NAALADL2-AS2 functions as a competing endogenous RNA to regulate apoptosis and drug resistance in DLBCL

ABSTRACT

To explore role of NAALADL2-AS2 as ceRNA in DLBCL. Fluorescence in situ hybridization was used to determine location of NAALADL2-AS2 in cells and to verify its expression in DLBCL tissues. The miRNAs interacting with NAALADL2-AS2 and related regulatory genes were identified by small interfering RNA (siRNA) assay, luciferase reporter assay, fluorescent quantitative polymerase chain reaction, western blotting. DLBCL cells transfected with NAALADL2-AS2 siRNA or control siRNA were treated with doxorubicin, rituximab at different concentrations alone or in combination. The growth curves, drug sensitivity changes of cells before and after transfection were detected by MTT assay, ATP-TCA drug sensitivity test. Cell proliferation was detected by BrdU cell proliferation assay, and apoptosis was detected by Annexin V-fluorescein isothiocyanate/propidium iodide staining. The effects and mechanisms of NAALADL2-AS2 on proliferation, apoptosis, drug resistance of DLBCL cells were studied at cellular level. We confirmed expression of NAALADL2-AS2 in both cytoplasm and nuclei of DLBCL cells. Additionally, we observed elevated levels of NAALADL2-AS2 in DLBCL tissues. We discovered that NAALADL2-AS2 functions as ceRNA to inhibit expression of miR-34a, miR-125a, whereas overexpression of NAALADL2-AS2 indirectly upregulates expression of BCL-2. Interfering with NAALADL2-AS2 promoted apoptosis in DLBCL cells, resulting in approximately a 40% increase in sensitivity to doxorubicin and rituximab. In vivo experiments further confirmed that targeting NAALADL2-AS2 effectively suppressed tumor growth, leading to upregulation of miR-34a and miR-125a, downregulation of BCL-2, and enhanced apoptosis in DLBCL cells, which significantly improved their sensitivity to doxorubicin and rituximab by approximately 50%. These results indicate that NAALADL2-AS2/miR-34a, miR-125a/BCL-2 networks hold promise as therapeutic targets for treatment of DLBCL.

Introduction

Diffuse large B-cell lymphoma (DLBCL) is a prevalent malignancy marked by its significant heterogeneity.¹ While notable advancements have been made in the research of certain pathological subtypes of DLBCL, such as germinal center B cell-like (GCB) and activated B cell-like (ABC) subtypes, as well as focused treatments such as rituximab-based chemo-immunotherapy,^2–4 approximately 35%–40% of patients exhibit challenges, including high aggressiveness and resistance to first-line treatment, or early relapse. Consequently, there is an urgent need to delve deeper into the underlying pathogenesis and drug resistance mechanisms affecting these patients. Recent research has identified many DLBCL patients with elevated levels of MYC and/or BCL-2 proteins, which are associated with a poor prognosis.⁵ The simultaneous overexpression of MYC and BCL-2, known as “double-expression” DLBCL, accounts for approximately 29%–45% of newly diagnosed DLBCL cases.⁶ It is widely agreed that these patients have unfavorable outcomes following R-CHOP treatment, leading to significantly shortened survival times, independent of other factors. Therefore, there is a pressing need for novel treatment strategies to address this specific subgroup of DLBCL patients.

MYC is an important transcription factor that plays a key role in signal amplification during cell growth, differentiation, apoptosis, and proliferation.^7–9 As one of the most critical drivers of lymphoma, it is expressed in 70%–100% of Burkitt lymphoma cases, 30%–40% of DLBCL cases, and ~ 5% of normal germinal center-derived B cells.¹⁰ MYC has a dual role; it not only induces cell proliferation by affecting cyclin-dependent kinases, but also mediates cell apoptosis by increasing the expression of TP53 and the pro-apoptotic protein BIM.¹¹ On the other hand, BCL-2 is an important gene for suppressing apoptosis, and its expression is elevated to varying degrees in lymphoid hematopoietic tumors and other malignant tumors.^12–14 As a relatively “mild” oncogene, lymphocytes with BCL-2 overexpression require additional genomic alterations for progression to lymphoma, which is critical in MYC-driven lymphoma. MYC-induced genetic instability, DNA damage, and energy stress induce BIM protein expression. In the absence of BCL-2 expression, BIM binds to effector proteins BAX or BAK to induce mitochondrial depolarization and apoptosis. However, when BCL-2 is overexpressed, it sequesters several BIM proteins, inhibiting apoptosis.¹⁵

