COPB2 as a key regulator of cell growth in human osteosarcoma cells: Potential therapeutic target and prognostic indicator

Highlights

• •High COPB2 in osteosarcoma patients links to worse survival, suggesting its use as a prognostic biomarker.
• •Silencing COPB2 reduces osteosarcoma cell growth and colony formation, underscoring its role in tumor progression.
• •Downregulation of COPB2 led to cell cycle arrest in the G2 phase and an increase in apoptosis.
• •COPB2 silencing was shown to inhibit tumor growth and lung metastases in vivo.
• •COPB2 may regulate cell growth via kinase phosphorylation changes, warranting further mechanistic study.

Abstract

Purpose

Coatomer protein complex subunit beta 2 (COPB2) is a crucial component of the coatomer protein complex I, responsible for vesicle transport. Previous studies have indicated that COPB2 is highly expressed in malignant tumors and is involved in cell proliferation and apoptosis. However, the role of COPB2 in osteosarcoma and its underlying mechanisms remain unclear. This study aimed to investigate the impact of COPB2 on proliferation, apoptosis, and colony formation in human osteosarcoma cells, as well as to explore potential mechanisms.

Methods

Kaplan-Meier survival analysis was conducted to assess the association between COPB2 expression and the prognosis of osteosarcoma patients using data extracted from the Cancer Genome Atlas (TCGA) database. Additionally, COPB2 expression was examined in osteosarcoma tissue samples and four osteosarcoma cell lines using immunohistochemistry and quantitative real-time PCR (qRT-PCR). COPB2 expression was downregulated using siRNA in U2OS and SAOS-2 human osteosarcoma cells. Cell proliferation and colony formation were assessed using Cellomics/Celigo and Giemsa staining, respectively. Flow cytometry was used to evaluate cell cycle distribution and apoptosis. Tumor growth was evaluated in vivo model. Furthermore, the regulation mechanism of COPB2 on osteosarcoma cells was investigated using the Human Phospho-Kinase Array Kit.

Results

Patients with high COPB2 expression exhibited shorter overall survival and disease-free survival compared to those with low COPB2 expression. COPB2 was found to be highly expressed in osteosarcoma tissue samples and cell lines. Silencing of COPB2 significantly inhibited cell proliferation and colony formation. Additionally, COPB2 silencing altered the cell cycle distribution, leading to cell cycle arrest in the G2 phase, and promoted cell apoptosis in osteosarcoma cells. Further investigations revealed that COPB2 silencing inhibited tumor growth and lung metastases of osteosarcoma cells in vivo, and its effects on cell proliferation and apoptosis may be mediated through the regulation of kinase phosphorylation levels.

Conclusions

COPB2 expression is increased in osteosarcoma cells and plays a crucial role in cell growth regulation. Silencing of COPB2 inhibits cell proliferation, colony formation, and promotes cell apoptosis. Furthermore, COPB2 silencing inhibits tumor growth in vivo, suggesting its potential as an important therapeutic target in treating osteosarcoma.

1. Background

Osteosarcoma is indeed one of the most prevalent types of bone cancers, primarily affecting children and adolescents [1]. Due to its aggressive nature, a combination of chemotherapy and radical surgical resection is typically required for treatment [2]. Osteosarcoma tends to develop distal metastases, spreading to other parts of the body beyond the primary tumor site, and the prognosis for osteosarcoma greatly depends on the presence or absence of metastases [3,4]. Patients without metastases have a 5-year survival rate of no more than 70 %. However, for patients who have developed metastasis or experienced recurrence, the 5-year survival rate drops significantly to lower than 20 % [5,6]. These statistics highlight the challenges associated with osteosarcoma and emphasize the importance of early detection, prompt treatment, and the development of more effective therapeutic strategies to improve the overall survival rates for patients with this aggressive bone malignancy.

Coatomer protein complex subunit beta 2 (COPB2), initially identified by Stenbeck et al. [7] in 1993, is an integral component of the COPI coatomer complex, which orchestrates the retrograde transport of proteins from the Golgi apparatus to the endoplasmic reticulum [8]. COPB2 facilitates intracellular protein trafficking and sustains cellular homeostasis through its interaction with dilysine motifs and its association with Golgi-derived nonclathrin-coated vesicles. Emerging evidence has highlighted the overexpression of COPB2 in a spectrum of malignancies, including breast [9], lung [10], and gastric cancers [11]. Mechanistically, COPB2 has been shown to modulate critical cellular processes such as apoptosis and cell cycle progression in these cancers [[12], [13], [14], [15]], while also regulating multiple signaling pathways implicated in tumorigenesis [13]. These findings underscore the potential of COPB2 as both a therapeutic target and a prognostic biomarker in oncology. Despite these advances, the functional role and clinical significance of COPB2 in osteosarcoma, the most prevalent primary malignant bone tumor, remain unexplored, representing a critical gap in our understanding of its oncogenic potential in skeletal malignancies.

