| Background and objectiveRheumatoid arthritis(RA)is a common disease in the Department of Orthopedics and Rheumatology.It can occur at any age,especially in young adults.The incidence of female is significantly higher than that of males.RA is a systemic autoimmune disease characterized by erosion of the joint structure and chronic synovial inflammation.The main symptoms are symmetry and progressive polyarthritis of chronic inflammation.Most of the early onsets are mainly small joints such as hands and wrists.In the middle and late stages,large knees,hips,and other joints can be involved,resulting in joint swelling,pain,deformity,and adverse activities.The pathological features of rheumatoid arthritis are synovial cell hyperplasia.Vascular vasculogenesis,When lesions invading synovial cartilage and bone tissue it can cause joint destruction and function loss.Long course,Long course can lead to heart,lung,kidney and other organs and nervous system involvement.RA can not be cured and high morbidity make RA a huge threat on health and quality of life.The patient had to bear the pain of decades of illness,The pathogenesis of RA has remained unclear,and its pathogenesis is a complex process with multiple links and multi-factor involvement.Some scholars have suggested that foreign antigens or their own potential antigenic variation may stimulate primary immune cells and fibroblasts in synovial tissue.Synovial cells,etc.,induce inflammation in the synovial tissue,and this inflammatory response is transiently reversible;however,there are some factors that are susceptible to rheumatoid arthritis in patients with genetic background of RA family susceptibility.Afterwards,the immune tolerance of the individual to the autoantigen is lost,and an adaptive immune response against the autoantigen is induced,so that the inflammatory response is sustained and degenerated into a chronic course.In the past decade or more,although researchers have gone deep into the cellular,molecular,and even genetic levels of RA pathogenesis,they have made some progress.The discovery of a large number of RA-related genes or pathogenic factors has enabled RA to achieve therapeutic methods.Continuous progress has,to a certain extent,controlled the occurrence and development of RA and improved the quality of life of patients.However,the etiology of RA is still not elucidated,which makes it difficult to find specific therapeutic methods.It is particularly important to continue to explore the pathogenesis of RA.Studies have showed that synovial tissue hyperplasia is not only the first link,but also one of the main features throughout the disease process.Excessive proliferation of synovial cell is the main reason for the synovial tissue hyperplasia,which can continue even after detaching from the inflammatory environment intervention.The mechanism of synovial cell hyperproliferation-is complex,and at present it is thought to be associated with active proliferation and apoptosis inhibition.Systematic studies on the mechanisms that may induce synovial cell hyperproliferation and apoptosis decrease are of great help in elucidating the etiology of RA and finding effective treatments.Epigenetics is a branch of the genetics that studies the inheritable gene expression changes without nucleotide sequences change.Non-coding RNA-regulated target gene modifications belongs to the generalized epigenetic category,of which microRNA is the most studied.In previous experiments we found miR-143-3p was high expressed in synovial tissue of RA patients,suggesting that miR-143-3p is likely associated with RA.It is predicted that the insulin-like growth factor 1 receptor(IGF1R)and insulin-like growth factor 5(IGFBP5)are the downstream target genes of miR-143-3p.There have been no reports about GF1R and GFBP5 and the proliferation and apoptosis of synovial cells.This study intends to take this as an entry point to study whether miR-143-3p can play a role in the proliferation of RA synovial tissue by regulating the expression of IGF1R or IGFBP5,promoting synovial cell proliferation and inhibiting apoptosis.RAS-MAPKs is one of the most important signaling pathway in RA.It is mainly induced to activate by binding of TNF-? to cell surface receptors,which is one of the important mechanisms involved in the signal transduction of synoviocytes.RAS-MAPKs and TNF can form a positive feedback regulation,causing a persistent inflammatory response.Therapy stratiges targeting p38 MAPK signaling pathway is very promising in the future.In this study,we also explore the impact of miR-143-3p on the expression of Ras/p-p38/p38 in RA synovial cells and study the relationship between miR-143-3p and Ras-p38 MAPK signaling pathway,which will provide the basis for further study on the regulatory mechanism of miR-143-3p on Ras-p38 MAPK pathway in RA.Methods1.Quantitative PCR was used to detect the expression of miR-143-3p in synovial tissue and TNF-? induced RA synovial cells in RA patients to verify the results of preliminary experiment results.Next,miR-143-3p inhibitor was transfected into cell lines,MTT assay and flow cytometry were used to detect the proliferation and apoptosis of RA synovial cells,quantitive PCR ? Western Blot was used to detect the expression of Bcl-2,Bax,pro-casepase-3 and active caspase-3 expressions.2.A double luciferase reporter assay system was used to detect whether there is interaction between