Screening of microRNA and the targets prediction in mice with iron-induced osteopenia

Abstract

Objective To explore the correlation between bone loss and the differential expression of microRNA (miRNA, miR) in mice with iron accumulation. Methods 12-week-old male ICR mice (n=12) were randomly divided into two groups: FAC group and Control group. Intraperitoneal injections of iron citrate amine (FAC) and equivalent saline were conducted in these two groups. Serum ferritin level was detected after 8 weeks of intervention. Micro-CT was used to detect bone volume and bone structure parameters in mice. The whole blood of mice were collected and RNA from white blood cells were extracted. Differential expression profiles of miRNA chips was conducted, and the result was verified by real-time fluorescence quantitative polymerase chain reaction(FQ- PCR) . Targetscan and miRDB were used to predict the downstream target genes of miRNA. Bioinformatics analysis of target gene was conducted, including celluar component, biologic process, molecular function and kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. Results Serum ferritin (218.2±29.5) μg/L was significantly higher than that in the control group (30.6±9.9) μg/L after 8 weeks. Micro-CT analysis showed a destruction of bone structure in iron accumulation mice. Bone mass, trabecular thickness (Tb. Th) and bone volume fraction (BV/TV) decreased significantly (P=0.001, 0.005, 0.021) in iron accumulation mice, while trabecular separation/spacing (Tb. Sp) increased. miRNA chip results: 20 miRNAs up regulated: mmu-miR-29b-3p, mmu-miR-324-3p, mmu-miR-133a-5p, mmu-miR-214-5p, mmu-miR-22-3p, mmu-miR-34a-5p, mmu-miR-31-5p, mmu-miR-143-5p, mmu-miR-423-3p, mmu-miR-223, mmu-miR-155, mmu-miR-106a, mmu-miR-2861, mmu-miR-148a, mmu-miR-96, mmu-miR-449a-5p, mmu-miR-423-5p, mmu-miR-204-5p, mmu-miR-211, mmu-miR-23b and 7 miRNAs down regulated: mmu-miR-18a-3p, mmu-miR-223-3p, mmu-miR-199a-5p, mmu-miR-196a-5p, mmu-miR-30c-5p, mmu-miR-15b, mmu-miR-130b were screened out. The difference of mmu-miR-423-5p is the most significant.The result of FQ-PCR on miR-423-5P had a high concordance with the result of microarray.91 downstream target genes of miR-423-5p were predict by Targetscan and miRDB tools. Bioinformatics analysis showed that target genes are clustered on phosphatidylinositol 3 kinase (PI3K)-protein kinase B (Akt), Rap1, Ras and mitogen-activated protein kinase (MAPK) signaling pathways. Conclusion The iron-induced bone loss may be related to the differential expression of miR-423-5p. Key words: Iron accumulation; Osteoporosis; MicroRNA