Functional Mechanisms Of GWAS SNP Rs1026364/miR-345-5p/Usf3 In Osteoporosis

Osteoporosis is a complex polygenic skeletal disease characterized by low bone mass,increased bone fragility and high risk of fractures.It is clinically defined through the measurement of bone mineral density(BMD)which is highly heritable with the heritability estimates of 0.5-0.8.Genome-wide association studies(GWAS)is a major approach to search for susceptibility genes and genetic variants that contribute to complex diseases in human.Over the last decade,GWAS have identified numerous SNPs(Single-Nucleotide Polymorphisms)that are associated with low BMD,osteoporosis,and osteoporotic fractures.The future challenge is to explore the functional mechanism of these SNPs in the 'post-GWAS' era.MiRNA is a kind of endogenous non-coding 20 to 25 nucleotides in eukaryotes,and it can negatively regulate the expression of its target genes at post-transcriptional level and is involved in various physiological processes.Objectives of present study are(1)To explore whether Usf3 rs 10263 64(C/A)affect the binding of miR-345-5-5p;(2)To investigate the function of miR-345-5p in osteogenesis;(3)To identify the target genes of miR-345;(4)To study the role of transcription factor Usf3 in osteogenesis.We systematically screened GWAS BMD associated SNPs which potentially affect miRNA-mRNA interaction through bioinformatics tools.Usf3 rs 1026364(p=4.10E-10)A allele was found to generate a binding site of miR-345-5p.Rank rs884205(p=9.40E-09)C allele was found to create a binding site of miR-3658.The transcription activity of Usf3 was decreased when the allele was A,and the activity of A allele was significantly lower than that of C allele in cell lines 293T,U-20S and Saos-2.When miR-345 was knocked down,the activity of A allele was restored to the activity of C level in Saos-2.The expression level of Usf3 was declined by 28%after overexpression of miR-345 in U937 cells with C/A genotype.The interaction of miR-3658 and Rank was not affect by SNP rs884205.Transfection of miR-345-5p agomir in U-20S significantly decreased bone mineralization,ALP activity and the expression of marker genes Runx2,Osterix,Alp,Collal and Osteopontin.In contrast,transfection of miR-345-5p antagomir increased bone mineralization,ALP activity and the expression of marker genes Runx2,Osterix,Alp,Collal and Osteopontin.Runx3,Smad,Klf10 and Mapkl genes were predicted as the potential targets of miR-345 by TargetScan6.2/7.0.The transcription activity of Runx3 and Smadl were significantly decreased when binding to rniR-345-5p.However,the activity of Klf10 and Mapkl were not changed.Overexpression of miR-345 downregulated the expression of Runx3 and Smadl.BLASTP analysis shows that Usf3 is unique in vertebrates.When Usf3 was suppressed by specific shRNA,the expression of Runx2,Osterix and Alp decrease in U-20S.Moreover,Usf3 positively regulates the promoter activity of Runx2.Our study have revealed a novel molecular pathological mechanism that GWAS SNP rs1026364(C/A)was associated with osteoporosis by influencing the interaction between miR-345-5p and Usf3 and regulating the expression of Usf3.MiR-345 negatively regulated osteoblast differentiation via targeting Usf3,Runx3 and Smadl.Usf3 might promote the osteogenic differentiation.