MicroRNA‑21 regulates the viability and apoptosis of diffuse large B‑cell lymphoma cells by upregulating B cell lymphoma‑2
- Authors:
- Published online on: August 24, 2017 https://doi.org/10.3892/etm.2017.5021
- Pages: 4489-4496
Abstract
Introduction
Diffuse large B-cell lymphoma (DLBCL) is a prevalent type of non-Hodgkin lymphoma (NHL) that occurs in developed countries (1) and the survival rate of untreated patients is only 50% for <1 year (1). Remarkable progress has been made in exploring the biological mechanism of DLBCL during the past decade, and results have indicated that environmental factors (2,3), dietary factors (4,5), genetic factors and clinical conditions may influence the risk of DLBCL (6,7). However, the pathology and mechanism of DLBCL still remains to be elucidated.
Major studies have started to focus on microRNA (miRNA), which are small non-coding RNAs composed of 20–22 nucleotides. miRNA have an important role in the lymphoid system, which is critical to the differentiation and malignant transformation of B-cells. A selection of miRNA function as regulators in oncogenic or tumor-suppressive pathways in lymphoma (8,9). Moreover, miR-21, miR-155 and miR-17-92 clusters have been acknowledged as oncogenic miRNA, which are believed to target tumor-suppressive molecules in various types of tumor, including glioblastomas, cholangiocarcinomas, lung cancer, breast cancer and colon cancer (10). Furthermore, overexpression of miR-21, miR-155 and miR-17-92 may be observed in lymphomas derived from B cells, T cells or natural killer cells (8,11,12). Notably, miR-21 has a vital role in regulating the chemosensitivity of DLBCL cells (13) and Bcl-2, a tumor-associated and anti-apoptotic molecule, has a key role in the chemoresistance of NHL and has been considered as a prognostic biomarker for DLBCL (14). However, the role of miR-21 in regulating the expression of Bcl-2 in DLBCL remains unclear, and there are no in-depth studies on the relationship between miR-21 and Bcl-2 in DLBCL.
The aim of the present study was to analyze the association between miR-21 and Bcl-2 expression levels in DLBCL cells. Furthermore, cell transfection, MTT, and flow cytometry analysis were used to investigate whether miR-21 has an important role in modulating DLBCL cells.
Materials and methods
Patients and tissue samples
Specimens were obtained from 55 patients with DLBCL (30 men and 25 women) diagnosed using hematology at the First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and Technology (Luoyang, Henan, China) between November 2012 and December 2014. The age of included patients ranged from 16 to 89 years, with a median age of 62. Histologic diagnoses were established according to the classification system outlined by the World Health Organization (15). According to the immune markers of cluster differentiation (CD)-10, Bcl-6, multiple myeloma oncogene-1 and Hans type principles (16), 55 patients with DLBCL were divided into germinal center B cell-like (GCB)-type (19 cases) and non-GCB type (36 cases) groups, with adjacent healthy lymph node tissues from the same patients as the control group. Tissue samples were frozen in liquid nitrogen immediately following surgery and stored at −80°C. A portion of the tumor tissues were fixed in 10% formalin and embedded with paraffin. Sections of 4 µm thickness were examined with immunohistochemistry. The present study was approved by the Ethics Committee of the First Affiliated Hospital and College of Clinical Medicine, Henan University of Science and Technology, and all participants gave their written informed consent.
Detection of miR-21 with reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA from tissue samples was extracted using TRIzol reagent and purified using a miRNeasy Mini Kit (Qiagen GmbH; Hilden, Germany). Genomic DNA was removed with DNase treatment and quantified using NanoDrop ND-2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA). A ratio of OD260/OD280 between 1.7 and 2.1 indicated a higher purity of RNA which was considered to be satisfactory for follow-up experiments. The Omniscript reverse transcription kit (Qiagen GmbH) was used to reverse transcribe total RNA into cDNA according to the manufacturer's protocol. Expression levels of miR-21 were detected using the QuantiTect SYBR Green PCR Kit (Qiagen GmbH). The primer sequences for miR-21 were: Sense, 5′-GCGCGTCGTGAAGCGTTC-3′; antisense, 5′-GTGCAGGGTCCGAGGT-3′. The total reaction volume was 10 µl, containing the following: MirVana 56 RT buffer (2 µl), miR-21 RT primer (1 µl), ArrayScript Enzyme Mix (0.4 µl), total RNA (0.2 µl), and H2O (6.4 µl). The cycling conditions were as follows: 95°C for 30 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 30 sec. All assays were repeated three times. The relative expression quantity of miR-21 was calculated using the 2−ΔΔCq method (17) and normalized to the expression of U6 snRNA.
