Study On Relationship Between Polymorphism Of Tim-3Gene And AMLin Hubei Han Population

PartⅠThe polymorphism and Haplotypestudy between Tim-3gene and AMLObjective:To study Tim-3gene promoter-574and-1516and Exon3+4259region SNP polymorphism, linkage associations and haplotype with AML in a Chinese Han population from Hubei.Method:Case-control study.200cases of AML patients and173health controls were collected from Wuhan Union Hospital. DNA was extracted to amplify by using pairs of designed respective primers by using AS-PCR. The results were analyzed by SPSS software19.0, figures were took out by using GraphPad Prism5.0as the linkage and haplotypes were analyzed by SHEsis.Results:①The allele frequencies of-1516G/T(rs10053538)、4259G/T(rs1036199) were accord to Hardy-Weinberg equilibrium.-574T/G (rs10515746) SNP has central tendency.②Minor allelic frequencies of Tim-3-1516G/T、-574T/G and4259G/T were24.5%(T),22.5%(T),1.5%(G).③The linkage association between-574and4259SNPs, D’value was0.49.④Haplotyepl GGT P=0.029, Odd ratio=0.714; Hap2GTT P>0.05; Hap3TGT P=0.034, OR1.554; Hap4TTT P>0.05.Conclusion:①-1516G/T allele SNP may have the susceptible relationship to AML.②A slight connection linkage between-574T/G and4259G/T SNP may exist.③ Hapl GGG has a lower risk(OR=0.714) in AML while Hap3TGT may be used for AML susceptibility prediction in Hubei Han population. Part IIExpression levels of human Tim-3and associated molecules (PD-1, Lag-3, Gal-9) mRNA in AML PBMCsObjective:To observe human Tim-3and other associated molecules (PD-1, Lag-3, Gal-9) mRNA expression levels in AML PBMCs, for further revealing the relationships between AML and Tim-3.Methods:Total RNA was extracted by Trizol reagent from47cases of AML patients (including25none-treated cases and22complete response cases) and40health controls. RNA was then reversely transcribed into cDNA, amplified by reverse transcription PCR and real time quantitative PCR. The results were calculated as2-ΔΔCT and analyzed by using SPSS software19.0.Results:①Tim-3、PD-1、Lag-3、Gal-9mRNA in PBMC were not significant different between cases and controls.②Lag-3and PD-1mRNA levels in CR groups were significant higher than none-treated groups.③The four molecules in AML groups (except M3) did not change significantly compared to health controls.④Compared to M2, Lag-3was significantly higher and Tim-3was slightly higher (P value was0.0539, closed to0.05)⑤PD-1and Gal-9were lower in cases than control among males. Lag-3was also lower than control among females. Part ⅢConstruction of Tim-3promoter (including-574SNP) luciferase reporter plasmidObjective:To construct luciferase reporter plasmid of human Tim-3promoter (including-574SNP), lay the foundation for the following study focused on Tim-3negative regulation in AML.Method:DNA was used as No.140samples of AML showed by above Part Ⅱ. Tim-3promoter sequence (including-574G/G SNP) was amplified by PCR and ligated to T4vectors respectively, then transformed into E.co.li. T4vectors were cloned for culturing overnight and plasmids were then extracted. The extracted plasmids were double digested with the restriction enzymes of Kpn Ⅰ and Hind Ⅲ. The positive clones were then ligated with reporter vector pGL3-amp-luc and cloned. Furthermore, reporter plasmids were extracted and digested with Kpn Ⅰ and Hind Ⅲ. To confirm the reporter plasmid, DNA sequencing also was used.Results:Tim-3promoter gene fragement(594bp) was cloned with DNA attracted from AML PBMC, including SNP genetype GG in Tim-3promoter-574. Then digested with Kpn Ⅰ and Hind Ⅲ as well as pGL3, Tim-3was ligated into reporter vector pGL3to form Tim-3-pGL3plasmid successfully.Conclusion:We successfully constructed Tim-3promoter (including-574SNP) reporter luciferase plasmid. This will help us in further studying focus on Tim-3pathways and the role of negative regulations in AML.