Functional Analysis Of GWAS Locus 1p36.12 And Identification Of LncRNAs Associated With Osteoporosis

Osteoporosis is a skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue,thereby leading to increased bone fragility and susceptibility to fracture.Osteoporosis is defined by bone mineral density,the heritability of which is 50%-85%.Genome-wide association study(GWAS)is an effective method to identify susceptibility loci for complex genetic diseases.GWASs have discovered lots of osteoporosis trait-associated single nucleotide polymorphisms(SNPs).The major challenge of osteoporosis genetics in post-GWAS era is to explore the biological function and molecular mechanisms underlying osteoporosis susceptibility loci.LncRNAs are proved to play important regulatory roles in disease development.A few lncRNAs related to osteoporosis have been identifed and well-characterized.Our study aims to investigate the regulatory function of osteoporosis GWAS locus 1p36.12,and to identify lncRNAs associated with osteoporosis.1.Functional study of the osteoporosis GWAS locus 1p36.12Genome-wide linkage and association studies have identified an osteoporosis-associated locus at 1p36.12 that harbors GWAS SNPs rs7521902,rs7524102,rs34920465,rs6426749.The lp36.12 locus also comprises the WNT4 gene with known role in bone metabolism and functionally unknown ZBTB40/lncRNA ZBTB40-IT1 genes.Computational analysis showed SNPs rs7521902,rs34920465 could differentially bind transcription factor JUN::FOS,rs7524102 and rs6426749 could differentially bind transcription factors TCF7L2 and CREB1 respectively.Rs7521902 and rs7524102 are eQTL of WNT4 and ZBTB40 respectively.SNPs rs7521902 and rs7524102 are enhancer SNPs.Dual-luciferase assay demonstrated that SNP rs7521902 alleles could not differentially bind transcriptional factor JUN::FOS,SNP rs7524102-A allele could bind transcriptional factor TCF7L2,which efficiently elevated the enhancer activity and increased ZBTB40-IT1 and ZBTB40 expression.SNPs rs34920465-G and rs6426749-C alleles could respectively bind transcription factors JUN::FOS and CREB1,and upregulated ZBTB40-IT1 expression.In U-2OS and hFOB1.19 cell lines,lncRNA ZBTB40-IT1 was able to significantly decrease the expression of osteogenesis related genes WNT4,RUNX2,OSX,COL1A1 and ALP,suppressing osteogenesis.However,lncRNA ZBTB40-IT1 could also inhibit osteoclastogenesis related gene OPG expression and increase osteoclastogenesis related gene RNAKL expression,RANKL/OPG ratio>1,promoting osteoclastogenesis.LncRNA ZBTB40-IT1 had no effect on ZBTB40 expression.Whereas ZBTB40 played an opposite role in bone metabolism.In U-2OS cell lines,ZBTB40 significantly elevated the expression of WNT4,RUNX2,OSX,COL1A1 and ALP,enhancing osteogenesis.ZBTB40 could increase OPG expression and reduce RNAKL expression,RANKL/OPG ratio<1,inhibiting osteoclastogenesis.However,ZBTB40 could suppress lncRNA ZBTB40-IT1 expression.PTH treatment significantly downregulated the expression of ZBTB40-IT1,upregulated the expression of WNT4,and had no effect on ZBTB40 expression.2.Identification and computational analysis of lncRNAs at osteoporosis GWAS lociUp to December 2018,524 osteoporosis GWAS SNPs with P<5 × 10-8 were reported.A total of 350 lncRNAs were located in GWAS loci,including 153 antisense lncRNAs,143 lincRNAs,38 pseudogenes.The conversation analysis of lncRNAs by Annolnc website found 43 lncRNAs with more than 20 conserved elements in primates,mammals,vertebrates.Subcellular localization analysis of lncRNAs by the iLoc-LncRNA website showed that 54.86%lncRNAs were located in cytoplasm,23.14%lncRNAs were located in cell nucleus,10.29%located in exosome and 11.71%located in ribosome.Analysis of lncRNA-protein interactions by the Annolnc website showed that 89 lncRNA were predicted to interact with proteins.CLIP data demonstrated that 117 lncRNAs mostly interacted with FMR1,PTBP1,CSTF2,HNRNPU and ELAVL1.Analysis of lncRNA-miRNA interactions through the LncRNASNP website identified miRNAs closely related to bone metabolism,such as hsa-miR-214,hsa-miR-107,hsa-miR-103a,hsa-miR-34a-3p,hsa-miR-19a-3p,hsa-miR-125a-3p.3.Identification and computational analysis of lncRNAs response to PTHRNA-seq yielded 24.42 Gb Clean Data in PTH treated Saos-2 cells.A total of 3321 lncRNAs were identified,including 2154 lincRNAs,486 antisense lncRNAs,534 intronic lncRNAs and 147 sense lncRNAs.These lncRNAs are characterized by short transcript length,short ORF length,less number of exon,simple structure,less isoform number,low expression level and widely distributed.A total of 1147 differentially expressed lncRNAs were obtained through differential expression analysis,including 527 up-regulated and 620 down-regulated lncRNAs.Cis-or trans-target genes of lncRNAs were mainly enriched in biological processes categories including cellular process,single-organization process,biological regulation and metabolic process,and cellular component categories including cell,cell part and organelle,and molecular function categories including binding.Cis-or trans-target genes of lncRNAs were mainly enriched in WNT,HIPPO,MAPK and TGF-?signaling pathway.Among these target genes of differentially expressed lncRNAs,there are 115 BMD GWAS genes.Protein-protein interaction analysis for the BMD GWAS gene was constructed by STRING website,and identified 15 hub genes,such as BMP4,LRP5,AXIN1,AXIN2,DKK1,SOX9 and JAG1.These hub genes were mainly involved in WNT and HIPPO signaling pathway.Comparative analysis of lncRNA identified from RNA-seq and osteoporosis GWAS loci showed that 6 lncRNAs shared between the two data sets,including 3 unaltered lncRNAs,1 upregulated lncRNA and 2 downregulated lncRNAs.