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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 66
| Issue : 4 | Page : 276-283 |
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Knockdown of neurotrophin receptor-interacting melanoma-associated antigen homolog inhibits acute myeloid leukemia cell growth via the ERK pathway
Hongxia Zhang1, Guangsheng Wu1, Beili Chen2
1 Department of Hematology, First Affiliated Hospital of Shihezi University, Shihezi, Xinjiang Uygur Autonomous Region, China
2 Department of Hematological, Affiliated Hospital of Guilin Medical University, Guilin, Guangxi Zhuang Autonomous Region, Guangxi, China
| Date of Submission | 19-Dec-2022 |
| Date of Decision | 06-Mar-2023 |
| Date of Acceptance | 23-Mar-2023 |
| Date of Web Publication | 04-Jul-2023 |
Correspondence Address:
Dr. Beili Chen
Department of Hematological, Affiliated Hospital of Guilin Medical University, No. 15, Lequn Road, Guilin, Guangxi Zhuang Autonomous Region, Guangxi 541001
China
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/cjop.CJOP-D-22-00162
Neurotrophin receptor-interacting melanoma-associated antigen homolog (NRAGE), a type II melanoma-associated antigen, plays a critical role in cell processes that are involved in the tumorigenesis of various cancers. However, the effect of NRAGE on acute myeloid leukemia (AML) is rarely reported. The expression of NRAGE in AML tissues and the survival rates between different AML groups were obtained from the GEPIA tool. Human AML cell lines were cultured and transfected with siRNA targeting NRAGE. The ability of AML cells to proliferate and cell cycle were examined. Western blotting was performed to detect the activity of the extracellular signal-regulated kinase (ERK) signaling pathway in AML cells. NRAGE expression was enhanced in AML tissues relative to control tissues, and the high NRAGE expression in AML patients is associated with a poor prognosis. The capacity of AML cells to survive and proliferate was significantly decreased and its cell cycle was arrested at the G1 phase after NRAGE was silenced. Furthermore, silencing NRAGE induced the inactivation of the ERK signaling pathway. Furthermore, supplement of tert-Butylhydroquinone, an ERK activator, improved the reduced ability of AML cell survival and proliferation as well as cell cycle arrest induced by NRAGE knockdown. In this study, NRAGE was identified as a tumor promoter in AML, which had an effect on cell proliferation, cell survival, and cell cycle through the ERK signaling pathway in AML cells.
Keywords: Acute myeloid leukemia, cell cycle, cell growth, extracellular signal-regulated kinase signaling pathway, neurotrophin receptor-interacting melanoma-associated antigen homolog
How to cite this article:
Zhang H, Wu G, Chen B. Knockdown of neurotrophin receptor-interacting melanoma-associated antigen homolog inhibits acute myeloid leukemia cell growth via the ERK pathway. Chin J Physiol 2023;66:276-83 |
How to cite this URL:
Zhang H, Wu G, Chen B. Knockdown of neurotrophin receptor-interacting melanoma-associated antigen homolog inhibits acute myeloid leukemia cell growth via the ERK pathway. Chin J Physiol [serial online] 2023 [cited 2023 Sep 26];66:276-83. Available from: https://www.cjphysiology.org/text.asp?2023/66/4/276/380364 |
| Introduction | |  |
Acute myeloid leukemia (AML) is a type of leukemia characterized by abnormal myeloid differentiation and the accumulation of leukemia stem cell precursors in the hemopoietic system.[1],[2] As the most frequent acute leukemia in adults (accounting for 80% in all cases), its incidence is increasing with age which ranges from 1.3/100,000 in people under 65 years to 12.2/100,000 in people over 65 years.[1],[3] At present, conventional chemotherapy, allogeneic hematopoietic cell transplantation, and chimeric antigen receptor-T-cell therapy have made some improvements in the treatment response and prognosis for AML, but the treatment effect is still generally poor.[4],[5],[6] Therefore, the treatment options of AML remain restricted, and more molecular studies are required to better understand the disease and find new therapeutic targets.
