Conceived and designed the experiments: XLG XW. Performed the experiments: XLG XL LLF JMG. Analyzed the data: XLG XQL NC. Contributed reagents/materials/analysis tools: LLF JMG XQL. Wrote the paper: XLG NC XW. Obtained permission for use of cell line: XLG.
The authors have declared that no competing interests exist.
Metadherin (MTDH) has been demonstrated as a potentially crucial mediator of various types of human malignancies. However, the expression and role of MTDH in diffuse large-B-cell lymphoma (DLBCL) have not been reported yet. This study aimed to illuminate the role of MTDH in the pathogenesis of DLBCL.
A remarkable elevation of MTDH on mRNA level was detected in DLBCL tissues by quantitative polymerase chain reaction (PCR). Using Western-blot analysis we found that the expression of MTDH protein was significantly upregulated in DLBCL cell lines and DLBCL tissues compared with peripheral blood mononuclear cells (PBMCs) from healthy samples and tissues from patients of reactive hyperplasia of lymph node. The results showed high expression of MTDH in 23 of 30 (76.67%) DLBCL tissues by using immunohistochemical analysis and the over expression of MTDH was strongly correlated to the clinical staging of patients with DLBCL (P<0.05). Furthermore, the finding suggested that the increase of MTDH in DLBCL cells could distinctly enhance cell proliferation and inhibit cell apoptosis; meanwhile, inhibition of MTDH expression by specific siRNA clearly enhanced LY8 cell apoptosis. Upregulation of MTDH elevated the protein level of total β-catenin and translocation of β-catenin to the nucleus directly or indirectly. Knockdown of MTDH decreased the level of total, cytoplasmic β-catenin and reduced nuclear accumulation of β-catenin protein. This indicated that the function of MTDH on the development of DLBCL was mediated through regulation of Wnt/β-catenin signaling pathway.
Our results suggest that MTDH contributes to the pathogenesis of DLBCL mediated by activation of Wnt/β-catenin pathway. This novel study may contribute to further investigation on the useful biomarkers and potential therapeutic target in the DLBCL patients.
Diffuse large-B-cell lymphoma (DLBCL) is an aggressive malignancy of mature B lymphocytes and is the most common type of lymphoma in adults
Metadherin (MTDH, also known as astrocyte elevated gene-1/AEG-1 and Lyric) was first cloned as an HIV- and TNF-α–inducible gene in primary human fetal astrocytes (PHFAs)
For the first time, our present manuscript focuses on illuminating the role of MTDH and the relationship between MTDH and Wnt/β-catenin pathway in the pathogenesis of DLBCL. We demonstrate the overexpression of MTDH and β-catenin in DLBCL and the effect of MTDH expression on biological behavior of DLBCL cell lines. We also provide evidences for the link between MTDH and Wnt/β-catenin pathway in DLBCL.
Expression of MTDH was detected by Western blot, with the single band size of 75 kDa, in peripheral blood mononuclear cells (PBMCs) from healthy samples, human DLBCL cell lines LY1 and LY8, and MCL cell lines Jeko-1, Mino, and SP53. The protein level of MTDH was much lower in PBMCs from healthy samples compared with all the human DLBCL and MCL cell lines (
(A) Expression of MTDH (75 kDa) and β-catenin (94 kDa) were detected in the indicated cell lines. PBMCs represent peripheral blood mononuclear cells from healthy samples. Expression of β-actin was used as loading control. (B) Subcellular protein fractionation using the cell lysates of 2 DLBCL cell lines revealed that MTDH and β-catenin were localized in the nucleus (N) and possibly also in the cytoplasm (C) and. The expression of β-actin in the cytoplasm and H3 in the nucleus served as controls for the efficiency of subcellular fractionation.
