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Abstract：
T-2 toxin and selenium deficiency are considered important etiologies of Kashin-Beck disease (KBD), although the exact mechanism is still unclear. To identify differentially expressed microRNAs (DE-miRNAs) in the articular cartilage of rats exposed to T-2 toxin and selenomethionine (SeMet) supplementation, thirty-six 4-week-old Sprague Dawley rats were divided into a control group (gavaged with 4% anhydrous ethanol), a T-2 group (gavaged with 100 ng/g·bw/day T-2 toxin), and a T-2 + SeMet group (gavaged with 100 ng/g·bw/day T-2 toxin and 0.5 mg/kg·bw/day SeMet), respectively. Toluidine blue staining was performed to detect the pathological changes of articular cartilage. Three rats per group were randomly selected for high-throughput sequencing of articular cartilage. Target genes of DE-miRNAs were predicted using miRanda and RNAhybrid databases, and the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway were enriched. The network map of miRNA-target genes was constructed using Cytoscape software. The expression profiles of miRNAs associated with KBD were obtained from the Gene Expression Omnibus database. Additionally, the DE-miRNAs were selected for real-time quantitative PCR (RT-qPCR) verification. Toluidine blue staining demonstrated that T-2 toxin damaged articular cartilage and SeMet effectively alleviated articular cartilage lesions. A total of 50 DE-miRNAs (28 upregulated and 22 downregulated) in the T-2 group vs. the control group, 18 DE-miRNAs (6 upregulated and 12 downregulated) in the T-2 + SeMet group vs. the control group, and 25 DE-miRNAs (5 upregulated and 20 downregulated) in the T-2 + SeMet group vs. the T-2 group were identified. Enrichment analysis showed the target genes of DE-miRNAs were associated with apoptosis, and in the MAPK and TGF-β signaling pathways in the T-2 group vs. the control group. However, the pathway of apoptosis was not significant in the T-2 + SeMet group vs. the control group. These results indicated that T-2 toxin induced apoptosis, whereas SeMet supplementation antagonized apoptosis. Apoptosis and autophagy occurred simultaneously in the T-2 + SeMet group vs. T-2 group, and autophagy may inhibit apoptosis to protect cartilage. Compared with the GSE186593 dataset, the evidence of miR-133a-3p involved in apoptosis was more abundant. The results of RT-qPCR validation were consistent with RNA sequencing results. Our findings suggested that apoptosis was involved in articular cartilage lesions induced by T-2 toxin, whereas SeMet supplementation antagonized apoptosis, and that miR-133a-3p most probably played a central role in the apoptosis process.
摘要：
T-2毒素和硒缺乏被认为是大骨节病（KBD）的重要病因，虽然确切的机制尚不清楚。为了鉴定暴露于T-2毒素和硒代蛋氨酸（SeMet）补充的大鼠关节软骨中差异表达的microRNAs（DE-miRNAs），将36只4周龄的SpragueDawley大鼠分为对照组（用4％无水乙醇灌胃），T-2组（用100ng/g·bw/dayT-2毒素灌洗），和一个T-2+SeMet组(用100ng/g·bw/dayT-2毒素和0.5mg/kg·bw/daySeMet)，分别。甲苯胺蓝染色检测关节软骨的病理变化。每组随机抽取3只大鼠进行关节软骨高通量测序。使用miRanda和RNA杂交数据库预测DE-miRNA的靶基因，并丰富了基因本体论和京都百科全书的基因和基因组途径。使用Cytoscape软件构建miRNA靶基因的网络图。从基因表达综合数据库获得与KBD相关的miRNA的表达谱。此外,选择DE-miRNA进行实时定量PCR(RT-qPCR)验证。甲苯胺蓝染色显示T-2毒素损伤关节软骨,SeMet能有效缓解关节软骨损伤。T-2组共50个DE-miRNA（28个上调，22个下调）与对照组,T-2+SeMet组的18个DE-miRNA（6个上调，12个下调）与对照组,和25个DE-miRNA（5个上调和20个下调）在T-2+SeMet组确定T-2组。富集分析显示DE-miRNAs的靶基因与细胞凋亡相关,在T-2组的MAPK和TGF-β信号通路中与对照组。然而,T-2+SeMet组的凋亡途径与对照组。这些结果表明T-2毒素诱导细胞凋亡,而SeMet补充拮抗细胞凋亡。T-2+SeMet组细胞凋亡和自噬同时发生T-2组,自噬可能通过抑制细胞凋亡来保护软骨。与GSE186593数据集相比，miR-133a-3p参与细胞凋亡的证据更为丰富。RT-qPCR验证结果与RNA测序结果一致。我们的发现提示T-2毒素诱导的关节软骨损伤与细胞凋亡有关。而SeMet补充剂拮抗细胞凋亡，miR-133a-3p很可能在细胞凋亡过程中发挥了核心作用。