The mitogen-activated protein kinase (MAPK) signaling pathway performs a pivotal role in signal transduction, which is related to numerous cellular biological processes.[7] Upon activation, MAPKs coordinate and regulate various cellular activities such as cell proliferation, cell motility, cell survival, and cell apoptosis.[8],[9] Extracellular signal-regulated kinase (ERK) is a crucial member of the MAPK family. Moreover, dysregulation of the ERK signaling pathway has been reported to be correlated with malignant phenotypes of AML cells.[10]
Melanoma-associated antigen (MAGE) is a member of cancer/testis antigen family comprising more than fifty kinds of proteins.[11] The MAGE family can be classified into two groups based on various gene architectures and tissue-specific expression patterns: MEGA-1 and MEGA-2.[11] Aberrant activation and expression of MAGEs were found in various cancers.[12],[13] Neurotrophin receptor-interacting melanoma-associated antigen homolog (NRAGE, also called as MAGED1) is one of the MAGE families.[11] Prior studies have shown that NRAGE can promote cancer in various tumors.[14] NRAGE facilitates cancer invasion and chemoresistance, acting as a tumor promoter in gastric cancer.[15] NRAGE expression was significantly higher in hepatocellular carcinoma tissues, which accelerated the malignant progress of hepatocellular carcinoma.[16] Furthermore, NRAGE significantly promotes the radiation resistance of esophageal cancer cells.[17] However, the effect of NRAGE on AML remains unidentified.
In this report, the expression of NRAGE in AML and the survival analysis with NRAGE subgroups were analyzed by bioinformatic approach. The capability of AML cell viability and apoptosis and cell cycle were also assessed. Moreover, the activity of the ERK signaling pathway in AML cells was also detected. The present research attempted to investigate the influence of NRAGE on cellular functions of AML cells, which might provide new ideas for the treatment of AML.
| Materials and Methods | | |
Bioinformatic analysis
The transcript levels of NRAGE in tumor samples and control samples from 173 AML patients and 70 controls were obtained, and then, the survival analysis with NRAGE subgroup in AML patients was analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA), which is a web-based tool (http://gepia.cancer-pku.cn/index.html) to deliver fast and customizable gene expression datasets based on the TCGA and GTEx database.[18] Transcript per million normalization was used to measure gene or transcript expression levels of RNA sequencing data.
Cell culture and transfection
In this study, human bone marrow stromal cell line (HS-5) and four human AML cell lines (OCI-AML-5, KG-1, HL-60, and MOLM-14) which were all obtained from American Type Culture Collection (Manassas, VA, USA) were cultured in RPMI 1640 medium containing 10% FBS (Thermo Fisher Scientific, Waltham, Massachusetts, USA) with 1% penicillin and streptomycin (Thermo Fisher Scientific) at 37°C with 5% CO2 in a humidified incubator.
KG-1 and HL-60 cells were transfected with NRAGE-short interference RNA (siRNA) and negative control (NC)-siRNA (Thermo Fisher Scientific) as previously described.[19] When cells were 60%–80% confluence, cells were incubated with Lipofectamine transfection reagent (Thermo Fisher Scientific) for 24 h according to the manufacturer's protocol. After transfection, the ability of cell survival and cell proliferation was examined. Besides, AML cells were treated with tert-Butylhydroquinone (tBHQ) (MedChemExpress, Shanghai, China) at a dose of 5 μM for 15 min to activate the ERK signaling pathway.
Colony formation assay
Cells were inoculated into 12-well plates and cultured for 2 weeks at 37°C. Then, cells were fixed with methanol and stained with crystal violet for 10 min. After staining, cell colonies were photographed and counted.
Cell viability assay
Cell viability was assessed using a Cell Counting Kit-8 (CCK-8) assay (Sigma, St. Louis, MO, USA) as directed by the manufacturer.[20] A total of 5 × 103 cells were seeded into 96-well plates and then 10 μL CCK-8 solution was added for 2-h incubation. At a wavelength of 450 nm, optical density values were determined using a microplate reader (Bio-Rad, Richmond, VA, USA).
