SNPs Mutation of Tropoelastin Gene Affects Tropoelastin mRNA and Elastin Expressions in Human Aortic Smooth Muscle Cells

Objectives: We aimed to explore the effects of single nucleotide polymorphisms (SNPs) in tropoelastin gene on tropoelastin mRNA and elastin expressions in human aortic smooth muscle cells (HASMCs). Methods: Two SNP loci, rs2071307(G/A) and rs1785598(G/C), were selected to construct recombinant lentivirus vector carrying wild-type and mutant tropoelastin gene. Recombinant plasmids, pWSLV-02-ELN, pWSLV-02-ELN-mut1 and pWSLV-02-ELN-mut2, were constructed, prior to amplified by polymerase chain reaction (PCR) and sequenced. The prepared plasmids and the packaging plasmids (pVSV-G and psPAX2) were co-transfected into HEK293T cells, to obtain recombinant lentivirus carrying tropoelastin gene. Afterwards, HASMCs were infected with recombinant lentivirus and the positive cells sorted by flow cytometry were amplified. Four stable HASMC cell lines, including pWSLV-02-ELN, pWSLV-02-ELN-mut1, pWSLV-02-ELN-mut2 and pWSLV-02 vector, were constructed. The expressions of tropoelastin mRNA and elastin in HASMCs were detected by real-time quantitative reverse transcription-PCR (RT-qPCR) and western blot, respectively. Results: Recombinant plasmids, pWSLV-02-ELN-mut1, pWSLV-02-ELN-mut2 and pWSLV-02-ELN, were successfully constructed. Recombinant lentiviruses carrying tropoelastin gene were obtained via lentivirus packaging. After infection for 24 h, 3 d and 5 d in HASMCs, tropoelastin mRNA expressions in pWSLV-02-ELN-mut1 and pWSLV-02-ELN-mut2 groups were significantly lower than that of pWSLV-02-ELN group. Besides. After infection for 24 h, elastin levels in pWSLV-02-ELN-mut1 and pWSLV-02-ELN-mut2 groups were significantly lower than that in pWSLV-02-ELN group. Conclusions: SNPs mutation of tropoelastin gene affected the expression of tropoelastin mRNA and elastin, suggesting that the polymorphisms of rs2071307 and rs17855988 in tropoelastin gene might be important factors for AD development. Keywords: aortic dissection; human aortic smooth muscle cells; single nucleotide polymorphisms; lentivirus vector; tropoelastin gene; elastin.

研究目的：本研究旨在探讨弹性蛋白原基因（tropoelastin gene）中的单核苷酸多态性（single nucleotide polymorphisms, SNPs）对人主动脉平滑肌细胞（human aortic smooth muscle cells, HASMCs）内弹性蛋白原mRNA及弹性蛋白表达的影响。
研究方法：选取rs2071307(G/A)与rs1785598(G/C)两个单核苷酸多态性位点，构建携带野生型与突变型弹性蛋白原基因的重组慢病毒载体。成功构建重组质粒pWSLV-02-ELN、pWSLV-02-ELN-mut1及pWSLV-02-ELN-mut2，随后通过聚合酶链式反应（polymerase chain reaction, PCR）扩增并进行测序验证。将构建完成的重组质粒与包装质粒pVSV-G、psPAX2共转染至HEK293T细胞，以获得携带弹性蛋白原基因的重组慢病毒。此后，用该重组慢病毒感染人主动脉平滑肌细胞，并通过流式细胞术分选获得阳性细胞并扩增，最终构建pWSLV-02-ELN、pWSLV-02-ELN-mut1、pWSLV-02-ELN-mut2及空载体pWSLV-02共4株稳定转染的人主动脉平滑肌细胞系。分别采用实时定量反转录聚合酶链式反应（real-time quantitative reverse transcription-PCR, RT-qPCR）与蛋白质印迹法（western blot）检测细胞内弹性蛋白原mRNA及弹性蛋白的表达水平。
研究结果：成功构建pWSLV-02-ELN-mut1、pWSLV-02-ELN-mut2及pWSLV-02-ELN重组质粒，通过慢病毒包装获得携带弹性蛋白原基因的重组慢病毒。将重组慢病毒感染人主动脉平滑肌细胞后，分别于感染后24h、3d及5d检测发现，pWSLV-02-ELN-mut1组与pWSLV-02-ELN-mut2组的弹性蛋白原mRNA表达水平均显著低于pWSLV-02-ELN组。此外，感染后24h时，pWSLV-02-ELN-mut1组与pWSLV-02-ELN-mut2组的弹性蛋白水平同样显著低于pWSLV-02-ELN组。
研究结论：弹性蛋白原基因的单核苷酸多态性突变可影响弹性蛋白原mRNA及弹性蛋白的表达，提示弹性蛋白原基因中rs2071307与rs17855988位点的多态性可能是主动脉夹层（aortic dissection, AD）发生的重要危险因素。
关键词：主动脉夹层；人主动脉平滑肌细胞；单核苷酸多态性；慢病毒载体；弹性蛋白原基因；弹性蛋白。