(A) Real-time quantitative PCR analysis of MTDH expression in DLBCL (n = 21) and control (n = 25). PCR products were confirmed as a single product at the desired size on agarose gels (1–3: DLBCL; 4–5: Control). Control, reactive hyperplasia of lymph node. β-actin was used as a loading control. (B) Expression of MTDH protein (75 kDa) in DLBCL (1–3) and reactive hyperplasia of lymph node tissues (4–6). Expression levels were normalized with β-actin. (C) Detection of MTDH expression in DLBCL by IHC. (D) Detection of MTDH expression in reactive hyperplasia of lymph node by IHC. Original magnification, ×400. Points indicate mean of triplicate determinations, bars, SD. ****p<0.0001 versus control.
To further determine whether MTDH protein overexpression is associated with clinicopathological characteristics of DLBCL, a cohort of paraffin-embedded, archived DLBCL tissues (n = 30) were examined by immunohistochemical staining with an antibody against human MTDH. As shown in
Statistical analyses were done to examine the correlation between the expression of MTDH protein by immunohistochemistry analysis and the clinical features of DLBCL. As shown in
Characteristics | MTDH expression | Fisher’s Exact Test P value | |
Negative no.(%) | Positive no.(%) | ||
Age(years) | |||
<60 | 2 (12.5) | 14 (87.5) | 0.204 |
>60 | 5 (35.7) | 9 (64.3) | |
Gender | |||
Male | 5 (22.7) | 17 (77.3) | 1.000 |
Female | 2 (25.0) | 6 (75.0) | |
Clinical stage | |||
I | 4 (57.1) | 3 (42.9) | 0.047 |
II | 2 (28.6) | 5 (71.4) | |
III | 1 (20.0) | 4 (80.0) | |
IV | 0 (0.0) | 11(100.0) | |
B symptoms | |||
Yes | 3 (23.1) | 10 (76.9) | 1.000 |
No | 4 (23.5) | 13 (76.5) |
It is previously reported that TNF-α up-regulates MTDH expression at both the mRNA and protein level in the human cervical carcinoma cell line HeLa
(A) LY1 and LY8 cells were either untreated or treated with 250 pg/mL of TNF-α for 48 hours. The expression of MTDH protein was analyzed by Western blot. (B) DLBCL cells were treated with TNF-α at the indicated concentration for 48 hours, and cell proliferation was determined by 3H-TdR incorporation assay. Columns indicate mean of triplicate determinations; bars, SD. (C) LY1(left panel) and LY8(right panel) cells were treated with TNF-α (250 pg/mL for 48 hours) and cell apoptosis was detected by flow cytometer. Early apoptotic cells were defined as Annexin-V-FITC-positive, PI-negative cells. Columns indicate mean of triplicate determinations; bars, SD. *p<0.05 versus control.
To further investigate the potential function of MTDH gene in DLBCL pathogenesis, cell apoptosis was evaluated in MTDH knockdown cells (
LY8 cells were transfected with MTDH siRNA or negative control siRNA and cell apoptosis was detected by flow cytometer. Early apoptotic cells were defined as Annexin-V-PE-positive, 7-AAD-negative cells. Columns indicate mean of triplicate determinations; bars, SD. *p<0.05 versus control; ▴p>0.05 versus control.
Between the LY8 cells untreated and treated with TNF-α and siRNA towards MTDH, there was little change in their apoptosis rate when the expression of MTDH was detected to be unchanged (
(A) LY8 cells transfected with control siRNA and MTDH siRNA were either untreated or treated with 250 pg/mL of TNF-α for 48 hours. Then cell apoptosis was detected by flow cytometer. (B) Detection of total β-catenin proteins by Western blot analysis in transfected LY8 cell line untreated and treated with TNF-α at the indicated concentration and exposure time. Data expressed as mean±SD. *p<0.05 versus control; ▴p>0.05 versus control.
