MicroRNA-208a-3p promotes osteosarcoma progression via targeting PTEN

Abstract

Osteosarcoma (OS) is a malignant bone tumor with a poor prognosis. Accumulated evidence has suggested that microRNAs (miRNAs/miRs) may function as either oncogenes or tumor suppressors, which are associated with tumorigenesis and the progression of different types of cancer. In the present study, the role of miR-208a-3p in OS was investigated. The expression levels of miR-208a-3p in OS tissues and cell lines were determined via reverse transcription-quantitative PCR (RT-qPCR). MTT and colony formation assays were performed to verify the proliferation rate of OS cells. In addition, the effects of miR-208a-3p on the migration and invasion of OS cells were revealed using wound-healing and Transwell assays, respectively. Furthermore, the association between miR-208a-3p and phosphatase and tensin homolog (PTEN) 3'-untranslated region was determined via luciferase reporter assays, western blot and RT-qPCR analysis. The results indicated that miR-208a-3p was upregulated in OS tissues and cell lines compared with adjacent normal tissues and human osteoblastic cells, respectively. miR-208a-3p overexpression promoted and miR-208a-3p knockdown inhibited OS cells proliferation and metastatic potential. Additionally, PTEN was validated as a direct target of miR-208a-3p and its expression was negatively associate with that of miR-208a-3p in OS cells. Taken together, these results may suggest that miR-208a-3p promoted OS cells proliferation and metastatic potential via targeting PTEN. Therefore, miR-208a-3p may be considered as a diagnostic biomarker for OS.

Keywords: invasion; microRNA-208a-3p; migration; osteosarcoma; phosphatase and tensin homolog; proliferation.

Figure 1

Expression of miR-208a-3p in OS tissues and cell lines. (A) RT-qPCR analysis of miR-208a-3p expression levels in 10 pairs of OS tissues and adjacent normal tissues. (B) RT-qPCR was performed to detect the expression levels of miR-208a-3p in osteoblast cells (hFOB 1.19) and OS cell lines SaOS-2, U2OS and MG-63. ^**P<0.01 and ^***P<0.001. RT-qPCR, reverse transcription-quantitative PCR; miR, microRNA; CTL, control; OS, osteosarcoma.

Figure 2

Overexpression of miR-208a-3p promotes proliferation in osteosarcoma cells. (A) The proliferation of SaOS-2, U2OS and MG-63 cells following overexpression of miR-208a-3p was determined via MTT assays at different time points. (B) Colony formation assays were performed to detect the proliferation of SaOS-2, U2OS and MG-63 cells following overexpression of miR-208a-3p. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. mimic NC. NC, negative control; miR, microRNA; OD, optical density.

Figure 3

Overexpression of miR-208a-3p facilitates osteosarcoma cell invasion and migration. (A) The invasion of SaOS-2, U2OS and MG-63 cells following overexpression of miR-208a-3p was detected via Transwell assays (magnification, x100). (B) The migration of SaOS-2, U2OS and MG-63 cells following overexpression of miR-208a-3p was examined via wound-healing assays (magnification, x40). NC, negative control; miR, microRNA.

Figure 4

Knockdown of miR-208a-3p inhibits osteosarcoma cell proliferation. (A) MTT assays were performed to detect the proliferation of SaOS-2, U2OS and MG-63 cells following knockdown of miR-208a-3p at different time points. (B) The proliferation of SaOS-2, U2OS and MG-63 cells following knockdown of miR-208a-3p was examined via colony formation assays. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. inhibitor NC. NC, negative control; miR, microRNA; OD, optical density.

Figure 5

Knockdown of miR-208a-3p suppresses the invasion and migration of OS cells. (A) Transwell assays were performed to detect the invasion of OS cells following knockdown of miR-208a-3p (magnification, x100). (B) Wound healing assays were performed to detect the migration of OS cells following knockdown of miR-208a-3p (magnification, x40). NC, negative control; miR, microRNA; OS, osteosarcoma.

Figure 6

PTEN is a direct target of miR-208a-3p. (A) Potential binding site between miR-208a-3p and the 3'-UTR of PTEN. (B) A luciferase reporter assay was conducted to verify that PTEN was directly targeted by miR-208a-3p. ^***P<0.001. (C) The mRNA expression levels of PTEN in OS cells was detected via reverse transcription-quantitative PCR. ^*P<0.05. (D) Western blotting was performed to measure the protein expression levels of PTEN in OS cells. ^**P<0.01 and ^***P<0.001 vs. NC groups. UTR, untranslated region; NC, negative control; miR, microRNA; OS, osteosarcoma; wt, wild-type; mut, mutant.