Fc receptor-like 1 (FCRL1) is a novel biomarker for prognosis and a possible therapeutic target in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin’s lymphoma, which can involve various types of mature B-cells. Considering that the incidence of DLBCL has increased, additional research is required to identify novel and effective prognostic and therapeutic molecules. Fc receptor-like 1 (FCRL1) acts as an activation co-receptor of human B-cells. Aberrant expression of this molecule has been reported in a number of B-cell-related disorders. Moreover, the clinical significance and prognosis value of FCRL1 in DLBCL are not completely identified. In this study, the expression levels of FCRL1 were determined in thirty patients with DLBCL and 15 healthy controls (HCs). In addition, the correlation between FCRL1 expressions with clinicopathological variables of DLBCL patients were examined. Then, the potential roles of FCRL1 in proliferation, apoptosis, and cell cycle distribution of B-cells from DLBCL patients were determined using flow cytometry analysis, after knockdown of this marker using retroviral short hairpin RNA interference. Quantitative real time-PCR, western blotting, and enzyme-linked immunosorbent assay were also used to identify the possible effects of FCRL1 knockdown on the expression levels of BCL-2, BID, BAX, intracellular signaling pathway PI3K/p-Akt, and p65 nuclear factor‐kappa B (NF-κB) in the B-cells of DLBCL. Statistical analysis revealed higher levels of FCRL1 expression in the B-cells of DLBCL patients compared to HCs at both protein and mRNA levels. A positive correlation was observed between the FCRL1 expression and some clinicopathological parameters of DLBCL patients. In addition, FCRL1 knockdown significantly decreased cell proliferation and stimulated apoptosis as well as G1 cell cycle arrest in the B-cells of DLBCL patients. The levels of p65 NF-κB and PI3K/p-Akt expressions were markedly reduced after knockdown of FCRL1 expression. These results suggested that FCRL1 could be a potential novel biomarker for prognosis and/or a possible effective therapeutic target for treatment of patients with DLBCL.


Introduction
Non-hodgkin's lymphoma (NHL) is a diverse group of cancers that has been classified into more than 50 subtypes based on cellular origin. About 90% of NHL types originate from B-cells [1]. Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL, with a broad spectrum of immunophenotypic, morphological, and clinical features, accounting for approximately 80% of aggressive lymphomas and 30 to 40% of adult NHL cases [2,3]. DLBCL can involve various types of mature B-cells and it is developed in both nodal and extra-nodal sites. The gene expression profiling studies have introduced three distinct molecular subtypes of DLBCL based on cell origin and overall survival rates including germinal-center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, and primary mediastinal B-cell lymphoma (PMBL) [4][5][6]. The GCB type of DLBCL is derived from germinal center (GC) centroblasts and demonstrates better diagnosis and survival rate compared to the ABC group. In addition, GCB DLBCLs can be treated with a combination of current therapeutic methods such as chemotherapy and monoclonal antibodies [7].
In recent years, numerous studies have been performed on the pathogenesis and molecular biology of DLBCL disorder to improve the overall survival rates of patients with this type of B-cell hematological malignancy [8,9]. However, the incidence rate of DLBCL has been increased in both genders and all age groups. Approximately 40 to 60% of the patients with DLBCL could be cured and they respond to the available therapeutic methods [2,7,10,11]. Therefore, additional research studies are required to identify novel prognostics and effective therapeutic molecules, which might improve prognosis, treatment, and survival rates of patients with DLBCL.
Fc receptor-like (FCRL) proteins (also known as FcRH, IRTA, IFGP, SPAP, and BXMAS) belong to a subgroup of lymphocyte receptors with immunoregulatory potential that are preferentially expressed by B lineage cells. FCRL genes are located on human chromosome 1q21-23 and encode two cytoplasmic proteins FCRL-A & B, and six transmembrane glycoproteins (FCRL1-6) with varying numbers of Ig-like domains on their extracellular regions and immunoreceptor tyrosine-based activating/inhibitory motifs (ITAM/ ITIM) in their cytoplasmic domain [12][13][14][15]. The structural properties and expression profile of FCRL molecules have proposed the regulatory potential of these receptors on human B-cell responses [15][16][17]. Moreover, several groups of researchers have revealed a clinical relevance between the aberrant expression of these proteins and different disorders such as autoimmune diseases [18][19][20], infections [21,22], and various types of B-cell-related hematological malignancies [23][24][25][26][27][28][29][30]. Among these receptors, FCRL1 is a pan B-cell marker with two ITAM-like sequences in cytoplasmic domain and glutamic amino acids with negative charges in the transmembrane region. This receptor is differentially expressed during B-cell development [31]. FCRL1 has been introduced as an activation co-receptor with a great potential to improve the function of human B-cells [32][33][34]. The aberrant expression of this molecule has been reported in several NHL cell lines and many B-cell related leukemia and lymphoma disorders including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), and hairy cell leukemia (HCL) [23,27,28,30]. However, there is very little research on the clinical significance and prognostic value of FCRL1 in DLBCL.
This study was performed to evaluate the levels of FCRL1 expression in patients diagnosed with DLBCL compared to healthy controls (HCs). For this purpose, the correlation between the expression levels of FCRL1 and clinicopathological features of these patients were determined. In addition, the potential effects of FCRL1 were investigated in development of DLBCL through evaluation of apoptotic cell death, proliferation, cell cycle distribution, and expression levels of important nuclear transcription factor (NF)-κB and intracellular signaling pathway phosphoinositide 3-Kinase (PI3K)/phosphorylated (p)-Akt in B-cells of DLBCL patients, following knockdown of FCRL1 expression.