Detection of Bcl-2 with immunohistochemistry
Bcl-2 expressed in DLBCL tissues was analyzed using immunohistochemical streptoavidin-biotin peroxidase. Tissues were fixed with 4% formalin at 4°C for 12 h. Formalin-fixed paraffin-embedded tissues obtained from 55 DLBCL tissues and 12 normal lymphoma tissues were cut into 4-µm slices. Subsequently, the following procedures were applied: Conventional dewaxing, graded ethanol dehydration, antigen retrieval were performed, and membranes were incubated with 3% hydrogen peroxide to block endogenous peroxidase (4°C for 10 min), and normal sheep serum (Cappel Laboratories, Cochranville, PA, USA) to block the tissue sample (27°C for 20 min). Primary mouse anti-human Bcl-2 monoclonal antibody (1:500; cat. no. IS61430, Dako, Glostrup, Denmark) was applied, incubated at 4°C overnight and stored at room temperature for 20 min. Membranes were washed three times with Tris-buffered saline with Tween 20 (TBST) for 10 min and subsequently incubated with the secondary antibody (anti-IgG; 1:2,000 dilution; cat. no. P0448, Dako) that was labeled with biotin (Dako), and the streptomycin-avidin that was labeled with horseradish peroxidase (Dako) for 30 min at room temperature. Membranes were washed again with TBST three times for 10 min, staining was performed using diaminobenzidine and slices were counterstained with hemalum. Phosphate-buffered saline instead of a primary antibody was considered as a negative control (NC) and a known positive antibody anti-CD38 (1:100; cat. no. TA353695, Origene Technologies, Inc., Rockville, MD, USA) was set as a positive control. Moreover, the product of staining intensity (3, brown; 2, yellow; 1, light yellow; and 0, colorless) and the percentage of positive cells (4, >75%; 3, 51–75%; 2, 26–50%; 1, 6–25%; and 0, <5%) were complied with the integral calculation method for Bcl-2. Cells were randomly selected from five high power fields under a light microscope (magnification, ×400) in each slice and 100 cells were counted in each field. Based on the two types of scores, the integral levels were evaluated as: Negative (−), 0 points; weak positive (+), 1–2 points; positive (++), 3–5 points; strongly positive (+++), >5 points. The slides were independently evaluated by two blinded pathologists.
miRNA target prediction and 3 untranslated region (UTR) luciferase-reporter assay
MiRNA targets were predicted using the TargetScan database version 7.1 (http://www.targetscan.org/vert_71/). Wild-type and mutant-type Bcl-2 3′UTR luciferase reporter vectors were constructed. miR-21 mimics or control were co-transfected with constructed wild-type or mutant-type luciferase reporter vector into DLBCL OCI-LY3 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific Inc.). The pRL-TK control vector (Promega Corporation; Madison, WI, USA) was transfected and served as a control. Subsequently, luciferase activity was analyzed using the Dual-Luciferase Reporter Assay System (E1910; Promega Corporation) following cell transfection for 48 h.
Cell culture and cell transfection
OCI-LY3 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). Cells were cultured in an incubator containing 5% CO2 at 37°C and the OCI-LY3 cell line was provided by the Chinese Academy of Sciences (Guangzhou, China). Cells were divided into five different groups: Control, NC, miR-21 mimics, miR-21 inhibitor, and Bcl-2 siRNA groups. Cells without any treatment were in the control group, while the other four groups were transfected with negative control (empty vector), miR-21 mimics, miR-21 inhibitor and Bcl-2 siRNA, respectively. The corresponding vectors were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China). Cells were transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific Inc.) and cultured in an incubator containing 5% CO2 at 37°C. Complete medium was replaced every 6 to 8 h until the culture process was completed.
MTT assay
MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide] assays were used to evaluate cell viability. Transfected cells, which were washed twice with PBS, were cultured to a confluence of 80%, digested with trypsin and constructed into cell suspensions and the number of cells were counted manually. OCI-LY3 cells were inoculated into 96-well plates at a cell density of 3–6×103 cells/well. Six wells were replicated. Cells were detected following transfection for 24, 48, 72, and 96 h, respectively. MTT (20 µl; 5 mg/ml; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) was added and the cell culture was sustained for 4 h at 37°C in an incubator with 5% CO2. Subsequently, DMSO (150 µl) was added into each well and the cells were lightly shaken for 10 min to dissolve the crystals. Samples were detected using a microplate reader (SpectraMAX Plus; Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 490 nm. An MTT curve was drawn with the absorbance value as the vertical axis and the time interval as abscissa.