Therefore, this study aimed to investigate the effect of COPB2 on osteosarcoma cells and explore potential mechanisms. In the present study, the expression of COPB2 in human osteosarcoma was investigated, and the siRNA technique was used to silence the expression of COPB2 in cell lines of osteosarcoma. The effect of COPB2 on cell proliferation, cell cycle, cell apoptosis, and cell colony formation were investigated in vitro. The vivo model was used to detect the effects of COPB2 on tumor growth. In addition, Human Phospho-Kinase Array Kit was used to explore the regulation mechanism of COPB2 on osteosarcoma.

2. Materials and methods

2.1. Sample collection and IHC analysis

In this study, a total of 10 patients with conventional osteosarcoma were included from the Peking University First Hospital. The diagnosis of osteosarcoma was confirmed through pathological examination. Tissue sections were prepared from formalin-fixed osteosarcoma samples as well as adjacent tissues. To achieve deparaffinization, the sections were immersed in a xylene solution for 30 min, followed by dehydration using gradient ethanol for 60 min. Subsequently, the endogenous peroxidase activity was blocked for 10 min using 3 % H₂O₂ in PBS. After antigen retrieval, the sections were cooled to room temperature, and non-specific binding was blocked with 10 % serum in TBS for 30 min. Next, the sections were incubated overnight with Rabbit anti-COPB2 antibody (1:100, SIGMA) in PBS. Following washing with TBS solution, the sections were incubated with a secondary antibody labeled with HRP. Finally, staining was achieved by adding DAB and hematoxylin sequentially, and the sections were observed under an inverted microscope after dehydration and covering. The Ethics Committee Board of Peking University First Hospital approved the study protocol. This study was conducted following the ethical standards in the 1964 Declaration of Helsinki.

2.2. Analysis of the cancer Genome Atlas (TCGA) dataset

In this study, we investigated the association between COPB2 expression and survival prognosis in patients with sarcoma using the powerful GEPIA tool [16]. A comprehensive cohort of 262 patients with complete RNA-Seq information and clinical data as of March 1, 2023, was meticulously selected for analysis. To evaluate the impact of COPB2 on overall survival and disease-free survival, we employed Kaplan–Meier survival analysis. Gene expression level was assessed using the transcripts per million (TPM) metrics.

2.3. Cell culture

U-2OS, MG-63, HOS, and SAOS-2 cell lines were obtained from Shanghai Genechem Co., Ltd. The cells were cultured in petri dishes using DMEM medium (Corning, USA) supplemented with 1 % antibiotic and 10 % fetal bovine serum (Ausbian, Australia) under standard conditions. The culture medium was refreshed every 3 days. Cell passaging was performed when the cells reached approximately 80 % confluency in the petri dish, and the collected cells were used for subsequent experiments.

2.4. Lentivirus production and transfection

To specifically silence COPB2 expression, a COPB2 siRNA sequence (TCAGACTATTCAGCACAAT) was designed based on the COPB2 gene sequence. A non-specific siRNA sequence (TCCTCCGAACGTGTCACGT) was used as a negative control. Both siRNAs were inserted into the AgeI/EcoRI site of the GV115-GFP plasmid vector (GeneChem, Shanghai, China). The vector contained an internal CMV promoter, and a green fluorescent protein (GFP) was used as a biomarker for the plasmid. The integrated plasmid was then transfected into osteosarcoma cells using a transfection reagent (GeneChem, Shanghai, China). Lentiviral particles expressing specific or non-specific siRNA were collected 48 h after transfection and stored at −80℃. After 1 day, the culture medium was aspirated, and both lentiviruses were added to each plate at a ratio of MOL 10:1. The culture medium was replaced 12 h after infection. Osteosarcoma cells were infected using the procedure, and the expression of GFP was observed under a fluorescence microscope (Olympus, Japan) 3 days after infection. The efficiency of COPB2 silencing was further confirmed using RT-qPCR and western blot.

