MicroRNA-181a Inhibits Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Progression by Repressing CARD11

We investigated the role of miR-181a in diffuse large B-cell lymphoma (DLBCL) and its potential target genes. miR-181a levels were lower in activated B-cell- (ABC-) like DLBCL cells than that in germinal center B-cell- (GCB-) like DLBCL cells. Overexpression of miR-181a in ABC-like DLBCL cell lines (OCI-LY10 and U2932) resulted in G0/G1 cell cycle arrest, increased apoptosis, and decreased invasiveness. miRNA target prediction programs (miRanda, TargetScan, and miRDB) identified caspase recruitment domain-containing protein 11 (CARD11) as a putative miR-181a target. CARD11 mRNA and protein levels were higher in the ABC-like DLBCL than that in GCB-like DLBCL. Moreover, CARD11 mRNA and protein levels were downregulated in the OCI-LY10 and U2932 cell lines overexpressing miR-181a. Dual luciferase reporter assays confirmed the miR-181a binding site in the CARD11 3′UTR region. OCI-LY10 and U2932 cells transfected with a CARD11 expression vector encoding miR-181a with a mutated binding site showed higher CARD11 protein levels, cell viability, G2/M phase cells, and invasiveness compared to those transfected with a wild-type CARD11 expression vector. Nude mice xenografted with OCI-LY10 cells with overexpressed wild-type miR-181a generated smaller tumors compared to those with overexpressed mutated binding site of CARD11 3′UTR and miR-181a. These results indicate that miR-181a inhibits ABC-like DLBCL by repressing CARD11.


Introduction
Diffuse large B-cell lymphoma (DLBCL) is a malignant lymphoma that is highly heterogeneous. Despite greater understanding of the DLBCL pathological subtypes and effectiveness of rituximab-based chemoimmunotherapy, 35%-40% of patients show diminished treatment efficacy with rapid emergence of drug resistance [1,2]. erefore, there is an urgent need for in-depth understanding of DLBCL pathogenesis and the mechanisms that lead to drug resistance in order to improve survival rates.
MicroRNAs (miRNAs) are involved in tumorigenesis of many malignant tumors including DLBCL. ey regulate cell differentiation, proliferation, apoptosis, and other basic cellular processes and play an important role in tumor diagnosis and prognosis [3,4]. miR-181a/b plays an important role in the early development of B-lymphocytes, positive selection of T-cells and development of NK cells. e miR-181 family is involved in immune regulation, angiogenesis, tumor resistance to chemotherapy, and prognosis evaluation [5][6][7][8]. miR-181a/b suppresses the malignant transformation of B-lymphocytes by inhibiting cytidine deaminase (AID), BCL-6, FOXP-1, and other key genes that are involved in malignant transformation and differentiation of B-cells [5,9,10].

Cell
Culture. SU-DHL4, U2932, OCI-Ly19, and OCI-LY10 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). SU-DHL4 and U2932 cells were grown in RPMI 1640 with 10% FBS; OCI-Ly19 and OCI-LY10 were grown in IMDM with 20% human serum at 37°C in a humidified incubator with 5% CO 2 .
e cell lines were authenticated by single nucleotide polymorphism profiling (fingerprinting).
e resulting lentiviral vector with miR-181a precursor or CARD11 was confirmed by PCR and sequencing.
To generate lentivirus, the HEK293 cell line was grown in the DMEM medium ( ermoFisher) supplemented with 10% fetal calf serum ( ermoFisher) and transfected with 1.8 ml DNA solution containing 10 μg Lv-miR-181a or Lv-CARD11, 10 μg psPAX, and 10 μg pMD2G. All virus stocks were produced by calcium phosphate-mediated transfection. Cell supernatants containing viral particles were collected at 48 h after transfection and were filtered using the 0.45-μm Steriflip vacuum filtration system (Millipore) and concentrated by ultracentrifugation at 25,000 rpm at 4°C. e titer of the virus was determined by the GFP expression. e day before infection, the OCI-LY10 and U2932 cells were seeded in dishes at a concentration of 10 6 /ml. On the day of infection, the OCI-LY10 and U2932 cells were infected by the packaged lentivirus with a MOI of 100, whereas uninfected OCI-LY10 and U2932 cells were cultured in parallel. After 72 h, the cells were selected by growing in the selection medium containing puromycin (300 mg/ml) for 2 weeks.

Quantitative Real-Time PCR (qRT-PCR).
Total RNA was extracted from cell lines using the miRNeasy isolation kit (Qiagen) and was quantified by NanoDrop 2000 ( ermo Fisher Scientific, MA, US). For miRNA quantification, 5 μl of total RNA (1 ng/μl) was mixed with 10 μl TaqMan Micro-RNA Reverse Transcription Kit reagent with specific miRNA primers and reverse transcribed at 16°C for 30 minutes, 42°C for 30 minutes, and 95°C for 5 minutes.
To quantify CARD11 mRNA expression, 1 μg RNA was reverse transcribed with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol with a minor modification (1 U/ul RNase inhibitor (Applied Biosystems) was added). e reaction was carried out at 25°C for 10 minutes and 37°C for 60 minutes. Real-time PCR was performed as follows: 94°C for 5 min and 40 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s in the ABI PRISM 7900HT Sequence Detection System Instrument (Applied Biosystems). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 were used as internal controls of CARD11 and miR-181a expression, respectively. Data were analyzed by the comparison Ct (2 − ΔΔCt ) method and expressed as fold change relative to GAPDH or U6. e primer sequences are shown in Table 1. Each sample was analyzed in triplicate.

