|Year : 2018 | Volume
| Issue : 4 | Page : 136-141
Expression and tumor-promoting effects of caprin-1 in human glioma
Li Zhang1, Hui Gui1, Xiang-Jun Tang1, Zhuo-Shun Yang1, Dan-Dan Zou1, Jun-Ti Lu1, Li-Dong Yan1, Long-Jun Dai2, Jie Luo1, Bin Wang3
1 Department of Neurosurgery, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, China
2 Department of Neurosurgery, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, China; Department of Surgery, University of British Columbia, Vancouver, Canada
3 Department of Neurosurgery, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, China; Biomedical Research Centre, University of British Columbia, Vancouver, Canada
|Date of Web Publication||30-Aug-2018|
Dr. Bin Wang
Biomedical Research Centre, The University of British Columbia, 2222 Health Sciences Mall, Vancouver, BC, V6TZ3, Canada
Dr. Jie Luo
Department of Neurosurgery, Taihe Hospital, Hubei University of Medicine, 32 South Renmin Road, Shiyan 442000, Hubei
Dr. Long-Jun Dai
Department of Surgery, University of British Columbia, 400-828 West 10th. Avenue, Vancouver, BC, V5ZL8, Canada
Source of Support: None, Conflict of Interest: None
Background: Cytoplasmic activation/proliferation-associated protein-1 (caprin-1) is a newly discovered RNA-binding protein and is now recognized as one of the putative oncogenes. This study was performed to reveal its presence in human gliomas and its oncogenic functions in human glioblastoma-derived Denver brain tumor research group 05 (DBTRG-05MG) cells. Materials and Methods: Clinical glioma samples were cumulatively collected for the identification of caprin-1 using immunoblot analysis and immunofluorescence detection. DBTRG cells transfected with caprin-1-specific small interfering RNA (siRNA) were used to verify caprin-1's oncogenic function using a real-time cell analyzer (RTCA) and scratch assay. Results: Seven of eight collected glioma samples were identified as positive for caprin-1 expression. siRNA dose-responsive inhibition of cell proliferation was observed in DBTRG cells with RTCA, and cell migration rate was significantly reduced by siRNA transfection (P < 0.05). Conclusion: The present study identified the higher expression of caprin-1 in human glioblastoma-derived DBTRG cells. Its oncogenic functions, mainly enhanced cell proliferation and promoted cell migration capacity, were also verified in these cells. This study provided fundamentals for developing caprin-1 as a therapeutic target for the treatment of gliomas.
Keywords: Caprin-1, gliomas, migration, PI3K/AKT/mTOR pathway, proliferation
|How to cite this article:|
Zhang L, Gui H, Tang XJ, Yang ZS, Zou DD, Lu JT, Yan LD, Dai LJ, Luo J, Wang B. Expression and tumor-promoting effects of caprin-1 in human glioma. Glioma 2018;1:136-41
|How to cite this URL:|
Zhang L, Gui H, Tang XJ, Yang ZS, Zou DD, Lu JT, Yan LD, Dai LJ, Luo J, Wang B. Expression and tumor-promoting effects of caprin-1 in human glioma. Glioma [serial online] 2018 [cited 2023 Jun 4];1:136-41. Available from: http://www.jglioma.com/text.asp?2018/1/4/136/240233
| Introduction|| |
Cytoplasmic activation/proliferation-associated protein-1 (caprin-1) is a well-conserved cytoplasmic phosphoprotein. In human, its gene is located in the chromosome 11 (11p13) encoding 709 amino acids with the molecular weight of 116 Kda., Caprin-1 colocalizes with G3BP1 (Ras GTPase-activating protein-binding protein 1) within messenger ribonucleoprotein particles (mRNPs) in the cytoplasmic RNA granules associated with microtubules. Since the carboxyl-terminal region of caprin-1 selectively binds to c-Myc or cyclin D2 mRNAs, caprin-1/G3BP1 complex is believed to selectively regulate the transport and translation of mRNAs whose protein products are involved in proliferation and migration of multiple cell types., Caprin-1 is ubiquitously expressed, and its phosphorylation is required for normal cell cycle progression from the G1 to S phase. Early studies indicate that as an RNA-binding protein (RBP), caprin-1 participates in the proliferation process of lymphocytes., Levels of caprin-1 proteins increased when resting splenic T- or B-lymphocytes were stimulated to divide. Subsequent studies revealed that caprin-1 is selectively involved in the transcription of coding mRNAs and noncoding mRNAs (i.e., microRNAs) of all types of proliferating cells. Because of its close relation to the cell cycle control, caprin-1 has attracted great research interest to explore the potential relationship between caprin-1 and tumorigenesis. The promoting effects of caprin-1 on breast cancer were recently demonstrated by Qiu et al. using shRNA technique and Gong et al. using caprin-1-specific microRNA (miR-223). Caprin-1 also facilitates the tumor growth and migration of osteosarcoma, which was verified by Sabile et al. using tissue microarray. However, the role of caprin-1 in the development of gliomas, one of the most devastating human cancers, has not been investigated yet.