2.5. qRT-PCR assay

Total RNA was extracted from the four target cells using the Trizol kit (Pufei Corp., Shanghai, China). The RNA was then reverse transcribed into cDNA using M−MLV reverse transcriptase (Promega, Madison, WI) according to the manufacturer's instructions. All RT-qPCR reactions were carried out using the SYBR Green Real-Time PCR assay kit (Takara, Japan) on an Agilent Mx3000P instrument (America), with GAPDH serving as the endogenous control. The amplification conditions for COPB2 were as follows: an initial denaturation step at 95 °C for 30 s, followed by 45 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. Finally, a melting curve analysis was performed with temperature steps at 95 °C for 15 s, 55 °C for 30 s, and 95 °C for 15 s. The primers for COPB2 (211 bps) and GAPDH (121 bps) were obtained from Borui Corp. (Guangzhou, China), and their sequences are summarized in Supplementary Table S1. The relative mRNA expression levels were determined using the 2-△△Ct method.

2.6. Western blot analysis

Cells were harvested by centrifugation and resuspended in a cooled lysis buffer (100 mM Tris-HCl, pH 6.8, 4 % SDS, 20 % glycerol, and 2 % mercaptoethanol) for 15 min. The cells were then sonicated for 4 cycles to facilitate cell lysis. After centrifugation at 12,000g for 15 min at 4 °C, the supernatant was collected for protein concentration determination using the BCA assay (Beyotime, Jiangsu, China). Equal amounts of denatured cell protein (20 µg) were separated on 10 % SDS-polyacrylamide gels by electrophoresis. Following electrophoresis, the proteins were transferred to a PVDF membrane using a transfer electrophoresis apparatus (Bio-Rad, Richmond, CA). The PVDF membrane was blocked with a TBST solution containing 5 % skim milk for 1 h. Subsequently, the PVDF membrane was incubated overnight with the primary antibody. Mouse anti-flag antibody (1:2000, Sigma) and Mouse anti-GAPDH antibody (1:2000, Santa Cruz) were used as the primary antibodies in this study. After three washes with TBST, the PVDF membrane was incubated with a horseradish peroxidase-conjugated goat anti-mouse antibody (1:2000, Santa Cruz) as the secondary antibody for 2 h. Finally, the blots were visualized using an ECL Western Blotting Substrate assay (Pierce).

2.7. Cell proliferation assay

Cells were seeded in a 96-well plate at a density of 2000 cells/well for U-2OS and 1000 cells/well for SAOS-2, and cultured under standard conditions. The cell number was assessed daily for five consecutive days using automated imaging systems such as Celigo (Nexcelom) and Cellomics (ArrayScan VTI, Thermo Fisher Scientific).

2.8. Colony formation assay

Cells were cultured in a 96-well plate for 15 days, with a medium change every 4 days. After the culture period, cells were fixed with 4 % paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd, China) and stained with Giemsa (Shanghai Dingguo, China) for 20 min. Subsequently, the colonies were counted and photographed.

2.9. Cell cycle assay

Cells were collected in a 5 ml centrifuge tube along with the medium, ensuring that the number of cells exceeded 10^6 per tube. The cells were then centrifuged at 1300 rpm for 5 min at 4 °C. After centrifugation, the cells were washed with D-Hank’s solution and fixed in 75 % ethanol. The staining solution was prepared by mixing 40x propidium iodide (2 mg/mL, Sigma), 100x RNase (10 mg/mL, Fermentas), and 1x D-Hanks in a ratio of 25:10:1000. The fixed cells were resuspended in the staining solution and thoroughly mixed. The cell suspension was analyzed using flow cytometry (Millipore, Guava easyCyte HT instrument) to determine the cell cycle distribution.

2.10. Apoptosis assay

Cells were collected in a 5 ml centrifuge tube as described above, ensuring that the number of cells exceeded 5 × 10^6 per tube. Cell apoptosis was detected using the Annexin V-APC method (eBioscience) through flow cytometry, as mentioned earlier.