Western blotting
The collected cells were lysed in the RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific). A BCA protein detection kit (Thermo Fisher Scientific) was utilized to evaluate the protein concentrations of supernatant.[21] Protein was loaded, separated, and then transferred to PVDF membranes. After blocking, the blots were incubated with primary antibodies. Second antibodies (Thermo Fisher Scientific) were added for 1-h incubation. Following that, ECL was applied to develop the blots. The primary antibodies used in Western blotting are present in [Table 1].
Cell apoptosis assay
Cell apoptosis was detected using an Annexin Apoptosis Detection Kit (Thermo Fisher Scientific) according to the manufacturer's protocol.[22] Briefly, cells were incubated with 5 μL Annexin V reagent and 5 μL propidium iodide reagent for 5 min in the dark. Then, cells were enumerated and data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).
Cell cycle analysis
Cells were collected and fixed with 70% cold ethanol at 4°C overnight. Then, cells were stained with 1 mL of prepared DNA staining solution (Thermo Fisher Scientific) and 10 μL permeabilization solution (Thermo Fisher Scientific) in the dark at 37°C for 30 min. Following that, cells were enumerated and then data were analyzed using FlowJo software.
Statistical analysis
In this study, the GraphPad Prism software (version 8.2, San Diego, CA, USA) was used for all data analysis and visualization. The mean ± standard deviation was employed to express the data. To ascertain the statistical difference, the student's t-test or one-way analysis of variance was applied. Kaplan–Meier plotter was applied to assess the survival rates between different NRAGE expression groups. The hazard ratio of 95% confidence interval was displayed in the graph, and the log-rank P < 0.05 was considered statistically different.
| Results | | |
The increased expression of NRAGE is correlated with a poor prognosis in AML patients
Rising research reports that NRAGE plays a vital role in multiple cancers.[14],[15],[16],[17] We first explored the expression level of NRAGE in AML patients using public database. As shown in [Figure 1]a, GEPIA platform indicated that the expression of NRAGE in AML tissues was higher than that in control tissues. Further analysis was carried out to compare the relationship between NRAGE expression and the survival rates of AML patients. Moreover, AML patients with high NRAGE expression exhibited a poor prognosis in relation to those with low NRAGE levels [Figure 1]b. A set of 100 samples was divided into the low NRAGE expression group and the high NRAGE expression group. In [Table 2], we compared clinicopathological characteristics between various groups. However, there was no difference in age or gender between the two groups [Table 2]. In comparison to human bone marrow stromal cell line (HS-5), the protein levels of NRAGE were also elevated in human AML cell lines (OCI-AML-5, KG-1, HL-60, and MOLM-14), especially KG-1 and HL-60, which were then chosen for further cell experiment [Figure 1]c. These data imply that NRAGE is highly expressed in AML tissues and is linked to overall survival in AML patients. | Figure 1: The increased expression of NRAGE is linked to a poor prognosis in AML patients. (a) The transcript level of NRAGE in AML tissues (n = 173) and normal tissues (n = 70) was obtained from GEPIA tool. (b) Kaplan–Meier curve showed the survival rates of the high NRAGE expression group (n = 53) and low NRAGE expression group (n = 53). (c) Western blotting showed the protein levels of NRAGE in human AML cell lines (OCI-AML-5, KG-1, HL-60, and MOLM-14) (n = 3) and human bone marrow stromal cell line (HS-5) (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 versus the normal tissue group or HS-5 cells. NRAGE: Neurotrophin receptor-interacting melanoma-associated antigen homolog, AML: Acute myeloid leukemia.
