Molecular Epidemiology Study On Hypoxia-inducible Factor1α In The Pathogenesis Of Systemic Lupus Erythematosus

BackgroundSystemic lupus erythematosus (SLE) is a typical chronic and systemic autoimmunedisease characterized by a range of autoantibodies production, which leading to tissueinjury with multiple organ involvement. The majorities of the inflammatory cells inaffected organs are T lymphocytes, which probably play a crucial role in the diseaseprocess. Activated T helper (Th) cells react to B cell differentiation and autoantibodyproductions, immune complex of autoantibodies and inflammatory cytokines result intissue and organ damages. Although the pathogenesis of SLE remains unclear, Th17cells and regulatory T (Treg) cells represent an intriguing issue to be addressed in thepathogenesis of SLE and potential therapeutic intervention, because Th17/Tregimbalance appears to play an important pathogenic role in the disease onset. Treg cellsdisplay suppressive activity towards autoreactive lymphocytes thus preventing the onsetof aberrant self-immune response. Conversely, Th17cells accumulate in target organscontributing to local IL-17production and eventually tissue damage.It has been alreadyreported that SLE flares may occur as a consequence of cytokine imbalance andeventually of the Th17/Treg ratio in lupus-prone mice.Hypoxia-inducible factor1(HIF-1) is a key transcription factor, which is aheterodimer with two subunits: the highly regulated oxygen-sensitive α-subunit and theconstitutively present β-subunit. In hypoxic conditions, induction of HIF-1is mediatedby HIF protein stabilization leading to an augmentation of adenosine triphosphate (ATP)production and oxygen tissue delivery defaults.Previous studies found that HIF-1enhances Th17development through direct transcriptional activation of RORγt and viatertiary complex formation with RORγt and p300recruitment to the IL-17promoter, thereby regulating Th17signature genes. Concurrently, HIF-1attenuates Tregdevelopment by binding to transcription factor Foxp3and targeting it for proteasomaldegradation. Importantly, this regulation occurs under both normoxic and hypoxicconditions. To date, the molecular mechanisms involved in HIF-1α expression andHIF-1(α/β heterodimer)-transcription factor activities are scarcely known in SLE. Onthe basis of these facts, we conclude that HIF-1might be involved in the pathogenesisof SLE by mediating imbalance of Th17/Tregs.In this study, the RNAi study will be conducted to preliminary explore themolecular mechanisms of HIF-1α in the development of SLE. Then, we will collectsamples form SLE patients and normal controls, we use ELISA to detect HIF-1αexpression in serum level. We also carried out a case-control association study toexamine the single nucleotide polymorphisms in HIF-1by using the SequenomMassARRAY SNP detection technology and investigate whether or not allele andgenotype frequencies of HIF-1gene confer susceptibility to SLE in the Chinesepopulation.The completion of this study would help to clarify the role of HIF-1genefunctions and mutations in the pathogenesis of SLE, and provide a scientific basis forlooking for new diagnostic and therapeutic targets in SLE.The study has three parts. Part1Inhibiting of gene HIF-1by RNA interference in vitroObjective The plasmid containing short hairpin RNA (shRNA) of HIF-1α is constructedso as to suppress the expression of HIF-1α and to observe the effects of the proliferationin SLE patients’ peripheral blood mononuclear cell, provided for exploring theHIF-1gene function.Method The recombinant plasmid Lv-shRNA-HIF were constructed and transfectedinto SLE patients PBMC, the expression of HIF-1α in PBMC were observed byRT-PCR and Western Blot, the level of HIF-1and IL-17were examined by ELISA.Results The restriction enzyme digestion and sequencing result showed that therecombinant plasmid of Lv-shRNA-HIF had been constructed successfully. After ittransfecting into PBMC, the genetic transcription and protein expression of HIF-1αwere inhibited significantily compared with blank controls. The HIF-1α and IL-17experessions were reduced significantly(p<0.001, respectively). Moreover, HIF-1αlevels were related to IL-17levels(r=0.959, p<0.001).Conclusio: It has been initially proved that the recombinant plasmid Lv-shRNA-HIFcan inhibit the genetic transcription and protein expression of HIF-1α, and inhibit theproliferation and induce the apoptosis of SLE patients PBMC, which is a basicfoundation for the oncogene therapy in future. Part2Increased Serum Hypoxia-inducible Factor1α in Patients with SystemicLupus ErythematosusObjective To determine serum hypoxia-inducible factor1α (HIF-1α) levels in patientswith systemic lupus erythematosus (SLE).Methods Serum HIF-1α levels were examined by ELISA in149patients with SLE,46 with rheumatoid arthritis (RA),50with primary sjogren’s syndrome (PSS),26withsystemic sclerosis (SSc) and102healthy individuals. Serum IL-17levels were alsomeasured in SLE patients.Results We found that serum HIF-1α levels were significantly higher in SLE patients(32.76±12.60pg/ml) than in healthy individuals (23.04±11.35pg/ml; p<0.0001), andpatients with SSc patients (26.15±13.92pg/ml; p=0.03), but no significant differenceswith RA patients (28.71±10.88pg/ml; p=0.09), and PSS patients (30.08±23.57pg/ml;p=0.25). Among SLE subgroups, HIF-1α levels in patients with nephritis (35.06±11.64pg/ml) and patients without nephritis (32.03±12.86pg/ml) were increased comparedwith healthy individuals (p<0.0001, respectively), but there were no differencesbetween the two disease subgroups. Moreover, HIF-1α levels were related to SLEdisease activity (r=0.23; p=0.004). HIF-1α levels also were correlated with the IL-17expression in SLE patients (r=0.53; p<0.0001).Conclusions Serum HIF-1α level was increased in patients with SLE and was associatedwith disease activity. The transcription factor HIF-1α may be implicated in thepathogenesis of the disease. Part3The association between the polymorphisms of HIF1A gene and SLEsusceptibility in a Chinese populationObjective Hypoxia-inducible factor1(HIF-1) introduced the immune imbalancebetween Th17and Treg cells, which may play an important role in the pathogenesis ofsystemic lupus erythematosus (SLE). The aim of the present study was to determinewhether the HIF1A gene influence the susceptibility to SLE. A study on this relationship has not been conducted to date.Methods A total of3,793subjects (1,497SLE patients and2,296controls) wereincluded in this study. The genotyping of five SNPs (rs11549465, rs12434438,rs1957757, rs1951795, rs7143164) was determined by Sequenom MassARRAYtechnology. The statistical analysis was conducted by chi square test. Odds ratio (OR)with95%confidence interval (CI) was calculated using unconditional logisticregression with adjusting age and sex.Results The allele frequencies were not associated with the disease. No significantdifferences in genotype frequencies existed between the patients with SLE and thecontrols in all five SNPs. It is worth mentioning that, the allele T at rs11549465, locatedat exon sequence, revealed a trend but not significant difference towards more frequentin SLE than controls (C versus T: OR=1.206,95%CI=0.972-1.495, p=0.088). Thegenotype effect of recessive, dominant and codominant model were observed, howeverno significant evidence for association was detected.Conclusions Our findings suggest that the gene polymorphisms of HIF1A might notcontribute to SLE susceptibility in the Chinese population. However, further studiesare needed on independent cohort from different genetic backgrounds to confirmHIF1A as an SLE genetic factor.