Isolation And Identification Of Exosomes From Fibroblast Like Synoviocytes In Rheumatoid Arthritis And Analysis Of MiRNAs Expression Profile

Objective: In this study,the fibroblast like synoviocytes in Rheumatoid arthritis(RA-FLS)and normal FLS(HFLS)were taken as the objects,which exosomes were obtained.The mi RNAs expression profile of their exosomes was analyzed by sequencing technology,Meanwhile the heterogeneously(differentially)expressed mi RNAs were obtained and verified,with new mi RNA obtained by prediction.GO enrichment analysis method was used to predict the target gene of new mi RNA.KEGG enrichment analysis was used to predict the possible signal pathway.They were used to clarify the difference of mi RNAs expression in exosomes from RA-FLS and HFLS and to provide a new strategy for RA disease diagnosis and prognosis research.Methods:(1)Isolation and identification of exosomes from RA-FLS and HFLS: three groups of RA-FLS and HFLS were used as experimental subjects.After cell resuscitation and subculture,exosomes of FLS were obtained by ultracentrifugation.The morphological characteristics of exosomes were observed by transmission electron microscope after dyeing with uranyl acetate / lead acetate.The particle size was calculated dynamically by particle size analyzer.The concentration and purity were measured by enzyme marker.The surface markers(CD81,CD63,CD9)were identified by Western blot.(2)Sequencing analysis and comparison of the mi RNAs of exosomes isolated from RA-FLS and HFLS: total small RNA was extracted by kit method.Enzyme labeling instrument was used to test its concentration and quality.The qualified mi RNA was constructed and sequenced by Illumina Hi Seq-4000 sequencing.We make statistics of the length distribution and copy number of the raw data,and then compare it with the mi RNA gene library to obtain clean reads.The mi RNAs annotated and analyzed through database were defined as known mi RNAs,whereas the rest ones were defined as new mi RNAs.Differential expression of the new mi RNAs was analyzed through the DESeq algorithm,bioinformatics analysis was used to screen the mi RNAs expressed by the traveling heterosexual,and then RT-q PCR was used to detect those mi RNAs' expression level.(3)Bioinformatics analysis of differentially expressed mi RNA: We take the differentially expressed mi RNAs as our research object.The potential target genes were predicted by GO enrichment analysis,and the signal pathways were predicted by KEGG enrichment analysis.Results:(1)Exosome extraction: the exosomes of RA-FLS and HFLS were obtained by ultracentrifugation,and they were observed as double-layer capsule structure with a saucer type and hemispherical structure of one side depression by transmission electron microscopy.The ranges of the diameters of the samples measured by NTA were among 30-150 nm.The Western blot was used to determine the expression of CD9,CD63 and CD81 in samples.(2)Sequencing and analysis of micro RNAs of synovial exosomes: the concentration and quality of mi RNA were qualified.The original sequence from Illumina hiseq-4000 sequencing,raw reads,were compared and filtered to get clean reads,with a peak length distribution of about 21-25 nt.The known mi RNAs were compared with different databases and annotated.The statistical results of base preference showed that the first base of the sequence was biased to the base U,meanwhile,No.2-4 was short of U,with both No.7 and No.10 inclined to A.The total length of the sequence was 15-25 nt.Mi RNAs without comparison were new mi RNAs.(3)Analysis and verification of differential expression of mi RNAs from exosomes extracted from FLS: Significant differential expressions were found in 128 mi RNAs,among which 59 mi RNAs were up regulated.On the contrary,the rest 69 mi RNAs were down regulated after DESeq calculation.Bioinformatics analysis showed that five mi RNAs(mi R-100-5p,mi R-34a-5p,mi R-199a-3p,mi R-29a-3p,hsa-let-7b-5p)were over expressed in exosomes which extracted from FLS and were quantified by RT-q PCR.The expression level of the above five mi RNAs were consistent with the sequencing results,which proved that the sequencing results were reliable.(4)Functional analysis of differentially expressed mi RNAs: GO enrichment analysis showed that differentially expressed mi RNA participated in the binding process of specific substances,such as protein binding.Cell component analysis showed that differentially expressed mi RNAs were closely related to the composition of cytoplasm?cell membrane?Golgi matrix and organelle.In terms of biological processes,they were involved in the regulation of transcription,transcription and DNA template,protein phosphorylation,positive regulation of biological processes and development of nervous system.The results of KEGG enrichment analysis showed that the signal pathways involved in differentially expressed mi RNA were endocytosis,calcium signaling pathway,insulin signaling pathway,Erb B signaling pathway,neurotrophin signaling pathway,adrenergic signaling pathway,etc.Conclusion:(1)The exosomes of RA-FLS and HFLS were successfully isolated and identified,and there were different expression patterns of mi RNAs in RA-FLS and normal FLS exosomes.(2)The mi RNAs in exosomes derived from RA-FLS may be involved in the regulation of RA,which is related to the biological function of exosomes.(3)The purpose of this study is to investigate the differential expression of micro RNAs in exosomes of FLS,and to analyze their possible target genes and signal pathways,which provides new research strategies for the diagnosis,treatment and prognosis of RA.