Study On The Relationship Between CEACAM1 And Systemic Lupus Erythematosus

Part IThe expression levels of CEACAM1 and associated molecules(TIM-3,PD-1,Galectin-9,IFN?) m RNAs in SLE PBMCs and its relevance with clinical indexesObjective: To detect the expression levels of CEACAM1 and associated molecules(TIM-3,PD-1,Galectin-9,IFN?) m RNAs in SLE PBMCs, and then find out its relevance with clinical indexes.Methods: Using Trizol reagent to extract the total RNA from 38 cases of SLE patients and 40 cases of health controls. SLEDAI is the score of SLE disease activity. Then the RNA was reversely transcribed into c DNA by reverse transcription PCR so it can become detectable by q RT-PCR. The clinical data of SLE patients was collected and analyzed in detail. Fixing all date for statistical analysis and find out its relevance with clinical indexes.Results: The SLEDAI of 38 SLE patients was 5.89±6.25 in a manner of Means ± Standard Deviation, and the majority of this data was 0. Only the expression levels of CEACAM1 and Galectin-9 m RNAs were significant different between SLEs and controls, and both of them were all upregulated in SLEs compared to controls. In SLEDAI-H(SLEDAI?10) groups, all expression levels of these m RNAs were downregulated, but only TIM-3 and PD-1's changes were statistically significant. Only the trend between decreased ESR value and increased expression level of CEACAM1 m RNA was statistically significant.Conclusion: The expression level of CEACAM1 m RNA was generally upregulated in SLEs but downregulated in SLEDAI-H ones distinctively. CEACAM1 may be a member of negative feedback regulation in SLE, so CEACAM1-related inhibition of immune cells may play a role in SLE self-defense.Part II The relevance between the polymorphism of CEACAM1 gene and SLE in Hubei Han populationObjective: To study CEACAM1 gene rs8102519, rs8103285 and rs10402677 single nucleotide polymorphism, linkage associations, haplotype with SLE in Hubei Han population, and then find out its relevance with clinical indexes.Methods: DNA was extracted from peripheral venous blood samples, the samples was from 177 cases of SLE patients and 210 case of health controls. Then these DNA was amplified to target gene fragment by using pairs of specific primers. These target gene fragment was detected by gene sequencing. The clinical data of SLE patients was collected and analyzed in detail. Using SPSS software 23.0 or SHEsis to fix all date for statistical analysis.Results: The allele frequencies of A(rs8102519), C(rs8103285) and T(rs10402677) were all 0.096 in SLEs, and 0.057 in health controls, P=0.041, OR=1.753, 95% CI 1.019-3.017. Haplotype GTC, P=0.039, OR=0.563, 95% CI 0.324-0.978; Haplotype ACT, P=0.039, OR=1.777, 95% CI 1.023-3.087. The D' and r2 value of linkage association between any two of these SNPs were all more than 0.9. Only ESR and C3 showed the statistically significant relevance with CEACAM1 gene SNP.Conclusion: A strong linkage disequilibrium among CEACAM1 gene rs8102519, rs8103285 and rs10402677 SNP may exist. The allele A(rs8102519), C(rs8103285) and T(rs10402677) may be involved in SLE susceptibility. Haplotype ACT may develop the risk of SLE. SLE patients with allele A(rs8102519), C(rs8103285) and T(rs10402677) may occur some restricted inhibition of immune cells activity and function.Part III Construct CEACAM1 promoter(including rs8102519, rs8103285 SNP) luciferase reporter plasmidObjective: To construct CEACAM1 promoter(including rs8102519, rs8103285 SNP) luciferase reporter plasmid(Wild-type and Homozygous mutant-type), contribute to further research on CEACAM1 regulation in SLE.Methods: CEACAM1 promoter target gene fragment(including rs8102519, rs8103285 SNP) was extracted by using pairs of specific primers,then target gene fragment were ligated to plasmid p GL3. The recombinant plasmids we named it p GL3-CEACAM1-1985. At last we have selected human 293 T cell strain for transfection, then we detect the expression level of luciferin.Results: The recombinant plasmids p GL3-CEACAM1-1985 show two different bands after double digested, one is above 500 bp, the other is above 4800bp; gene sequencing show the same results. The expression level of luciferin of 293 T cell which was transfected by the recombinant plasmids p GL3-CEACAM1-1985(Homozygous mutant-type) was significantly higher than Wild-type ones.Conclusion: We successfully constructed CEACAM1 promoter(including rs8102519, rs8103285 SNP) luciferase reporter plasmid. This homozygous mutant in CEACAM1 promoter can enhance its activity, it may promote the expression of CEACAM1.