CCK-8 Cell Viability Assay.
e cell viability was monitored by the Cell Counting Kit-8 (CCK8) assay kit (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer's protocol. In brief, cells were seeded into 96-well plates at a density of 1 × 10 4 cells/well. Detection was done in 6 wells per group, and blank controls were also detected. At 0, 24 h, 48 h, 72 h, and 96 h, 10 μl of CCK-8 solution was added, followed by incubation for 3 h at 37°C. e optical density (OD) was measured at 450 nm to reflect the cell viability. e experiments were repeated thrice.

Transwell Matrigel Cell Invasion
Assay. Transwell invasion assay was performed in 24-well BD Matrigel invasion chambers (BD Biosciences) according to the manufacturer's instructions. A total of 5 × 10 4 cells per well were seeded in the upper well with the DMEM medium without serum, whereas the lower chamber was filled with the DMEM medium containing 10% FBS. After 24 hours of incubation, the noninvading cells in the top well were removed with a cotton swab and the cells in the bottom well were fixed with 3% paraformaldehyde. Subsequently, the cells were stained with 0.1% crystal violet, extracted with 33% acetic acid and quantified in a standard microplate reader at 570 nm.

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Journal of Oncology

Flow Cytometry Analysis of Cell Cycle and Apoptosis.
For cell cycle analysis, single cell suspensions were fixed with 70% ethanol for 30 min at 4°C followed by RNA digestion with RNAase (0.5 mg/ml). en, the permeabilized cells were labeled with propidium iodide (5 mg/ml; Sigma-Aldrich, MO, USA). Subsequently, DNA content was assessed by using an Epics xL flow cytometer (Beckman Coulter, U. K.). For the cell apoptosis assay, the cells were stained with APC conjugated anti-AnnexinV antibody and propidium iodide (PI) according to the manufacturer's protocol manufacturer (BioVision Inc., Milpitas, CA, USA). e percentage of AnnexinV + PI + cells were determined by using an Epics xL flow cytometer (Beckman Coulter, UK).

Tumor Growth in the Xenograft Mouse Model.
e animal experiments were performed according to the recommendations in the Guide for the Care and Use of Laboratory Animals of the ird Affiliated Hospital of Soochow University, and the animal studies were approved by the ethics committee of the ird Affiliated Hospital of Soochow University. Six SCID mice per group were injected subcutaneously with 1 × 10 7 OCI-LY10 cells (200 μl PBS) that were transfected with (1) vector control, (2) lentiviral vectors with miR-181a plus wild-type CARD11, and (3) lentiviral vectors with miR-181a plus mutant CARD11. e xenograft tumors were measured every 5 days to determine tumor volume as (1/2) × length × width 2 . e mice were sacrificed on the 30 th day, and tumors were harvested and weighed to determine the tumor weights.

Statistical
Analysis. Data were analyzed using SPSS 16.0 software package and expressed as mean ± SEM. Statistical significance was determined by ANOVA or repeated ANOVA for multiple comparisons or repeated measurements. Significant differences between two mean values were estimated using Student's t-test. P < 0.05 was considered statistically significant.

Decreased miR-181a Expression in ABC-DLBCL Cell
Lines. As shown in Figure 1, qRT-PCR analysis showed decreased miR-181a levels in the ABC-like DLBCLs (OCI-LY10 and U2932) than that in GCB-like DLBCLs (OCI-Ly19 and SU-DHL-4) ( Figure 1). However, miR-181b levels were similar between GCB-and ABC-like DLBCL cell lines. is suggested that miR-181a expression may be associated with the differential development of the different DLBCL subgroups. We chose OCI-LY10 and U2932 cells for further studies.

Effects of miR-181a Overexpression in OCI-LY10 and U2932 Cells on Cell Cycle, Viability, and Invasion.
e CCK-8 assay showed decreased proliferation of OCI-LY10 and U2932 cells transfected with miR-181a overexpressing lentiviral vector than that in controls (P < 0.05; Figure 2(a)). e miR-181a overexpressing OCI-LY10 and U2932 cells showed higher G0/G1 and lower G2/M phase cells than that in the controls, suggesting G0/G1 cell cycle arrest (Figure 2(b)). FACS analysis with AnnexinV/PI staining showed increased apoptosis in miR-181a overexpressing OCI-LY10 and U2932 cells than that in the controls (Figure 2(c)). Transwell matrigel assays showed that miR-181a overexpression in OCI-LY10 and U2932 cells led to less invasiveness compared to the controls (Figure 2(d)). ese data demonstrated that miR-181a acted as a tumor suppressor in ABC-DLBCL cells.

