Abstract：
This study aims to investigate the impact of T-2 toxin on the regulation of downstream target genes and signaling pathways through exosome-released miRNA in the development of cartilage damage in Kashin-Beck disease (KBD). Serum samples from KBD patients and supernatant from C28/I2 cells treated with T-2 toxin were collected for the purpose of comparing the differential expression of exosomal miRNA using absolute quantitative miRNA-seq. Target genes of differential exosomal miRNAs were identified using Targetscan and Miranda databases, followed by GO and KEGG enrichment analyses. Validation of key indicators of chondrocyte injury in KBD was conducted using Real-time quantitative PCR (RT-qPCR) and Immunohistochemical staining (IHC). A total of 20 exosomal miRNAs related to KBD were identified in serum, and 13 in chondrocytes (C28/I2). The identified exosomal miRNAs targeted 48,459 and 60,612 genes, primarily enriched in cell organelles and membranes, cell differentiation, and cytoskeleton in the serum, and the cytoplasm and nucleus, metal ion binding in chondrocyte (C28/I2). The results of the KEGG enrichment analysis indicated that the Ras signaling pathway may play a crucial role in the pathogenesis of KBD. Specifically, the upregulation of hsa-miR-181a-5p and hsa-miR-21-3p, along with the downregulation of hsa-miR-152-3p and hsa-miR-186-5p, were observed. Additionally, T-2 toxin intervention led to a significant downregulation of RALA, REL, and MAPK10 expression. Furthermore, the protein levels of RALA, REL, and MAPK10 were notably decreased in the superficial and middle layers of cartilage tissues from KBD. The induction of differential expression of chondrocyte exosomal miRNAs by T-2 toxin results in the collective regulation of target genes RALA, REL, and MAPK10, ultimately mediating the Ras signaling pathway and causing a disruption in chondrocyte extracellular matrix metabolism, leading to chondrocyte injury.
摘要：
本研究旨在探讨T-2毒素通过外泌体释放的miRNA在大骨节病(KBD)软骨损伤发展过程中对下游靶基因和信号通路调控的影响。为了使用绝对定量miRNA-seq比较外泌体miRNA的差异表达,收集来自KBD患者的血清样品和来自用T-2毒素处理的C28/I2细胞的上清液。使用Targetscan和Miranda数据库鉴定差异外泌体miRNA的靶基因,其次是GO和KEGG富集分析。使用实时定量PCR（RT-qPCR）和免疫组织化学染色（IHC）对KBD软骨细胞损伤的关键指标进行验证。在血清中鉴定出20个与KBD相关的外泌体miRNAs,和软骨细胞中的13（C28/I2）。鉴定的外泌体miRNA靶向48,459和60,612个基因，主要富集在细胞器和细胞膜中，细胞分化，和血清中的细胞骨架，细胞质和细胞核，软骨细胞中的金属离子结合(C28/I2)。KEGG富集分析结果表明，Ras信号通路可能在KBD的发病中起着至关重要的作用。具体来说,hsa-miR-181a-5p和hsa-miR-21-3p的上调，随着miR-152-3p和hsa-miR-186-5p的下调，被观察到。此外,T-2毒素干预导致RALA显著下调，REL,和MAPK10表达。此外，RALA的蛋白质水平，REL,在KBD的软骨组织的表层和中层中，MAPK10显着降低。T-2毒素诱导软骨细胞外泌体miRNAs的差异表达导致靶基因RALA,REL,和MAPK10，最终介导Ras信号通路并导致软骨细胞细胞外基质代谢中断，导致软骨细胞损伤。
