Human umbilical cord mesenchymal stem cell-derived exosomes deliver miRNA-451a targeting ATF2 to inhibit rheumatoid arthritis [RNA-seq]
Background and aim: Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints. Synovial fibroblasts (SFs) play a key role in the pathogenesis of RA. Dysregulation of MicroRNAs (miRNAs) expression in SFs mediates the development of RA. Exosomes (Exos) can transfer miRNAs between cells and have been validated as carriers for the delivery of therapeutic molecules.This study aimed toinvestigate the therapeutic potential of human umbilical cord mesenchymal stem cells (hUCMSC)-Exos deliveringmiR-451a to regulate ATF2 for RA both in vivo and in vitro. Methods: In this study, hUCMSC and RASF were isolated and identified, and hUCMSC-EXOs were extracted and characterized. The influence of hUCMSC-Exos on RASF was detected. hUCMSC-Exos RNA was labeled and hUCMSC-Exos targeting RA-SFs was traced. AGO2 promoted miRNA maturation, and hUCMSCKD-AGO2 was prepared by knocking down AGO2 in hUCMSC. hUCMSCKD-AGO2-Exos was extracted and characterized,and their influence on RASFs was detected. mRNA and miRNA profiles before and after hUCMSC-Exos intervention in RASFs were mapped to identify differential miRNAs. RT-qPCR was used to verify the differential miRNAs, with miR-451a finally selected as the target gene.The effect of miR-451a on SFs was detected. The latent binding of miR-451a to activating transcription factor 2 (ATF2)was analyzed. The effect of hUCMSC-ExosmiR-451a on SFs was detected, and the expression of miR-451a and ATF2 was measured by RT-PCR.In vivo, hUCMSC-ExosmiR-451a was injected into the ankle joint of CIA rats, and arthritis index, joint imaging and synovial pathology were assessed. The expression of miR-451a and ATF2 in synovial tissue was detected. Finally, the safety of hUCMSC-ExosmiR-451a in CIA rats was evaluated. Results: This study revealed that hUCMSC-Exos can inhibit RASF proliferation, migration and invasion through miRNAs. High throughput sequencing detected 13 miRNAs that could be transmitted from hUCMSC to RASF via hUCMSC-EXOs. MiR-451a inhibited RASF proliferation, migration and invasion by regulating ATF2. hUCMSC-Exos loaded with miR-451a targeted ATF2 to inhibit RASF proliferation, migration and invasion, and improve joint inflammation and imaging findings in CIA rats. Conclusions: This study demonstrates that miR-451a carried by hUCMSC-Exos canplay a role ininhibiting RASF biological traits and improving arthritis in CIA rats by inhibiting ATF2. The findings study suggest a promising treatment for RA and provide insights into the mechanism of action of hUCMSC-Exo in RA. Overall design: To investigate which miRNAs were delivered to RA SFs by hUCMSC-Exos, we divided 6 RASFs derived from various genotypes into the blank control group and the hUCMSC-Exos intervention group. The blank control group was treated with complete medium while the hUCMSC-Exos intervention group was treated with 150µg/ml hUCMSC-Exos. After 48 hours of intervention, RASFs were collected. We identified the miRNAs in hUCMSC-Exos and detected the miRNAs and mRNAs in the RASFs of both the blank control group and the hUCMSC-Exos intervention group. Subsequently, the gene expression profiles were analyzed using RNA-seq data. To identify differentially expressed genes, a comparative analysis of the RNA-seq data was conducted. We intersected miRNAs that were differentially upregulated in RASFs after hUCMSC-Exos intervention with miRNAs in hUCMSC-Exos. The resulting differentially expressed miRNAs were further used to predict target genes. From the predicted target genes, we selected the ones that overlapped with the down-regulated mRNAs identified through transcriptome sequencing. Finally, GO function annotation and KEGG pathway analysis were performed for the selected intersection mRNAs.