Flow cytometric analysis
Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kits (BD Biosciences, San Jose, CA, USA) were used to evaluate the apoptosis of OCI-LY3 cells. Following 48-h transfection, cells were washed twice with cold PBS and re-suspended with binding buffer to a density of 0.5–1×106/ml. Subsequently, cell suspensions (100 µl) were incubated with 5 µl of annexin V-FITC and PI in the dark for 15 min at room temperature. Binding buffer (400 µl) was added to each tube and cells were analyzed using flow cytometry (Beckman FC 500 MCL/MPL; Beckman Coulter, Inc., Brea, CA, USA).
Detection of caspase-3 activity
Caspase-3 activity was detected using the caspase colorimetric assay kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), following cell transfection for 48 h. Cells were lysed in lysis buffer on ice for 20 min in order to detect the activity of caspase-3. Following centrifugation at 28,341 × g for 5 min at 4°C, supernatants were incubated with the caspase substrate in the reaction buffer at 37°C for 4 h. Samples were detected with a SpectraMAX Plus microplate reader at the wavelength of 405 nm. Relative caspase-3 activity was calculated as the percentage of A405 values in the experimental samples over those in the control groups.
RT-qPCR for detecting the expression levels of Bcl-2 mRNA
The extraction of cellular total RNA was conducted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was reverse transcribed from RNA using the Omniscript RT kit (Qiagen GmbH). qPCR detection of Bcl-2 mRNA was performed using the QuantiTect SYBR Green PCR Kit (Qiagen GmbH). Primers used for Bcl-2 (Invitrogen; Thermo Fisher Scientific Inc.) were as follows: Bcl-2 sense, 5′-CTGTGCTGCTATCCTGC-3′ and antisense, 5′-TGCAGCCACAATACTGT-3′. Relative expression levels of Bcl-2 were calculated using the 2−ΔΔCq method (17) and β-actin was set as the corresponding control.
Western blotting assay
Bcl-2 expression was detected by western blotting. Cellular proteins were extracted after 48-h transfection and the bicinchoninic acid method was used to evaluate the protein density. Furthermore equal quantities of protein (50 µg) from each group were loaded and separated by 10% SDS-PAGE, transferred onto polyvinylidene fluoride membranes, and blocked with 5% non-fat milk for 1 h at room temperature. Membranes were incubated with primary mouse anti-human Bcl-2 monoclonal antibody (1:500) and GAPDH antibody (1:1,000; cat. no. 5174, Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight. Furthermore, membranes were washed three times with TBST for 10 min each and incubated with horseradish-peroxidase-linked anti-IgG (1:2,000; Origene Technologies, Inc.) at room temperature for 1 h. Membranes were washed again with TBST three times (10 min each) and signal detection was performed using a Super Enhanced Chemiluminescence Plus Detection Reagent (Applygen Technologies Inc., Beijing, China). The samples were quantified using Lab Works software version 4.5 (Mitov Software, Moorpark, CA, USA) with GAPDH as an internal control.
Statistical analysis
All statistical analyses were performed using SPSS 19.0 software (IBM SPSS, Armonk, NY, USA) and P<0.05 was considered to indicate a statistically significant difference. Significant differences in continuous data (mean ± standard deviation) were analyzed using the analysis of variance with Student Newman-Keuls post-hoc tests for comparisons between groups, and differences in continuous data between two groups were analyzed using unpaired Student's t-tests. Furthermore, results of immunohistochemistry for Bcl-2 protein were analyzed by the rank sum test. The association between miR-21 and Bcl-2 protein expression was analyzed using the Spearman rank correlation.
Results
miR-21 and Bcl-2 protein expression levels in DLBCL clinical specimens
RT-qPCR was used to evaluate the expression level of miR-21 in 19 cases with GCB-DLBCL, 26 cases without GCB and 20 normal lymph node tissues (Fig. 1). The expression of miR-21 in DLBCL tissues was significantly increased when compared to that in normal tissues (P<0.01) and the expression level of miR-21 in non-GCB DLBCL tissues was significantly higher than GCB-DLBCL tissues (P<0.01; Fig. 1). In addition, positive Bcl-2 expression was predominantly detected in the cell cytoplasm or cell membrane with yellow granules. Bcl-2-positive cells appeared to be focally gathered or unevenly distributed (Fig. 2). Bcl-2 expression levels were significantly increased in non-GCB DLBCL tissues when compared with normal tissues (P<0.01) and GCB-DLBCL tissues (P<0.05); the positive expression of Bcl-2 in GCB-DLBCL tissues was significantly increased when compared with normal tissues (P<0.05; Table I).