Gliomas account for the majority (almost 80%) of primary malignant brain tumors. Glioblastoma multiforme (GBM) is characterized by high proliferative activity and is the most common and aggressive type of glioma associated with very poor survival rate. The mainstay of GBM treatment has been surgical resection to the extent feasible, followed by chemotherapy and radiotherapy. Although enormous advances in treating other solid cancers have been achieved, median survival for GBM stayed nearly the same over the last half-century, averaging around 1 year. Since GBM infiltrates surrounding tissues and sometimes locates in privileged sites, its complete resection is impossible. In addition, the existence of the blood–brain barrier and local hypoxic microenvironment makes tumor cells resistant to or escape from standard radiation and chemotherapy. Thus, it is of special significance to explore an effective and most suitable therapeutic modality for GBM. In the clinical practice, activation of the PI3K pathway is significantly associated with increasing tumor grade, decreased levels of apoptosis, and with adverse clinical outcome in human gliomas. The majority of collected GBM biopsies (>80%) exhibited enhanced activity of PI3K/Akt/mTOR pathway, which was mainly induced by overexpression of EGFR (including EGFRvIII) and/or reduced activity of PTEN.,, PI3K/Akt/mTOR pathway is a major determinant regulating cell cycle through controlling its downstream proliferation-related proteins, thereby maintaining high proliferation activity of tumor cells. Apparently, there may exist an interrelation between caprin-1 and PI3K/Akt/mTOR pathway in terms of cell-cycle control. The studies on identifying caprin-1 in gliomas and investigating the possible underlying mechanisms may lead to finding a novel potent therapeutic target toward effective treatment of gliomas. In the present study, clinical specimens were used to identify the expression of caprin-1 in human gliomas, and the tumor-promoting effects of caprin-1 were verified in Denver brain tumor research group (DBTRG) glioma cells.
| Materials and Methods|| |
Human glioma specimen collection
A total of 8 cumulative patients underwent surgical resection of glioma from September 22, 2015 to July 21, 2016 in Department of Neurosurgery, Taihe Hospital, Hubei University of Medicine. All enrolled patients were diagnosed as gliomas according to the World Health Organization (WHO) classification published in 2007. Eight glioma specimens were collected for the analysis of Caprin-1 expression with written consent from each individual before surgery. Tumor specimens (around 0.5–2.0 g/each) were obtained within 5 min of resection and immediately stored in ice-cold sterilized saline. Collected samples were kept in ice and delivered to the laboratory in 10 min. The procedures of cryosection and protein extraction were commenced within 30 min of tumor resection. The protocol for this study was approved by the Ethics Committee of Taihe Hospital (2015KS039).
Denver Brain Tumor Research Group cell culture and small interfering RNA transfection
DBTRG cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). DBTRG cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, 2 mM L-glutamine and 1% penicillin-streptomycin solution (all from Invitrogen, Shanghai, China) and incubated at 37°C in a humidified atmosphere with 5% CO2. Subculture was performed once newly grown cells reached subconfluence. The DBTRG cells used in this study were limited to 5–7 passages, counted from the initial culture in our institute.