Subjects
In this research, according to previous studies and using StataCorp. 2003, Stata Statistical Software: Release 8, College Station, TX: StataCorp LP, the minimum sample sizes were determined to be 28 and 14 for patients and HCs groups, respectively. In total, thirty newly diagnosed patients with DLBCL were included in the study. Two-thirds of the patients were male and 10 patients were female (mean age = 63.7 ± 15.5). According to the immunohistochemistry (IHC) results and their medical records, 10 out of 30 patients (33.3%) had GCB-DLBCL and other patients (66.7%) had non-GCB subtypes of DLBCL. The clinical characteristics of these patients are listed in Table 1. In DLBCL patients, the biopsy specimens were collected from different sources, including 24 lymph node cases, 2 soft tissue cases, 2 tonsil cases, 1 spleen case, and 1 gastrointestinal tract case. The specimens were placed inside a sterile conical tube containing DMEM as the transport medium. In addition, 15 cases with reactive hyperplastic lymph nodes without any familial history of various malignancies were included in the study as HCs including nine males and six females (mean age = 59.3 ± 16.8).

CD19 + B cells isolation
The tissue disaggregation method and magnetic-activated cell sorting (MACS) approach were performed to prepare human B-cells from the tissue samples, as previously mentioned [35,36]. Briefly, each tissue sample was transferred into a sterile petri dish (Nunc-Nalgene, USA), cut into 2-3 mm diameter pieces, and incubated for 60 min at 37 °C with a combination of serum-free DMEM-high glucose medium (Gibco), collagenase (Sigma-Aldrich, Germany), trypsin (Gibco), and DNAse (Sigma-Aldrich). Then, residual activity of trypsin was neutralized and single-cell suspensions were prepared by injection of complete DMEM-high glucose medium (Gibco) into the solid tissues with a pushing and pulsing action, until cells were released. The cell suspensions were collected and centrifuged for 5 min at 1300 rpm. To remove the red blood cells (RBCs), the cell pellet was resuspended in 5 ml ammonium chloride solution and incubated for 5 min at room temperature. Human CD19 + B-cells were isolated from cell suspensions using CD19 positive selection kit and AutoMACS Pro Separator (Miltenyi Biotech, USA) according to the kit instructions. The cell counts and percentage of cell viability were elucidated using a hemocytometer and the trypan blue dye exclusion method. In addition, purity of the isolated B-cells was determined using flow cytometry assay and fluorescein isothiocyanate (FITC)-labeled anti-human CD19 antibody. In each sample, cell viability and purity ≥ 90% were acceptable for further investigation. Then, the isolated B-cells were cultured in DMEM-high glucose medium supplemented with 1% penicillin-streptomycin, 2 mM L-glutamine, 1 × non-essential amino acids, 15% FBS (all from Gibco), and 0.25 M B-class CpG oligodeoxyribonucleotide (CpG-ODN) (Miltenyi Biotech) for 5-7 days, until B-cell aggregation and increase in the number of cells were visible.

