Mechanism Of MiR-9-5p Loss In Rheumatoid Arthritis Complicated With Peripheral Neuropathy

Rheumatoid arthritis(RA)is a systemic inflammatory autoimmune disease with joint as the main target of injury which is eventually causes joint destruction.The prevalence of rheumatoid arthritis is 0.34%-0.36%and the disability rate of non-regular treatment is 50-70%for 2-3 years.The main pain in patients with rheumatoid arthritis is joint pain and its loss of function.In addition to severe joint inflammation,peripheral nerve injury has also been found to play an important role in causing pain and loss of joint function in recent years.Rheumatoid arthritis neurological damage can involve peripheral nerves,central nervous system,autonomic nerves and muscles.Studies have found that the mechanism of peripheral nerve injury in rheumatoid arthritis may be different from the pathogenesis of arthritis,so the study of rheumatoid arthritis complicated with peripheral neuropathy is of great significance for the diagnosis and treatment of rheumatoid arthritis complicated with peripheral neuropathy.MicroRNAs(miRNAs)are a class of non-coding,single-stranded and small-molecule RNAs that are widely found in eukaryotes.They could regulate the translation or degradation of target mRNAs by complementary binding of nucleic acid sequences to specific target mRNAs.It also plays a negative regulatory role.At present,studies have shown that miRNAs are involved in several physiological processes such as growth,development,differentiation,and immune response.Their expression and dysfunction may lead to a variety of pathological phenomena such as tumors,autoimmune/inflammatory diseases,and neurodegenerative diseases.At present,at least 10 kinds of miRNAs are abnormally expressed in rheumatoid arthritis,and our scholars have used miRNA chip technology to screen 9 specifically expressed miRNAs in plasma of patients with rheumatoid arthritis.Among them,miR-122-3p,miR-3926,miR-9-5p,miR-3925-3p,miR-342-3p and miR-219-2-3p expressions were down-regulated,while miR-4764-5p,miR-181d and miR-4634 were up-regulated.Of them,some miRNAs are found associated with inflammatory factors.miRNA plays an important role in myelination and differentiation of Schwann cells.However,there are few studies focus on the relationship between miRNA and rheumatoid arthritis complicated with peripheral neuropathy.To explore the relationship between miRNA and rheumatoid arthritis complicated with peripheral neuropathy is conducive to the diagnosis and treatment of the disease.In this study,we used qRT-PCR to screen out miRNAs that may be involved in rheumatoid arthritis complicated with peripheral neuropathy,predict and analyze target genes related to miRNAs in rheumatoid arthritis complicated with peripheral neuropathy,and construct Schwann cell models by different inflammatory conditions.The damage of Schwann cells is tested to verify the role of the miRNA in rheumatoid arthritis complicated with peripheral neuropathy.Further analysis of the downstream gene regulated by the miRNA and construction of the miRNA regulating network for rheumatoid arthritis complicated with peripheral neuropathy will provide a new method for clinical diagnosis and treatment of rheumatoid arthritis peripheral neuropathy.Part ? Expression analysis of microRNAs in rheumatoid arthritis complicated with peripheral neuropathyObjective:Detection the expression levels of miR-9-5,miR-342-3p,miR-122-3p,miR-219-2-3p and miR-181a in clinical samples of patients with rheumatoid arthritis.Methods:1.Samples collection,including rheumatoid arthritis complicated with peripheral neuropathy group and rheumatoid arthritis group;2.Real-time PCR was used to detect the expression of miR-9-5p,miR-342-3p,miR-122-3p,miR-219-2-3p and miR-181a in clinical samples of patients with rheumatoid arthritis.Results:1.A total of 15 cases of rheumatoid arthritis complicated with peripheral neuropathy and 16 cases of rheumatoid arthritis were collected;2.Compared with patients with rheumatoid arthritis without peripheral neuropathy,the expression levels of miR-9-5p and miR-342-3p were significantly decreased in peripheral blood of rheumatoid arthritis complicated with peripheral neuropathy.The expression levels of miR-122-3p and miR-219-2-3p did not change significantly,while the expression level of miR-181a was significantly increased.Conclusion:The expressions of miR-9-5p and miR-342-3p were negatively correlated with rheumatoid arthritis complicated with peripheral neuropathy,especially for miR-9-5p.The expression of miR-181a was positively correlated with rheumatoid arthritis complicated with peripheral neuropathy.Part ? Functional analysis of miR-9-5p in rheumatoid arthritis complicated with peripheral neuropathyObjective:To verify the role of miR-9-5p in Schwann cell injury model and its regulatory target gene.Methods:1.Construction of Schwann cell model under inflammatory conditions by TNF-? combined with IL-6 stimulation;2.CCK-8 kit was used to detect the