| Background:Rheumatoid arthritis(RA)is a chronic,systemic autoimmune disorder characterized by synovial hyperplasia of the joint,resulting in joint destruction,deformity,and loss of function.Under the stimulation of inflammatory factors,fibroblast like synoviocytes(FLS)in RA proliferate,migrate and invade,and play a key role in joint cartilage and bone erosion.Therefore,the search for targets for RA-FLS is of great research significance.Mesenchymal stem cells(MSCs)originate from a variety of tissues and have biological properties such as self-renewal,multidirectional differentiation potential,immunomodulation,and anti-inflammation.Current evidence supports MSC’s ability to reduce joint inflammation,bone erosion and destruction,which are key points in the treatment of RA.MSCs carry and deliver nucleic acids and proteins mainly through paracrine exosomes(Exos).Among them,miRNAs have been shown to play a mediating role in angiogenesis,antigen presentation,inflammatory response,and immune regulation.Human umbilical cord mesenchymal stem cells(hUCMSCs)have the advantages of wide source and convenient access to materials,and are relatively easy to obtain.Human umbilical cord mesenchymal stem cell-derived exosomes(hUCMSC-Exos)are mainly transmitted to recipient cells as lipid bilayer vesicles,which have the advantages of no immunogenicity and no risk of tumorigenesis.Our group has previously confirmed that hUCMSC-Exos can improve joint inflammation in animal models of RA in vivo,and inhibit synovial hyperplasia,cartilage and bone destruction;In vitro,miRNA may be transmitted to RA-FLS,which inhibits its migration and invasion ability,and promotes apoptosis.However,the key miRNAs and specific mechanisms of hUCMSC-Exos in the therapeutic role are not well understood.Therefore,based on the efficacy of hUCMSC-Exos,this article intends to further explore the molecular mechanism of hUCMSC-Exos in the treatment of RA and identify the relevant miRNA pathways.Objective:1.This study aims to validate the molecular mechanism of miRNA delivered from hUCMSC-Exos to RA-FLS in the treatment of RA.2.This study aims to investigate the effect of miR-451a and hUCMSCs-ExosmiR-451a on the biological characteristics of RA-FLS.Methods:1.Screen miRNAs delivered from hUCMSC-Exos to RA-FLS(1)Extraction and identification of primary RA-FLS and hUCMSC-Exos:primary FLS derived from synovial tissue and hUCMSCs derived from normal human sources in RA patients were extracted by pancreatic digestion and tissue adhesion,respectively;hUCMSC-Exos was extracted by differential centrifugation and identified by transmission electron microscopy,particle size analysis,WB and BCA methods.(2)Detection of miRNAs with significant differences in expression after intervention in RA-FLS by RT-qPCR:13 target miRNAs were obtained by mining the previously described miRNA sequencing(miRNA-seq)data,respectively,with hUCMSC-Exos,The exosome inhibitor GW4869 after intervention with hUCMSC intervened in the cell supernatant(CdMGW4869)and the cell supernatant of normal MSCs(CdMMSC)to intervene in RA-FLS,and the miRNAs with significant differential expression before and after the intervention of CdMGW4869 and hUCMSC-Exos were screened by RT-qPCR.2.Effects of overexpression of miR-451a on the biological traits of RA-FLS(1)Construction and verification of RA-FLS model overexpressing miR-451a:The plasmid overexpressing miR-451a was transfected with RA-FLS(FLSmiR-451a),and the mRNA expression level of miR-451a and downstream target gene ATF2 was detected by RT-qPCR.(2)Effects of overexpression of miR-451a on the biological traits of RA-FLS:CCK-8 method,scratch experiment and Transwell invasion experiment were used to detect the proliferation,migration and invasion ability of FLSmiR-451a,respectively.3.Effect of hUCMSC-ExosmiR-451a on the biological traits of RA-FLS(1)Construction of hUCMSC-Exos overexpressing miR-451a:miR-451a was loaded onto hUCMSC-Exos,hUCMSC-ExosmiR-451a was constructed,and the mRNA expression levels of hUCMSC-Exos and hUCMSC-ExosmiR-451a and downstream target gene ATF2 were detected by RT-qPCR after intervention in RA-FLS.