The Level Of Vitamin D In Peripheral Blood Of Patients With Rheumatoid Arthritis And The Mechanism Of Active Vitamin D Regulating The Function Of THP-1 Cell Through LncRNA HOTAIR

Background:Rheumatoid arthritis（RA）is a highly disabling autoimmune disease caused by the interaction of genetic and environmental factors,with a global prevalence of up to 1%.It is well known that active vitamin D has a wide range of immunomodulatory effects and may be involved in the pathogenesis of RA as an important immunomodulatory factor.Recent years,studies have shown that over expression of HOX transcript antisenseRNA（HOTAIR）could lead to macrophage activation and inflammatory response.Therefore,the regulation of HOTAIR may be a new target for RA treatment.However,it is unclear whether 1,25（OH）₂D₃influences the function of THP-1 cell through HOTAIR expression.Objective:To investigate the level of VitD in patients with RA and the correlation between endogenous VitD level and serum cytokine,Th1 cells,Th2 cells,Th17 cells,Treg cells and disease activity.On this basis,further to clarify the mechanism by which supplementation of exogenous 1,25（OH）₂D₃affects THP-1 cell function and elucidate the role of HOTAIR.Methods:1.Fifty-four patients with RA and 54 age-and sex-matched healthy controls（HCs）were enrolled in this study.The expression level of 25（OH）D₃was detected by ELISA.The absolute number of T cell subsets was detected by flow cytometry（FCM）.The levels of serum cytokines were detected using CBA,and disease activity was evaluate using the Disease Activity Score 28.2.The THP-1 cells were infected by lentiviral vector overexpressing HOTAIR or lentiviral vector silencing HOTAIR,and a cell culture system was established in vitro.This study were divided into the enhanced HOTAIR expression group,HOTAIR overexpression negative control group,the HOTAIR interference group,HOTAIR interference negative control group and the blank control group.The state of THP-1 cells was observed by Deltavision Ultra,and the efficiency of infection was determined by FCM.Cells were screened with puromycin hydrochloride and the stablely infected cell lines were expanded and cultured.Each group was treated with different doses（10,30,100 n M）of 1,25（OH）₂D₃and cultured in an incubator at 37℃with 5%CO₂.The control group was given equal amount of ethanol.Cells were collected after 4,24 and 48 hours.The levels of HOTAIR,VDR,NF-κB and IL-1βwere detected by real-time PCR,and the effect of 1,25（OH）₂D₃on THP-1 cell function was analyzed.Results:1.The levels of 25（OH）D₃was significantly lower in RA patients than in HC（p<0.001）.Relative to HCs,the percentages and absolute values of Th1 and Th17 cells were higher（p<0.05）,while the lower percentages and absolute values of Th2 and Treg cells were found in RA patients（p<0.01）.Meanwhile,the levels of IL-2,IL-4,IL-6,IL-10,IL-17,TNF-αand IFN-γin patients with RA were significantly elevated（p<0.001）.Furthermore,25（OH）D₃levels were negatively correlated with DAS28,ESR,CRP,Ig M,IL-6,IL-17,respectively（p<0.05）,but it was positively associated with with CD4⁺Th cells and CD4+CD25+FOXP3+Treg cells,respectively（p<0.05）.2.After 48h of infection,the infected cell was observed by Deltavision Ultra,we found that the cells were in good condition and expressed green fluorescent protein（GFP）.After screening with puromycin hydrochloride,flow cytometry results showed that the expression efficiency of GFP was more than 90%.HOTAIR mRNA level of THP-1 cells in HOTAIR enhanced group was found to be significantly higher than that in control group and HOTAIR interference group（p<0.05）.A lower level of HOTAIR mRNA in HOTAIR inhibition group was found than that in control group and HOTAIR overexpression group（p<0.05）.There were no significant difference in VDR and NF-κB mRNA between HOTAIR enhanced group and HOTAIR inhibited group and blank group（p>0.05）.Notably,the level of IL-1βmRNA in HOTAIR expression enhanced group was found to be significantly higher than that in HOTAIR expression inhibited group and control group（p<0.05）.In blank control group,the expression of HOTAIR,VDR and IL-1βmRNA levels were regulated by 1,25（OH）₂D₃,and were correlated with the concentration and duration of stimulation.However,no significant difference in NF-κB mRNA level was found（p<0.05）.In addition,1,25（OH）₂D₃decreased the expression of HOTAIR,VDR and IL-1βmRNA in the enhanced HOTAIR group（p<0.05）,but the expression of NF-κB did not change significantly（p>0.05）.After interfering with HOTAIR,1,25（OH）₂D₃decreased the expression of HOTAIR and VDR mRNA levels（p<0.05）,but the regulation of IL-1βwas weakened.Conclusions:Vitamin D may be an important factor in the occurrence and development of RA,and1,25（OH）₂D₃regulated the function of THP-1 cell and inhibited the expression of cytokine through HOTAIR.