2.11. Tumor growth in vivo

Osteosarcoma cells were collected in a 5 ml centrifuge tube with D-Hank’s solution, ensuring a concentration of 1 × 10^7 cells/ml. A 200 µl cell suspension was subcutaneously injected into the armpit of the right forearm of female BALB/c nude mice (obtained from Jiangsu Jicai Yaokang Biotechnology Co., Ltd.). The tumor volume was measured on days 5, 10, 15, 20, and 25 after inoculation using the formula: tumor volume = π/6 × L × W × W (where π = 3.14, L represents the long diameter, and W represents the short diameter). On the 25th day, all nude mice were euthanized, and tumor tissues were collected for weighing and histological observation, including HE staining and Ki67 staining. In addition, six-week-old male C57BL/6 mice were inoculated with luciferase-labeled osteosarcoma cells (Control vs. COPB2 silencing) via paratibial injection. Tumor progression was monitored every 5 days using the IVIS Spectrum Imaging System (PerkinElmer, Waltham, MA, USA), beginning 5 days post-implantation. Prior to imaging, animals were anesthetized by ether inhalation. Bioluminescent signals were acquired over an exposure period and quantified as radiance (photons/sec/cm2/steradian) using Living Image® Software (v4.5.2, PerkinElmer).

2.12. Human phospho-kinase array

To investigate the regulatory mechanism of COPB2 in osteosarcoma, the Human Phospho-Kinase Array Kit (R&D Systems®, Inc. USA) was utilized following the manufacturer’s instructions. This array kit allows for the simultaneous detection of phosphorylation levels of 37 kinase phosphorylation sites and 2 related total proteins. Osteosarcoma cells were transfected and three days after transfection, the cells were analyzed using the Human Phospho-Kinase Array.

2.13. Statistical analysis

The data are expressed as mean ± standard deviation. A student t-test was performed to compare the differences between groups. In addition, repeated-measures analysis of variance (ANOVA) was used to compare the measured parameters at different time points between the two groups. A p-value less than 0.05 was considered statistically significant (two-sided test). All statistical analyses were conducted using IBM SPSS Statistics 21.

3. Results

3.1. The expression of COPB2 in osteosarcoma cell lines

IHC was performed to measure the expression of COPB2 in the tissue of human osteosarcoma (Fig. 1A), and it demonstrated that the expression of COPB2 was significantly upregulated in the osteosarcoma tissue in comparison to the carcinoma adjacent normal tissue (Fig. 1B). The baseline characteristics of the 10 patients with osteosarcoma are shown in Supplementary Table S2. The qRT-PCR demonstrated that the mRNA level of COPB2 was remarkably expressed in the four human cell lines of osteosarcoma (Fig. 1C), and the western blot analysis also confirmed the obvious expression of COPB2 protein in these cell lines (Fig. 1D). The above data indicated that COPB2 was highly expressed in human tissue and cell lines of osteosarcoma.

Fig. 1.

The expression of COPB2 in osteosarcoma and survival analysis of patients stratified by the expression level of COPB2. A. Immunohistochemical analysis of COPB2 in osteosarcoma patients (The osteosarcoma adjacent normal tissue was used as the control, original magnification × 400); B. Qualitative analysis of COPB2 expression in osteosarcoma; C. The mRNA expression of COPB2 in the four human osteosarcoma cell lines; D. The protein expression of COPB2 in the four human osteosarcoma cell lines. E. Overall survival analysis based on the cut-off value of median; F. Overall survival analysis based on the upper and lower quartile (>75 % vs. < 25 %); G. Disease-free survival analysis based on the cut-off value of median; H. Disease-free survival analysis based on the upper and lower quartile (>75 % vs. < 25 %).

3.2. COPB2 was related to the prognosis of patients

A total of 262 patients were classified into two groups based on their COPB2 gene expression levels. The analysis revealed that patients with high COPB2 expression had a significantly shorter overall survival (P = 0.035) compared to those with low COPB2 expression (Fig. 1E). Furthermore, when the cut-off value was set at the upper and lower quartile (>75 % vs. < 25 %) for high and low COPB2 expression, there was a significant difference in overall survival (P = 0.031) between the high and low COPB2 groups (Fig. 1F). Similarly, patients with high COPB2 expression had a shorter disease-free survival (P = 0.082) compared to those with low COPB2 expression (Fig. 1G). This trend was maintained when the cut-off value was set at the upper and lower quartile (>75 % vs. < 25 %), although it did not reach statistical significance (P = 0.06) (Fig. 1H). These findings suggest that high COPB2 expression may be associated with a poorer prognosis in patients with this type of cancer.

