摘要
The gasotransmitter role of H2S in mammalian has been extensively studied, and cystathionine gammalyase （CSE） is the major H2S-producing enzyme in vascular system. Dysregulation of CSE/H2S system was found in various pathophysiological conditions. MicroRNA （miRNA） and short interfering RNA （siRNA） are known to inhibit gene expression by mRNA degradation and/or translation repression. The regulation of CSE expression by miRNA and siRNA has been reported recently, but the off-target effect of miRNA and the lower knockdown efficiency of siRNA have shadowed the application of these approaches. In the present study, we designed CSE-specific miRNAs based on the rules of miRNA-mRNA complementary and human CSE cDNA sequence. The CSE-specific miRNAs significantly inhibited CSE expression and H2S production, increased reactive oxygen species generation, and induced proliferation of human aorta smooth muscle cells （HASMCs）. The designed CSE-specific miRNAs specifically targeted on CSE gene as evidenced by the direct inhibition of luciferase activity from reporter gene containing human CSE 3t-UTR sequence. The expression of other genes, such as estrogen receptor a, heme oxygenase 1, specificity protein 1, and 3-mercaptopyruvate sulfurtransferase, was not affected by the CSE-specific miRNAs.Compared with CSE-siRNAs, CSE-specific miRNAs dis- played significantly higher efficacy in suppressing CSE expression and H2S production, miR-143, a highly expressed miRNA in vascular system, down-regulated the expressions of CSE as well as other genes, such as insulin-like growth factor binding protein 5 and kruppel-like factor 4. miR-143 suppressed H2S production but had no effect on HASMC proliferation. In conclusion, CSE-specific miRNAs designed in our study provide a highly effective research tool for regulating CSE expression and H2S production. These CSE-specific miRNAs have potential as being novel therapeutic agents for treating vascular disorders related to abnormal oxidative stress and SMC growth.
基于miRNA:mRNA互补原则和CSE cDNA序列,设计了CSE特异的miRNAs.这些CSE特异的miRNAs显著地抑制CSE蛋白表达和H2S产生,增加活性氧的生成,并且诱导人类主动脉平滑肌细胞的增殖.通过构建包含CSE 3′-UTR序列报告基因和抑制荧光素酶活性实验,证实CSE特异的miRNA通过碱基互补结合CSE基因序列.CSE特异的miRNA没有影响其他基因的表达.与CSE特异的siRNAs相比,CSE特异miRNAs在抑制CSE基因表达和H2S的产生方面,展现出显著的高效率.miR-143是一个在心血管组织中高表达的miRNA,下调了CSE基因和其他基因的表达.miR-143抑制H2S的产生,但是对人类主动脉平滑肌细胞的增殖没有影响.本文设计的CSE特异miRNAs为研究调控CSE表达和H2S产生提供一个高效率的工具.这些CSE特异miRNAs具有成为治疗有关氧化应激异常和平滑肌细胞增长相关的心血管疾病的新型药物的潜在可能.
基金
supported by a grant-in-aid from the Heart and Stroke Foundation of Canada