To determine the activity of Wnt/β-catenin pathway in DLBCL, we presently assessed the expression of β-catenin, the chief downstream effector of Wnt/β-catenin pathway, in 2 DLBCL, 3 MCL cell lines. As illustrated in
Since Wnt/β-catenin pathway was found to be aberrant in DLBCL cells and is probably involved in the pathogenesis of DLBCL, we focused on discussing whether Wnt/β-catenin pathway is overactivated by MTDH using western blot analysis. Total β-catenin protein level was increased in TNF-α-treated cells (P<0.05,
(A) Detection of total β-catenin proteins by Western blot analysis in LY1 and LY8 cell lines untreated and treated with TNF-α at the indicated concentration and exposure time. (B) Analysis of nuclear β-catenin protein expression using subcellular fractionation and Western blot. The expression o f β-actin in the cytoplasm(C) and H3 in the nucleus (N) served as controls for the efficiency of subcellular fractionation. Data expressed as mean±SD. *p<0.05 versus control.
To more immediately study the relationship between MTDH and Wnt/β-catenin signaling in DLBCL, we examined the expression of β-catenin in LY8 cells infected with MTDH-RNAi lentivirus or negative control lentivirus. The protein level of total β-catenin was decreased in MTDH-siRNA-treated cells (
(A) Analysis of total β-catenin protein by Western blot analysis in LY8 cells transfected with negative control siRNA and MTDH siRNA. (B) and (C) Expression of cytoplasmic(B) and nuclear (C) β-catenin protein was analyzed in LY8 cells. The expression of β-actin in the cytoplasm and H3 in the nucleus served as controls for the efficiency of subcellular fractionation. Data expressed as mean±SD. *p<0.05 versus control; **p<0.01 versus control; ▴p>0.05 versus control.
Between the LY8 cells untreated and treated with TNF-α and siRNA towards MTDH, there was almost no change of β-catenin expression when the expression of MTDH was detected to be unchanged (
These findings indicate that MTDH upregulation could directly or indirectly increase the protein level of β-catenin in the nucleus and thus play a significant part as an upstream activator for Wnt/β-catenin signaling pathway.
In recent years MTDH has been demonstrated as a potentially crucial mediator of various types of human malignancies. Its expression is significantly higher in melanoma, breast, esophageal, gastric, hepatocellular and prostate cancers, renal cell carcinoma, neuroblasoma and malignant glioma cell lines compared with their normal counterparts
Studies are ongoing to further clarify the biological impact of MTDH overexpression on DLBCL tumor cells, in which we investigated gain-of-function through MTDH upregulation induced by TNF-α and loss-of-function through MTDH knockdown by small interfering RNA in DLBCL cells. We observed that the upregulation of MTDH in DLBCL cells could promote cell proliferation, which is in accordance with the inhibitory effect on cell apoptosis. Considering the possibility that the effect on the proliferation and apoptosis occurs directly after TNF-α treatment, we treated the cell lines with TNF-α and control siRNA or siRNA towards MTDH simultaneously and detected the apoptosis rate and β-catenin expression. The results suggested that there was almost no change of apoptosis rate and β-catenin expression between treated and untreated cells when the expression of MTDH was detected to be unchanged. However, cell apoptosis was inhibited and the protein level of total β-catenin was increased in the cells treated with TNF-α and control siRNA compared with the cells treated with TNF-α and MTDH siRNA. This probably suggested that the effect on the cell biological behavior and Wnt/β-catenin signaling occurred after the expression change of MTDH rather than directly after TNF-α treatment. The results also show that the reducing of MTDH in DLBCL cells could enhance cell apoptosis. Taken together, these data suggest that MTDH upregulation is likely associated with the pathogenesis of DLBCL.