3.3. Development of COPB2 silencing cells

COPB2 siRNA was used to interfere with COPB2 expression to develop COPB2 silencing cells. Three days after transfection, the vast majority of cells of U2OS and SAOS-2 cells expressed GFP under fluorescence microscopy (Fig. 2A), and the percentages of transfection cells were 88.92 % and 83.91 %, respectively (Fig. 2B). Additionally, RT-qPCR showed that the mRNA of COPB2 in cells of COPB2 silencing group decreased by 84.0 % (Fig. 2C) and 76.4 % (Fig. 2D), respectively. As showed in Fig. 2E and Fig. 2F, the protein of COPB2 was decreased SAOS-2 and U-2OS cells in the COPB2 silencing group after two days of transfection. The above results suggested that the specific COPB2 siRNA could effetely downregulate the expression of COPB2, and COPB2 silencing cells were successful developed and could be used for next studies.

Fig. 2.

COPB2 siRNA was used to interfere with COPB2 expression. A. Observation of GFP positive cells under fluorescence microscopy; B. Percentage of successful transfection cells; C. RT-qPCR showed that the mRNA of COPB2 was downregulated in SAOS-2; D. RT-qPCR showed that the mRNA of COPB2 was downregulated in U-2OS; E. Western blot analysis showed the protein of COPB2 was downregulated in SAOS-2; F. Western blot analysis showed the protein of COPB2 was downregulated in U-2OS.

3.4. COPB2 silencing inhibited cell proliferation and colony formation

Celigo and Cellomics were performed to determine the effects of COPB2 silencing on cell proliferation of the SAOS-2 (Fig. 3A) and U-2OS (Fig. 3B) cells. Cell counts analysis demonstrated that the number of cells in the COPB2 silencing group grew slowly, whereas the control group had a more rapid growth of cells in the SAOS-2 (Fig. 3C) and U-2OS (Fig. 3D) cells. In detail, after COPB2 silencing, the cell number decreased up to 72.8 % and 71.5 % for SAOS-2 and U2OS cells, respectively. Next, the clonal formation test further revealed that the number of colony formation in the COPB2 silencing group was significantly less than that in the control group in SAOS-2 (22 ± 3 vs. 50 ± 4, Supplementary Fig. 1A and Supplementary Fig. 1B) and U2OS (27 ± 3 vs. 108 ± 5, Supplementary Fig. 1C and Supplementary Fig. 1D) cells. In addition, MTT analysis confirmed COPB2 silencing significantly decreased the cell proliferation in both the SAOS-2 (Supplementary Fig. 1E) and U2OS (Supplementary Fig. 1F) cells. The above results revealed that COPB2 silencing could significantly inhibit cell proliferation and the ability of colony formation in cells of osteosarcoma.

Fig. 3.

COPB2 silencing inhibited cell proliferation of U2OS and SAOS-2 cells. A. SAOS-2 cells were plated in a 96-wall plate from1 day to 5 days; B. U2OS cells were plated in a 96-wall plate from1 day to 5 days; C. Celigo and Cellomics showed the number of SAOS-2 cells; D. Celigo and Cellomics showed the number of U2OS cells.

3.5. COPB2 silencing altered cell cycle and induced apoptosis

The effects of COPB2 on the cell cycle in SAOS-2 and U-2OS cells were analyzed. The cell count of G1 phase was significantly reduced in COPB2 silencing group in the both SAOS-2 (41.98 % ± 0.21 % vs. 54.92 % ± 0.12 %, Fig. 4A and Fig. 4B) and U-2OS cells (48.09 % ± 0.59 % vs. 54.47 % ± 0.24 %, Fig. 4C and Fig. 4D), while the cell count of G2 phase was significantly increased (SAOS-2: 27.33 % ± 0.75 % vs. 17.23 % ± 0.84 %; U-2OS: 26.09 % ± 0.57 % vs. 19.62 % ± 0.87 %). In addition, annexin V-APC was performed to measure cell apoptosis of SAOS-2 and U2OS cells. The cell apoptosis rate of COPB2 silencing group was significantly increased in both SAOS-2 (13.95 % ± 0.47 % vs 4.43 % ± 0.12 %, Fig. 4E and Fig. 4F) and U-2OS cells (11.16 % ± 0.15 % vs. 4.65 % ± 0.15 %, Fig. 4G and Fig. 4H). These results suggested that COPB2 silencing altered the cell cycle and induced apoptosis of U2OS and SAOS-2 cells. Cells were arrested at the G2 phase after COPB2 silencing.

