Osteoporosis genome‐wide association study variant c.3781 C>A is regulated by a novel anti‐osteogenic factor miR‐345‐5p

Abstract

Upstream transcription factor family member 3 (USF3) c.3781C>A (rs1026364) in the 3′-untranslated region (3′-UTR) has been firmly associated with bone mineral density (BMD) in genome-wide association study (GWAS). However, the molecular mechanism by which it influences BMD and osteoporosis is unknown. Bioinformatics analyses suggested that the risk c.3781A allele creates a target site for hsa-miR-345-5pbinding. Luciferase assay validated that the c.3781A allele displayed significantly lower luciferase activities than the c.3781C allele in the human osteoblast cell line hFOB1.19, osteosarcoma cell lines U-2OS and Saos-2, and embryonic kidney cell line 293T. Furthermore, hsa-miR-345-5p regulated USF3 expression on both messenger RNA and protein levels in hFOB1.19 and U937 cells with heterozygous A/C genotype.Transfection of hsa-miR-345-5p antagomiR in heterozygous hFOB1.19 cells significantly increased the expression of osteogenic marker genes RUNX2, OSTERIX, COL1A1, ALP, OPN, OCN, and alkaline phosphatase activity and matrix mineralization level. Importantly, we found that hsa-miR-345-5p also inhibits osteoblast maturation in cell lines U-2OS with hsa-miR-345-5p nonbinding C/C genotype by targeting RUNX3 and SMAD1. Our findings uncovered a novel pathogenetic mechanism of osteoporosis by GWAS variant c.3781C>A-mediated disruption of hsa-miR-345-5p binding at the 3′-UTR of USF3 and the functional role of hsa-miR-345-5p in osteogenic differentiation. K E Y W O R D SGWAS SNP, miR-345-5p, osteoporosis, RUNX3, SMAD1, USF3

Identification of key serum biomarkers for the diagnosis and metastatic prediction of osteosarcoma by analysis of immune cell infiltration

“…No similar relationships were found for COL1A1. However, in hFOB1.19 cells heterozygous for the c.3781A allele and the c.3781C allele, the expression of COL1A1 was regulated by hsa-miR-345-5p, affecting ALP activity and substrate mineralization levels [ 68 ]. Hawkins et al .…”

Construction of a circRNA-miRNA-mRNA network based on competitive endogenous RNA reveals the key pathways and central genes of osteosarcoma in vivo

“…At the mRNA level of the ceRNA network, Wang et al found that in hFOB1.19 cells which were heterozygous for the c.3781A allele and the c.3781C allele, the expression of COL1A1 was regulated by hsa-miR-345-5p and affected alkaline phosphatase activity and substrate mineralization level [30] . Research by Hawkins et al on Ewing's sarcoma found that activation of Wnt/beta-catenin signaling affects the expression level of COL1A1 [31] .…”

Identification of the Key Serum Biomarkers to Diagnose and Predict Metastasis of Osteosarcoma Based on the Analysis of Immune Cell Infiltration Characteristics

“…As for COL1A1, no statistically signi cant results were shown in serum samples. But, it was reported that in hFOB1.19 cells which were heterozygous for the c.3781A allele and the c.3781C allele, the expression of COL1A1 was regulated by hsa-miR-345-5p and affected alkaline phosphatase activity and substrate mineralization level [64] . In addition, Hawkins et al found that activation of Wnt/beta-catenin signaling affected the expression level of COL1A1 on Ewing's sarcoma [65] .…”