Spearman correlation analysis suggested that there was a positive correlation between miR-21 and Bcl-2 expression levels in both GCB-DLBCL (rs=0.528, P=0.02) and non-GCBDLBCL tissues (rs=0.708, P<0.01), whereas no significant correlation between miR-21 level and Bcl-2 protein level was detected in normal tissues.
Targeting Bcl-2 by miR-21
A putative conserved binding site for miR-21 at nucleotide position 720–726 of human Bcl-23′UTR was predicted using TargetScan. Perfect base pairing was observed between the seed sequence of mature miR-21 and the 3′UTR of Bcl-2 mRNA (Fig. 3A). Dual luciferase reporter gene assays revealed that miR-21 significantly promoted the luciferase activity of Bcl-2 wild-type with an upregulation of 61% (P<0.01); however, there was no significant effect on the luciferase activity of Bcl-2 mutant-type 3′UTR (Fig. 3B). Collectively, these findings indicated that Bcl-2 was likely to be a direct target for miR-21.
Proliferation of DLBCL cells
Results of MTT assays suggested that decreased proliferation of OCI-LY3 cells was observed in groups transfected with miR-21 inhibitor and Bcl-2 siRNA for 48 h when compared with the control and NC groups (all P<0.05). This trend was more pronounced as the length of time extended. miR-21 inhibitor appeared to have a stronger ability than Bcl-2 siRNA with respect to the inhibition of cell proliferation; however, this distinction did not reach statistical significance. The cell number of the control group at each time point was not significantly different from that of the NC group. Compared with the other four groups, the proliferation capacity of OCI-LY3 cells significantly increased after the transfection of miR-21 mimics for 48 h (all P<0.05; Fig. 4).
Apoptosis assay
Results of flow cytometry analysis indicated that the apoptosis percentages of cells transfected with miR-21 inhibitor and Bcl-2 siRNA were 20.10±1.16 and 19.15±0.91%, with no significant difference observed. However, the apoptosis percentages of miR-21 inhibitor and Bcl-2 siRNA groups were significantly higher than the miR-21 mimic, control and NC groups (P<0.01; Fig. 5). Furthermore, there was no significant difference observed in the apoptosis percentage between the control (5.71±0.62%) and NC (5.86±0.32%) groups. The apoptosis rate (mean ± standard deviation) of the miR-21 mimic group was 2.73±0.48%, which was significantly lower than those of the other four groups (P<0.01; Fig. 5). These findings indicated that miR-21 may inhibit the apoptosis of OCI-LY3 cells and the downregulation of miR-21 or Bcl-2 may increase the apoptotic ability of OCI-LY3 cells.
Caspase-3 activity assay
The caspase-3 activity in cells transfected with miR-21 mimics was 0.47±0.05, indicating a significant difference when compared with the other four groups (all P<0.01; Fig. 6). Caspase-3 activity levels for cells transfected with miR-21 inhibitor and Bcl-2 siRNA were 2.6±0.24 and 2.47±0.15, with no significant difference. However, the caspase-3 activities in miR-21 inhibitor and Bcl-2 siRNA groups were significantly increased compared with the control, NC and miR-21 mimic groups (P<0.01; Fig. 6). These results suggest that miR-21 may restrain the activity of caspase-3 and the downregulation of miR-21 or Bcl-2 may increase the caspase-3 activity.
Bcl-2 mRNA and Bcl-2 protein expression levels in DLBCL cells
Results from RT-qPCR and western blotting demonstrated that the expression levels of Bcl-2 mRNA and Bcl-2 protein were significantly decreased when compared to the control and NC groups after 48 h transfection with the Bcl-2 siRNA or miR-21 inhibitor (P<0.01; Figs. 7 and 8, respectively). No significant difference in Bcl-2 mRNA and Bcl-2 protein expression levels were indicated between the control and NC group (P>0.05). Bcl-2 expression levels in cells transfected with miR-21 mimics were significantly upregulated when compared with the other four groups (P<0.01). These findings indicated that miR-21 may target and regulate the expression of the Bcl-2 gene in OCI-LY3 cells.