Caprin-1 small interfering RNA (siRNA) (sc-72785) and control siRNA (sc-36869) were provided by Santa Cruz Technology (Dallas, TX, USA). The transfection of siRNA into DBTRG cells was carried out with TranIT-mRNA (Mirus), as per manufacturer's instructions. Briefly, siRNA was diluted in Opti-MEM-based medium (Invitrogen) and then, boost reagent and TransIT-mRNA were added sequentially. After 2 min incubation at room temperature, the RNA-lipid complexes were delivered to culture media in the culture plates. The plates were then returned to the incubator for 24 h and the cells were used for cell viability assessment with real-time cell analyzer (RTCA) and scratching determination.
Immunoblotting analysis of caprin-1
Immunoblotting analysis was used to detect the expression of caprin-1 in clinical glioma specimens. Briefly, fresh human glioma specimens (200–400 mg) were kept in 2 volumes of ice-cold cell lysis buffer (Beyotime Biotechnology, Haimen, China) and smashed with syringe by repeated aspiration. Tissue lysates were incubated on ice for 30 min followed by centrifugation at 1300 g for 15 min. Equal amounts of protein (50 μg/each sample) were loaded on each lane and separated by electrophoresis in 8% sodium dodecyl sulfate-polyacrylamide gel and electrotransferred onto nitrocellulose membranes. The membrane was placed in blocking buffer containing 5% bovine serum albumin for 1 h at room temperature followed by overnight incubation at 4°C with rabbit anti-caprin-1 antibody (Proteintech Group, Rosemont, IL, USA). The blots were rinsed with Tris Buffered Saline with Tween 20 (TBST) three times and incubated with horseradish peroxidase-conjugated secondary antibody (1:1000) for 60 min and detected by chemiluminescence using ECL Hyperfilm.
Immunofluorescence detection of caprin-1 in glioma specimens
Human glioma specimens were collected during surgery, frozen in Tissue-Tek OCT compound (Sakura Finetek USA, Torrance, CA, USA), and subjected to immunofluorescent staining. Cryosections (20 μm thick) were mounted onto slides, dried for 20 min in air, fixed in ethanol/ether at room temperature for 10 min, washed with phosphate-buffered saline (PBS) three times, permeabilized with 0.5% Triton X-100 for 10 min, and then preblocked for 10 min with PBS containing 10% normal goat serum. Sections were stained with rabbit anti-caprin-1 antibody (1:1000) and mouse antiglial fibrillary acidic protein (anti-GFAP) antibody (1:1000) overnight at 4°C, followed by PE-labeled goat anti-rabbit immunoglobulin G (IgG) (1:1000) and FITC-labeled goat anti-mouse IgG (1:1000) at 37°C for 30 min in dark, and then stained with Hochest 33258 for 10 min at room temperature. The slides were finally mounted with Mount-G (SouthernBiotech, Birmingham, AL, USA) and the digital images were captured by a digital camera mounted on a Leica microscope (DMI8) operated by LAS V4.8 software (Leica Microsystems Inc., Buffalo Grove, IL, USA).
Cell viability assessment with real-time cell analyzer
The cell viability was detected by real-time assessment using the xCELLigence RTCA, Roche, Indianapolis, IN, USA as previously described. Cell index (CI) value is defined as relative change in measured impedance to background impedance and represents cell status. CI is directly proportional to the quantity, size, and attachment forces of the cells reflecting cell viability or proliferation.
The scratch assay was used to detect the effects of Caprin-1 on cell migration capability. DBTRG cells were cultured in 6-well plates (1 × 105 cells/well). When the cells reached complete confluency, a line was scratched in the middle of the wells using a 200 μL pipette tip. The plates were washed with PBS for three times. Cells were then incubated with fresh serum-free DMEM medium at 37°C with 5% CO2 for 18 h. As described in the previous study,, the width of the scratched line was measured and the percentage of narrow down was compared at 4 h, 12 h, and 18 h, respectively. The experiment was performed in triplicates.
