The Molecular Mechanism Of ASAP1 Gene Polymorphism Regulating Susceptibility To Tuberculosis In Vitro

Tuberculosis(TB)is one of the most important infectious Zoonosis in the world,which poses a huge threat to public health in humans.A large-scale GWAS(genome wide association study)study has found that rs10956514 located in intron 4 of ASAP1 and rs4236749 located in 3’UTR of ASAP1 are associated with the active TB,but the mechanism underlying the regulation of TB susceptibility in humans by these two SNPs(single nucleotide polymorphism)is totally unclear.In this study,A549 and THP1 were used as cell models to elucidate the molecular mechanism by which rs10956514 and rs4236749 affect the development and progression of TB through their regulation of gene expression.For rs10956514,software was first used to predict whether different base types of rs10956514 would affect transcription factor binding affinity,and double luciferase reporting system was applied to detect whether target sequences containing the SNP would affect gene expression with different levels by base alteration at this SNP site.The nucleoproteins of macrophages(MΦ)were extracted and the probes of different base types were used for EMSA(electrophoretic mobility shift assay)to detect the effects of different base types on the binding of nucleoproteins to genomic DNA.For rs4236749,software was used to predict and screen mi RNA bound to rs4236749,and dual luciferase reporting system was applied to verify the interaction between mi RNA and target sequences with different levels by base changes at the SNP site.The regulation of mi RNA on ASAP1 expression was detected by Western blot and q RT-PCR.Finally,the effects of mi RNA expression levels on intracellular bacterial load in MΦ,cellular cytoskeleton remodeling and cell migration were tested.Results are as follows:1)The bioinformatic analysis predicted that the mutation of A to G at rs10956514 would cause the reduction of transcription factor binding affinity,and the results of double luciferase report showed that the sequence of rs10956514 played the function of gene expression silencer,and the A-G conversion would cause the increase of gene expression level.At the same time,EMSA verified that the mutation of A to G would lead to the reduction of nucleoprotein binding affinity.2)The software predicted that the mutation of rs4236749 affected the binding affinity of mi R-4474-3p,and the double luciferase report showed that the mutation of rs4236749 G to A enhanced the binding affinity of mi R-4474-3p to the target gene.The regulatory effect of mi R-4474-3p on the expression of ASAP1 was further confirmed by Western blot and RT-q PCR analysis.3)After Mtb infected with MΦ differentiated from THP1,the expression of mi R-4474-3p was significantly up-regulated.Overexpression of mi R-4474-3p increased the infection rate of MΦ to Mtb.Overexpression of mi R-4474-3p caused cytoskeletal remodeling,impaired cell migration,and inhibited inflammatory responses.4)Overexpression of mi R-4474-3p also caused cytoskeletal remodeling in A549.It is concluded that different base types of SNP rs10956514 located in the intron of ASAP1 can regulate the expression of ASAP1 by affecting the binding of nuclear protein,thus affecting the host susceptibility to TB.The SNP rs4236749 located in the 3 ’UTR of ASAP1 can affect the binding of mi R-4474-3p,which can regulate the expression of ASAP1 and affect the TB occurrence and development.At the same time,the expression level of mi R-4474-3p changes after Mtb infection.This study revealed the molecular mechanism by which rs10956514 and rs4236749 affect the human susceptibility to TB through their regulation of ASAP1 expression,which enriches our understanding of human susceptibility to TB and expands our knowledge of TB pathogenesis,and provides a scientific basis for the research and development of novel strategies for precision prevention and control of host susceptibility to TB and host-directed therapy.Meanwhile,mi R-4474-3p maybe acts as a potential biomarker for the diagnosis,and be used for screening the susceptible population,prevention and treatment of human TB.