Potential mechanism by which iroquois homeobox 3 regulates the browning of perivascular adipose tissue in vascular injury

doi:10.12307/2026.169

Abstract

Abstract: BACKGROUND: Vascular injury-related diseases have garnered significant attention in the medical field, and the browning of perivascular adipose tissue is closely linked to these diseases. However, the regulatory mechanisms of specific genes involved in this process remain unclear.
OBJECTIVE: To investigate the potential mechanism by which iroquois homeobox 3 regulates the browning of perivascular adipose tissue in vascular injury.
METHODS: The perivascular adipose tissue-related single-cell sequencing data matrix GSE275779 was analyzed to investigate the expression levels and functions of iroquois homeobox 3 in various cell subpopulations. In conjunction with adipocyte-related microarray and sequencing data GSE44059, GSE7032, GSE185518, and GSE168387, differentially expressed genes were identified, and the expression level of iroquois homeobox 3 during the differentiation of browning adipocytes was validated. The downstream target genes of iroquois homeobox 3 were screened using the msigdb database and the ChIP-seq database GTRD. By disrupting iroquois homeobox 3 and overexpressing retinol saturase in adipocyte precursor cells, the mRNA and protein expression levels of adipocyte browning-related genes were detected using quantitative polymerase chain reaction and western blot analysis.
RESULTS AND CONCLUSION: Bioinformatics analysis results showed that adipocyte characteristic factors PR domain containing 16, cell death-inducing DFFA-like effector A, and uncoupling protein 1 were significantly downregulated in diabetic perivascular adipose tissue, and these genes were all involved in fat browning. Combined with high-throughput sequencing data analysis, iroquois homeobox 3 was found to be highly expressed in brown adipose tissue and to play a role in the differentiation process of brown adipocytes. Retinol saturase was identified as a downstream target gene of iroquois homeobox 3, and its expression levels differed during the differentiation of brown adipocytes. In mature brown adipocytes, knocking down iroquois homeobox 3 resulted in a decrease in the expression of retinol saturase and adipocyte browning-related markers (uncoupling protein 1, peroxisome proliferator-activated receptor gamma coactivator 1 alpha, PR domain containing 16). In the retinol saturase rescue experiment, overexpressing retinol saturase substantially upregulated the protein levels of adipocyte browning-related markers, but did not affect the expression of iroquois homeobox 3. This study preliminarily reveals the potential mechanism by which iroquois homeobox 3 regulates the browning of perivascular adipose tissue in vascular injury.

Key words: vascular injury, perivascular adipose tissue, bioinformatics, single cell sequencing, browning, iroquois homeobox 3

CLC Number:

Hu Xiaoyong, Song Qianhua, Yang Zhaoying, Tang Rui, Li Hongjian. Potential mechanism by which iroquois homeobox 3 regulates the browning of perivascular adipose tissue in vascular injury[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(22): 5671-5681.