Previous studies have demonstrated that MTDH promotes tumor initiation and progression by modulating multiple downstream oncogenic pathways, such as NF-κB, PI3K/Akt and Wnt/β-catenin pathways
In summary, our findings suggest that the over expression of MTDH is associated with the pathogenesis of DLBCL for the first time. We demonstrate that MTDH was markedly overexpressed in both DLBCL cell lines and tissues and β-catenin was upregulated, with its nuclear localization, in DLBCL cell lines compared with their counterparts. We found that the over expression of MTDH was strongly correlated to the clinical staging of patients with DLBCL. Furthermore, we determined that apoptosis of DLBCL cell lines was distinctly inhibited while cell proliferation was enhanced according with MTDH upregulation after treatment with TNF-α. Cell apoptosis was also promoted in MTDH knockdown cells in comparison to those transfected with negative control siRNA. Moreover, MTDH promotes growth and survival of DLBCL cells via regulating Wnt/β-catenin signaling pathway. The upregulation of MTDH induced by TNF-α could increase the protein level of total β-catenin and its nuclear translocation directly and indirectly, whereas, MTDH silencing could reduce the level of total β-catenin protein, facilitate the degradation of cytoplasmic β-catenin and decrease its nuclear translocation. Further efforts are needed to expound the particular molecular mechanism of MTDH upregulating Wnt/β-catenin pathway and understand the roles of MTDH in DLBCL development, which may enable MTDH to be a useful biomarker and potential therapeutic target for DLBCL.
Paraffin-embedded archived samples, including thirty cases of DLBCL diagnosed between January 2008 and December 2010 according to the WHO criteria
All cell lines were maintained at 37°C in 5% carbon dioxide. The human DLBCL cell lines LY1 and LY8 were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA)
Rabbit anti-MTDH polyclonal antibody was purchased from Invitrogen (Carlsbad, CA, USA). Rabbit anti-β-catenin polyclonal antibody and mouse anti-β-actin monoclonal antibody was from Abcam (Cambridge, MA). Rabbit anti-histone 3 (H3) polyclonal antibody was obtained from Beyotime (Shanghai, China). 3H-thymidine was purchased from Perkin-Elmer (Waltham, MA). TNF-α was purchased from PeproTech (Rocky Hill, NJ, USA). For studies, it was dissolved in 0.1% of BSA and aliquoted as a stock solution. To prepare working solutions, aliquots were further diluted in Iscove’s modified Dulbecco’s medium (IMDM; Hyclone, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT, USA) immediately before each experiment.
RNAi target sequence to human MTDH gene was
Name | Sequence |
MTDH-F |
|
MTDH-R |
|
β-catenin-F |
|
β-catenin-R |
|
β-actin-F |
|
β-actin-R |
|
Total RNA was extracted from tissues using Trizol (Invitrogen). Then reverse transcription reaction was conducted by means of TaKaRa reverse transcription reagents (TaKaRa, Dalian, China). The reaction was incubated at 37°C for 15 minutes, and 85°C for 5 seconds. Amplification reactions were performed using SYBR Premix Ex Taq (Perfect Real Time) (TaKaRa, Dalian, China) on ABI 7500 Real-Time quantitative PCR System with cycling as follows: an initial cycle for 2 minutes at 95°C, followed by 40 bi-phasic cycles of 15 seconds at 95°C and 1 minute at 60°C. PCR products were confirmed as a single product at the desired size on agarose gels and visualized by ethidium bromide staining. Specific primers for RT-PCR were obtained from Biosune (Shanghai, China), and the primer sequences are listed in
Total protein was extracted from DLBCL tumors, reactive hyperplasia of lymph node tissues, LY1, LY8, Jeko-1, Mino, SP53 cell lines and PBMCs of healthy samples using RIPA and 1% PMSF (Shenergy Biocolor, Shanghai, China). For cytoplasmic and nuclear extracts, cells were washed with phosphate-buffered saline (PBS) and were lysed in NE-PER extraction reagent (Pierce) according to the manufacturer's protocol. The protein concentration of the samples was determined by the BCA assay (Shenergy Biocolor). Cell lysates were then electrophoresed on 10% SDS-polyacrylamide gels, while cytoplasmic and nuclear lysates were electrophoresed on 12% SDS-polyacrylamide gels, and next transferred onto nitrocellulose membranes. After the membranes were blocked with 5% skim milk in Tris-saline buffer with 0.1% Tween-20, they were subsequently probed with primary antibodies at 4°C overnight. After washings with TBST, secondary antibody conjugated with the horseradish peroxidase (Zhongshan Goldenbridge, Beijing, China) was added to the membranes. After washings with TBST, proteins were detected using the chemiluminescence detection kit (Millipore, Massachusmetts, USA). Antibodies used in this study included anti-MTDH (1 µg/ml), anti-β-catenin (1∶5000), anti-β-actin (1∶10000) and anti-H3 with a dilution of 1∶1000. Western blot results were analyzed using the Las-4000 Image software and Multi Gauge Ver.3.0 software (Fujifilm Life Science, Japan).