cell viability in different Schwann cell model groups at Oh,24h,48h and 72h after TNF-? combined with IL-6 stimulation;3.Flow cytometry was used to detect the cells of different cell model groups by TNF-?combined with IL-6 stimulation after 24h;4.The expression levels of miR-9-5p and REST were detected by Real-time PCR and western blot;5.Using luciferase reporter system to detect the targeted regulation of REST gene by miR-9-5p.Results:1.Compared with the control group,the cell viability of TNF-? combined with IL-6 stimulation group was significantly decreased at 24h,48h and 72h after TNF-? combined with IL-6 stimulation and the apoptosis was significantly increased after 24h stimulation;2.Compared with the control group,the expression of miR-9-5p was significantly decreased in the TNF-? combined with IL-6 stimulation group;3.Compared with the control group,the expression level of REST in the cells of RA+PNP group was significantly increased;the expression level of REST in RA+PNP group was negatively correlated with the expression level of miR-9-5p;The expression level of REST in Schwann cells of TNF-? combined with IL-6 stimulation group was significantly higher than that in the control group;4.Compared with the control group,the cell viability of TNF-? combined with IL-6 +miR-9-5p mimics group and TNF-? combined with IL-6+ si-REST group was significantly decreased in 48h and 72h after TNF-? combined with IL-6 stimulation and apoptosis was significantly increased at 24h.Compared with TNF-? combined with IL-6 + transfected control group,TNF-? combined with IL-6 + miR-9-5p mimics group and TNF-a combined with IL-6+ si-REST group,the cell viability was significantly increased in 48h and 72h after TNF-? combined with IL-6 stimulation,while the apoptosis decreased.5.Compared with the control,the relative activity of LUC was significantly decreased after application of miR-9-5p mimics in wild-type REST cells,while the relative activity of LUC was not observed obvious change after application of miR-9-5p mimics in mutant REST cells;6.Compared with TNF-a combined with IL-6 + transfected control group,miR-9-5p expression was significantly increased in TNF-? combined with IL-6 + miR-9-5p mimics group;The expression levels of REST in TNF-? combined with IL-6 + miR-9-5p mimics group and TNF-a combined with IL-6 + si-REST group were significantly reduced.Conclusion:1.The change of miR-9-5p expression has an important effect on the activity and apoptosis of Schwann cells;2.REST is negatively correlated with miR-9-5p,and REST acts as a target of miR-9-5p in inflammation-induced peripheral nerve injury;3.Overexpression of miR-9-5p or interference with REST can reduce the damage of Schwann cells under inflammatory conditions,indicating that miR-9-5p affects the state of peripheral nerve cells by regulating the expression of REST gene,which may be one of the mechanisms of rheumatoid arthritis complicated with peripheral neuropathy.Part ? Functional analysis of miR-132 in rheumatoid arthritis complicated with peripheral neuropathyObjective:To demonstrate the function and molecular pathway of miR-132 in rheumatoid arthritis complicated with peripheral neuropathy by establishing an in vitro cell model.Methods:1.Using qRT-PCR and western blot to detect the expression of miR-132,EP300,PTEN and FOXO3 in different cell models;2.CCK-8 kit was used to detect the cell viability in different cell model groups at Oh,24h,48h and 72h after TNF-? combined with IL-6 stimulation.3.Flow cytometry was used to detect the cells of different cell model groups by TNF-a combined with IL-6 stimulation after 24h.Results:1.The expressions of miR-132 in Schwann cells were significantly increased in TNF-a combined with IL-6 + miR-9-5p mimics group and TNF-a combined with IL-6 + si-REST group;However,the expression levels of EP300,PTEN and FOXO3 were significantly reduced;2.Compared with TNF-a combined with IL-6 + transfected control group,the cell viability of Schwann cells was significantly increased in TNF-a combined with IL-6 + miR-132 mimics group and TNF-a combined with IL-6 + si-REST at 48h and 72h after induction and apoptosis was decreased.On the contrary,the cell viability in TNF-a combined with IL-6 + si-REST group + miR-132 inhibitor group was significantly decreased while apoptosis was increased;3.Compared with TNF-a combined with IL-6 + transfected control group,the protein expressions of EP300,PTEN and FOXO3 were significantly reduced in TNF-a combined with IL-6 + miR-132 mimics group and TNF-a combined with IL-6 + si-REST group;However,the expressions of EP300,PTEN and FOXO3 were significantly increased in TN F-a combined with IL-6 + si-REST + miR-132 inhibitor group.Conclusion:Overexpression of miR-9-5p or interfering with REST can promote the expression of miR-132.The high expression of miR-132 inhibits the expressions of EP300,PTEN and FOXO3,which may be involved in the protection of nerve cells and prevent rheumatoid arthritis complicated with peripheral neuropathy.