Minichromosome maintenance protein 2 and 3 promote osteosarcoma progression via DHX9 and predict poor patient prognosis

Abstract

A label free quantitative proteomic approach (SWATH™ experiment) was performed to identify tumor-associated nuclear proteins that are differentially expressed between osteosarcoma cells and osteoblast cells. By functional screening, minichromosome maintenance protein 2 (MCM2) and minichromosome maintenance protein 3 (MCM3) were found to be related to osteosarcoma cell growth. Here, we show that knockdown of MCM2 or MCM3 inhibits osteosarcoma growth in vitro and in vivo. In co-immunoprecipitation and co-localization experiments, MCM2 and MCM3 were found to interact with DExH-box helicase 9 (DHX9) in osteosarcoma cells. A rescue study showed that the decreased growth of osteosarcoma cells by MCM2 or MCM3 knockdown was reversed by DHX9 overexpression, indicating that MCM2 and MCM3 activity was DHX9-dependent. In addition, the depletion of DHX9 hindered osteosarcoma cell proliferation. Notably, MCM2 and MCM3 expression levels were positively correlated with the DHX9 expression level in tumor samples and were associated with a poor prognosis in patients with osteosarcoma. Taken together, these results suggest that the MCM2/MCM3-DHX9 axis has an important role in osteosarcoma progression.

Keywords: DHX9; MCM2; MCM3; osteosarcoma; proliferation.

Figure 1. Experimental workflow for the SWATH™ quantitative proteomics analysis

(A) Flowchart of the proteomic analysis. (B) Numbers of identified and quantified nuclear proteins in the SWATH™ analysis. (C) Numbers of upregulated and downregulated nuclear proteins in osteosarcoma cells. (D) A heat map of dysregulated proteins in osteosarcoma. (E, F) The mRNA expression levels of 18 upregulated and 5 downregulated nuclear proteins in MNNG/HOS and U2OS cells compared with hFOB 1.19 cells were verified by qRT-PCR. (G) Representative blots display the protein expression levels of 11 upregulated and one downregulated nuclear proteins. β-actin was used as an internal control.

Figure 2. Functional screening of 9 candidate nuclear proteins in the MNNG/HOS cell line

A CCK-8 assay was used to detect the proliferation of MNNG/HOS cells after transfection with siRNA. *P < 0.05.

Figure 3. Knockdown of MCM2 or MCM3 inhibits osteosarcoma cell proliferation

in vitro. (A–H) The mRNA and protein expression levels were validated after MCM2-specific or MCM3-specific siRNA transfection by qRT-PCR and western blotting in MNNG/HOS cells and U2OS cells. (I–L) CCK-8 assays were performed after siRNA transfection in MNNG/HOS cells and U2OS cells. (M–P) Colony formation assay for MCM2-silenced or MCM3-silenced osteosarcoma cells and control cells. Data are representative of results from three independent experiments. *P < 0.05.

Figure 4. Knockdown of MCM2 or MCM3 inhibits osteosarcoma cell growth in vivo

(A) Representative blots display the protein expression of MCM2 and MCM3 in MNNG/HOS cells stably expressing sh-MCM2 or sh-MCM3. β-actin was used as an internal control. (B) The upper photograph shows tumor-bearing mice and the lower photograph shows tumors when mice were euthanized. (C) Growth curve drawn by measuring tumor volumes on the indicated days. (D) The diagram shows tumor weights in the sh-control, sh-MCM2, and sh-MCM3 groups. (E, F) Representative images of ki-67 staining in the sh-control, sh-MCM2, and sh-MCM3 groups. Magnification, ×50, ×200. *P < 0.05.

Figure 5. MCM2 and MCM3 interact with DHX9 in osteosarcoma cells

(A) The silver staining for proteins separated by SDS-PAGE after IgG, MCM2, or MCM3 pull-down in 293T cells. (B) Whole-cell lysates were immunoprecipitated with the anti-MCM2 antibody or anti-MCM3 antibody followed by immunoblotting with anti-MCM2, MCM3, and DHX9 antibodies in the 293T cell line and the indicated osteosarcoma cell lines. IgG was used as a negative control. (C) Whole-cell lysates were immunoprecipitated with the anti-DHX9 antibody followed by immunoblotting with anti-MCM2, MCM3, and DHX9 antibodies in the MNNG/HOS cell line. IgG was used as a negative control. (D) An immunofluorescence study was performed using anti-MCM2, MCM3, and DHX9 antibodies in the MNNG/HOS cell line. DAPI was used as a control for nuclear staining.

Figure 6. Knockdown of MCM2 or MCM3 inhibits osteosarcoma cell proliferation via DHX9

(A, C) A CCK-8 assay was used to detect tumor cell proliferation after transfection with pcDNA 3.1-DHX9 plus si-MCM2 or si-NC. (B, D) CCK-8 assay was used to detect tumor cell proliferation after transfection with pcDNA 3.1-DHX9 plus si-MCM3 or si-NC. (E, G) The mRNA and protein expression levels were validated after DHX9-specific siRNA transfection by qRT-PCR and western blotting in MNNG/HOS and U2OS cells. (F, H) A CCK-8 assay was used to detect tumor cell proliferation after transfection with si-DHX9 in MNNG/HOS and U2OS cells. (I, J, K, L) Colony formation assay for DHX9-silenced osteosarcoma cells and control cells. Data are representative of three independent experiments. *P < 0.05.

Figure 7. Clinical significance of MCM2 and MCM3 in osteosarcoma patients

(A–C) Representative IHC images of the expression levels (negative: score = 0, low: score = 1, middle: score = 2, and strong: score = 3) of MCM2, MCM3, and DHX9 in osteosarcoma tissues. Original magnification: 50×, 200×. (D–G) The impact of MCM2 and MCM3 on TFS and OS for patients with osteosarcoma.