單核細胞趨化蛋白-1(MCP-1)之rs13900 微小核糖核酸單一核苷酸多態性體外分析實驗

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Title:	單核細胞趨化蛋白-1(MCP-1)之rs13900 微小核糖核酸單一核苷酸多態性體外分析實驗 In Vitro Study of miRSNP (rs13900) on MCP-1 Gene
Authors:	莊淑靜
Contributors:	生物科技研究所
Keywords:	單核細胞趨化蛋白-1 微小核糖核酸單一核苷酸多態性微小核醣核酸
Date:	2017
Issue Date:	2017-04-07 10:26:17 (UTC+8)
Abstract:	研究目的：趨化因子家族近年來被認為是與免疫球蛋白參與免疫反應的一群蛋白分子。單核細胞趨化蛋白-1(Monocyte Chemoattractant Protein-1；MCP-1；CCL2)在各種器官患有疾病時皆有表達，而當細胞遭受細胞激素或內毒素的刺激時就會分泌MCP-1。而MCP-1所引起的相關疾病有：粥狀動脈硬化、肺部發炎及纖維化及風濕性關節炎等。而微小核糖核酸單一核苷酸多態性（miRSNP）為一種具有功能性之單一核苷酸多態性（Single Nucleotide Polymorphism；SNP），可影響微小RNA（miRNA）與訊息RNA（mRNA）結合，進而調節基因表現。本實驗利用電腦預測發現rs13900為MCP-1基因上的一個miRSNP，可與微小核醣核酸miR-624-3p以及miR-609結合，因此想進一步探討此miRSNP是否因為此2種miRNA而調節MCP-1基因的表現。研究方法：實驗方法主要利用基因選殖將含此SNP不同之變異基因型的3端非轉譯區(3’ untranslated region；3’UTR) 之序列轉入螢光載體，再將載體、miR-624-3p及miR-609 微小核糖核酸模擬物利用轉染送入HEK-293細胞株後，測定螢光蛋白Luciferase的表現量。結果：結果顯示，miR-624-3p在濃度為100nM時，帶有C等位基因和T等位基因的組別與控制組相比皆有明顯的下降(C等位基因p=0.04/ T等位基因p=0.03)。而再將C等位基因與T等位基因在濃度為100nM的組別相比較，T等位基因組有明顯的降低(p<0.05)。結論：目前結果顯示rs13900的多態性會影響miRNA miR-624-3p與mRNA的結合，而帶有等位基因T的基因序列與miRNA結合強度較C等位基因佳，與預測結果相符。接下來還會以西方墨點法以及逆轉錄聚合酶連鎖反應分別測定蛋白質和基因表現，期望能提供MCP-1相關疾病新的治療方式。 Purpose: Chemokines family is considered be involved in immune response immunoglobulin in recent years. Monocyte chemoattractant protein -1 ( MCP-1; CCL2) is expression at various organs with disease, and it secreted when cells stimulation by cytokines or endotoxin. The diseases including atherosclerosis, pulmonary inflammation, fibrosis, and rheumatoid arthritis are associated with MCP-1. Polymorphisms in microRNA target sites (miRSNP) as a functional single nucleotide polymorphism (SNP), can affect microRNA (miRNA) and mRNA in combination, and then regulation of gene expression. In this study, we predicted that rs13900 is a miRSNP in MCP-1 gene in silico, and it can regulate the activity for binding with miRNA hsa-miR-624-3p and hsa-miR-609. Therefore, we want to further investigate whether this miRSNP regulated MCP-1 gene expression because hsa-miR-624-3p and hsa-miR-609. Method: We cloned the sequences which containing the variant alleles in 3’ untranslated region (3’UTR) into the luciferase vector, and co-transfected with miRNA mimic hsa-miR-624-3p into HEK293 cells. Their activities further characterized by in vitro reporter assays. Result: The reporter assays showed that luciferase activity significantly decreased by rs13900-G or T-containing 3’ UTRs for hsa-miR-624-3p miRNAs then scramble control miRNA (p = 0.04 for C allele and p = 0.03 for T allele, respectively). Further, miR-624-3p at a concentration of 100 nM showed significant decrease in the rs13900-T- then C-containing 3’ UTRs (P =0.034). Conclusion: The result shows that rs13900 polymorphism affects miRNA miR-623-3p binding with mRNA. The rs13900-T allele has better binding activity with miRNA then C allele, consistent with the prediction. Western blot and reverse transcription-PCR will perform in next step for detection the protein and gene expression of MCP-1. It hope that this study will provide a new treatment for MCP-1 related disease.
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