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 | Table 2: The clinical characteristics of acute myeloid leukemia patients between the different NRAGE expression groups
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Silencing NRAGE inhibits AML cell survival and growth
We further investigated the effect of NRAGE on the capacity of AML cells. KG-1 and HL-60 cells were transfected with siNRAGE to downregulate the expression of NRAGE. After transfection, NRAGE expression was dramatically lower in the siNRAGE groups compared to the siNC group [Figure 2]a. Furthermore, the ability of AML cell survival and proliferation was examined by both CCK-8 assay and colony formation assay. We observed that NRAGE knockdown reduced AML cell viability using a CCK-8 assay [Figure 2]b. Furthermore, colony formation assay demonstrated that silencing NRAGE weakened the proliferation ability of AML cells [Figure 2]c. As a result, silencing NRAGE downregulates the capacity of AML cell survival and growth. | Figure 2: Silencing NRAGE inhibits AML cell survival and growth. (a-c) KG-1 and HL-60 cells were transfected with siNRAGE or siNC. (a) Western blotting showed the protein levels of NRAGE in the siNC and siNRAGE groups (n = 3). (b) CCK-8 assay showed cell viability of KG-1 and HL-60 cells in the siNC and siNRAGE groups (n = 3). (c) Colony formation assay showed the ability of cell proliferation of KG-1 and HL-60 cells in the siNC and siNRAGE groups (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 versus the siNC group. NRAGE: Neurotrophin receptor-interacting melanoma-associated antigen homolog, AML: Acute myeloid leukemia, CCK-8: Cell Counting Kit-8.
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Silencing NRAGE induces AML cell cycle arrest at G1 phase
We next explored the impacts of NRAGE on AML cell cycle progression, and the cell cycle phase distribution of AML cells with or without NRAGE knockdown was examined by flow cytometry. NRAGE knockdown led to AML cell cycle arrest at G1 phase compared to the siNC group [Figure 3]a. Cyclin D1 and cyclin-dependent kinase 4 (CDK4) are vital players in the G1-S cell cycle transition which are overexpressed in multiple cancers.[23] Silencing NRAGE decreased both cyclin D1 and CDK4 production in AML cells [Figure 3]b and [Figure 3]c. Altogether, these data suggest that NRAGE knockdown results in AML cell cycle arrest by regulating cyclin D1 and CDK4 expression. | Figure 3: Silencing NRAGE induces AML cell cycle arrest at G1 phase. (a-c) KG-1 and HL-60 cells were transfected with siNRAGE or siNC. (a) Flow cytometry showed the cell cycle phase distribution of KG-1 and HL-60 cells in the siNC and siNRAGE groups (n = 3). (b) Western blotting showed the protein levels of cyclin D1 of KG-1 and HL-60 cells in the siNC and siNRAGE groups (n = 3). (c) Western blotting showed the protein levels of CDK4 of KG-1 and HL-60 cells in the siNC and siNRAGE groups (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 versus the siNC group. NRAGE: Neurotrophin receptor-interacting melanoma-associated antigen homolog, AML: Acute myeloid leukemia, CDK4: Cyclin-dependent kinase 4.
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NRAGE controls AML cell apoptosis through regulating Bcl-2 expression
Whether NRAGE had a role in AML cell apoptosis needs to be identified, and flow cytometry and Western blotting were applied to detect the ability of AML cell apoptosis. Flow cytometry indicated that the number of apoptotic AML cells was significantly increased after the depletion of NRAGE [Figure 4]a. Prior research has evidenced that B-cell lymphoma-2 (Bcl-2) is crucial for the survival of AML cells.[24] Western blotting was used to determine the protein levels of Bcl-2 and Bcl-2-associated X protein (Bax) in order to confirm the hypothesis that NRAGE modulates AML cell apoptosis by Bcl-2. Comparing the siNRAGE group to the siNC group, we observed that the levels of Bax were higher and the levels of Bcl-2 were lower [Figure 4]b. These results indicate that NRAGE has a regulatory role in AML cell apoptosis through regulating Bcl-2 expression. | Figure 4: NRAGE controls AML cell apoptosis through regulating Bcl-2 expression. (a and b) KG-1 and HL-60 cells were transfected with siNRAGE or siNC. (a) Flow cytometry showed the number of apoptotic KG-1 and HL-60 cells in the siNC and siNRAGE groups (n = 3). (b) Western blotting showed the protein levels of Bcl-2 and Bax of KG-1 and HL-60 cells in the siNC and siNRAGE groups (n = 3). **P < 0.01 and ***P < 0.001 versus the siNC group. NRAGE: Neurotrophin receptor-interacting melanoma-associated antigen homolog, AML: Acute myeloid leukemia, Bcl-2: B-cell lymphoma-2, Bax: Bcl-2-associated X protein.