Fig. 4.

COPB2 silencing altered the cell cycle and induced apoptosis of the SAOS-2 and U2OS cells. A. Cell cycle analysis in the SAOS-2 cells; B. Percentage of cells in G1, S, and G2 in the SAOS-2 cells; C. Cell cycle analysis in the U-2OS cells; D. Percentage of cells in G1, S, and G2 in the U-2OS cells; E. Cell apoptosis analysis in the SAOS-2 cells; F. Percentage of apoptosis in the SAOS-2 cells; G. Cell apoptosis analysis in the U-2OS cells; H. Percentage of apoptosis in the U-2OS cells.

3.6. COPB2 silencing inhibit tumor growth in vivo

The effect of COPB2 on the tumor growth in vivo were analyzed. The tumor volume (Fig. 5A) and weight (Fig. 5B) of the control group were significant increased compared with the COPB2 silencing group. The IHC staining showed that the expression of Ki67 in the control group were significantly higher than that in the COPB2 silencing group (Fig. 5C and Fig. 5D). Additionally, bioluminescence imaging of parosteal osteosarcoma mouse model demonstrated weaker tumor-derived signals in the COPB2-silenced group (Fig. 5E), further confirming its suppressive effect on osteosarcoma growth (Fig. 5F). Notably, metastatic progression was also impaired by COPB2 silencing. At days 20 and 25 post-implantation, both groups exhibited detectable pulmonary bioluminescent signals, indicating lung metastasis. However, the control group displayed significantly stronger metastatic signals compared to the COPB2-silenced group, suggesting that COPB2 knockdown not only inhibits primary tumor growth but also attenuates metastatic dissemination.

Fig. 5.

COPB2 silencing inhibit tumor growth and lung metastases of osteosarcoma cells in vivo. A. The volume of tumors in nude mice tumor model; B. The weight of tumors; C. Immunohistochemical showed the expression of Ki67; D. The comparison of the expression of Ki67 in the two groups; E. Bioluminescence imaging of the parosteal osteosarcoma mouse model between the two groups; F. Quantitative analysis of bioluminescence imaging in the parosteal osteosarcoma mouse model.

3.7. Human Phospho-Kinase Array

Human Phospho-Kinase Array (Fig. 6A and Fig. 6B) was used to explore the regulation mechanism of COPB2 on osteosarcoma. The expression of 15 kinase phosphorylation sites in the COPB2 silencing group were significantly upregulated (Fig. 6C), including CREB (S133), eNOS (S1177), Fgr (Y412), p53 (S15), p53 (S46), JNK1/2/3 (T183/Y185, T221/Y223), Lyn (Y397), Msk1/2 (S376/S360), p38α (T180/Y182), PDGF Rβ (Y751), STAT2 (Y689), STAT5a/b (Y694/Y699), WNK1 (T60), β-Catenin, Hsp60, and 15 kinase phosphorylation sites were significantly downregulated, including Akt1/2/3 (T308), Akt1/2/3 (S473), Chk-2 (T68), c-jun (S63), GSK-3β (S9), p53 (S392), p70 S6K (T389), PYK2 (Y402), RSK1/2 (S221/S227), RSK1/2/3 (S380/S386/S377), Yes (Y426), STAT1 (Y701), STAT3 (Y705), STAT3 (S727), and STAT6 (Y641). These results indicated that COPB2 might affect the proliferation and apoptosis of human osteosarcoma cells by regulating the level of kinase phosphorylation. Western blot analysis (Fig. 6D) further demonstrated that the protein expression of STAT3 significantly increased in the COPB2 silencing group (Fig. 6E), and p-STAT3 significantly decreased (Fig. 6F). In addition, the p53 (Fig. 6G) and β-Catenin (Fig. 6H) both increased in the COPB2 silencing group. Based on bioinformatic analysis, COPB2 was found to be significantly associated with STAT3 (Fig. 6I), CREB1 (Fig. 6J), and AKT1 (Fig. 6K).

Fig. 6.

Human Phospho-Kinase Array and validation. A. Effect of COPB2 silencing of kinase phosphorylation in osteosarcoma cells; B. The map of kinase phosphorylation sites of Human Phospho-Kinase Array Kit; C. Analysis of the expression of phosphorylation sites; D. Validation of the expression of phosphorylation sites; E. Analysis of STAT3; F. Analysis of p-STAT3; G. Analysis of p53; H Analysis of β-Catenin; I. Association between COPB2 and STAT3; J. Association between COPB2 and CREB1; K. Association between COPB2 and AKT1.