In brief, formalin-fixed, paraffin-embedded tissue sections of 4-µM thickness were deparaffinized and hydrated. High-pressure antigen retrieval was performed using citrate buffer (pH6). Endogenous peroxidase was quenched with 3% hydrogen peroxide in methanol for 15 minutes, followed by incubation with normal serum to block non-specific staining. Rabbit anti-MTDH (1∶25) antibody was then incubated with the sections overnight at in a humidified chamber 4°C; the second antibody was from SP reagent kit (Zhongshan Goldenbridge Biotechnology Company, Beijing, China). After washing, the tissue sections were treated with biotinylated anti-rabbit secondary antibody, followed by further incubation with streptavidin-horseradish peroxidase complex. Stained with diaminobenzidine Kit (DAB, Zhongshan Goldenbridge Biotechnology Company, Beijing, China), the sections were counterstained with hematoxylin and mounted. Immunohistochemical staining of samples and negative controls occurred simultaneously, and the primary antibody was replaced with PBS for negative controls.
Immunohistochemical stainings were assessed in a series of randomly selected 5 high-power fields, which were believed to be representative of the average in tumors at×400 magnification, by two independent observers who were blinded to all clinical data. The sections were scored according to the proportion of positively stained tumor cells. Tumors displaying staining in 30% or more of the cells were categorized as positive cases. In the meanwhile, tumors displaying staining less than 30% of the cells were categorized as negative cases.
LY1 and LY8 cell lines (5×103, respectively) were seeded into 96-well plates, treated with TNF-α (250 pg/ml) and cultured for 48 hours. 3H-thymidine (1 µCi/well) was added into the cultures 16 hours before the end of the experiment. Then cell proliferation was evaluated using 3H-TdR incorporation method as described previously
After treatment of LY1 and LY8 cells with TNF-α (250 pg/ml) for 48 h, cell apoptosis and necrosis were determined using an annexin V- fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit (Neobioscience, Shenzhen, China), according to the manufacturer's instructions. Briefly, an aliquot of 106 cells was incubated with annexin V-FITC and PI for 10 minutes at room temperature in the dark. Cells were then immediately analyzed with FACScan flow cytometer (Beckman Coulter, Chicago, USA). Viable cells are not stained with annexin V-FITC or PI. The necrotic cells were annexin V-FITC and PI-positive, whereas apoptotic cells were annexin V-FITC-positive and PI-negative
All statistical analyses were performed by using the statistics software SPSS 13.0 for Windows. The numerical data were statistically analyzed by 2-tailed Student’s t test t-test. Fisher’s exact test was used to analyze the relationship between the level of MTDH expression and clinicopathological features. Bivariate correlation between two independent variables was calculated by Spearman’s rank correlation coefficient. Statistically significance was defined as P<0.05.
(TIF)
The authors sincerely thank B. Hilda Ye (Albert Einstein College of Medicine, NY) for LY1 and LY8 cells, and also thank Michael Wang (Department of Lymphoma and Myeloma, The University of Texas MD Anderson Cancer Center, Houston) for Jeko-1, Mino, and SP53 cell lines.