Functional Analyses Of Rs6426749 And Rs34920465 In The 1p36 Locus

Osteoporosis is a complex disease characterized by reduced bone mass and increased risk of fracture in bone tissue.Bone mineral density(BMD)is the most important indicator for predicting fracture risk which is high heredity with the heritability estimation of 0.5-0.8.Genome-wide association study(GWAS)is an effective method for revealing susceptibility genes for complex diseases and gene mutations affecting complex traits in human.In the past decade,GWAS has found a large number of single nucleotide polymorphisms(SNPs)that are significantly associated with complex diseases The study of the function and mechanism of these SNPs is a major task in the post-GWAS era SNPs rs6426749 and rs34920465 located in upstream of the genes ZBTB40 and WNT4 are significantly associated with osteoporosis.The purpose of this study:(1)To explore whether rs6426749(G/C)and rs34920465(A/G)can differentially bind transcription factor CREB1 and JUN::FOS,respectively.(2)To explore the regulation of the genes ZBTB40 and WNT4 by rs6426749 and rs34920465.JASPAR database predicted that rs6426749(G/C)can bind to the transcription factor CREB1 differentially.Dual luciferase reporter gene assays showed that the luciferase activity of recombinant vector C increased significantly compared to recombinant vector G,when transfected U-2OS cell by CREB1 overexpression and interference carrier vector.The activity of C was higher than that of G in 293T and U-2OS lines.The results of qPCR showed that the expression of ZBTB40 was raie by 2.7 fold When CREB1 was overexpression in U-2OS cells with G/C.When transfected U-2OS cell by CREB1shRNA,the transcriptional activity of endogenous ZBTB40 was declined by 43%.When transfected U-2OS cell by the SNP of the upstream of WNT4 rs6426749-C and rs6426749-G groups the luciferase activity changes were not significant.But the results of qRT-PCR were not same,When we transfected U-20S cell by CREB1 overexpression,the transcriptional activity of endogenous WNT4 was upregulated significantly,when transfected U-2OS cell by CREB1shRNA,the transcriptional activity of endogenous WNT4 was down-regulated significantly.JASPAR database predicted that rs34920465(A/G)can bind to the transcription factor JUN::FOS differentially.Dual luciferase reporter gene assays showed that the luciferase activity of recombinant vector G increased significantly compared to recombinant vector A,when transfected U-2OS cell by JUN::FOS overexpression and interference carrier vector.Furthermore,the activity of G was higher than A in 293T and U-2OS cell lines.We transfected Saos-2 cell by JUN::FOS overexpression,JUN's endogenous expression is 25 times that of the control group,the endogenous expression level of ZBTB40 was upregulated by 2.4 times.There was no significant change in the endogenous expression of ZBTB40 when the endogenous expression of JUN was downregulated to 50%of the control group.When transfected U-2OS cell by the SNP of the upstream of WNT4 rs34920465-T and rs34920465-C groups the luciferase activity changes were not significant,there was no significant change in the transcriptional activity of WNT4.Our study have revealed a novel molecular mechanism for the first time that GWAS SNP rs6426749(G/C)and rs34920465(A/G)regulated ZBTB40 expression by affecting the binding of CREB1 and JUN::FOS.