The effects of TRAF6 on proliferation, apoptosis and invasion in osteosarcoma are regulated by miR-124

Abstract

The present study aimed to verify tumor necrosis factor receptor‑associated factor 6 (TRAF6) as the target gene of microRNA-124 (miR-124). In addition, the expression of miR‑124 was investigated in osteosarcoma tissues and cells, and its effects on the biological characteristics of osteosarcoma cells were determined, in order to provide an experimental and theoretical basis for the application of TRAF6 in the treatment of osteosarcoma. A fluorescence reporter enzyme system was used to verify TRAF6 as a target gene of miR‑124, and western blotting was used to detect the effects of miR‑124 on the protein expression levels of TRAF6 in cells. The expression levels of miR‑124 were detected in osteosarcoma tissues and an osteosarcoma cell line (MG‑63) by quantitative polymerase chain reaction (qPCR). In addition, a total of 48 h post‑transfection of MG‑63 cells with a miR‑124 mimic, qPCR was used to detect the expression levels of miR‑124, and the effects of miR‑124 on the viability of MG‑63 human osteosarcoma cells was determined using the MTT method. The effects of miR‑124 on the cell cycle progression and apoptosis of MG‑63 cells were analyzed by flow cytometry, whereas the effects of miR‑124 on the migration of MG‑63 cells was detected using the Transwell invasion chamber analysis method. A TRAF6 recombinant expression plasmid (pcDNA3.1‑TRAF6) was also constructed, and MG‑63 cells were transfected with the recombinant plasmid and a miR‑124 mimic, in order to further validate the biological role of miR‑124 via the regulation of TRAF6. The results of the present study indicated that, compared with in the normal control group, the expression levels of miR‑124 were significantly increased in MG‑63 cells transfected with a miR‑124 mimic (P<0.01). In addition, the luciferase reporter gene system demonstrated that, compared with in the control group, relative luciferase activity was significantly reduced in the miR‑124 mimic group (P<0.01). The results of MTT analysis indicated that cell viability was also significantly reduced in response to the overexpression of miR‑124 in MG‑63 cells (P<0.01). Flow cytometric analysis demonstrated that the proportion of cells in S phase and G2/M phase was significantly decreased (P<0.01) in cells overexpressing miR‑124, and the number of apoptotic cells was significantly increased (P<0.01). Furthermore, the results of the Transwell invasion assay suggested that the number of invasive cells was significantly decreased following enhanced expression of miR‑124 (P<0.01). In MG‑63 cells overexpressing miR‑124 and TRAF6, the results of MTT, flow cytometric and Transwell assay analyses demonstrated that the overexpression of TRAF6 had the opposite biological effects compared to miR‑124 overexpression. In conclusion, the present study indicated that the expression levels of miR‑124 were downregulated in human osteosarcoma tissues and cells, and that miR‑124 is associated with negative regulation of TRAF6 expression; therefore, the role of TRAF6 in primary osteosarcoma may be regulated by miR‑1...

<p>Knockdown of lncRNA HOXA-AS2 Inhibits Viability, Migration and Invasion of Osteosarcoma Cells by miR-124-3p/E2F3</p>

“…Here, we first validated HOXA-AS2 as a decoy of miR-124-3p in OS cells by luciferase reporter assay, RIP and RNA pull-down analyses. Former works suggested that miR-124, the precursor miRNA of miR-124-3p, could suppress proliferation, migration and invasion of OS by targeting receptor tyrosine kinase-like orphan receptor 2, B7 homolog 3, sphingosine kinase 1 or tumor necrosis factor receptor-associated factor 6 19–22. More particularly, miR-124-3p was reported as a tumor suppressor in OS by regulating snail family zinc finger 2 or Rho-associated kinase 1 23,24.…”

The relationship between TRAF6 and tumors

“…miR-643 has recently been proved to targeting TRAF6 in human endometrial epithelial cells (HEECs) that significantly reduces the expression of TRAF6 [78]. miR-124 has also been reported to be related to negative regulation of TRAF6 expression in human osteosarcoma cells and colorectal cancer cells [79,80].…”

Genome-wide methylation analyses identifies Non-coding RNA genes dysregulated in breast tumours that metastasise to the brain

“…3) suggesting that MIR124 is dysregulated in BBM. Recent studies have reported that MIR124-2 suppresses proliferation, aggressiveness, apoptosis, and invasion of osteosarcoma 47 , 48 . MIR124 is abundantly expressed in the nervous system where it contributes to regulation of alternative splicing and plays a crucial role in the differentiation of progenitor neuronal cells 49 , 50 .…”