Evaluation of FCRL1 protein expression
The flow cytometry assay was used to evaluate the levels of FCRL1 protein expressions in the prepared CD19 + B-cells. To determine the expression levels of FCRL1 in malignant B-cells of the patients diagnosed with DLBCL compared to HCs, the cultured CD19 + B-cells were stained with a combination of

Preparation of retrovirus packaging
To determine the potential roles of FCRL1 on DLBCL development, the retrovirus expression system was selected for knockdown of this molecule in the B-cells of the patients with DLBCL. A high-level and more efficient FCRL1 retrovirus is essential for efficient transduction of human B-cells in vitro. For this purpose, a potent packaging retroviral cell line Plat-A and retroviral GFP vectors (OriGene Technologies, USA, Locus ID 115350) were used in this experiment. This vector contained four unique 29-mer shRNA constructs against different splice variants of the FCRL1 genes. Moreover, a non-targeting scrambled shRNA in the pGFP-V-RS vector was used as negative control to assess the efficiency of FCRL1 knockdown process. The shRNA constructs were transformed in Escherichia coli (E. coli) strain DH5α and selected on antibiotic plates to detect resistance plasmids [37]. The antibiotic-resistance bacteria were grown overnight in LB-agar media, and accuPrep Plasmid Maxi-Prep DNA Extraction Kit (Bioneer, Korea) was used to extract the E. coli DH5α plasmids, according to the kit instruction. The calcium phosphate (CaPO4) precipitation method was performed to transient transfection Plat-A cells and produce retroviral particles, due to the ease and safety of this method [38]. The transfection efficiency was determined based on the GFP signals under the fluorescence microscopy. The supernatant of the Plat-A cells containing retroviral particles was collected at 48 and 72 h posttransfection, it was filtered with 0.45 μm syringe filters (Millipore, USA) to remove the cell debris, and stored at − 80 °C until used for viral transduction in the human B-cells.

Retroviral transduction
Spin infection is the most efficient method for viral transduction of suspended human blood cells such as B-cells. Transduction duration, use of 8 μg/ml polybrene, and suitable density of the target cells are crucial factors for increasing the efficiency of this protocol. The wells of 24-well cell culture plates were seeded with a combination of 1 ml retrovirus solution, 10 μg/ml goat F(ab')2 anti-human IgG/IgM (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), 8 μg/ml polybrene (Santa Cruz Biotechnology, Dallas, Tx), 0.25 µM B-class CpG-ODN (Miltenyi Biotech), and 3 × 10 6 prepared B-cells of the DLBCL patients. The plates were centrifuged at 2500 rpm for 90 min, and maintained in a CO 2 incubator with 95% humidity at 37 °C. The efficiency levels of transduction and knockdown of FCRL1 expressions were determined using quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting after 48 and 72 h of incubation.
Then, the treated cells and control cells were indicated to describe the isolated B-cells that had been transduced with retroviral particles harboring FCRL1-targeting DNA or control vector DNA.