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The capacity of AML cell growth and apoptosis is modulated by NRAGE through the ERK signaling pathway
Emerging evidence reveals that the ERK/MAPK signaling pathway modulates AML cell proliferation and G1/S transition.[25] We investigate whether NRAGE has an involvement in cell proliferation, cell survival, and cell cycle through the ERK/MAPK signaling pathway. After NRAGE was knockdown, the phosphorylation of ERK was inhibited in AML cells [Figure 5]a. tBHQ, an ERK activator, was then applied to activate the ERK signaling pathway.[26] We observed that tBHQ treatment restored the reduced cell viability in AML cells with siNRAGE transfection [Figure 5]b. Moreover, the levels of cell cycle progression regulators (cyclin D1 and CDK4) were enhanced by tBHQ treatment in the siNRAGE group [Figure 5]c and [Figure 5]d. tBHQ treatment also decreased the levels of Bax in AML cells, which was increased by NRAGE knockdown [Figure 5]d. Moreover, the downregulated levels of Bcl-2 in the siNRAGE group were upregulated by tBHQ treatment [Figure 5]d. These findings suggest that NRAGE regulates the activation of the ERK signaling pathway, thereby controlling AML cell growth and cell cycle. | Figure 5: The capacity of AML cell growth and apoptosis is modulated by NRAGE through the ERK signaling pathway. (a) KG-1 and HL-60 cells were transfected with siNRAGE or siNC. Western blotting showed the protein levels of phosphorylated ERK of KG-1 and HL-60 cells in the siNC and siNRAGE groups (n = 3). (b-d) KG-1 and HL-60 cells were transfected with siNRAGE or siNC and treated with the ERK activator, tBHQ. (b) CCK-8 assay showed cell viability of KG-1 and HL-60 cells in the siNC, siNRAGE, and siNRAGE with tBHQ treatment groups (n = 3). (c) Western blotting showed the protein levels of cyclin D1 of KG-1 and HL-60 cells in the siNC, siNRAGE, and siNRAGE with tBHQ treatment groups (n = 3). (d) Western blotting showed the protein levels of CDK4, Bcl-2, and Bax of KG-1 and HL-60 cells in the siNC, siNRAGE, and siNRAGE with tBHQ treatment groups (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 versus the siNC group. #P < 0.05, ##P < 0.01 and ###P < 0.001 versus the siNRAGE group. NRAGE: Neurotrophin receptor-interacting melanoma-associated antigen homolog, AML: Acute myeloid leukemia, CCK-8: Cell Counting Kit-8, CDK4: Cyclin-dependent kinase 4, Bcl-2: B-cell lymphoma-2, Bax: Bcl-2-associated X protein, tBHQ: Tert-Butylhydroquinone.
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| Discussion | | |
AML is a genetically heterogeneous disorder of blood system. Increasing evidence shows a variety of genetic and epigenetic alterations in oncogenes and tumor suppressors in patients with AML.[27],[28],[29] Despite considerable advances in understanding the molecular landscape of AML and its effect on disease pathophysiology and the invention of new drugs, standard medical interventions have not changed significantly over the past decades.[4],[5],[6] In this study, we observed that NRAGE functioned as a tumor promoter in AML, regulating cell survival, cell apoptosis, and cell cycle through the ERK1/2 signaling pathway.