Numerical data were expressed as mean ± standard error. Statistical differences between the means for the different groups were evaluated with Prism 4.0 (GraphPad Software, La Jolla, CA, USA) using the Student' s t-test. Significance was assumed at P = 0.05 or less.
| Results|| |
Basic information of patients with gliomas
The information of all involved patients who donated the biopsies is listed in [Table 1]. The corresponding brain tumor histology is presented in [Figure 1].
|Figure 1: Brain tumor histology of involved patients: The digits represent the number of patients, which corresponds to the patient's number indicated in Table 1. The corresponding histological slices with H and E staining were collected from patient's files. The original magnification was ×200|
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Identification of caprin-1 expression in glioma specimens
The identification of caprin-1 expression in glioma specimens was performed by immunoblotting and immunofluorescence assay, respectively. As shown in [Figure 2]A, expression of caprin-1 was detected by immunoblotting analysis in seven of eight cumulative specimens. The representative immunofluorescence detection is illustrated in [Figure 2]B.
|Figure 2: The identification of caprin-1 in human glioma tissues. (A) Immunoblotting analysis of caprin-1 in glioma specimens: The expression of caprin-1 in glioma specimens was examined by immunoblotting analysis. Protein extractions (50 μg/lane) from glioma specimens were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by caprin-1 antibodies. Protein sample isolated from Denver brain tumor research group cells were used as a positive control. The detection of glyceraldehyde-3-phosphate dehydrogenase was used as an indicator of sample loading control. (B) Immunofluorescence detection of caprin-1 in glioma specimens: Frozen sections derived from patient glioma specimens were stained with (i) anti-caprin-1 and (ii) antiglial fibrillary acidic protein antibodies, followed with PE-labeled goat anti-rabbit immunoglobulin G (red) and FITC-labeled goat anti-mouse immunoglobulin G (green), and then with (iv) Hochest 33258 for counterstaining nuclei (blue).(iii) is the merge of (i) and (ii) (yellow)|
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Tumor-promoting effects of caprin-1 in Denver brain tumor research group cells
Tumor-promoting effects of caprin-1 were verified by RTCA and scratch test in DBTRG cells. Since high expression of caprin-1 was revealed in wild-type DBTRG cells, caprin-1-specific siRNA was used to detect its effects on cell proliferation and migration. CI was inhibited by caprin-1-specific siRNA in a dose-dependent pattern [Figure 3]A, and the migration capacity of DBTRG cells was significantly lowered by the application of siRNA [Figure 3]B.
|Figure 3: The functional characterization of caprin-1 in Denver brain tumor research group cells. (A) Real-time assessment of caprin-1-induced cell viability changes in Denver brain tumor research group cells. Denver brain tumor research group cells were pretransfected with caprin-1-specific small interfering RNA at indicated concentrations. Mock-transfected cells were used as control. Cell index was automatically recorded with the xCELLigence real-time cell analyzer every 10 min until the end of the experiment (45 h). Each tracing represents an average of three parallel assessments. (B) The effect of caprin-1 on migrating capacity in Denver brain tumor research group cells. Left panel: the cultures of Denver brain tumor research group cells were observed and photographed by phase-contrast microscopy (×100) at the indicated time points after scratching, scale bar, 200 μm. The top row illustrates representative images from control Denver brain tumor research group cells and bottom row presents representative images from DBTRG cells transfected with 40 nM small interfering RNA. Right panel: cell migration into the scratch wound after treatment was quantified and presented as a percentage of wound healing. It was calculated by dividing migrated distance by scratched distance. *P < 0.05 vs control (n = 3)|
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| Discussion|| |
Cancer is caused by genomic alterations that occur in tumor suppressor genes, microRNA genes, and oncogenes. The majority of oncogene products function as components of the signaling pathways that regulate cell cycle and proliferation. Since “oncogene theory” was first proposed by Huebner and Todaro in 1969, a few hundreds of oncogenes have been identified, including viral and cellular oncogenes that contribute to the uncontrolled proliferation of cancer cells. Recently, it has been known that posttranscriptional regulation of gene expression is important to control cell proliferation, differentiation, invasion, metastasis, apoptosis, and angiogenesis, all of which are involved in the initiation and progression of tumor. Regulating transcribed messenger RNA is an efficient and rapid way to control the gene expression and plays a crucial role in tumorigenesis. RBPs are the key regulators of posttranscriptional gene expression, which are expected to be closely related to cancer development. Previously, we found caprin-1 as a novel, highly conserved phosphoprotein that is dramatically upregulated when lymphocytes are activated. Caprin-1 can selectively bind to c-Myc and cyclin D2 mRNAs and other mRNAs which are involved in cell proliferation and migration through its conserved RNA-binding domains and motifs. By knocking out both copies of caprin-1, we showed that the absence of caprin-1 results in decrease in proliferation, cloning efficiency and clone-size, and a delay in transition from G1 to S phases of the cell cycle. The regulation of tumor cell cycle by proliferation-related genes is an important part of the research on mechanisms underlying tumorigenesis, and furthermore, any oncogene-related study will potentially lead to the development of effective therapy of cancer.