Quantitative real-time PCR
To assess the expression levels of the genes, total RNAs were extracted from 1 to 1.5 × 10 6 target cells by RNAx plus solution (Cinagen Company, Iran) according to the manufacturer's protocols. The quality and concentration of the extracted RNAs were determined using NanoDrop 1000 spectrophotometer (Thermo Scientific, USA). The same concentrations of the extracted RNAs were reverse transcribed to first-strand complementary deoxyribonucleic acid (cDNA) using random hexamer and one-step SYBR PrimeScript RT Reagent Kit (Takara, Japan) according to the manufacturer's guidelines. The qRT-PCR was performed on a rotor-gene 6000 instrument (Qiagen, Germany) in a 20 µl reaction mixture for each sample containing 2 µl of the template cDNA, 10 µl of SYBR Green PCR Master Mix (Takara), and appropriate amounts of each primer and DNase-RNase free water. Each reaction was incubated at 95 °C for 30 s, followed by 45 cycles (FCRL1), 30 cycles (BCL2, BID), and 35 cycles (BAX, PI3K) of 95 °C for 5 s as well as 60 °C (FCRL1, BCL2 and BID) or 61 °C (PI3K and BAX) for 30 s. The Primers were designed using Alle-leID 7.0 software (Premier Biosoft, USA). Sequences of the primers are shown in Table 2. Human β-actin was used as an endogenous control to normalize the expression level of each gene. Relative expression levels of the target genes were calculated using Relative Expression Software Tool 2009 (REST 2009) and were reported as 2 −ΔΔCt [39,40].

Western blotting
Western blotting was performed to detect the expression levels of FCRL1 and phosphorylated Akt (p-Akt) proteins in the prepared B-cells of the DLBCL patients. The cells were collected and washed three times with phosphate-buffered saline (PBS, pH 7.2), 72 h post retrovirus transduction. The target cells were lysed in RIPA buffer with protease and phosphatase inhibitors and their protein concentration was determined using a bicinchoninic acid (BCA) kit (Sigma-Aldrich, Germany). The protein samples (40 µg per sample) were separated by 12-15% SDS-PAGE gel, transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore), and blocked with 5% skimmed milk in Tris Buffered Saline with Tween (TBST) at room temperature for 2 h. The membranes were incubated with primary antibodies against FCRL1 (0.5 µg per ml, catalog number MAB20491, R&D Systems, USA), p-Akt (S473) (0.2 µg per ml, catalog number MAB887, R&D Systems), and β-actin (1:4000, catalog number 66009-1-LG, Proteintech) at 4 °C overnight with gentle shaking. They were washed three times with TBST and incubated with a horseradish-peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Finally, the expression levels of the target proteins were assessed using an enhanced chemiluminescence (ECL) assay.

Detection of intracellular proteins by flow cytometry
The expression levels of intracellular proteins p-Akt (S473), BCL2, and BCL6 were determined by flow cytometry analysis according to the established protocols. Briefly, the target cells were fixed with 0.01% formaldehyde for 20 min at room temperature and permeabilized with Tween 20 (0.1% v/v in PBS, pH 7.2) for 15 min in the dark at room temperature. Then, intracellular staining of the cells was performed with a combination of Alex Flour® 647-labeled anti-BCL2 (Biolegend; clone: 100), APC anti-human/ mouse BCL6 (Miltenyi Biotech; clone: REA373), and fluorescein isothiocyanate (FITC)-labeled anti-human p-Akt (S473) (eBioscience; clone CA) antibodies or matched isotype control antibodies for 30-45 min at 4 °C. The expressions of these proteins were also determined by FACSCalibur flow cytometry (BD Biosciences) and then analyzed using FlowJo software.

Cell proliferation
Effects of FCRL1 knockdown on the rate of B-cell division was evaluated using cell division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE) (Biolegend) according to the manufacturer's instructions. In brief, the cultured B-cells of DLBCL patients (1 × 10 6 ) were re-suspended in PBS and incubated with 0.5 µM CFSE (Biolegend) for 8 min in the dark at room temperature, followed by extensive washing of the cells with complete DMEM high-glucose medium. The CFSE-labeled B-cells were transduced with FCRL1-retroviral particles based on the protocol described in the "transduction of B-cells" section, and they were incubated in a CO 2 incubator at 37 °C. The cells were harvested at days three and five post-transduction and washed three times in PBS. The percentages of B-cells proliferation were determined using FACSCalibur flow cytometry (BD Biosciences) and assessed by FlowJo software based on the fluorescent intensity of CFSE dye. The non-labeled B-cells were used to exclude cell autofluorescence.

