Abstract
We investigated the mechanisms by which microRNA (miR)-182 promotes apoptosis and inhibits proliferation in human osteosarcoma (OS) cells. Levels of miR-182 and Homeobox A9 (HOXA9) expression were compared between human OS and normal cells. Subjects were divided into OS and normal groups. We analyzed the target relationship of miR-182 and Homeobox A9 (HOXA9). Cells were then assigned into blank, negative control, miR-182 mimics, miR-182 inhibitors, siRNA-HOXA9, or and miR-182 inhibitors + siRNA-HOXA9 groups. Cell function was assayed by CCK-8, flow cytometry and wound healing assay. Additionally, we analyzed OS tumor growth in a xenograft mouse model. Dual-luciferase reporter assays indicated miR-182 directly targets HOXA9. Reverse transcription quantitative PCR and western blotting revealed elevated expression of miR-182, WIF-1, BIM, and Bax, and reduced expression of HOXA9, Wnt, β-catenin, Survivin, Cyclin D1, c-Myc, Mcl-1, Bcl-xL, and Snail in osteosarcoma cells treated with miR-182 mimic or siRNA-HOXA9 as compared to controls. Osteosarcoma cells also exhibited decreased cell proliferation, migration, and tumor growth, and increased apoptosis when treated with miR-182 mimic or siRNA-HOXA9. Correspondingly, in a xenograft mouse model, osteosarcoma tumor volume and growth were increased when cells were treated with miR-182 inhibitor and decreased by miR-182 mimic or siRNA-HOXA9. These results indicate that miR-182 downregulates Wnt/β-catenin signaling, inhibits cell proliferation, and promotes apoptosis in osteosarcoma cells by suppressing HOXA9 expression.
Highlights
Osteosarcoma (OS) is the most common primary bone tumor in children
These results indicate that miR-182 downregulates Wnt/β-catenin signaling, inhibits cell proliferation, and promotes apoptosis in osteosarcoma cells by suppressing Homeobox A9 (HOXA9) expression
We found that relative luciferase activity in OS cells transfected with a wild-type HOXA9 construct (HOXA9wt) was reduced in the miR-182 mimic compared to negative control (NC) group (P < 0.05)
Summary
Osteosarcoma (OS) is the most common primary bone tumor in children. There are limited treatments and the prognosis of patients with metastatic OS is poor [1]. OS arises from primitive bone-forming mesenchymal cells. The incidence rates are 3.5–4.6 per million in children aged 0–14 years and 4.6–5.6 in children aged 0–19 years each year [2]. The molecular mechanisms underlying OS recurrence and metastasis have not been elucidated [3]. MicroRNAs (miRs) are non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. Differential expression of 177 miRs was reported in 19 human OS cell lines [4]. The Wnt/β-catenin pathway has been shown to play a role in skeletal development and the specification of the osteoblast lineage and may have a key role in OS [5]
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