Caprin-1 also binds with other RBPs to affect the gene expression of their specific target mRNAs. Besides G3BP-1, Caprin-1 has been reported to directly interact with Fragile X mental retardation protein (FMRP), another RBP. FMRP is the most intensively studied RBP because mutations in the gene cause the most common genetic case of mental retardation. Caprin-1/G3BP1 colocalizes with FMRP in RNA transport granules and stress granules, and FMRP is coprecipitated with caprin-1 or G3BP1. As FMRP also coprecipitates with Argonate 1 (Ago1), Ago2, miRNAs, and RNA-induced silencing complex (RISC) and is also involved in RNA interference, the caprin-1/G3BP1 is linked with the RISC and miRNA function. Owing to its close relation with cell cycle control, caprin-1 is now considered as a putative oncogene. The identification and oncogenic characterization of caprin-1 in human gliomas will definitely contribute to understanding its pathogenesis and developing effectively targeted therapy for this most devastating human cancer.
In the present study, the expression of caprin-1 was identified by immunoblotting and immunofluorescence in clinical glioma specimens [Figure 2]A and [Figure 2]B. Caprin-1 expression was detected in seven of eight cumulative specimens, although it requires adequate samples to achieve accurate expression rate. Almost all glial originated cells (GFAP + cells) showed positive expression of caprin-1. As indicated in [Table 1] and [Figure 1], the human glioma samples were cumulatively collected from a single clinical center. They cover both male and female patients and included the WHO Grade I to Grade IV tumors. In the present study, the relationship between caprin-1 expression and WHO grade of glioma was not analyzed, even though the expression of caprin-1 was undetectable in the WHO Grade I sample (patient # 5).
The oncogenic function of caprin-1 was investigated in DBTRG cells using RTCA and scratch assay. Enhanced proliferation and facilitated migration capacity are the major characteristics of cancer cells. Since considerable amount of endogenous caprin-1 exists in DBTRG cells, a specific siRNA was used to reveal the oncogenic function of caprin-1. As shown in [Figure 3]A, a dose-responsible inhibition of DBTRG cell proliferation suggests that caprin-1 plays an important role in cell cycle control. Cell migration plays an important part in tumor metastasis. Scratch assay is a well developed and easy method for analyzing cell migration in vitro. Under the current experimental condition, transfection of caprin-1-specific siRNA inhibited the migration capacity of DBTRG cells [Figure 3]B, indicating the migration-promoting effect of caprin-1 in this type of cells. Actually, the observed changes of migration capacity were also attributed to caprin-1-induced enhancement of cell proliferation. In terms of exploring a novel and effective therapy of gliomas, further in vitro and in vivo studies are guaranteed to clarify caprin-1's up- and down-stream regulators, the relationship between caprin-1 and mRNPs, and underlying mechanisms of its oncogenic functions.
The present study identified the positive expression of caprin-1 in human gliomas. As a RBPs, caprin-1 plays an essential part in the process of cell cycle control through specifically regulating cell proliferation-related proteins. Its oncogenic functions, mainly enhanced cell proliferation and promoted cell migration ability, were also verified in human glioblastoma-derived DBTRG cells. This study provided fundamentals for caprin-1 be developed as a therapeutic target for the treatment of gliomas.
The authors would like to thank Drs. Gang Cao (Department of Neurosurgery, Taihe Hospital), Tie-Yan Wang, Xian-Bin Tang (Department of Pathology, Taihe Hospital), Jing-Bo Feng, and Shi-Nan Ma (Hubei Key Laboratory of Stem Cell Research, Taihe Hospital, Hubei University of Medicine, Shiyan, China) for their technical assistance.
Financial support and sponsorship
This study was financially supported by the Key Projects of Precision Medicine (2016JZ01), Education Department of Hubei Province (Q20162102), and Research and Technology Development Projercts of Shiyan (17Y06).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]