| BackgroundNon-coding RNA(nc RNA)is the RNA transcribed from genes that don't code protein.According to its length,nc RNA can be divided into long non-coding RNA(lnc RNA)and micro RNA(mi RNA).Lnc RNA,with more than 200 nucleotides,can specifically bind to RNA and DNA through base complementary pairing principle.Mi RNA,generally with 18-24 nucleotides,can specifically bind to corresponding messenger RNA(m RNA)of protein coding gene,and regulate gene expression by inhibiting translation process or degrading m RNA.Lnc RNA,mi RNA and m RNA can be binding to each other through the base complementary pairing.They can regulate the expression level of protein coding genes through the mechanism of competing endogenous RNA(ce RNA),and play important role S in the occurrence and development of various diseases,including tumors,cardiovascular diseases and autoimmune diseases.Ankylosing spondylitis(AS)is a systemic inflammatory autoimmune disease that mainly endangers the sacroiliac joint.The main pathological characteristics are chronic progressive inflammation of tendon and ligament attachment point and new bone formation.Studies have shown that mi RNAs can participate in the pathogenesis of AS by regulating the expression of Dickkopf related protein 1(DKK-1).However,studies rarely explored the role of their potential upstream lnc RNAs in the pathogenesis and development of AS.ObjectivesThe purpose of this study is to explore the role of lnc RNA IFNG-AS1 in the development of AS.Firstly,a case-control study was conducted to investigate the expression levels of IFNG-AS1 and DKK-1 in AS patients and health controls.And then,PBMCs of AS patients were cultured with small interfering RNA(si RNA)interfering the expression of IFNG-AS1 to explore the changes of mi RNAs and DKK-1 expression.MethodsThe main purpose of this study is to explore the interaction network of lnc RNA,mi RNA and m RNA regulating the expression of DKK-1.Firstly,we tested the expression levels of relevant lnc RNA and cytokines in AS patients and health controls.Based on detailed literature reading and bio-information analysis,we selected IFNG-AS1 and OIP5-AS1 as potential lnc RNAs and mi R-29 a and mi R-181 c as mi RNAs.Quantitative real-time reverse transcription polymerase chain reaction(q RT-PCR)was used to tested the expression levels of lnc RNAs in PBMCs of patients and controls,and enzyme linked immunosorbent assay(ELISA)was used to test the expression levels of DKK-1 and related cytokines in serum,and their relationships with clinical characteristics were analyzed.The second part of the experiment is to knock down IFNG-AS1 using si RNA in PBMCs of AS patients to verify the signal pathway.PBMCs of AS patients were collected for cell culture and IFNG-AS1 was knocked down using si RNA transfection to explore the transcription or expression levels of mi R-29 a,mi R-181 c,DKK-1 and corresponding cytokines.ResultIn the first phase,49 AS patients and 49 health controls were included.The expression of IFNG-AS1 in PBMCs of AS patients was significantly increased(Z =-2.451,P = 0.014),the expression levels of OIP5-AS1 and DKK-1 in AS patients was similar with health controls(Z =-0.978,P = 0.328;Z =-1.536,P = 0.125).The levels of DKK-1,IL-6 and TNF-? in serum of AS patients and health controls were tested by ELISA.We found that the level of IL-6 in serum of AS patients increased significantly(Z =-2.491,P = 0.013),but there was no significant difference between DKK-1 and TNF-? in serum of AS patients and health controls.Compared with the negative control group,the transfection efficiency of si RNA of 13 samples was more than 40%.The relative expression level of IFNG1 was 0.479(0.326,0.575).Compared with the negative control group,the relative expression level of mi R-29 a in the si RNA transfected group was significantly(Z =-2.494,P = 0.013)increased to 1.782(1.162,2.289).The relative expression levels of mi R-181 c in si RNA transfected group was 1.873(0.860,2.978),and there was no significant difference between the two groups(Z =-0.356,P = 0.722).The relative expression of DKK-1 in si RNA transfected group was 0.873(0.728,1.358),and there was no significant difference compared with negative control group(Z =-1.781,P = 0.075).The expression of DKK-1,IL-6,TNF-? in the supernatant of culture solution was tested by ELISA.The expression level of IL-6 decreased(t = 2.763,P = 0.011),and the expression levels of DKK-1 and TNF-? were not significantly changed(t = 1.846,P = 0.077;t = 1.783,P = 0.087)in si RNA transfected group.Conclusion(1)Our study found that the expression levels of IFNG-AS1 in AS patients were significantly higher than that of health controls in PBMCs,indicating that IFNG-AS1 may be involved in the pathogenesis of AS.(2)After the transcription in the PBMCs of AS patients,the RNA relative expression levels of mi R-29 a was increased and DKK-1 in PBMCs were decreased,suggesting that IFNG-AS1 may regulate the expression of DKK-1 through mi R-29 a to participate in the pathogenesis of AS. |
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