SOST Expression Regulation And Evolution

Sclerostin(SOST),a cysteine-knot glycoprotein predominately expressed by osteocytes,is a negative regulator of bone formation by binding to LRP5/6 and inhibiting canonical Wnt/?-catenin signaling.SOST monoclonal antibodies have been clinically used to treat osteoporosis.Protein-protein interaction network analysis of osteoporosis associated genes showed that SOST is a hub protein.Objectives of this study are to explore the evolution of SOST,and to characterize SNP function in the SOST gene by computional approach;and to investigate the regulation of miRNA and hormones on the SOST expression.BLAST and MEGA4 are used to explore the evolution of the SOST gene.Phylogenetic analysis showed that SOST evolved from sostdc1a and first appeared in chordates.The protein sequence alignment revealed that it first expressed in the amphioxus(Cephalochordata).SOST specifically expressed in chordates and highly conserved in vertebrate species.A total of 463 SNPs in SOST gene region was found by NCBI database.We use JASPAR to predict that twelve SNPs in the upstream region of the SOST gene influence the binding of osteogenesis transcription factors such as RUNX2.Two SNPs(rs372515234,rs200581535)were identified to potentially affect SOST protein structure,function and activity by SIFT,PolyPhen,I-Mutant Suite.Thirteen SNPs may affect the internaction of miRNA with SOST by MirSNP,PolymiRTS and miRNASNPv2.0.Four miRNA binding sites were identified in SOST 3'UTR by TargetScan.The dual-luciferase reporter assay system demonstrated that rs75901553(C/T)in SOST 3'UTR can mediate miR-98 binding to SOST.The activity of C allele(binding miR-98)was lower significantly than that of T allele in U-20S cell.When 293T with C/C genotype was transfected with the plasmid of different SNP alleles,we detected the difference of SOST mRNA level by qRT-PCR.Then 293T was transfected with the expression vector of miR-98,SOST expression is reduced.These results indicated that the change of rs75901553 from C to T attenutes affinity ability between miR-98 and SOST 3'UTR and increased the SOST expression.When U-20S cells was transfected with miR-98 agomir,the expression of SOST was inhibited,transfected with miR-98 antagomir SOST increased.So miR-98 can target SOST.When 10 nM E2 treated SAOS-2 cells for 9 h and 12 h respectively,the expression level of miR-98 raised 4.82 and 1.73 times respectively by qRT-PCR.while 50 nM PTH treated SAOS-2 cell for 12 h and 16 h respectively,the expression of miR-98 raised 2.58 and 5.64 times respectively.These results suggested that estrogen and parathyroid hormone could suppress the SOST expression in a short time by increasing the expression of miR-98.