Figures/Tables 5

2.1 单细胞测序分析发现损伤血管周围脂肪组织褐变减少血管损伤是糖尿病的常见并发症[20]。GSE275779数据集展示了正常和糖尿病大鼠血管周围脂肪组织中单细胞景观。经Seurat降维得到28个细胞亚群(图1A)，然后根据细胞标志物的表达特异性进一步降维，得到13个细胞亚群，分别为成纤维细胞、脂肪细胞、T细胞、巨噬细胞、B细胞、间皮细胞、内皮细胞、NKT细胞、神经施万细胞、肥大细胞、中性粒细胞、树突状细胞、单核细胞(图1B)。对脂肪细胞进一步分析，表达排名前5的特征因子包括脂肪酸结合蛋白4、过氧化物酶体增殖物激活受体γ、脂联素、长链酰基辅酶A合成酶1和细胞死亡诱导DFFA样效应蛋白C(图1C)，其中脂肪酸结合蛋白4、过氧化物酶体增殖物激活受体γ的统一流形逼近与投影聚类图谱如图1D所示。研究发现与对照组相比，PR结构域蛋白16、细胞死亡诱导DFFA样效应蛋白A和解偶联蛋白1等在糖尿病大鼠的血管周围脂肪组织中显著下调，且这些基因均参与脂肪褐变(图1E)[23]。结果表明，糖尿病血管损伤伴随着血管周围脂肪组织褐变的抑制。 2.2 IRX3在脂肪细胞中特异性表达并且在棕色脂肪中表达上调对单细胞测序数据GSE275779中对照和糖尿病大鼠脂肪细胞中差异基因(∣logFC∣> 1，adjusted P.value < 0.05)进行筛选(图2A)及功能富集发现差异基因富集到脂肪褐变这一过程(图2B)，表明具有血管损伤特性的糖尿病大鼠血管周围脂肪组织确实存在脂肪褐变的改变。进一步分析发现IRX3只在脂肪细胞中特征表达，并且在对照组血管周围脂肪组织的脂肪细胞中高表达，在糖尿病大鼠的血管周围脂肪组织的脂肪细胞中下调(图2C，D)，提示血管损伤时血管周围脂肪组织褐变减少可能与IRX3表达减少有关联。同样，在棕色脂肪相关芯片表达数据(GSE44059和GSE7032)中，也发现相较于白色脂肪组织，IRX3在棕色脂肪组织中显著高表达(图2E，F，P < 0.05)。 2.3 IRX3在脂肪褐变过程中表达上调使用GSE185518数据，包括诱导多潜能干细胞、分化过程中的棕色脂肪以及胚胎棕色脂肪组织验证，发现随着棕色脂肪细胞的成熟，IRX3的mRNA表达显著提高，并且在棕色脂肪组织中表达最高(图3A)。使用GSE168387展示了未分化的胚胎干细胞(Undiff-Stem cells)、中胚层期(Mesoderm stage)、棕色脂肪前体细胞(Brown adipocytes precursor)以及成熟的棕色脂肪细胞(Brown adipocytes)这一棕色脂肪诱导分化过程中基因表达数据，同样也观察到类似"

结果，从未分化的干细胞到成熟褐色脂肪细胞分化过程中IRX3的mRNA表达显著提高，其中以棕色脂肪前体细胞的表达量最高(图3B)。总之，棕色脂肪细胞分化过程中IRX3的mRNA表达显著提高。进一步基于GSE168387所有样本数据得到与IRX3表达具有相关性的基因(∣r∣> 0.95，P < 0.05)，并对其进行功能富集分析(图3C，D)和KEGG通路富集分析(图3E，F)，结果表明，正相关基因大多参与多潜能分化调控以及心肌细胞发育调控，而负相关基因参与能量平衡代谢。以上结果表明，IRX3可能参与棕色脂肪细胞分化过程。 2.4 IRX3可能介导RETSAT参与血管周围脂肪组织褐变 IRX3作为IRX家族的转录因子调控基因表达，为了进一步探讨IRX3参与棕色脂肪细胞分化的可能机制。此研究筛选IRX3分化过程中的下游靶基因。从msigdb (https://www.gsea-msigdb.org/)得到了IRX3的靶基因(IRX3_TARGET_GENES)，其中55个基因经ChIP-seq数据库GTRD鉴定，在其启动子区(TSS-1000，+100 bp) 含有1个或多个IRX3蛋白(UniProt:P78415) 结合位点。将IRX3的靶基因与GSE168387数据中和IRX3表达具有显著相关性的基因取交集(r > 0.95，OR < -0.95，P < 0.05)，发现核糖体蛋白S13、RETSAT可能是IRX3的靶基因(图4A)。RETSAT和IRX3表达也具有显著的正相关性(图4B)。进一步研究结果显示，在棕色脂肪细胞分化测序数据GSE185518和GSE168387中，观察到RETSAT的mRNA水平随着棕色脂肪细胞分化程度增加而有不同程度的升高(图4C，D，P < 0.05)，说明IRX3极有可能介导RETSAT参与棕色脂肪细胞的分化过程。 2.5 IRX3可上调RETSAT参与脂肪细胞褐变为了进一步验证IRX3/RETSAT是否调控脂肪褐变，检测3T3-L1脂肪前体细胞诱导成棕色脂肪细胞后IRX3表达变化，发现IRX3 mRNA和蛋白表达水平都显著升高(图5A)。在3T3-L1细胞中敲低IRX3并进行棕色脂"

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