Screening And Functional Study Of Long Non-coding RNAs In Rheumatoid Arthritis And The Effect Of Astragalosides

Rheumatoid arthritis（RA）,a systemic and autoimmune disease,is characterized by chronic inflammation,synovial hyperplasia,joint swelling and joint tenderness.It mainly occurs in individuals 35 to 50 years old and it is estimated that the morbidity rate of RA in women is 2³ times higher than that in men,especially in older women.These pathological changes resulted in functional limitations,working disability,and poor quality of life.A number of studies have demonstrated that RA is potentially related to immune genetic,environmental factors and hereditary factor,etc.Despite the intensive investigation of RA pathogenesis,the precise molecular mechanism of RA remains unclear.Therefore,it is vitally important to explore the molecular characteristics of RA,which could help find new diagnostic targets and find new mechanisms for the pathogenesis of RA.Previous studies on mechanisms of RA mainly focused on protein-coding genes.Recently,accumulating evidence suggested that rather than being transcriptional noise,many non-coding RNAs（nc RNAs）serve as master regulators that affect expression levels of dozens or even hundreds of target genes.Among them,long non-coding RNAs（Lnc RNAs）,a recently discovered class of nc RNAs,are m RNA-like transcripts longer than 200 nucleotides that have no or little protein-coding capacity and play important roles in a variety of biological processes,such as transcription,splicing,and translation.In addition,Lnc RNAs are also known to be associated with the pathogenesis of different kinds of diseases.However,the expression of Lnc RNAs and their biological functions in RA still remain unknown.In clinical practice,one of the important therapeutic approaches for RA in Western medicine is to alleviate pain,a strategy of symptomatic treatment.However,the pain-control strategy is not satisfied in clinic,and side effects occur.In this regard,traditional Chineses medicine has made significant contributions to the findings of new therapeutic products for the treatment of RA.The Astragalus membranaceous（Fisch.）,also known as Huang Qi,is one of the most commonly used herbal medicines in traditional Chinese medicine and exerts its functions of “Xing Zhi Tong Bi,Bu Qi Sheng Yang,Li Shui Xiao Zhong”.Astragalus and its compound have been widely used for treatment of RA for thousands of years in China.Astragalosides（AST）is the effective element extracted from astragalus,mainly containing astragaloside I^VIII,etc.Accumulating evidence demonstrated that AST had many biological effects,including anti-inflammatory,anti-oxidation,immunological regulation and anti-aging.However,to our knowledge,little attention has been paid to the differentially expressed Lcn RNAs in experimental arthritis after AST treatment.In this study,in order to advance our understanding of the potential molecular mechanism of experimental arthritis pathogenesis and the possible treatment targets for AST from the perspective of Lnc RNAs,we will carry out three lines of studies:（1）Lnc RNAs expression in adjuvant-induced arthritis rats revealed a potential role of Lnc RNAs in rheumatoid arthritis pathogenesis and in mediating the effect of astragalosides;（2）construction and analysis of Lnc RNA-mi RNA-m RNA network for identification of key Lnc RNAs in experimental arthritis;（3）Lnc RNA S56464.1 targeting mi R-152/Wnt pathway induced synovioeytes proliferation and was involved in the effect of astragalosides.Part Ⅰ Lnc RNAs expression in adjuvant-induced arthritis rats revealed a potential role of Lnc RNAs in experimental arthritis pathogenesis and in mediating the effect of astragalosidesObjectives: To identify the differentially expressed genes in experimental arthritis using gene chips,and explore the possible molecular mechanisms of experimental arthritis pathogenesis and the possible targets of astragalosides（AST）.Methods:The genes in adjuvant arthritis（AA）rats were measured by Arraystar.The differentially expressed genes with a fold change >1.5 and a P-value <0.05 were selected and analyzed.The coding-non-coding genes co-expression network was drawn based on the correlation analysis between the differentially expressed Lnc RNAs and m RNAs.Meanwhile,the real-time PCR（RT-q PCR）was performed to verify the differentially expressed critical Lnc RNAs.Results: According to the gene expression profiles,up to 260 Lnc RNAs（170 up-regulated and 90 down-regulated Lnc RNAs in the model group）and 675 m RNAs（422 up-regulated and 253 down-regulated m RNAs in the model group）,which differentially expressed in normal and model groups were found.GO analysis manifested that 4163 GO terms were enriched（2494 GO terms were significantly enriched among the up-regulated genes and 1669 GO terms were significantly enriched among the down-regulated genes）.Pathway analysis manifested that 42 pathway were enriched（25 pathways were significantly enriched among the up-regulated genes and 17 pathways were significantly enriched among the