Functional Study Of Osteoporosis GWAS SNPs Rs7220711 And Rs1107748

Osteoporosis is a common multigenic disease characterized by a decrease of bone mass,a deterioration in bone microarchitecture,and a increase in fracture risk.Genome-wide association study(GWAS)had identified 129 SNPs with P-value<10-8 and 107 genes with osteoporosis.Current challenge is to further study the pathogenic mechanism of these SNPs and susceptibility genes,and translate genetic discovery into the the knowledge of pathophysiology.Osteoporosis GWAS lead SNPs rs7220711 and rs1107748 located in a 52 KB fragment deleted in the downstream of sclerosteosis and van Buchem patient’s SOST gene and their functions are unknown.The purpose of this study:(1)Explore if rs7220711(A/G)and rs1107748(C/T)can differentially bind transcription factor MAFF/MAFK and CTCF.(2)Study the function of MAFF/MAFK and CTCF in the osteogenesis system.The JASPAR database pridicted that rs7220711(A/G)can bind transcription factor MAFF/MAFK differentially.When U-20S,Saos-2 and 293T cells were transfected by the rs7220711-A and rs7220711-G carrier,dual luciferase reporter gene assays showed that the luciferase activity of recombinant vector A decreased significantly compared to recombinant vector G.When transfected U-20S cell by MAFF/MAFK overexpression and interference carrier vector,the luciferase report gene activity of rs7220711-A+MAFF/MAFK overexpression group was lower than the luciferase activity of rs7220711-G + MAFF/MAFK overexpression group.The luciferase report gene activity of rs7220711-A + MAFF/MAFK shRNA was higher than the luciferase activity of rs7220711-G MAFF/MAF+ shRNA group.The results of qRT-PCR were same,illustrating that MAFF/MAFK can bind rs7220711(A/G)differentially and inhibit the SOST expression.Rs 1107748(T/C)can differentially bind CTCF through the prediction and sequence alignment in the wedsite.When U-20S,Saos-2 and 293T cells were transfected by the rs1107748-T and rs1107748-C carrier,dual luciferase reporter gene assays showed that the luciferase activity of recombinant vector T decreased significantly compared to recombinant vector C.When transfected U-20S cell by CTCF overexpression and interference carrier vector,the luciferase report gene activity of rs 1107748-T + CTCF overexpression group was lower than the activity of rs1107748-C + CTCF overexpression group.The luciferase report gene activity of rs1107748-T + CTCF shRNA was higher than the luciferase activity of rs rs1107748-C+ CTCF shRNA group.The results of qRT-PCR were same,illustrating that CTCF can bind rs1107748(T/C)differentially and promote the SOST expression.This study suggested that GWAS lead SNPs rs7220711 and rs1107748 are associated with osteoporosis by affecting the binding of MAFF/MAFK and CTCF.