Serum tsncRNAs Reveals Novel Potential Therapeutic Targets of Salvianolic Acid B on Atherosclerosis

Objective: Salvianolic Acid B (SalB) extracted from the roots and rhizomes of Salvia Miltiorrhiza Bunge has been utilized to delay the progression of atherosclerosis (AS). Its therapeutic mechanisms remain unclear. Pre-transfer RNA and mature transfer RNA (tsncRNAs) is a novel class of short non-coding RNA possessing potential regulating functions. In this study the novel therapeutic targets of SalB by tsncRNAs-sequencing were explored. Methods: Mice were randomly divided into three groups: control group (CON) AS model group (MOD) and SalB-treated group (SAB). The weight change aortic sinus plaque and inflammatory cytokines (IL-6 IL-10 TNF-) were assessed. Total RNA was extracted from serum to obtain tsncRNAs followed expression profiling by the Illumina NextSeq instrument. Subsequently tRNAscan and TargetScan were used to predict tsncRNAs targets. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were used to predict the bioinformatics analysis. Quantitative real-time PCR (qPCR) was the method to validate potential tsncRNAs and related targets. Results: A total of 8 tsncRNAs (fold change >1.5 and P-value <0.05) were significantly decreased in MOD group compared to CON group. Among them tRNA-Glu-CTC-014 and tRNA-Gly-GCC-074 were markedly increased by SalB treatment and validated with quantitative real-time PCR. GO analysis revealed that the altered target genes of the selected tsncRNAs were most enriched in protein binding and cellular process. Moreover KEGG pathway analysis demonstrated that altered target genes of tsncRNAs were most enriched in mitogen-activated protein kinase (MAPK) signaling pathway. Conclusion: In conclusion tRNA-Glu-CTC-014 and tRNA-Gly-GCC-074 were the potential therapeutic targets of SalB on AS treatment. Comparative gene expression profiling analysis of RNA-seq data for blood of mice (control group AS-model group and SalB-intervention group).

目的：从丹参（Salvia Miltiorrhiza Bunge）根及根茎中提取的丹酚酸B（Salvianolic Acid B, SalB）已被证实可延缓动脉粥样硬化（atherosclerosis, AS）的进展，但其具体治疗机制尚未明确。转运前RNA与成熟转运RNA（tsncRNAs）是一类具有潜在调控功能的新型短链非编码RNA。本研究通过tsncRNAs测序技术，探究丹酚酸B治疗动脉粥样硬化的潜在新型治疗靶点。
方法：将小鼠随机分为三组：对照组（CON）、动脉粥样硬化模型组（MOD）以及丹酚酸B干预组（SAB）。检测各组小鼠的体重变化、主动脉窦斑块情况以及炎症因子[白细胞介素-6（IL-6）、白细胞介素-10（IL-10）、肿瘤坏死因子-α（TNF-α）]水平。从血清中提取总RNA以获取tsncRNAs，随后通过Illumina NextSeq测序仪进行表达谱分析。后续采用tRNAscan与TargetScan工具预测tsncRNAs的靶基因，并通过基因本体论（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路分析开展生物信息学预测。采用实时定量聚合酶链反应（quantitative real-time PCR, qPCR）验证潜在的tsncRNAs及其相关靶基因。
结果：与对照组相比，模型组中共筛选出8个表达量显著下调的tsncRNAs（倍数变化>1.5且P值<0.05）。其中，tRNA-Glu-CTC-014与tRNA-Gly-GCC-074的表达经丹酚酸B干预后显著上调，并通过实时定量聚合酶链反应得到验证。基因本体论分析显示，所筛选tsncRNAs的差异靶基因最显著富集于蛋白质结合与细胞过程条目。此外，京都基因与基因组百科全书通路分析表明，tsncRNAs的差异靶基因最显著富集于丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）信号通路。
结论：综上，tRNA-Glu-CTC-014与tRNA-Gly-GCC-074可作为丹酚酸B治疗动脉粥样硬化的潜在治疗靶点。本研究通过对小鼠血液（对照组、动脉粥样硬化模型组与丹酚酸B干预组）的RNA测序数据进行比较基因表达谱分析完成相关研究。