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.