硝酸甘油耐受大鼠主动脉差异表达基因与长链非编码RNA的鉴定与分析

doi:10.5246/jcps.2021.09.060

中国药学(英文版) ›› 2021, Vol. 30 ›› Issue (9): 725-735.DOI: 10.5246/jcps.2021.09.060

硝酸甘油耐受大鼠主动脉差异表达基因与长链非编码RNA的鉴定与分析

曹春芽¹, 贺凯¹, 曾振华¹, 刘建新¹, 蔡伟¹^,²^,^*(), 吴卫华¹^,^*()

1. 湖南医药学院药学院新型抗体药物及其智能运输系统湖南省重点实验室, 湖南怀化 418000
2. 耶鲁大学医学院药理研究所, 美国康涅狄格州纽黑文 06520

收稿日期:2021-03-14 修回日期:2021-05-11 接受日期:2021-06-13 出版日期:2021-09-27 发布日期:2021-09-27
通讯作者: 蔡伟, 吴卫华
作者简介:
+ Tel.: +86-745-2381210, E-mail: caiyunrui357743447@163.com
+ E-mail: wwh815@hotmail.com
基金资助:
Scientific Research Fund of Hunan Provincial Education Department (Grant No. 17B188, 18A490).

Transcriptome-wide identification of differentially expressed genes and long non-coding RNAs in nitroglycerin-tolerant rat aorta

Chunya Cao¹, Kai He¹, Zhenhua Zeng¹, Jianxin Liu¹, Wei Cai¹^,²^,^*(), Weihua Wu¹^,^*()

1 Hunan Province Key Laboratory for Antibody-based Drug and Intelligent Delivery System, School of Pharmaceutical Sciences, Hunan University of Medicine, Huaihua 418000, Hunan, China
2 Department of Pharmacology, Yale University School of Medicine, New Haven 06520, CT, USA

Received:2021-03-14 Revised:2021-05-11 Accepted:2021-06-13 Online:2021-09-27 Published:2021-09-27
Contact: Wei Cai, Weihua Wu

摘要/Abstract

摘要：

硝酸甘油耐受极大地限制了其临床长期使用, 其发生机制尚未完全阐明。本研究分析了硝酸甘油耐受大鼠主动脉转录组的变化。通过RNA测序鉴定及检测mRNAs和lncRNAs, 利用IPA进行差异基因的通路分析, 使用RT-qPCR验证测序结果。在硝酸甘油耐受大鼠主动脉组织中, 我们共鉴定了22 788个基因(RPKM > 0.1, 其中有14 720个蛋白编码基因、4408个lncRNAs), 差异表达的mRNAs有115个(上调65个, 下调50个), 差异表达的lncRNAs有104个(上调56个, 下调48个)。IPA分析显示经典通路"Rho家族GTP酶信号通路"被抑制, 6个上游调控因子发生了改变, 11个基因与"血压异常"有关。通过对其邻近基因的分析来预测差异表达lncRNAs的顺式调控靶基因, 结果显示26个蛋白编码基因附近有28个差异表达 lncRNAs, 许多差异表达mRNAs和差异表达lncRNAs的顺式调控靶基因与血压或细胞收缩的调节有关。本研究表明, 失调的mRNAs和lncRNAs参与了硝酸甘油耐受性的发生, 有望从中发现预防和逆转硝酸甘油耐受的潜在新靶点。

关键词: 转录组, 硝酸甘油, 耐受性, 长链非编码RNA, 主动脉

Abstract:

Tolerance to nitroglycerin (GTN) greatly limits its long-time application, and the underlying mechanism remains largely unexplored. In the present study, we aimed to investigate the comprehensive changes in the transcriptome of rat aorta tolerant to GTN by analyzing lncRNA expression. We employed the RNA sequencing (RNA-seq) technique to identify mRNAs and lncRNAs. Ingenuity pathway analysis (IPA) was used for pathway and functional analysis. RT-qPCR was used to validate the RNA-seq results. We identified 22 788 genes (reads per kilobase million [RPKM] > 0.1, 14 720 protein-coding genes and 4408 lncRNAs), including 115 differentially expressed (DE) mRNAs (65 up-regulated and 50 down-regulated) and 104 DE lncRNAs (56 up-regulated and 48 down-regulated), in GTN-tolerant aortas. IPA revealed the inhibition of a canonical pathway "Signaling by Rho Family GTPases" and alteration in six upstream regulators. Functional analysis showed that 11 genes were related to "disorder of blood pressure". We predicted the cis-target genes of DE lncRNAs by the analysis of their neighboring genes. The results revealed the 28 DE lncRNAs adjacent to the 26 protein-coding genes. Many DE mRNAs and cis-target genes of DE lncRNAs have been implicated in the regulation of blood pressure or cell contraction. These results suggested that the dysregulated mRNAs and lncRNAs contributed to the development of GTN tolerance and could serve as potential targets to prevent and reverse GTN tolerance.

Key words: Transcriptome, Nitroglycerin, Tolerance, LncRNA, Aorta

Supporting:

曹春芽, 贺凯, 曾振华, 刘建新, 蔡伟, 吴卫华. 硝酸甘油耐受大鼠主动脉差异表达基因与长链非编码RNA的鉴定与分析[J]. 中国药学(英文版), 2021, 30(9): 725-735.

Chunya Cao, Kai He, Zhenhua Zeng, Jianxin Liu, Wei Cai, Weihua Wu. Transcriptome-wide identification of differentially expressed genes and long non-coding RNAs in nitroglycerin-tolerant rat aorta[J]. Journal of Chinese Pharmaceutical Sciences, 2021, 30(9): 725-735.

图/表 6

Figure 1. Experimental flow and transcriptome landscape. (A) Experimental flow. (B) Venn diagrams depicting the overlap of the expressed genes (RPKM > 0.1) in the aortas of control rats (blue) and GTN-tolerant rats (yellow).

Figure 2. DE genes after GTN tolerance. Volcano plots of DE mRNAs (A) and DE lncRNAs (B). |Log2FC| = 1.0 was used as a threshold.

Table 1. RNA-seq and RT-qPCR results of the selected genes in the aorta of GTN-tolerant rats as compared with the controls.

Table 2. IPA of the DE mRNAs.

Table 3. RNA-seq and RT-qPCR results of the top genes in IPA.

Table 4. Lists of lncRNAs within a reference gene intron or that overlapped with a reference gene on the opposite strand.

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硝酸甘油耐受大鼠主动脉差异表达基因与长链非编码RNA的鉴定与分析

Transcriptome-wide identification of differentially expressed genes and long non-coding RNAs in nitroglycerin-tolerant rat aorta

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