Association Of Single-nucleotide Polymorphisms From The Genes That Regulation Of MiRNA Maturation With Tuberculosis Susceptibility

Objective: The SNPs from the genes that regulation of miRNA maturation in signaling pathway may affect the transcriptional ability and protein function of genes,interfere with miRNA maturation,and regulate of miRNA silencing,that is manifested as the change of disease susceptibility.Further,it can be considered that SNPs from the miRNA maturation pathway are associated with diseases.On the basis of the above theory,combined with the high prevalence of Xinjiang Uygur tuberculosis,in this study the whole-exome sequencing was used to screen the susceptibility genes from the mature pathways of miRNA of Xinjiang Uygur tuberculosis patients,to study the correlation between the gene polymorphisms and the expression levels of corresponding target genes and Uygur tuberculosis patients,so as to infer the pathogenic effect of miRNA on the Uygur tuberculosis.Methods: 1)The peripheral blood of 3 Uygur tuberculosis patients and 3 control patients were collected respectively from hotan district.The whole-exome sequencing was performed on all samples by second-generation sequencing technology,so as to screen the candidate genes.Through reviewed the related literatures on the pathogenesis of tuberculosis,and combined with the methods of bioinformatics,Then,selected the relevant genes that regulation of miRNA maturation in signal pathways from the acquired mutated genes,finally identify the candidate genes;2)Used the International HapMap Project database,the dbSNP database and miRBase registry database,screened the qualified SNPs from the candidate genes.465 Uygur tuberculosis patients were collected from the hospitals of hotan region,aksu,kirgiz autonomous prefecture and kashi district in Xinjiang,310 healthy controls were collected with the gender,age matched roughly with tuberculosis patients,extracted the genomic DNA,detected the SNPs on 10 candidate genes from the mature pathway of miRNA by iMLDR technology,selected the different expressions of SNPs by statistical analysis;3)The other 21 tuberculosis patients and 21 health controls were collected from hotan region,used the double antibody clamp method to determination of target protein concentration,extraction of peripheral blood RNA,used the real-time fluorescent quantitative PCR technique to detect the mRNA expression levels of target genes.Combined with the results of SNPs and mRNA expressions,Finally,the positive locis were analyzed by eQTL.Results: 1)A total of 10 candidate genes on the miRNA mature pathway were determined namely DROSHA,DGCR8,C50rf22,RAN,DICER1,DDX20,AGO1,AGO2,GEMIN4,and PIWIL1;2)20 SNPs were screened from the candidate genes,respectively were rs1045481 rs1045491,rs2740349,rs3744741,rs7813,rs10719,rs642321,rs595961,rs636832,rs4961280,rs3742330,rs6877568,rs1106042,rs10773771,rs417309,rs720012,rs1640299,rs3757,rs197412,rs14035.Via the iMLDR detection,there were 6 SNPs were found to be correlated with the incidence of tuberculosis,which were derived from DICER1,GEMIN4,PIWIL1 and DROSHA genes;3)Individuals with GEMIN4 rs1045481 GA genotype,rs1045491 CT genotype and rs7813 AG genotype had an increased risk of active tuberculosis.Individuals with the rs3742330 AG/GG genotype on DICER1 had an increased risk of tuberculosis.Individuals with the rs642321 CT genotype on DROSHA had a reduced risk of active tuberculosis.Individuals with the gene PIWIL1 rs10773771 CC genotype had a reduced risk of tuberculosis;4)Finally through the eQTL analysis,it was found that rs116042 on gene PIWIL1 had a reverse regulatory effect on DICER1.Rs3744741 on gene GEMIN4 had a regulatory effect on PIWIL1 gene.Rs3742330 on gene DICER1 has a reverse regulatory effect on GEMIN4 gene.Rs2740349 on gene GEMIN4 has a regulatory effect on gene GEMIN4.Conclusion: The prevalence of tuberculosis was higher in Xinjiang Uygur population.In addition to geographical factors,gene polymorphism of host genetic factors was also involved in the occurrence of the tuberculosis.In this study,the differential expression of SNPs from the target genes that regulating miRNA maturation in the signaling pathway,confirmed it was involved in incidence of tuberculosis in Xinjiang Uygur,and it can be further confirmed by the differential expressions of the target genes.The analysis results of eQTL provided the clues of the regulatory effect of SNPs on genes.These results provided the clues for in-depth study of the regulatory role of miRNA in the pathogenesis of tuberculosis and its application value as a diagnostic molecular marker,also provided the scientific basis of further larger samples verification and optimal combination of molecular markers.