microRNA-146a调控脂联素基因表达的遗传多态网络机制

Mechanisms of microRNA regulation on adiponectin gene expression need to be explored furtherly. Based on the associations of genetic polymorphism in miR-146a gene with adiponectin level which we had detected in our previous study and the evidence that miR-146a could regulate adiponectin gene expression in Genecard, we hypothesised that miR-146a mediated the regulation of adiponectin gene expression. First of all, single nucleoside polymorphsims with minor allele frequency more than 10% and their haplotypes were chosen through sequence for miR-146a gene and adiponectin gene 3′UTR. From bioinformatics, cell aspect and population study, combining the data about the influence of genetic polymorphisms in adiponectin gene 5′UTR on the adiponectin gene expression which we had identified in our previous study, the regulation of genetic polymorphisms in miR-146a gene and adiponectin gene(3′UTR and 5′UTR) on the adiponectin gene expression will be explored using luciferase report gene assay, eukaryocyte transfection, quantitative real-time polymerase chain reaction(qRT-PCR) and Western blot technology. Our research will provide experimental and epidemiological evidences to individual precision medicine about the effects of microRNA on adiponectin level.

microRNA调控脂联素基因表达的机制还不十分清楚。基于我们前期研究发现miR-146a遗传多态与血清脂联素水平相关及Genecard数据库中提示miRNA-146a可调控脂联素基因表达的证据，提出miR-146a调控脂联素基因表达的假设。本研究首先通过基因组测序筛选miR-146a和脂联素基因3′UTR最小等位基因频率＞10%的多态位点及其单倍型作为研究对象，应用荧光素酶报告基因检测、真核基因转染、qRT-PCR及Western blot技术，结合我们前期脂联素基因5′UTR遗传多态影响脂联素基因表达的数据，从生物信息学、细胞和人群水平阐明miR-146a与脂联素基因（3′UTR和5′UTR）遗传多态相互作用共同调控脂联素基因表达的机制。研究结果为microRNA影响血清脂联素的个体化精准医疗提供实验及流行病学证据。

研究围绕影响糖脂代谢的关键蛋白质及调控microRNA，脂联素基因及miR-146a遗传多态为切入点展开。一方面，本项目结果显示，miR-146a调控脂肪细胞中的脂联素基因无表达差异，提示miR-146a在脂肪细胞中不能调控脂联素基因表达。进一步，在脂肪细胞中过表达miR-146a-3p后细胞表达谱分析，通过生物信息学分析及预测，双荧光报告基因实验确定miR-146a的靶基因为RPS6和TNFRSF10D；同时，转染携带rs2910164C等位基因表达载体的成熟miR-146a表达水平高于和G等位基因表达载体；且转染携带rs2910164C等位基因表达载体细胞中的RPS6和TNFRSF10D表达水平显著低于转染携带G等位基因表达载体。结果提示，miR-146a基因rs2910164可能是通过影响成熟miR-146a表达而进一步影响了miR-146a靶基因的表达水平。最后，本研究在人群研究中，证明miR-146ars2910164C等位基因可能增加中国人群2型糖尿病和代谢综合征发病风险。另一方面，本项目对脂联素基因5′非编码区进行测序分析，发现4个遗传多态位点：SNP-12140、SNP-11426、SNP-11377和SNP-110161，并证明脂联素基因SNP-12140其单倍型与中国人群2型糖尿病发病风险相关。对脂联素基因3′非编码区（3'UTR）进行测序分析，发现9个遗传多态位点：rs56354395、rs6773957、rs1063537、rs2082940、rs1063538、rs4686804、rs1063539、rs17846865和rs6414520；以上9个变异位点与中国人群2型糖尿病发病风险无关。但非糖尿病组中，与rs17846865C/C+C/T基因型个体相比，T/T基因型个体具有更低的空腹血糖水平和糖化血红蛋白水平，更高的低密度脂蛋白胆固醇水平。结果提示脂联素基因3'UTR rs17846865可能参与中国人群糖脂代谢。此外，对脂联素受体基因的研究提示，CDH13基因rs12596316AG杂合子可能增加中国人群2型糖尿病和代谢综合征的风险。结果为研究microRNA参与调控糖脂代谢机制及脂联素、脂联素受体在代谢性疾病中的作用奠定遗传学基础。