NRAGE is a type II melanoma-associated antigen that is required for numerous cell processes, such as cell survival and cell apoptosis.[14] NRAGE has been identified as a cancer-related protein with complex functions in various cancers.[14] Previous studies reported that NRAGE acted as a tumor suppressor in melanoma, pancreatic cancer, and breast cancer.[30],[31] Chu et al. reported that overexpression of NRAGE inhibited the mRNA expression and activity of matrix metalloproteinase 2 (MMP2), thus suppressing metastasis of melanoma and pancreatic cancer. Paradoxically, conflicting research has shown that NRAGE could function as a tumor promoter, which facilitates the tumorigenesis of hepatocellular carcinoma, esophageal carcinoma, and gastric cancer.[15],[16],[17] Prior research found that NRAGE depletion negatively regulated Bcl-2 and p-ERK and upregulated ZO-1 and p27 expression levels which then acted as a tumor promoter in gastric cancer by facilitating cancer invasion and chemoresistance. Furthermore, NRAGE subcellular localization is related to radiation resistance of esophageal carcinoma cells by regulating epithelial-mesenchymal transition process. However, the role of NRAGE in AML is barely identified. In the present study, an increase in the expression of NRAGE in AML tissue samples was observed. Moreover, NRAGE upregulation was linked to a poor survival of AML patients. Moreover, silencing NRAGE inhibited the ability of AML cell survival and cell proliferation and induced AML cell cycle arrest. Therefore, we identified NRAGE as a tumor promoter in AML patients.
The Bcl-2 family proteins have a central role in the mitochondrial apoptotic pathway.[32] The death effector, Bax, promotes cell apoptosis by activating mitochondrial membrane potential, while the antiapoptotic protein, Bcl-2, negatively regulates Bax to inhibit cell apoptosis.[33] We observed that silencing NRAGE reduced the levels of Bcl-2, whereas Bax expression was enhanced by NRAGE knockdown in AML cells, suggesting that NRAGE regulated cell survival through Bcl-2 family proteins.
Cyclins and CDKs are important proteins that are essential for the regulation and expression of the large number of components required for the passage through the cell cycle.[23] Cyclin D1 and CDK4 are the important G1/S checkpoint of the cell cycle.[34] In this study, we found that knockdown of NRAGE induced cell cycle arrest and decreased cyclin D1 and CDK4 expression, demonstrating that NRAGE controlled cell cycle through regulating the production of cyclin D1 and CDK4.
There are four specific cascades – ERK, Jun amino-terminal kinases, p38-MAPK, and ERK5 sharing the MAPK signaling pathway.[35] The ERK/MAPK signaling pathway is reported to be closely correlated with cellular biological processes such as cell proliferation, cell apoptosis, and cell cycle that were dysregulated in various cancers.[8],[36] In leukemia, progenitor cells were revealed to have abnormally activated ERK/MAPK signaling pathways, and decreasing ERK activity prevented primary AML cells from proliferating and resulted in cell apoptosis.[37] Furthermore, the improper MAPK signaling pathway activity is a poor predictor of overall survival in patients with AML.[38] In this report, silencing NRAGE inactivated the ERK signaling pathway. Moreover, in AML cells with NRAGE knockdown, tBHQ, an ERK activator, could restore the impaired ability of AML cell proliferation and survival as well as cell cycle arrest. Taken together, these data imply that NRAGE possesses a role in AML cell function through the ERK signaling pathway.
| Conclusion | | |
In the present study, an increase of NRAGE expression was observed in AML tissues, and high NRAGE expression showed a poor prognosis in AML patients. Moreover, NRAGE has a regulatory role in the capability of AML cell survival and proliferation as well as cell cycle through the ERK signaling pathway. Thus, we identified NRAGE as a tumor promoter in AML, which might be a therapeutic target for AML treatment in the future.
Availability of data and materials
All data generated or analyzed during this study are included in this published article. The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
Authors' contributions
All authors contributed to the study conception and design. Material preparation and the experiments were performed by Hongxia Zhang. Data collection and analysis were performed by Guangsheng Wu. The first draft of the manuscript was written by BeiLi Chen and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Financial support and sponsorship
This work was supported by the Shihezi University 2021 Corps financial science and technology projects (Grant No. 2021CA002) and Shihezi University 2021 independent funding support for university-level research projects (Grant No. ZZZC202186).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]
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