Target genes of miRNA in AIONFH cases
To further correlate the differentially expressed miRNAs with potential target genes involved in bone homeostasis, the mRNA transcripts in necrotic bone samples from the AIONFH patients were analyzed. As report, VEGFA was targeted by miR-127-3p or miR-1, and PDGFA was regulated by miR-1. These genes were upregulated in necrotic bone samples from AIONFH patients compared with their expression in samples from healthy control individuals. PTEN and RUNX2 regulated by miR-628-3p and IGF2 by miR-483-5p were downregulated, whereas the expression of SEMA3D, which was regulated by miR-885-5p, and SOD1, which was regulated by miR-1, was not significantly different in samples from AIONFH patients compared with that in samples from healthy control individuals.
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qRT-PCR system was used to identify miRNAs related to osteogenesis and angiogenesis. These include insulin-like growth factor 2 (IGF2), semaphorin 3D (SEMA3D), runt-related transcription factor 2 (RUNX2), platelet-derived growth factor subunit A (PDGFA), superoxide dismutase 1 (SOD1), telomerase-associated protein 1 (TEP1), and vascular endothelial growth factor A (VEGFA). cDNA from 10 necrotic bone tissue samples were used to compare with 10 control bone tissue samples from femoral neck fracture patients. The relative expression levels of each gene were determined by qRT-PCR using the 2-△△CT method, with β-actin as a normalized control. The RT reaction was performed under the following reaction conditions: 30 min at 25°C, 30 min at 42°C, and 5 min at 85°C, followed by maintenance at 4°C. The selected miRNAs were confirmed with SYBR Green I dye (Takara, Dalian, China) with an ABI PRISM 7300 Real-time PCR System (Applied Biosystems, USA) at 95°C for 3 min, followed by 40 cycles at 95°C for 12 s and 62°C for 40 s.