Apoptotic cell death and cell cycle analysis
The potential roles of FCRL1 on apoptosis and cell cycle distribution of the B-cells were examined using flow cytometry analysis. The percentage of apoptotic cell death was evaluated using FITC Annexin V apoptosis detection kit (BD bioscience). The cultured B-cells were collected and washed twice in PBS, 48 and 72 h after retroviral transduction. The cells were re-suspended with a solution for FITC Annexin V binding and stained with 5 µl Annexin V FITC and 5 µl propidium iodide (PI). The samples were incubated in the dark for 15 min at 4 °C, analyzed using a BD FACS-Calibur flow cytometry, and assessed using FlowJo software.
To determine the cycle phases of the cells, the B-cells were collected 72 h post-transduction, fixed in ice-cold 70% ethanol overnight, and incubated with 50 μg/ml PI (Sigma-Aldrich), 0.1% Triton X-100, and 10 μg/ml DNAse-free RNaseA (Sigma Aldrich) in the dark for 15 min at 37 °C. Cell cycle progressions were assessed by FlowJo software.

p65 NF-κB detection by enzyme-linked immunosorbent assay
The expression levels of p65 NF-κB were measured by NF-kB p65 (Total) Multispecies InstantOne Enzyme-linked immunosorbent assay (ELISA) Kit, (eBioscience) according to the instructions. Briefly, the B-cells were collected 72 h after the transduction procedure, washed twice in PBS, resuspended in 1 ml DMEM-high glucose medium containing 0.5% FBS, and incubated at 37 °C for 30 min with or without 20 ng/ml of tumor necrosis factor-α (TNF-α) (Miltenyi Biotech). Absorbance rate was determined at 450 nm with the ELISA reader and the concentration of this nuclear transcription factor was measured based on the standard curve and was adjusted by the dilution factor. Each assay was performed in duplicate wells and repeated three times independently.

Statistical analysis
Data analysis was performed using IBM SPSS 20 and Microsoft Excel 2016. Bonferroni adjustment and Dunn's post hoc tests were carried out on each pair of the groups. The unpaired t-test and Mann-Whitney U test were used to determine the source of significant variations between the two groups with normal and non-normal distributions, respectively. The Fisher's exact test was also used to identify the correlation between the FCRL1 expression and the clinical features of the patients. Data is presented as mean ± standard deviation (SD) and p value ≤ 0.05 is considered statistically significant.

Expression levels of FCRL1
Data showed a significant difference between the expressions of FCRL1 in malignant B-cells of the patients with DLBCL (CD19 + CD20 + BCL2 + BCL6 + ) compared to the B-cells of HCs (CD19 + CD20 + ) at protein (p < 0.0001) and mRNA (p < 0.001) levels (Fig. 1A, B). An example of gating strategy and flow cytometry analysis of FCRL1 protein expression in the B-cells of one healthy individual and one patient with DLBCL is demonstrated in supplementary Fig. 1A Table 2).
A positive correlation was found between the expression levels of FCRL1 and multiple clinicopathological features of newly-diagnosed patients with DLBCL including tumor size (p < 0.05), stage of disease (p < 0.01), performance status at diagnosis (p < 0.05), and IPI scores (p < 0.01). However, no significant differences were observed between the expression levels of FCRL1 and age, gender, lactate dehydrogenase (LDL) enzyme, and extra nodal status of the patients ( Table 2).

Knockdown of FCRL1 expression
Data analysis revealed a significant decrease in the levels of FCRL1 expression in the treated B-cells compared to control and uninfected cells at both mRNA and protein levels (p < 0.01, Fig. 2A, B).