4. Discussion

4.1. Principal findings

Our study provides evidence that COPB2 plays a critical role in regulating cell growth in human osteosarcoma. Silencing of COPB2 led to decreased cell proliferation, colony formation, and increased cell apoptosis. Additionally, COPB2 silencing inhibited tumor growth in vivo. These findings suggest that COPB2 may serve as an important therapeutic target for the treatment of osteosarcoma. Moreover, our in vivo experiments demonstrated that COPB2 silencing effectively inhibited tumor growth. This further emphasizes the therapeutic potential of targeting COPB2 in treating osteosarcoma.

4.2. COPB2 expression and its prognostic significance in osteosarcoma

In addition, our study demonstrated that the expression of COPB2 was negatively associated with overall survival and disease-free survival among patients of sarcoma. COPB2, with a molecular weight of 102 kDa, is involved in forming the coatomer protein complex I and responsible for vesicle transport between the Golgi apparatus and endoplasmic reticulum [7,17,18]. The encoding gene is located on human chromosome 3q23 and contains 23 exons [19]. The N-terminal third of COPB2 is made up of five repeated motifs typical of the WD-40. COPB2 is known to be involved in intracellular protein trafficking as a subunit of the coatomer protein complex I. This complex is responsible for vesicle transport within cells, facilitating the transport of proteins from the Golgi apparatus to the endoplasmic reticulum [8]. COPB2 specifically recognizes and binds to the dilysine motifs present on cargo proteins destined for retrograde transport [20]. The dysregulation of protein trafficking can have significant implications for cellular homeostasis, as it can disrupt essential cellular processes such as proliferation, apoptosis, and cell cycle regulation [11].

4.3. Mechanistic insights into COPB2 function in osteosarcoma

In our study, silencing of COPB2 resulted in a significant inhibition of cell proliferation and colony formation in osteosarcoma cells. This suggests that COPB2 is crucial for maintaining the proper balance of protein trafficking and cellular growth in osteosarcoma. Additionally, our findings indicate that COPB2 silencing altered the cell cycle distribution, causing cells to arrest in the G2 phase, and promoted cell apoptosis in osteosarcoma cells. These observations further support the notion that COPB2 is involved in the regulation of essential cellular processes related to cell growth and survival.

4.4. COPB2 and key signaling pathways in osteosarcoma

Tumor cell regulation by COPB2 involves intricate signaling pathways. COPB2 interacts with platelet-derived growth factor-beta receptors, triggering cell mitosis by activating cell proliferation signaling pathways [15]. Furthermore, COPB2 has been shown to influence cell-to-cell junctions and promote breast cancer metastasis through its regulation of N-cadherin [21]. Normal physiological cell function is governed by multiple signaling pathways, and the dysregulation of these pathways is a key mechanism in tumorigenesis. Our study revealed that COPB2 is associated with the phosphorylation of critical molecules in signal transduction pathways, including PI3K/Akt, MAPK, Wnt, and JAK/STAT. The dysregulation of the PI3K/Akt pathway, demonstrated by reduced Akt phosphorylation upon COPB2 silencing, is implicated in various pathological processes in osteosarcoma, such as cell proliferation, apoptosis, and metastasis [22]. Consistent with previous findings [13], we observed that the reduced phosphorylation of Akt in the COPB2 silencing group led to a significant downregulation of c-Myc expression, which in turn suppressed cell proliferation and invasion in osteosarcoma cells [23]. Additionally, treatment with an AKT inhibitor (Deguelin) attenuated the secretion of MMP-2 and MMP-9, thereby inhibiting cell migration and invasion in osteosarcoma cells. STAT3 is a key factor in the JAK/STAT pathway, and our study demonstrated a decrease in STAT3 phosphorylation upon COPB2 silencing. STAT3 is overexpressed in osteosarcoma and its expression is associated with prognosis [24]. The reduced phosphorylation of STAT3 promotes apoptosis and enhances chemosensitivity in human osteosarcoma [24]. Furthermore, β-Catenin plays a pivotal role in the Wnt pathway, and our study revealed an increase in β-Catenin phosphorylation in the COPB2 silencing group. Phosphorylated β-Catenin undergoes degradation via ubiquitination, thereby inhibiting Wnt pathway activity. Previous studies have shown that the inhibition of β-catenin activation significantly suppresses the proliferation, migration, and invasion abilities of osteosarcoma cells [25].

