Abstract
Down-regulation of p16INK4a and miR-146b-5p contributes to tumorigenesis in osteosarcoma (OS). However, the correlation between p16INK4a and miR-146b-5p in OS proliferation remains largely unknown. In the present study, we demonstrated that miR-146b-5p expression was positively correlated with p16INK4a in OS, but inversely correlated with TNF receptor associated factor 6 (TRAF6) expression. Overexpression of miR-146b-5p dramatically suppressed OS cell proliferation. Mechanistically, we validated TRAF6 as a direct functional target of miR-146b-5p and found that miR-146b-5p overexpression significantly decreased the level of phosphorylated PI3k and Akt, which are the pivotal downstream effectors of TRAF6. Moreover, TRAF6 expression was positively correlated with Ki-67 but inversely correlated with miR-146b-5p expression. In OS cells, silencing of TRAF6 mimicked the anti-tumor effects of miR-146b-5p. p16INK4a is an important tumor suppressor gene frequently down-regulated in OS. We found that this inhibitory effect is associated with the suppression of the miR-146b-5p, and is mediated via up-regulating TRAF6 expression. Our findings identified p16INK4a and miR-146b-5p as tumor suppressors, and suggested p16INK4a, miR-146b-5p and TRAF6 as potential therapeutic candidates for malignant OS.
Highlights
Osteosarcoma (OS) is a high-grade malignant cancer of skeleton, which originates from immature osteoid matrix deposition by spindle cells originated from mesenchymal tissue [1,2]
We found that transfection with miR-146b-5p inhibitor and TNF receptor associated factor 6 (TRAF6) uniformly significantly reduced Ki-67 expression, and OS cell proliferation, as measured by Western blotting (Figure 4A) and CCK8 proliferation assay (Figure 4B), respectively
A strong correlation between p16INK4a and miR-146b-5p expression was observed. This is demonstrated by Quantitative RT-PCR (qRT-PCR) analysis of p16 expressing cells (EH1) and p16-deficient cells (U2OS) (Figure 1), which clearly showed elevated expression of mature miR-146b-5p in EH1 cells as compared with U2OS cells
Summary
Osteosarcoma (OS) is a high-grade malignant cancer of skeleton, which originates from immature osteoid matrix deposition by spindle cells originated from mesenchymal tissue [1,2]. OS is the most prevalent bone cancer in children and adolescents [4]. It is bimodally distributed among different age groups of 10–14 years and 60 years [4]. One class of emerging therapeutic molecules against cancers is microRNA (miRNA). MiRNAs perform a broad spectrum of biological functions by binding with 3 UTR of genes [7,8]. They are involved in the other biological processes, such as apoptosis, metabolism, development, proliferation and cellular differentiation [8].
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