Analysis Of HLA-E Gene Full-length Sequence Polymorphism And Study On Expression Regulation Of Promoter Region Variation In Leukemia Cell Line

Objective:The correlation between HLA-E gene polymorphisms and leukemia is studied among 142 healthy blood donors and 111 leukemia patients,the difference in the frequency distribution of HLA-E gene of two groups is analyzed,and the activity of the wide type and mutation type is tested by a double luciferase reporter gene experiment,Real-time quantitative PCR and Western blotting are used to detect the transcription and protein expression levels of K562-HLA-E-W and K562-HLA-E-M cell lines constructed in vitro to verify whether mutation in the promoter region of HLA-E has a regulatory effect on their expression.Methods:1.A method for HLA-E full-length sequencing and typing based the sequence information of HLA-E gene in Genbank and IPD-IMGT/HLA database is established,Primer and Oligo software are used to design amplification primers and sequencing primers,the sequenced sequences are spliced,confirmed,and aligned,and finally the HLA-E allele is typed.2.The double luciferase reporter assay is used to detect the activity of the promoter region of the wide type(NT-26G)and the mutation type(NT-26T),the activity of the promoter region of the wide type is used as a control.3.K562-HLA-E-W(NT-26G)and K562-HLA-E-M(NT-26T)cell lines are constructed,Real-time quantitative PCR is used to detect the mRNA expression of two cell lines,Western blotting is used to detect the expression of HLA-E molecules on the surface of two cell lines.Results:1.There were 16 alleles and 32 genotypes in the HLA-E sequencing results of 142 healthy blood donors and 111 leukemia patients,among the 16 alleles,the HLA-E*01:03:01:01 allele had the highest frequency(31.23%),followed by HLA-E*01:03:02:01(28.46%)and HLA-E*01:01:01:01/02(19.37%),among the 32 HLA-E genotypes,HLA-E*01:03:01:01,*01:03:02:01(19.76%),HLA-E*01:01:01:01:01/02,*01:03:01:01(13.05%),HLA-E*01:03:01:01,*01:03:01:01(11.07%)were more frequency.And the information of a novel HLA-E allele(HLA-E*01:03:07)is submitted to the Genbank and IPD-IMGT/HLA Database(MK905081-HWS10054909).2.Through double luciferase reporter gene experiments,we found that compared with the wild type(NT-26T),the mutant type(NT-26G)can decreased promoter activity,and the difference was statistically significant(p=0.0242).3.qPCR and Western blot experiments are used to detect the transcription and expression levels of K562-HLA-E-W and K562-HLA-E-M cell lines constructed in vitro,the results showed that the relative expression of mRNA in K562-HLA-E-M cell line was significantly lower than that in K562-HLA-E-W cell line,and the difference was statistically significant(p=0.0003).the expression of HLA-E molecules in K562-HLA-E-M cell line was significantly lower than that in K562-HLA-E-W cell line,and the difference was statistically significant(p=0.0103).Conclusions:1.In the HLA-E sequencing results of 142 healthy blood donors and 111 leukemia patients,the information of a novel HLA-E allele(HLA-E*01:03:07)is submitted to the Genbank and IPD-IMGT/HLA Database(MK905081-HWS10054909),which proves that the polymorphism of HLA-E in Chinese needs further exploration.2.The activity of the mutant type in promoter region is significantly reducedcompared with the wild type(NT-26G),it proves that the mutation of a single nucleotide in the promoter region reduces the activity of the promoter region.3.The transcription level and protein expression level of K562-HLA-E-M cell line is significantly reduced compared with K562-HLA-E-W cell line.The variation in the promoter region(NT-26)significantly affect the activity of the promoter region.