miR‑9 depletion suppresses the proliferation of osteosarcoma cells by targeting p16

Abstract

Osteosarcoma (OS) is a common primary malignancy in adolescents and children. MicroRNAs (miRNAs or miRs) can regulate the progression of OS. Herein, we explored the target genes and effects of miR-9 in OS. Cell growth, colony formation and cell cycle were respectively examined using a cell counting kit-8 (CCK-8), crystal violet staining and flow cytometry. The target gene of miR-9 was predicted according to the MicroRNA.org website. Luciferase activity was examined using a dual luciferase reporter gene assay kit. The corresponding factors levels were analyzed by carrying out reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis. A mouse model of OS was also established and the volume and weight of the tumors of the mice with OS were measured. The levels of p16 in the mice with OS were detected by immunohistochemistry (IHC). The data revealed a high expression of miR-9 and a low expression of p16 in the OS tissue. p16 was found to be the target gene for miR-9 in OS. miR-9 depletion decreased the proliferation and colony formation of Saos-2 cells by arresting the cells at the G1 phase, accompanied by the downregulation of cyclin A, cyclin D1 and c-Myc expression levels. Moreover, miR-9 depletion inhibited the phosphorylation of p38, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). In vivo , miR-9 depletion decreased the tumor volume and weight and increased p16 expression in the mouse tumor tissues. Nevertheless, p16 silencing reversed the suppressive effects of miR-9 inhibitors on OS cells. On the whole, the findings of this study substantiate that miR-9 depletion suppresses cell proliferation by targeting p16 in OS and by mediating the activation of the ERK/p38/JNK pathway.

The impact of miR-9 in osteosarcoma

“…[ 21 ] In osteosarcoma, inhibition of miR-9 decreases the proliferation of tumor cells by targeting p16. [ 39 , 40 ] In addition, miR-9 was upregulated in spontaneous canine osteosarcoma and promoted the invasion and migration of osteosarcoma cells. [ 41 ] Meanwhile, miR-9 was an oncogene and promoted proliferation of osteosarcoma through targeting Grap2 and cyclin D interacting protein.…”

Inhibition of miR-9 decreases osteosarcoma cell proliferation

“…The validated target genes include nuclear factor kappa B subunit 1 (NFKB1), chromodomain helicase DNA binding protein 1 (CHD1), www.bjbms.org VEGFA, vimentin (VIM), MMP13, and BCL2 interacting killer (BIK) [8]. In a recent study, Gao et al showed the p16 CDKN2A gene to be another target gene of miR-9 in OS [23].…”

“…In their study, miR-9 depletion downregulated the extracellular signal-regulated kinase (ERK)/p38/c-Jun N-terminal kinase (JNK) pathway by targeting CDKN2A [23]. Abnormal cell cycle causes tumor growth and malignant cell proliferation [16][17][18].…”

Role of MicroRNAs in Human Osteosarcoma: Future Perspectives

“…Patients with highly expressed levels of miR-9 in osteosarcoma tissues present bigger tumor size, more advanced clinical stages, more frequent distant metastasis, and lower survival rates [ 74 ]. The effect that miR-9 has on OS progression seems to be related to it targeting p16 and mediating the activation of the ERK/p38/JNK pathways [ 75 ]. Interestingly, the high expression of miR-9, occurs not only in OS tissues, but also in the serum of patients [ 76 ].…”