CARD11 Is a miR-181a Target Gene in ABC-DLBCL.
We analyzed downstream targets of miR-181a with three miRNA target prediction programs, miRanda, TargetScan, and miRDB (Supplementary Table 1), that identified CARD11 as a putative target (Figure 3(e)). High CARD11 mRNA and protein levels in the ABC-DLBCL cell lines suggested negative correlation between miR-181a and CARD11 in DLBCL (Figures 3(a) and 3(b)). is was further corroborated by decreased CARD11 expression in miR-181a overexpressing OCI-LY10 and U2932 cells compared to the controls (Figures 3(c) and 3(d)). en, fragments of the 3′-UTR of CARD11 were cloned with the wild-type or mutated miR-181a binding site into a firefly luciferase reporter vector (Figure 3(e)). Luciferase activity was reduced by approximately 55% in OCI-LY10 and 51% in U2932 cells comparing to the controls, thereby confirming that CARD11 is a direct target of miR-181a in ABC-DLBCL cells (Figure 3(e)).

Discussion
DLBCL is the most common type of lymphoma accounting for 30%∼40% of non-Hodgkin lymphoma with significant heterogeneity in its clinical manifestation, pathogenesis, and prognosis. Based on gene expression profiling and tumor cell origins, DLBCL is classified into germinal center B-cell (GCB) type and activated B-cell (ABC) type [12,13]. e proportion of the ABC type is higher in Chinese patients than that in western countries and accounts for 70%-80% of DLBCL cases [14]. Chemoimmunotherapy combined with rituximab (R-CHOP) is the standard treatment for DLBCL, but the prognosis for ABC-type patients is worse than GBCtype ones [15,16].
miRNAs are noncoding RNAs that are involved in various biological processes through posttranscriptional regulation. Aberrant miRNA expression mediates the tumor proliferation and tumor drug resistance by targeting cell cycle, apoptotic, and other signaling pathways [17,18]. To date, there have been relatively few studies on the relationship between miRNA and prognosis, drug resistance, and proliferation in DLBCL. Alencar et al. showed that miR-181a was an independent prognostic factor in patients with DLBCL after standard R-CHOP chemotherapy; patients with low miR-181a expression had shortened progression-free survival (PFS), and high miR-181a expression inhibited FOXP-1 gene expression in the ABC-type of DLBCL [10]. de Yébenes et al. reported that miR-181b regulated the B-cell antibody-type conversion and recombination process and suppressed malignant transformation by inhibiting AID gene expression [9]. Zhang et al. showed that miR-181b was highly expressed in GCB-type B-cells compared to that in non-GCB-type cells (memory B-cells and plasma cells) [19]. is suggested that miR-181a/b was important for the B-cell differentiation process and was closely associated with the pathology of ABC/GCB-type DLBCL.     Most probably, miR-181a/b regulates genes related to the NF-κB and p38/MAPK pathways because aberrant activation and high expression of the proteins related to these two pathways are closely associated with cell survival and resistance of DLBCL cells to immunochemotherapy; their inhibition significantly increases the therapeutic effects of immune chemotherapy [20][21][22].
We observed reduced miR-181a expression was in ABC-type DLBCL cells, whereas miR-181b expression was similar in ABC and GCB cell lines. In vivo and in vitro studies showed that exogenous overexpression of miR-181a significantly inhibited tumor growth by inhibiting CARD11. CARD11 is the only lymphocyte-specific member of the membrane-associated guanylate kinase family that acts at the intersection of the BCR and TCR signaling pathways. CARD11 was mutated and highly expressed in ABC DLBCL, which further activated the NF-κB pathway leading to increased nuclear transcription of various target genes including BCL2, API2, and BCLX [23]. In a Middle Eastern population, high CARD11 mRNA and protein expression were related to the prognosis of DLBCL [24]. Shigeo et al. found that the overexpression of CARD11 and CARD9 was associated with gastric B lymphoma [25]. Ngo et al. performed loss of function screen of 2500 genes in DLBCL and found that CARD11, MALT1, and BCL-10 were critical genes for tumor cell survival and proliferation with CARD11 playing a key role in ABC-DLBCL [26]. Moreover, higher CARD11 mRNA levels were observed in ABC-DLBCL biopsy specimens than that in GCB-DLBCL specimens. It was postulated that CARD11 enabled cell survival through BCL10-dependent IKK activation in ABC-DLBCL. Furthermore, since CARD11 is only expressed in lymphoid tissues, it is an attractive therapeutic target for ABC-DLBCL [27].
In conclusion, our study reveals that miR-181a suppresses ABC-DLBCL by targeting CARD11 and has immense therapeutic potential. Data Availability e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.

Authors' Contributions
Danxia Zhu and Cheng Fang contributed equally to this work.