Guo Li, Guo Hua, Zhang Yuanyu, Chen Zhi, Sun Jian, Wu Gaige, Wang Yunfei, Zhang Yang, Wei Xiaochun, Li Pengcui
Shanxi Key Laboratory of Bone and Soft Tissue Injury Repair, Department of Orthopedics, the Second Hospital of Shanxi Medical University, Taiyuan, China.
Bone Joint Res. 2023 Jul 7;12(7):433-446. doi: 10.1302/2046-3758.127.BJR-2022-0279.R2.
To explore the novel molecular mechanisms of histone deacetylase 4 (HDAC4) in chondrocytes via RNA sequencing (RNA-seq) analysis.
Empty adenovirus (EP) and a overexpression adenovirus were transfected into cultured human chondrocytes. The cell survival rate was examined by real-time cell analysis (RTCA) and EdU and flow cytometry assays. Cell biofunction was detected by Western blotting. The expression profiles of messenger RNAs (mRNAs) in the EP and transfection groups were assessed using whole-transcriptome sequencing (RNA-seq). Volcano plot, Gene Ontology, and pathway analyses were performed to identify differentially expressed genes (DEGs). For verification of the results, the A289E/S246/467/632 A sites of were mutated to enhance the function of HDAC4 by increasing HDAC4 expression in the nucleus. RNA-seq was performed to identify the molecular mechanism of HDAC4 in chondrocytes. Finally, the top ten DEGs associated with ribosomes were verified by quantitative polymerase chain reaction (QPCR) in chondrocytes, and the top gene was verified both in vitro and in vivo.
HDAC4 markedly improved the survival rate and biofunction of chondrocytes. RNA-seq analysis of the EP and groups showed that HDAC4 induced 2,668 significant gene expression changes in chondrocytes (1,483 genes upregulated and 1,185 genes downregulated, p < 0.05), and ribosomes exhibited especially large increases. The results were confirmed by RNA-seq of the EP versus mutated groups and the validations in vitro and in vivo.
The enhanced ribosome pathway plays a key role in the mechanism by which HDAC4 improves the survival rate and biofunction of chondrocytes.
通过RNA测序（RNA-seq）分析探索组蛋白去乙酰化酶4（HDAC4）在软骨细胞中的新型分子机制。
将空腺病毒（EP）和过表达腺病毒转染至培养的人软骨细胞中。通过实时细胞分析（RTCA）、EdU和流式细胞术检测细胞存活率。通过蛋白质免疫印迹法检测细胞生物学功能。使用全转录组测序（RNA-seq）评估EP组和转染组中信使核糖核酸（mRNA）的表达谱。进行火山图、基因本体论和通路分析以鉴定差异表达基因（DEG）。为验证结果，将HDAC4的A289E/S246/467/632 A位点进行突变，通过增加HDAC4在细胞核中的表达来增强其功能。进行RNA-seq以确定HDAC4在软骨细胞中的分子机制。最后，通过定量聚合酶链反应（QPCR）在软骨细胞中验证与核糖体相关的前十个DEG，并且在体外和体内对 top 基因进行验证。
HDAC4显著提高了软骨细胞的存活率和生物学功能。EP组和HDAC4组的RNA-seq分析表明，HDAC4诱导软骨细胞中2668个显著的基因表达变化（1483个基因上调和1185个基因下调，p<0.05），核糖体的增加尤为显著。EP组与突变HDAC4组的RNA-seq以及体外和体内验证证实了该结果。
增强的核糖体途径在HDAC4改善软骨细胞存活率和生物学功能的机制中起关键作用。
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