4.5. COPB2 in other malignancies: Comparative insights

The dysregulation of COPB2 expression has been implicated in various malignant tumors, including breast cancer [9], lung cancer [10], and gastric cancer [11]. In these malignancies, high expression of COPB2 has been associated with poorer cell growth. Our study adds to this body of evidence by demonstrating a similar association between high COPB2 expression and poorer survival in osteosarcoma patients. The potential therapeutic implications of targeting COPB2 in cancer treatment have also been suggested in previous studies. For example, a study showed that knockdown of COPB2 significantly inhibited cell growth, colony formation, and promoted cell apoptosis in gastric cancer [13]. In vivo experiments showed that knockdown of COPB2 reduced tumor growth [13]. Furthermore, the study revealed that COPB2 knockdown inhibited the receptor tyrosine kinase signaling pathway and its downstream signaling cascade molecules [13]. In addition, the upregulation of COPB2 was associated with advanced clinical symptoms and reduced overall survival in patients with lung adenocarcinoma [14]. Knockdown of COPB2 led to increased cell apoptosis and decreased cell proliferation in lung adenocarcinoma cells. Additionally, COPB2 overexpression translocated Yes-associated protein 1 (YAP1) from the cytoplasm to the nucleus, promoting cell proliferation, tumorigenesis, and inhibiting cell apoptosis [14]. The effects of COPB2 on cell growth and tumor progression were dependent on the upregulation of YAP1 expression [14]. Furthermore, a review concluded that COPB2 has been identified as an oncogene in several cancer types, and it was involved in tumor cell proliferation, survival, invasion, and metastasis [11]. COPB2 plays a significant role in tumor progression and contributes to the development of hallmarks of cancer.

4.6. Limitations and future directions

While our study provides valuable insights into the role of COPB2 in osteosarcoma, there are some limitations that should be acknowledged. First, our study relied on in vitro and in vivo experiments using cell lines and mice models. Although these models provide valuable information and allow for the investigation of COPB2′s effects on cell growth and tumor progression, they may not fully recapitulate the complexity of the tumor microenvironment and the heterogeneity of osteosarcoma in human patients. Therefore, further studies using patient-derived xenograft models or clinical samples are needed to validate our findings. Second, our study focused on the impact of COPB2 on cell proliferation, colony formation, cell cycle distribution, and apoptosis. While these are important aspects of tumor growth and progression, there may be other cellular processes and signaling pathways that are influenced by COPB2, which were not investigated in our study. Additionally, our study primarily relied on COPB2 silencing to investigate its effects on osteosarcoma cells. While this approach demonstrated the importance of COPB2 in regulating cell growth, it would be valuable to further explore the effects of COPB2 overexpression and understand the potential dual roles of COPB2 in cancer. Lastly, our study focused on the association between COPB2 expression and patient prognosis in osteosarcoma. However, more comprehensive clinical data, including information on patient demographics, treatment regimens, and follow-up, are needed to fully evaluate the prognostic value of COPB2 in osteosarcoma patients.

5. Conclusions

COPB2 expression is increased in osteosarcoma cells and plays a crucial role in cell growth regulation. Silencing of COPB2 inhibits cell proliferation, colony formation, and promotes cell apoptosis. Furthermore, COPB2 silencing inhibits tumor growth in vivo, suggesting its potential as an important therapeutic target for the treatment of osteosarcoma and other malignancies. Further studies are warranted to elucidate the specific mechanisms by which COPB2 regulates cell growth in osteosarcoma and to explore its potential as a prognostic marker for this aggressive bone malignancy.

CRediT authorship contribution statement

Yunpeng Cui: Writing – original draft, Investigation, Formal analysis, Data curation. Xuedong Shi: Writing – review & editing, Writing – original draft, Validation, Resources, Project administration, Conceptualization. Qiwei Wang: Validation, Supervision, Software, Methodology, Data curation, Conceptualization. Wence Wu: Writing – review & editing, Writing – original draft, Visualization, Supervision, Investigation, Data curation. Yuanxing Pan: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Conceptualization. Bing Wang: Writing – review & editing, Writing – original draft, Validation, Resources, Investigation, Conceptualization. Mingxing Lei: Writing – review & editing, Writing – original draft, Visualization, Supervision, Project administration, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: