一个多态位点存在AEI并与高血压密切相关；同时该AEI在不同氧化压力下可能存在动态调控机制。生物信息学研究揭示CAT5'-UTR能与MIR147B结合，并且与种子区匹配位点存在多态，可能影响二者之间结合力；初步的分子生物学实验也证实了这一点。我们拟定量检测MIR147B与CAT基因AEI及疾病的相关性；检测不同培养条件及不同细胞周期中细胞株的MIR147B表达水平及AEI变化；在细胞学水平检测功能性SNP与MIR147B作用；期望建立miRNA调控AEI的模型，并阐明其作用机制。

传统认为miRNA与靶基因3'-UTR结合，在转录后水平降解mRNA或抑制翻译，而Lytle发现miRNA与5'-UTR结合同样可以有效降低mRNA表达；同时有研究表明相当数量的基因在个体水平上存在等位基因的不平衡表达（AEI），并且可能与某些疾病密切相关。本课题组前期发现过氧化氢酶基因一个多态位点存在AEI并与高血压密切相关；同时该AEI在不同氧化压力下可能存在动态调控机制。本课题首次发现了CAT 5'-UTR的1个SNP是MIR147B结合的关键位点，基因多态变化可以影响CAT 基因mRNA 与相关miRNA 的结合效率，CAT基因调控区存在两个单倍型CATH1(G-A-T)和CATH2(A-T-C)，我们构建到荧光素酶表达载体中，命名为pGL3-CATH1和pGL3-CATH2。在Hela细胞株中，当pGL3-CATH1与MIR147B前体结合后，表达下降到44.7％，而pGL3-CATH2下降到27.3％（P＝0.019）；在ECV-304细胞株中当pGL3-CATH1与mir-147b前体结合后，表达下降到56.1％，而pGL3-CATH2下降到44.3％（P＝0.005）；但是加入miRNA抑制剂没有显著变化，HepG2细胞中也有一致的结果，当pGL3-CATH1与mir-147b前体结合后，表达下降到100％，而pGL3-CATH2下降到55.1％（P＝0.005）但是在293细胞株没有看到差异。MIR147B对pGL3-CATH2调控程度较pGL3-CATH1大，与预测结果相一致。进一步我们利用PGA中的多态数据对CAT进行中性检验，结果发现：在非洲人群中Fu & Li's D*＝1.99399，P < 0.02；Fu & Li's F*＝1.567890，0.1> P > 0.05；在欧洲人群中Fu & Li's D*＝2.02971，P < 0.02，Fu & Li's F*＝2.02527，P < 0.02。CAT可能存在平衡选择。