miR-143-3p and IGF1R and IGFBP5;quantitative PCR and western blot was to detect the IGF1R and IGFBP5 expression in RA synovial cells;the correlation of miR-143-3p and IGF1R and IGFBP5 expression level was analyzed.3.MiR-143-3p inhibitor/mimic was transfected into RA synovial cells,quantitative PCR and western blot was carried out to assay the effect of miR-143-3p on the expression of IGF1R and IGFBP5.The miR-143-3p inhibitor and IGF1R siRNA or IGFBP5 siRNA were co-transfected into synovial cells to detect the changes of apoptosis and proliferation of synovial cells.4.The expression of Ras,p-p38,p38 was detected by quantitative PCR and western blot in cells transfected with miR-143-3p inhibitor to observe the effect of miR-143-3p on Ras/p-p38 MAPKs pathway in RA synovial cells.Results1.The mRNA level of miR-143-3p in synovial tissue of RA patients was 3.23 times of that in normal human synovial tissue(p<0.05),there was no significant difference of miR-143-3p level in synovial tissue of OA patients and normal people(P>0.05).The expression of miR-143-3p in RA fibroblast synovial cells induced by TNF-a was 2.91 times higher than that of normal fibroblasts(p<0.01).2.The cell survival rates(OD570)of TNF-induced RA fibroblast synovial cells at 24,48,72 and 96 hours after transfection with miR-143-3p inhibitor were 0.51,0.55,0.76 and 0.93,respectively,which were 0.50,0.78,0.99 and 1.47 in cells transfected with Inhibitor NC.The cell survival rates in Control group were 0.50,0.78,0.99 and 1.47,respectively.The cell survival rate in miR-143-3p inhibitor group was significantly lower than that in both Control group and Inhibitor NC group(p<0.05).3.The apoptosis rate of TNF-a induced RA fibroblast synovial cells transfected with miR-143-3p inhibitor was(13.56 ± 2.33)%,which was(7.48 ± 2.01%)and and(6.11 ± 1.01)%in miR-143-3p group and NC group.The apoptosis rate in miR-143-3p inhibitor group was significantly higher than that in Inhibitor NC group and Control group(p<0.01).4.The mRNA and protein expressions of Bcl-2 and pro-caspase-3 in RA fibroblasts transfected with miR-143-3p were significantly lower than that in Control group(p<0.01).The mRNA and protein levels of Bax and Active caspase-Group was significantly higher(p<0.01).There was no significant difference in the expression of Bcl-2,Bax and casepase-3 between Inhibitor NC group and Control group(p>0.05).5.Luciferase reporter assay showed that luciferase activity was significantly decreased after co-transfected with miR-143-3p mimic and 3'-UTR wild-type recombination reporter plasmid of IGF1R/IGFBP5 gene,indicating that IGF1R/IGFBP5 is a direct target of miR-143-3p.6.The level of IGF1R and IGFBP5 mRNA and protein were significantly lower in TNF-? induced RA fibroblasts synovial cells than that of normal fibroblasts cells(p<0.01).miR-143-3p was negatively correlated with IGF1R and IGFBP5 expression levels,r2 was 0.6359 and 0.5876,respectively,p<0.001.7.The mRNA and protein expressions of IGF1R and IGFBP5 in RA fibroblasts synovial cells in miR-143-3 inhibitor group were significantly higher than that in Control group(p<0.05);IGF1R And IGFBP5 mRNA expression levels were significantly lower in miR-143-3 mimic group than that in Control group(p<0.05,p<0.01).8.The survival rate of RA fibroblast synovial cells transfected with si-IGF1R or si-IGFBP5 were significantly higher than that of si-NC group(p<0.05).The survival rate of RA fibroblasts transfected with miR-143-3p inhibitor was significantly lower than that of Inhibitor NC group(p<0.05).The survival rates of cells co-transfected with miR-143-3p inhibitor and si-IGF1R or si-IGFBP5 were significantly lower than that of si-IGF1R group and si-IGFBP5 group(p<0.05).9.The apoptotic rate of RA fibroblasts transfected with si-IGF1R or si-IGFBP5 was significantly lower than that of si-NC groups(p<0.05).The apoptosis rate of RA fibroblast synovial cells transfected with miR-143-3p inhibitor was significantly higher than that of Inhibitor NC(p<0.05).The apoptosis rates of cells co-transfected with miR-143-3p inhibitor and si-IGF1R or si-IGFBP5 were significantly higher than those of si-IGF1R and si-IGFBP5(p<0.05).10.The mRNA levels of Ras and p-p38 in RA fibroblasts were significantly higher than that in normal synoviocytes(**p<0.01),which was significantly lower after transfected with miR-143-3p inhibitor(**p<0.01).11.Compared with normal synovial cells,the expression of Ras and p-p38 protein in RA fibroblast synovial cells was significantly increased and the level of p38 protein was decreased.The expression of Ras and p-p38 protein in RA fibroblast synoviocytes after miR-143-3p inhibitor treatment was significantly lower while p38 protein was increaseds.Conclusions1.miR-143-3p is a RA pathogenic factor which was highly expressed in RA synovial cells.Inhibition of miR-143-3p can inhibit the proliferation and promote apoptosis of RA fibroblast synovial cells.2.IGF1R and IGFBP5 are two downstream target genes of miR-143-3p,which are low expressed in RA synovial cells and negatively correlated with miR-143-3p.3.miR-143-3p inhibitor can up-regulate the expression of IGF1R and IGFBP5 and partially reverse the over-proliferation and apoptosis inhibition of RA fibroblast synovial cells induced by si-IGFIR and si-IGFBP5,which plays a protective role on RA.4.miR-143-3p inhibitor can inhibit Ras/p38 MAPK signaling pathway in RA fibroblast cells. |
PDF Full Text Request