| Backgroundheumatoid arthritis(RA)is an autoimmune disorder that is pathologically characterized by synovial hyperplasia,neovascularization,inflammatory cell infiltration and cartilage and bone destruction.The exact cause of RA remains unclear,while it is believed that one of the significant pathophysiological aspects of RA is synovial fibro-blasts(SFs)which plays a prominent role in the synovial hyperplasia,joint inflammatory,cartilage and bone destruction.Several studies previously reported that phosphoinositide 3-kinase/protein kinase B(PI3K/AKT)signaling pathway is widely found in SFs and manifests an abnormal activation state,which can give rise to the imbalance of SFs proliferation and apoptosis.The PIK3R2 is a negative regulator of PI3K/AKT signaling pathway.Recent study has demonstrated that the gene coding for the PIK3R2 is one of the targets of micro RNA-126(miR-126).MicroRNAs represent a class of about 22 nucleotide long,non-coding RNAs that are recognized as pivotal regulators of gene expression,which play critical roles in cell proliferation,differentiation and apoptosis.miR-126,encoded by the intron 7 of the epidermal growth factor-like domain 7 gene(EGFL7),regulates vascular functions and vascular integrity and are also shown to participate in the proliferation,invasion,migration,and drug resistance.Recently,miR-126-osteosarcoma association has become an intensive area of research,while less enough data is collected to demonstrate whether miR-126 is involved in the development and progression of RA.ObjectiveThe purpose of our study was to elucidate the impact of micro RNA-126(miR-126)targeting PIK3R2 gene on cell proliferation and apoptosis of rheumatoid arthritis synovial fibro-blasts(RASFs)by regulating PI3K/AKT signal pathway.METHODS1.The synovial tissue samples of this study were from 29 RA patients undergoing joint operation and 27 healthy people undergoing joint repair due to trauma.The RASFs were cultured for further usage.Detect the expression of miR-126 and PIK3R2 mRNA in synovial tissue of RA group and the control group by using real-time quantitative polymerase chain reaction(RT-PCR).Detect the expression of PIK3R2 protein in two groups of RASFs by Western blot.2.The target genes of miR-126 were collected by the TargetScan and PIK3R2 as the direct target gene of miR-126 was confirmed by dual-Luciferase reporter assay system.3.The RASFs in logarithmic growth phase performed cell transfection using electroporation method.Organization of groups in our study was as follows:(1)blank control group(no transfection with any sequence);(2)miR-126 mimic group;(3)miR-126 mimic control group;(4)miR-126 inhibitor group(5)miR-126 inhibitor control group.The effect of miR-126 on the proliferation of RASFs was detected by cell counting Kit(CCK-8)method.The effect of miR-126 on the apoptosis of RASFs was detected by flow cytometry.Detect the expression of for detecting PIK3R2 protein and PI3K/AKT signal pathway relevant proteins(PI3K,Akt and p-Akt)using Western blot method.RESULTS1.Compared with healthy individuals,the RA patients had increased miR-126,but decreased PIK3R2 mRNA expressions in the synovial tissues(miR-126: 1.93 ± 0.45 vs0.89 ± 0.22;t = 3.596,P = 0.023;PIK3R2 mRNA: 0.76 ± 0.16 vs 1.43 ± 0.27;t = 3.698,P= 0.021).As Western-Blot implied,the RA patients also had decreased PIK3R2 protein expression in the synovial tissues(0.61 ± 0.13 vs 1.27 ± 0.31;t = 3.401,P = 0.027).Pearson correlation analysis indicated that miR-126 expression was negatively correlated with PIK3R2 mRNA expression(r =-0.742,P < 0.001).2.PIK3R2 was the potential targeted gene of miR-126 after retrieval of TargetScan.The luciferase reporter indicated that the miR-126 mimic group co-transfected with pMIR/PIK3R2 has less active luciferase than other groups co-transfected with pMIR/PIK3R2(P < 0.05),while the groups co-transfected with pMIR/PIK3R2/mut exertedno significant inhibition on luciferase and there were also no significant difference in activity of luciferase between the miR-126 inhibitor groups co-transfected with pMIR/PIK3R2 or pMIR/PIK3R2/mut(P>0.05),suggesting that PIK3R2 was the direct targeted gene of miR-126.3.The results of CCK-8 revealed that the cells were grown in all groups after transfection as time goes on,while no significant difference in rate of cell proliferation was observed among the mimic control,inhibitor control and blank groups(all P > 0.05).Compared with the blank group,the miR-126 mimic and miR-126 inhibitor groups both showed no notable difference in rate of cell proliferation after 24 hours of transfection.However,after 48 hours of transfection,the rate of cell proliferation remarkably increased in the miR-126 mimic group,but decreased in the miR-126 inhibitor group also when compared with the blank group(all P < 0.05).4.The results of flow cytometry showed that there was no notable difference in cell cycle and apoptosis among the mimic control,inhibitor control and blank groups(all P >0.05).When compared with the blank group respectively,the miR-126 mimic group had raising cell proportions in S and G2/M phases with reduced rate of cell apoptosis,while the miR-126 inhibitor group had raising cell proportions in G0/G1 and G2/M phases with increased rate of cell apoptosis(all P < 0.05),indicating that miR-126 functions on cell cycle of RASFs from G0/G1 to S phases.5.As RT-qPCR results indicated,both miR-126 and PIK3R2 mRNA expressions exhibited no remarkable difference among the mimic control,inhibitor control and blank groups(all P > 0.05).Compared with the blank control group,the miR-126 mimic group had enhanced miR-126 expression with reduced PIK3R2 mRNA expression while the miR-126 inhibitor group had decreased miR-126 expression with increased PIK3R2 mRNA expression(all P < 0.05),which implied that miR-126 could target PIK3R2 and inhibit its mRNA expression.6.With regard to Western-Blot results,no significant difference in the expressions of PIK3R2 and PI3K/AKT pathway relevant proteins(PI3K,Akt and p-Akt)was examined among the mimic control,inhibitor control and blank groups(all P > 0.05).Compared with the blank control group,the miR-126 mimic group had declined expression of PIK3R2protein(P < 0.05)with ascended expression of PI3 K and p-Akt(both P < 0.05)and slightly increased expression of Akt(P > 0.05),while the miR-126 inhibitor group had increased expression of PIK3R2 protein(P < 0.05)with decreased expression of PI3 K and p-Akt(both P < 0.05)and slightly reduced expression of Akt(P > 0.05).These results revealed that miR-126 could up-regulate PI3K/AKT pathway by inhibition of PIK3R2 protein expression.ConclusionOur study confirmed that overexpressions of miR-126 in the RA synovial tissues.And our study provided evidence miR-126 targeting PIK3R2 can promote growth and resistance apoptosis of SFS by regulating PI3K/AKT signaling pathway in RA.Therefore,down-regulation of miR-126 may inhibit the PI3K/AKT signaling pathway to disrupt the imbalance between growth and death of RASFs by targeting PIK3R2,which may be clinically helpful to find therapeutic strategies directed toward restoring miR-126 function for RA. |
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