Downregulation of DEPTOR inhibits the proliferation, migration, and survival of osteosarcoma through PI3K/Akt/mTOR pathway

Abstract

Accumulating evidence reveals that DEP-domain containing mTOR-interacting protein (DEPTOR) plays pivotal roles in the pathogenesis and progression of many tumors. However, the expression level of DEPTOR and its function in the tumorigenesis of osteosarcoma (OS) remain unknown. In this study, we conducted quantitative real-time polymerase chain reaction, Western blot, and immunohistochemistry to detect DEPTOR expression level in human OS tissues and cell lines. To assess DEPTOR function, DEPTOR siRNA was designed and transfected into OS cells, which were then used in a series of in vitro assays. Our results indicated that DEPTOR was highly expressed in some OS tissues and cell lines. DEPTOR knockdown by siRNA dramatically inhibited cell proliferation, migration, invasion, and the formation of vasculogenic mimicry in OS cells. In addition, DEPTOR knockdown induced cell cycle arrest in the G0/G1 phase and apoptosis in the OS cell lines, MG63 and MNNG/HOS. Furthermore, we found that DEPTOR knockdown notably activated mTOR and inhibited the PI3K/Akt pathway. Taken together, these results suggest that DEPTOR overexpression is necessary for the proliferation, migration, invasion, formation of vasculogenic mimicry, and survival of OS cells and may be a potential target for the treatment of OS.

Keywords: DEPTOR; PI3K/Akt/mTOR pathway; apoptosis; osteosarcoma; proliferation.

Figure 1

Quantitative RT-PCR, immunohistochemical staining, and Western blot analysis of DEPTOR expression in osteosarcoma tissues, corresponding adjacent normal tissues, and cell lines. Notes: (A) qRT-PCR results of DEPTOR expression in osteosarcoma tissues and adjacent normal tissues. Results are presented as the fold change in tumor tissues relative to that in adjacent normal tissues within five pairs of cancer and adjacent tissues. (B) Immunohistochemical staining of DEPTOR expression in tumor samples and adjacent normal tissues. Original magnification: ×100 (upper images), ×400 (bottom images). (C) Western blot analysis of DEPTOR expression in tumor tissues and adjacent normal tissues; the results were normalized to β-actin expression. (D) Protein and mRNA expression of DEPTOR in different osteosarcoma cell lines and normal osteoblasts. qRT-PCR results are expressed as mean ± SD of three independent experiments. Results are presented as the fold change in osteosarcoma cell lines relative to that in the osteoblastic cell line hFOB1.19 (**P<0.01, ***P<0.001). Abbreviations: DEPTOR, DEP-domain containing mTOR-interacting protein; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 2

The DEPTOR siRNA efficacy and influence of DEPTOR knockdown on the proliferation of OS cells. Notes: (A) Relative expression of DEPTOR mRNA in MG63 and MNNG/HOS cells after transfection with NC siRNA or DEPTOR siRNA for 24 h. Results are presented as the fold change in DEPTOR siRNA transfected cells relative to that in NC siRNA transfected cells. (B) Western blot analysis of DEPTOR protein when transfected with DEPTOR siRNA or NC siRNA in MG63 and MNNG/HOS cells. (C, D) The proliferation of MG63 (C) and MNNG/HOS (D) cells transfected with NC siRNA or DEPTOR siRNA. Data are expressed as mean ± SD of three independent experiments (*P<0.05, **P<0.01, ***P<0.001 compared with nonspecific siRNA group). Abbreviations: DEPTOR, DEP-domain containing mTOR-interacting protein; NC siRNA, nonspecific siRNA; OS, osteosarcoma.

Figure 3

Colony formation capacity of osteosarcoma cells. Notes: (A) Representative images of crystal violet staining of MNNG/HOS and MG63 cell colony formation assays. (B) Colonies containing >50 cells were counted. Quantified results were expressed as a percentage to the NC siRNA group, setting at 100%. Data are expressed as mean ± SD of three independent experiments (**P<0.01 compared with NC siRNA group). Abbreviation: NC siRNA, nonspecific siRNA.

Figure 4

Influence of DEPTOR knockdown on the migratory capacity of osteosarcoma cells. Notes: (A) Micrographs of wound healing assays for MNNG/HOS and MG63 cells transfected with NC siRNA or DEPTOR siRNA. Images were obtained at 0, 24, and 48 h, original magnification: ×100. (B) Percentage of wound closure. Data are expressed as mean ± SD of three independent experiments. (C) Micrographs of the Transwell migration assay in the MNNG/HOS and MG63 cells, original magnification: ×100. (D) Number of migratory cells. Data are expressed as mean ± SD of three independent experiments (*P<0.05, **P<0.01, compared with NC siRNA group). Abbreviations: DEPTOR, DEP-domain containing mTOR-interacting protein; NC siRNA, nonspecific siRNA.

Figure 5

Effects of DEPTOR knockdown on the VM formation and invasion capacity of osteosarcoma cells. Notes: (A) Images of tube-like structures in MNNG/HOS and MG63 cells transfected with NC siRNA or DEPTOR siRNA, original magnification: ×100. (B) Mean number of tube-like structures in MNNG/HOS and MG63 cells. Data are expressed as mean ± SD of three independent experiments. (C) Micrographs of the Transwell invasion assay in the MNNG/HOS and MG63 cells, original magnification: ×100. (D) Number of invaded cells. Data are expressed as mean ± SD of three independent experiments (**P<0.01, ***P<0.001 compared with nonspecifc siRNA group). Abbreviations: DEPTOR, DEP-domain containing mTOR-interacting protein; NC siRNA, nonspecific siRNA; VM, vasculogenic mimicry.

Figure 6

DEPTOR knockdown induced cell cycle arrest and apoptosis in osteosarcoma cells. Notes: (A) Flow cytometric analysis of the percentage of cells transfected with NC siRNA or DEPTOR siRNA in various phases of the cell cycle. (B) Flow cytometric analysis of the percentage of cells in different phases of the cell cycle with three independent experiments. (C) Flow cytometric analysis of apoptotic cells by Annexin V-FITC/PI staining; in all four plots, the right upper and right lower quadrants show apoptotic cells. (D) The proportion of apoptotic cells in MG63 and MNNG/HOS cells. Data are expressed as mean ± SD of three independent experiments (**P<0.01, ***P<0.001 compared with nonspecific siRNA group). Abbreviations: DEPTOR, DEP-domain containing mTOR-interacting protein; FITC, fluorescein isothiocyanate; NC siRNA, nonspecific siRNA; PI, propidium iodide.

Figure 7

DEPTOR knockdown inhibits the progression of OS by regulating the PI3K/AKT/mTOR pathway. Note: After 48 h of transfection, the expression levels of PI3K/AKT/mTOR pathway-related proteins, PI3K, AKT, p-AKT, mTOR, p-mTOR, 4E-BP1, and p-4E-BP1, were determined by Western blot. Abbreviations: DEPTOR, DEP-domain containing mTOR-interacting protein; mTOR, mammalian target of rapamycin; OS, osteosarcoma.