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SRC1/SRC2双基因敲除小鼠胎肺转录组分析

禹娅琴¹, 陈怀艳¹, 刘媛媛¹, 高路^1,2,3,*

¹海军军医大学基础医学院生理学教研室，上海 200433；²上海市辅助生殖和生殖遗传重点实验室，上海 200433；³上海市胚胎源性疾病重点实验室，上海 200433

摘要

甾体激素受体共激活因子(steroid receptor coactivators, SRC)显著提高各种甾体激素受体的转录活性，在摄食、睡眠、应激反应和繁殖等多种生理功能中发挥着重要的调节作用。前期研究发现SRC1/SRC2双基因敲除(SRC1/2 double-knockout, SRC1/2 dKO)的小鼠会出现分娩启动的延迟，部分原因是由于胎肺的发育不全和肺表面活性物质相关蛋白A (pulmonary surfactant protein-A, SP-A)、血小板激活因子(platelet activating factor, PAF)分泌减少所引起。然而，对于SRC1/2 dKO小鼠胎肺基因表达的变化，尚缺乏全转录组层面的系统解析。本研究选用SRC1基因敲除(SRC1 knockout, SRC1KO)、SRC2基因敲除(SRC2 knockout, SRC2KO)、SRC1/2 dKO与野生型(wild-type, WT)小鼠孕18.5天胎肺样本，利用Illumina平台进行转录组mRNA测序，筛选差异表达基因，并进行GO和KEGG功能注释和富集分析。结果显示，所有样本的碱基质量值Q30占比均在92%以上，SRC1KO和SRC2KO与WT胎肺相比分别有61和32个差异基因；SRC1/2 dKO与WT、SRC1KO和SRC2KO胎肺相比分别有480、11和901个差异基因，其中Aspg、Crispld2、Eln、Ntsr2、Slc10a6和Vgll3为SRC1/2 dKO与其余基因型小鼠相比特有的差异表达基因，实时荧光定量PCR及蛋白质免疫印迹验证了转录组测序结果可靠。SRC1/2 dKO与WT小鼠胎肺差异基因的 GO 分析结果显示，差异基因在细胞组分上显著富集于细胞外空间、细胞外区域和细胞外基质等条目，在生物学过程大类中则与多器官的发育密切相关。KEGG 通路分析表明，差异基因主要富集于补体系统、细胞外基质-受体的相互作用和蛋白质消化与吸收等信号通路。本研究从转录组水平全面揭示了SRC1/2 dKO所致的胎肺基因表达的变化，为妊娠过程中胎肺的发育调控机制研究和影响分娩启动的胎源性因子的进一步筛选提供了新的理论依据。

关键词： 甾体激素受体共激活因子; 基因敲除; 胎肺发育; 分娩启动; 转录组测序

Transcriptome analysis in fetal lungs of SRC1/SRC2 double-knockout mice

YU Ya-Qin¹, CHEN Huai-Yan¹, LIU Yuan-Yuan¹, GAO Lu^1,2,3,*

¹Department of Physiology, College of Basic Medical Sciences, Naval Medical University, Shanghai 200433, China；²Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Shanghai 200433, China；³Shanghai Key Laboratory of Embryo Original Diseases, Shanghai 200433, China, Shanghai 200433, China

Abstract

Steroid receptor coactivators (SRCs) significantly increase the transcriptional activity of various steroid hormone receptors, and play an important regulatory role in a variety of physiological functions such as food intake, sleep, stress response and reproduction. Previous studies have found that pregnant mice carrying fetuses with SRC1/2 double-knockout (dKO) manifested delayed labor, partly due to the hypoplasia of fetal lungs and the decreased secretion of pulmonary surfactant protein-A (SP-A) and platelet activating factor (PAF). However, there is still a lack of systematic analysis of the changes in gene expression at the whole transcriptome level in the fetal lungs of SRC1/2 dKO mice. In this study, the SRC1KO, SRC2KO, SRC1/2 dKO and wild-type (WT) mouse fetal lung samples were collected at 18.5 days post coitus. The Illumina platform was employed for transcriptome mRNA sequencing, and then the differentially expressed genes (DEGs) were annotated and analyzed by GO and KEGG analysis. The results showed that the proportion of quality score of the sequencing data above Q30 in all samples was more than 92% and passed the quality control. Compared with WT fetal lungs, SRC1KO and SRC2KO fetal lungs had 61 and 32 DEGs, respectively; SRC1/2 dKO fetal lungs had 480, 11 and 901 DEGs compared with WT, SRC1KO and SRC2KO fetal lungs, respectively. Among these genes, Aspg, Crispld2, Eln, Ntsr2, Slc10a6 and Vgll3 were the unique DEGs of SRC1/2 dKO fetal lungs compared with other genotype mice. Real-time PCR and Western blotting verified the reliability of transcriptome sequencing results. The GO analysis of the DEGs between SRC1/2 dKO and WT mouse fetal lungs showed that the DEGs were significantly enriched in the extracellular space, extracellular region, and extracellular matrix in terms of cellular component. In the biological process, they were significantly enriched in the term of development of multiple organs. KEGG pathway analysis showed that the DEGs were mainly enriched in signaling pathways such as the complement system, extracellular matrix-receptor interactions, and protein digestion and absorption. In summary, this study comprehensively revealed the changes of gene expression in the fetal lungs of SRC1/2 dKO mice at the transcriptome level, which provides a new theoretical basis for the study of the developmental regulatory mechanism of the fetal lung during pregnancy, and the fetus-derived signals that affect the initiation of labor. 

Key words: steroid receptor coactivator; gene knockout; fetal lung development; parturition; transcriptome sequencing

收稿日期：　　录用日期：

通讯作者：高路　　E-mail: roadgao@163.com

DOI: 10.13294/j.aps.2022.0018

引用本文：

禹娅琴, 陈怀艳, 刘媛媛, 高路. SRC1/SRC2双基因敲除小鼠胎肺转录组分析[J]. 生理学报 2022; 74 (2): 246-254.

YU Ya-Qin, CHEN Huai-Yan, LIU Yuan-Yuan, GAO Lu. Transcriptome analysis in fetal lungs of SRC1/SRC2 double-knockout mice. Acta Physiol Sin 2022; 74 (2): 246-254 (in Chinese with English abstract).