Discussion
A number of studies have demonstrated that miRNA exhibited differential expression levels in various tumors, and they may interplay with tumor suppressor genes or oncogenes to affect tumorigenesis (18). Indeed, significant overexpression of miR-21 has been reported in glioma (19), pancreatic cancer (20), lung cancer (21), leukemia (22), and lymphoma (23). In the present study, we examined miR-21 expression levels in both DLBCL and normal lymph node tissues. The present results suggest that miR-21 was upregulated in DLBCL tissues and its expression in non-GCB tissues was higher when compared with GCB tissues. These findings were consistent with the results from previous studies (24,25).
Bcl-2 directly participates in cell apoptosis and functions as an anti-apoptotic substance (26); furthermore, it has been indicated that Bcl-2 exhibited low expression levels in apoptotic cells (27). Moreover, Bcl-2 expression is associated with the development of various types of cancer, including breast cancer (28), non-small cell lung cancer (29), nasopharyngeal cancer (30), gastric cancer (31) and non-Hodgkin B-cell lymphoma (32–34). Immunohistochemical results from the present study revealed that the Bcl-2 protein was overexpressed in DLBCL tissues and that Bcl-2 protein expression levels exhibited a significant difference between GCB-DLBCL and non-GCBDLBCL tissues. Therefore, we hypothesized that Bcl-2 was heterogeneously expressed in different histological subtypes.
Additionally, a positive correlation was determined between miR-21 and Bcl-2 expression levels in both GCB-DLBCL and non-GCBDLBCL tissues. The dual-luciferase reporter assay was conducted to illuminate the potential mechanisms. The results indicated that Bcl-2 was a target gene of miR-21 and the Bcl-2 expression levels may be increased through the direct binding of miR-21 to the 3′UTR of Bcl-2 mRNA. However, further comprehensive research is required to confirm these findings.
miR-21 is believed to be a multi-functional miRNA involved in the proliferation, differentiation and anti-apoptosis of cancer cells (35). A previous study has reported that miR-21 was able to increase cell growth and inhibit apoptosis in DLBCL (36). Our findings revealed that upregulation of miR-21 not only exacerbated cell proliferation, but also suppressed the apoptosis of DLBCL cells. However, the inhibition of miR-21 expression may inhibit the proliferation of DLBCL cells and promote the apoptosis of DLBCL cells. Consequently, the present study provides evidence that miR-21 may regulate cell viability and apoptosis in DLBCL and miR-21 mimics may increase Bcl-2 expression levels in DLBCL OCI-LY3 cells. Furthermore, downregulation of miR-21 may contribute to the significant decrease in Bcl-2 expression levels observed in both mRNA and protein levels in OCI-LY3 cells. Our data indicates that the expression of Bcl-2 was modulated by miR-21, which may decrease cell apoptosis through its anti-apoptosis effects via upregulating Bcl-2 gene expression.
Caspase-3 is a member of the caspase protease family and functions as a pivotal participant in cellular apoptosis (37). The activation of caspase-3 has been identified in various types of cells observed with apoptosis (38). Caspase-3 was previously revealed as a downstream molecule of the Bcl-2 family (37). The present study findings were consistent with these previous reports and suggested that higher caspase-3 activity along with increased apoptosis was reflected in cells transfected with Bcl-2 siRNA, whereas lower caspase-3 activity accompanied by decreased apoptosis was observed in cells transfected with miR-21 mimics. Therefore, our data provided evidence that caspase-3 may participate in cellular apoptosis and an underlying interaction may exist between Bcl-2 and caspase-3.
The present study provides evidence that miR-21 may exacerbate the viability of DLBCL cells and inhibit cell apoptosis by positively regulating Bcl-2 expression in DLBCL. Since only one DLBCL cell line (OCI-LY3) was used to detect interactive effects between miR-21 and its target gene, Bcl-2, on tumor proliferation and apoptosis in the present study, the molecular effect of miR-21 and Bcl-2 on tumorigenesis should be further studied.
In conclusion, miR-21 expression was upregulated in DLBCL tissues and the expression of miR-21 was positively correlated with Bcl-2 expression. These findings further demonstrated that miR-21 regulates Bcl-2 expression in a direct manner in DLBCL. Notably, the present study provided evidence that miR-21 may exacerbate the viability of DLBCL cells and inhibit cell apoptosis by targeting Bcl-2. Hence, both Bcl-2 and miR-21 are likely to serve as effective targets for developing alternative treatments for DLBCL.
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