(2)Effect of hUCMSC-ExosmiR-451a on the biological traits of RA-FLS:CCK-8 method,scratch experiment and Transwell invasion experiment were used to detect changes in cell proliferation,migration and invasion ability of hUCMSC-Exos and hUCMSC-ExosmiR-451a after intervention in RA-FLS,respectively.Results:1.Screen miRNAs delivered from hUCMSC-Exos to RA-FLS(1)Morphological identification of primary RA-FLS:cells crawled out about 3-7 days after bottle laying,and RA-FLS adherent growth under the microscope was visible,spindle-shaped or flat astrocytes,which gradually purified into single-morphological fibroblasts with increasing generation.(2)Morphological identification of primary hUCMSCs:cells can be seen crawling out about 7-14 days after bottle laying,and when they are transmitted to P3 generation,they can be observed under a microscope,and hUCMSCs can be seen to grow adherently,roughly spindle-shaped or spindle-shaped in morphology,and grow in parallel or vortex at high density.(3)Identification of hUCMSC-Exos:the morphology is "tea saucer-like" structure,the particle size is 116.5nm,the surface iconic proteins Alix,CD9,CD63 are expressed,and the concentration is 1.8ug/ul,which meets the exosome identification standards.(4)After the intervention of hUCMSC-Exos,CdMGW4869 and CdMMSC in RA-FLS48h,compared with the negative control CdMMSC group,13 miRNAs in the hUCMSC-Exos group were significantly upregulated,and significantly reduced in the CdMGW4869 group(P<0.05).Among them,the changes of miR-451a were obvious and of great research significance.2.Effects of overexpression of miR-451a on the biological traits of RA-FLS(1)Compared with RA-FLS in the blank control group,the expression of miR-451a in cells in the overexpression miR-451a(FLSmiR-451a)group was significantly increased,and ATF2 was significantly reduced(P<0.05),while there was no significant change in the miR-NC group(P>0.05).(2)Compared with RA-FLS in the blank control group,the proliferation,migration and invasion ability of FLSmiR-451a group was significantly reduced(P<0.05),while there was no significant change in the miR-NC group(P>0.05)。3.Effect of hUCMSC-ExosmiR-451a on the biological traits of RA-FLS(1)Compared with RA-FLS in the blank control group,the expression of miR-451a in the hUCMSC-Exos(150μg/ml)intervention group and the hUCMSC-ExosmiR-451a(150μg/ml)intervention group was significantly increased,and ATF2 was significantly reduced(P<0.05).The increase in the hUCMSC-Exos group was lower than in the hUCMSC-ExosmiR-451agroup(P<0.05).(2)Compared with RA-FLS in the blank control group,the proliferation,migration and invasion capacity of RA-FLS were significantly reduced in the hUCMSC-Exos(150μg/ml)intervention group and the hUCMSC-ExosmiR-451a(150μg/ml)intervention group(P<0.05).Compared with the hUCMSC-Exos group,the hUCMSC-ExosmiR-451a group had a more significant reduction effect(P<0.05).Conclusion:1.RT-qPCR detected the expression differences of 13 target miRNAs before and after CdMGW4869 and hUCMSC-Exos intervention in RA-FLS,and the results highlighted miR-451a as a potential delivery miRNA.2.A series of biological functional experiments were used to detect RA-FLS after FLSmiR-451a and hUCMSC-ExosmiR-451a intervention,and the results showed that both could upregulate miR-451a in RA-FLS and inhibit the mRNA expression level of ATF2,thereby inhibiting the proliferation,migration and invasion of RA-FLS.It was confirmed that hUMSC-Exos downregulated the expression level of ATF2 by delivering miR-451a to RA-FLS,resulting in a decrease in the proliferation,migration and invasion capacity of RA-FLS.This experiment provides a novel miRNA target for hUMSC-Exos for the treatment of RA. |
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