Effects of FCRL1 knockdown on proliferation, apoptosis, and cell cycle progression of DLBCL B-cells
Inhibition of B-cell proliferation was identified in the treated B-cells compared to control and uninfected cells, after 3 and 5 days of retroviral transduction procedure and knockdown of FCRL1 expression (p < 0.001-0.05, Fig. 3A, B).
The FCRL1 knockdown also reduced the expression level of anti-apoptotic BCL2 gene in the treated B-cells in comparison to the control and/or uninfected B-cells (p < 0.001, Fig. 4A), while the expression levels of proapoptotic BID and BAX genes were increased in the treated B-cells (p < 0.01-0.05, Fig. 4A).
Double staining of the cells with FITC Annexin V-PI and flow cytometry analysis showed a significant increase in the percentage of cell apoptosis in the treated B-cells compared to control and uninfected cells in 48 and 72 h after the transduction process and knockdown of FCRL1 expression (p < 0.05) (Fig. 4B, C).
The cell cycle assay revealed a statistically significant reduction in the percentage of the treated B-cells in the S and G2/M phases, after 72 h of transduction procedure. However, the percentage of the treated cells in the sub-G1 (apoptotic cells) and G1 phases were increased in comparison to control and uninfected cells (p < 0.01-0.05, Fig. 5).

Effects of FCRL1 knockdown on p65 NF-κB and PI3K/ p-Akt expressions
The results revealed that knockdown of FCRL1 expression significantly decreased the expression levels of phosphorylated-p65 NF-κB in the treated B-cells compared with control and uninfected cells in the presence of TNF-α, after 72 h of transduction procedure (p < 0.05, Fig. 6A). Similar results were found in the levels of total p65 NF-κB expression in absence of TNF-α, even though the reduction of the levels of this protein was not statistically significant (p = 0.072, Fig. 6A).
In addition, significant decreases were observed in the expression levels of PI3K mRNA and p-Akt (S473) protein in the treated compared to control and uninfected B-cells (p < 0.01-0.05, Fig. 6B-E).

