Analysis Of SNP Sites In Myeloid Leukemia And Construction Of Vector For Knocking Out ALOX5 Gene Using CRISPR/Cas9 Technolog

Leukemia is malignant proliferative disorder of hematopoietic stem and progenitor cells as well as a blockade of differentiation and apoptosis.At present,the occurrence and development of leukemia is related to the polygenic variation and multiple risk factors.The variety of responses to pathogenic factors of leukemia resulted in different people,different course as well as different outcomes,which implied it’s associated with individual’s genetic susceptibility to leukemia.Single nucleotide polymorphisms(SNPs)are the most common form of variation in the human genome,involving in disease susceptibility,which play an important role in prediction of early onset,prevention,treatment and prognosis of tumor.It is right now unclear the genotype frequencies of SNPs in arachidonate 5-lipoxygenase(ALOX5)gene,5-lipoxygenase activating protein(ALOX5AP)gene and let-7 microRNA as well as their genetic susceptibility to myeloid leukemia.Besides,in order to explore the biological functions of ALOX5 gene and its roles in molecular mechanism of leukemia,we construct the Px458-ALOX5-sgRNA plasmids and use the CRISPR/Cas9 system knock out human gene ALOX5.Objective:We investigate the genotypes and allelic frequencies of six SNPs in rs2029253,rs2228064,rs2228065 of ALOX5,the rs10507391,rs4769874 of ALOX5AP and the rs10877887 of let-7 genes closely related to myeloid leukemia stem cells among myeloid leukemia and healthy population.The generation of recombinant plasmid PX458-ALOX5 was constructed by CRISPR/Cas9 gene editing technology to target knock out ALOX5 gene.This result could lay a foundation and provid new technology for the research of the molecular pathogenesis,molecular mechanism of leukemia as well as the function of ALOX5 gene.Methods:Under the informed consent,the bone marrow specimens were collected from 150 patients with the myeloid leukemia patients as experimental group and 134 peripheral blood samples of healthy control admitted to the Yunnan Provincial First People’s Hospital.The genomic DNA was extracted from the samples.Then we analysis six SNPs in rs2029253、rs2228064、rs2228065、rs10507391、rs4769874、rs10877887 of gene ALOX5、ALOX5AP、let-7 employed by the direct sequencing of PCR products and the polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP).Data analysis was performed with the software of DNAStar and SPSS 17.0.In order to construct the plasmid vector of PX458-sgRNA by knocking out the ALOX5 gene,CRISPR-Cas9 technique was performed.The oligonucleotides(sgRNAs)targeting exon 6 of ALOX5 were designed and synthesized and then inserted into linearized plasmid PX458.The recombinant plasmid PX458-ALOX5 was transformed into competent E.Coli.DH5α,and identified by sequencing.293T cells were transfected with recombinant plasmid GFP-PX458-sgRNA-ALOX5 and transfected 293T cells after 24-48 h to verify the effect of knocking off.Results:(Ⅰ)The distribution of genotype frequencies of SNP in control group was in accordance with the Hardy-Weinberg equilibrium law states(P>0.05)by Chi-square test as representative samples.(Ⅱ)The AA,AG,GG genotype frequencies of SNP rs2029253 in ALOX5 gene were 32.2%,21.5%and 46.3%in myeloid leukemia,respectively.Its allele frequencies A,G were 43.0%and 57.0%.There existed significantly difference between in myeloid leukemia group and control group(X2=14.034,P=0.001;X2=4.417,P=0.036)in the distribution of AA genotype and A allele frequencies of SNP rs2029253.The AA genotype and the A allele frequencies appeared to be at increased risk of myeloid leukemia with P values of 0.001(OR,2.257;95%CI,1.434 to 4.562;and OR,1.440;95%CI,1.024 to 2.025(P<0.036,).However,no significant difference was observed for genotype and allele frequencies of SNP rs2228064,rs2228065 between the experimental group and the control group(P>0.05).(Ⅲ)The AA、AT、TT genotype frequencies of SNP rs10507391 in ALOX5AP in myeloid leukemia were 1.3%、58.7%and 40.0%,respectively.Statistical difference in the distribution of AA genotypes was observed between the experimental group and the control group(p=0.002).Genotypes AA was associated with a significantly reduced the risk of developing myeloid leukemia(OR:0.126,95%CI:0.028-0.568).However,there is no statistical difference in genotype and allele frequencies of rs4769874 between in the experimental group and the control group.(IV)The polymorphic sites at rs10877887 of let-7 gene were detected at TT(39.3%),TC(51.4%),CC(9.3%)in myeloid leukemia.There was no significant difference with the genotype and allele frequency of rs4769874 among the experimental group and the control group(X2=1.463,P=0.481;X2=1.243,P=0.265).(V)The sgRNA targeted nucleotide sequences of ALOX5 gene were successfully inserted into the expected sites of PX458 vector and the sequences were correct.It is mean the CRISPR/Cas9 system by knocking out the exon 6 of ALOX5 gene is successfully constructed.After 48 the GFP fluorescence signal was observed in the 293T cells,which suggesting that the recombinant plasmid is successfully transfected.Conclusion:(I)Statistical analysis indicated that AA genotype and A allele of rs2029253 in ALOX5 may improve the susceptibility of myeloid leukemia.(II)Genotyping AA of rs10507391 in ALOX5AP may relate to reduce the risk of myeloid leukemia.(Ⅲ)No significant difference was found between patients and control subjects with regard to genotype and allele of the SNP rs2228064,rs2228065 in ALOX5 gene,the rs4769874 in ALOX5AP gene and the rs10877887 of let-7 gene.(Ⅳ)The CRISPR/Cas9 plasmid targeting the exon 6 of ALOX5 gene has been successfully constructed.