down-regulated genes）.Six Lnc RNAs（XR₀08357,U75927,MRAK046251,XR₀06457,DQ266363 and MRAK003448）were selected as critical Lnc RNAs and the expression trend had a strong consistency between RT-q PCR results and microarray data.According to the gene expression profiles,up to 75 Lnc RNAs（41 up-regulated and 34 down-regulated Lnc RNAs in the model group）and 247 m RNAs（171 up-regulated and 76 down-regulated m RNAs in the model group）,which differentially expressed among the three groups were found.GO analysis manifested that 135 GO terms were enriched（117 GO terms were significantly enriched among the up-regulated genes and 18 GO terms were significantly enriched among the down-regulated genes）.Pathway analysis manifested that 17 pathways were enriched（14 pathways were significantly enriched among the up-regulated genes and 3 pathways were significantly enriched among the down-regulated genes）.Four Lnc RNAs（MRAK012530,MRAK132628,MRAK003448 and XR₀06457）were selected as critical Lnc RNAs and their expression trend had a strong consistency between RT-q PCR results and microarray data.Conclusion: This study suggests that differentially expressed LncRNAs are associated with the development of experimental arthritis,and AST has a therapeutic action on AA in rats,which might be partly associated its effects with differentially expressed Lnc RNAsPart Ⅱ Construction and analysis of Lnc RNA-mi RNA-m RNA network for identification of key Lnc RNAs in rheumatoid arthritisObjectives: To reveal functional Lnc RNAs and identify the key Lnc RNAs in experimental arthritis.Methods : mi RNAs expression data were downloaded from National Center for Biotechnology Information（NCBI）Gene Expression Omnibus and particularly were obtained from published studies,Lnc RNAs/m RNAs expression data were obtained from our previously published study.Differentially expressed m RNAs（DEMs）,Lnc RNAs（DELs）,mi RNAs（DEMis）were screened according to the P-values <0.05 and fold change >2.Target Lnc RNAs of mi RNAs were predicted by RNAhybrid,target m RNAs of mi RNAs were predicted by miranda and targetscan.DELs and DEMs were merged with the target Lnc RNAs and m RNAs of DEMis,respectively.The co-expression Lnc RNAs and m RNAs were selected.Then the DEMis,co-expression Lnc RNAs and m RNAs were mapped into the Lnc RNAmi RNA-m RNA network based on the competitive endogenous RNA（ce RNA）theory.Meanwhile,Gene Ontology（GO）and pathway analysis was performed with Cytoscape plug-in Bin GO and Database for Annotation,Visualization,and Integration Discovery（DAVID）,respectively.According to the node degrees and the first relationship pairs of Lnc RNA-mi RNA and the secondary relationship pairs of mi RNA-m RNA,the key Lnc RNAs were selected,then GO and pathway annotations for each of the key lnc RNA was performed by using their first m RNAs neighbors in the key lnc RNA-mi RNA-m RNA sub-networkResults: The Lnc RNA-mi RNA-m RNA co-expression network was composed of 7 Lnc RNA nodes,90 m RNA nodes,24 mi RNA nodes and 301 edges.Functional assay showed that 147 GO terms and 23 pathways,including various metabolic processes and Wnt signaling pathway were significantly enriched.In addition,three Lnc RNAs（S56464.1,XR₀06437.1,J01878）were highly related to experimental arthritis and thus were selected as critical Lnc RNAs.Conclusion: This study suggests that specific Lnc RNAs are associated with the development of experimental arthritis and three Lnc RNAs（S56464.1,XR₀06437.1,J01878）can be used as potential diagnostic biomarkers and therapeutic targets.Part Ⅲ Lnc RNA S56464.1 targeting mi R-152/Wnt pathway induced synovioeytes proliferation and was involved in the effect of astragalosidesObjectives: To determine the effect of Lnc RNA S56464.1 targeting mi R-152/Wnt pathway on synovioeytes proliferation and its involvement in astragalosides-induced effects.Methods:Synovial cells were derived from adjuvant arthritis（AA）rats and cultured by tissue fragment transplantation.The proliferation of fibroblast-like synovial cells（FLS）was assessed by MTT assay.The expression levels of Lnc RNA S56464.1,mi R-152,β-catenin,C-myc,Cyclin D1,glycogen synthase kinase（GSK）-3β,and Secreted Frizzled-Related Protein（SFRP）4 m RNAs were determined by real-time fluorescent quantitative PCR.Meanwhile,the protein expression levels of β-catenin,C-myc,Cyclin D1,GSK-3β,p-GSK-3β（Ser9）,and SFRP4 were determined by immunofluorescence histochemical staining and Western blot.Results: Compared with the model group,si RNA Lnc RNA S6464.1 group and/or astragalosides（AST）group not only significantly inhibited the proliferation of fibroblast-like synovial cells in AA rat,but also significantly attenuated m RNA of Lnc RNA56464.1,β-catenin,C-myc,Cyclin D1,and protein expressions of β-catenin,C-myc,Cyclin D1,and p-GSK-3β（Ser9）/GSK-3β,up-regulated the expressions of mi R-152,SFRP4 both at protein and/or m RNA levels.Conclusion: The AST effects might be partially attributed to decreasing LncRNA S6464.1 and regulating mi R-152/Wnt pathway,which led to inhibition on the excessive proliferation of synovial cells.