Discussion
DLBCL is an aggressive form of B-cell lymphoma with various pathogenesis, prognosis, and clinicopathological features. Approximately two-thirds of diagnosed patients with DLBCL can be treated with the current therapeutic approaches [41,42]. In addition, most patients are diagnosed in the late stages of the disease, which increases the mortality rate. Therefore, identification of the unique biomarkers that are associated with pathogenesis and development of DLBCL is essential for early diagnosis and effective treatment of patients with DLBCL. Recently, aberrant expression and potential roles of FCRL1 in the pathogenesis of multiple types of hematological malignancies including HCL, CLL, MCL, and FL have been reported by various studies [15,[23][24][25]27]. In addition, a recent study by the authors revealed higher levels of FCRL1 expression in Iranian patients with DLBCL, HCL, and Burkitt's lymphoma (BL) compared to HCs [30]. In another study, the authors  [14,27,28]. The results also revealed a positive relationship between FCRL1 expression levels and tumor size, stage, and IPI scores of the patients. These findings suggested that FCRL1 is an important molecule in the pathogenesis and development of DLBCL, and the expression profile of FCRL1 might serve as a suitable marker for DLBCL prognosis. Then, the biological activities of FCRL1 were investigated in DLBCL. According to the results obtained from a study, FCRL1 has the potential to amplify the B-cell receptor (BCR)-mediated B-cells proliferation using Flowjo software, 3 and 5 days after retroviral transduction procedure. DLBCL diffuse large B-cell lymphoma; *p < 0.05; **p < 0.01; ***p < 0.001; NS not statistically significant proliferation [34]. As expected, the findings of this study affirmed that knockdown of FCRL1 expression suppressed the proliferation of B-cells from DLBCL. This study also showed that FCRL1 knockdown changes the expression levels of important apoptosis-related molecules including BCL2, BID, and BAX as well as stimulation of G1 cell cycle arrest induced B-cells apoptosis in DLBCL. Moreover, these results were inconsistent with the findings of a related study that revealed that FCRL1 down-regulation had no effect on the expression of different anti-apoptotic and pro-apoptotic proteins [34]. This discrepancy in the potential effects of FCRL1 on B-cell apoptosis might be associated with the differences in the type and sensitivity of applied methods.
Having considered that, the results of the current study showed that FCRL1 might be involved in the pathogenesis or development of DLBCL by regulating various biological activities of B-cells such as proliferation, cell cycle, and apoptotic cell death.
However, additional investigation is required to determine the underlying molecular mechanisms of activation of co-receptor FCRL1 in human B-cells. Different oncogenic pathways are involved in the pathogenesis and development of various subtypes of DLBCL. The PI3K/p-Akt and transcription factor NF-κB have been reported as hallmarks of DLBCL biology. The deregulation of PI3K/p-Akt signaling pathway and constitutive activation of key transcription  Effects of FCRL1 knockdown on the relative expression levels of transcription factor p65 NF-κB, and PI3K/p-Akt. A The levels of total p65 NF-κB and phosphorylated p65 NF-κB in absence and presence of TNF-α, respectively. B The relative gene expression levels of PI3K in treated B-cells of DLBCL compared with control cells. C-D Expression levels of p-Akt protein following FCRL1 knockdown using flow cytometry analysis and western blotting assay. These processes were performed 72 h after transduction procedure. DLBCL diffuse large B-cell lymphoma; *p < 0.05; **p < 0.01; NS not statistically significant factor NF-κB play critical roles in the pathogenesis of DLBCL through inducing B-cell survival and inhibition of cell apoptosis [4,[43][44][45]. PI3K with activation and phosphorylation of serine/threonine kinase Akt at serine 473 (S473) induces TNF-α mediated NF-κB activation, through degradation of IκB inhibitor and phosphorylation of p65 subunit of NF-κB using p-Akt (S473) [43,44,46,47]. The cross talk between key transcription factor NF-κB and PI3K/p-Akt pathway by activation of several cell survival mediators and apoptosis-regulating genes in B-cells such as BCL2 genes family, may be involved in DLBCL progression [48]. In addition, recent studies on DLBCL point to activation of NF-κB as a critical mechanism for chemotherapy resistance in ABC-DLBCL, and suggest NF-κB pathway as an effective target for new therapeutic strategies [45,47,49,50]. Worse clinical outcomes and decreased overall survival rates have been reported in DLBCL patients with higher expression of p-Akt [49][50][51].
Regarding the potential effects of FCRL1, p65 NF-κB, and PI3K/p-Akt in DLBCL pathogenesis, there is a possible association between the levels of FCRL1 expression and these oncogenic factors evaluated in the B-cells of DLBCL tissues after FCRL1 knockdown. The results of this study showed that FCRL1 knockdown significantly reduced the levels of p-p65 NF-κB, PI3K, and p-Akt (S473) in the treated B-cells.
This study has some limitations: The small number of samples could be considered as an important limitation. DLBCL patients who were included in this study were recently diagnosed with DLBCL. The results could have been more reliable if treated DLBCL patients were used for examination of the expression patterns of FCRL1. In addition, evaluation of the possible association between the expression patterns of FCRL1 and other oncogenic molecules involved in the pathophysiology of DLBCL could have been improved if FCRL1 was used as a beneficial target for management of FCRL1 positive B-cell leukemia or lymphoma including DLBCL, which is something that should be investigated in future research studies.

Conclusions
One conclusion obtained from the results of the presented study is that higher expression levels of FCRL1 in malignant B-cells of DLBCL patients compared to HCs are associated with the pathophysiology of this disorder. In addition, the potential effects of FCRL1 knockdown on various activities of B-cells from DLBCL were evaluated, and surprisingly, the proliferation of B-cells and expression levels of antiapoptotic BCL-2 were decreased while apoptotic cell death, cell cycle arrest, and expressions of all the candidate molecules including pro-apoptotic genes BID and BAX as well as PI3K/p-Akt and NF-κB were decreased. These results suggest that as an activation co-receptor of human B-cells, FCRL1 can be used as a new target for early prognosis and immunotherapeutic intervention for patients with DLBCL. However, more investigation with larger sample sizes is required to gain a better understanding of this matter.