In a small subset of “double expression” DLBCLs, the high expression levels of MYC and BCL-2 result from the translocation of MYC and BCL-2 genes. This subtype is referred to as “double hit” B cell lymphoma, and it accounts for approximately 6% of DLBCL cases and associates with a poor prognosis.¹⁶ In some cases, the overexpression of MYC and BCL-2 is linked to functional gene mutations.¹⁷ Most DLBCLs have normal MYC and BCL-2 gene sequences; however, the functions of MYC and BCL-2 proteins are severely disrupted. For patients with normal gene sequences but abnormal protein functions, it is critical to regulate the expression of these two proteins at various levels because an earlier study has shown that simultaneous targeted inhibition of MYC and BCL-2 could significantly enhance the sensitivity of dual-expressing DLBCL to chemotherapy.¹⁸

Long-chain non-coding RNAs (lncRNAs) are a class of non-protein-coding RNA molecules with a transcript length exceeding 200 nucleotides. Unlike protein-coding RNAs, lncRNAs lack specific and complete open reading frames and they have no role in protein coding. LncRNAs have emerged as critical regulators of gene expression in various cancers, including hematological malignancies, where they modulate tumor growth, apoptosis, and chemoresistance.^19,20 Moreover, lncRNAs are increasingly being explored as potential biomarkers and therapeutic targets for cancer diagnosis and treatment.²¹ Extensive research has established that the regulatory mechanisms linking lncRNAs and epigenetic processes could serve as new indicators and targets for overcoming or reversing drug resistance in cancer therapy.²² In our previous study, we used high-throughput lncRNA chips to compare the differences in lncRNA expression profiles between DLBCL cell lines and normal B cells, and we singled out NAALADL2-AS2, which exhibited the most significant upregulation, for further study. Through a mining of chip data, we identified a potential association between NAALADL2-AS2 and the regulation of MYC expression.²³ Through bioinformatics prediction, we identified many binding sites on the NAALADL2-AS2 sequence that might interact with miRNAs capable of inhibiting MYC and/or BCL-2. Based on this foundation, we confirmed the expression of NAALADL2-AS2 in DLBCL tissues and identified the miRNAs that bind to NAALADL2-AS2. We studied the regulatory relationship between NAALADL2-AS2, miRNAs, and MYC/BCL-2 through cellular assays and an animal model, and investigated the impact of NAALADL2-AS2 on the resistance of DLBCL to doxorubicin and rituximab. Given the limited efficacy of R-CHOP in refractory cases, targeting NAALADL2-AS2 offers a novel approach for overcoming chemoresistance in this subset of patients. Our findings reveal a novel regulatory mechanism involving NAALADL2-AS2 in DLBCL and provide a valuable biological target for the reversal of drug resistance in DLBCL.

Materials and methods

Cell lines and cell culture

All cells used in this study were obtained from the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences. In brief, 293T, U2932, and OCI-Ly19 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, Melbourne, Australia) supplemented with 10% fetal bovine serum (Gibco) and employed for further studies.

siRNA and cell transfection

The siRNAs targeting NAALADL2-AS2 and the other genes were purchased from GenePharma (Shanghai, China). siRNA duplexes (100 nM), miR-RNA mimic, and corresponding negative control (NC) oligonucleotides were transfected into cells using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The siRNA sequences are provided in Supplementary Table S1.

Lentivirus production and infection

To establish individual stably transfected cells, we used lentivirus. Lentiviral particles were harvested from cell culture supernatants 72 h after lentiviral transfection of HEK293 cells. U2932 and OCI-ly19 cells were then infected with lentivirus. Stably transfected cells were selected by exposing them to puromycin (2 µg/mL, Sigma-Aldrich, St. Louis, MO, USA) for 1 week, starting 48 h after lentiviral infection.

Western blotting

Proteins were isolated from cells using RIPA buffer (Beyotime, Shanghai China) and quantified with a BCA assay (BioSharp, Shanghai China). Proteins were separated by 6% SDS-PAGE and electro-transferred onto nitrocellulose membranes. After blocking the membranes with 5% skim milk for 1.5 h, they were incubated with specific antibodies. The immunoreactive proteins were detected using secondary antibodies and visualized using enhanced chemiluminescence kit reagents (Service-Bio, Wuhan, China). The protein bands were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA). MYC (cat no. ab32072, Abcam, Cambridge, MA, USA) and BCL-2 (cat no. GB113375, Service-bio) antibodies were used at a concentration of 1:2000. The GAPDH antibody (cat no. GB15004, 1:5000, Service-Bio) served as the internal control.

RNA isolation and quantitative PCR (qPCR)

Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. First-strand cDNA was synthesized using Superscript II (Invitrogen), with 1 μg of total RNA used for each cDNA synthesis reaction. For qPCR, SYBR green Universal Master Mix reagents (Roche, Basel, Switzerland) and primers were used. The GAPDH gene served as the internal control. The miRNA sequences are provided in Supplementary Table S2.

The miRNA expression levels were measured using the All-in-One miRNA qPCR Detection kit (GeneCopoeia, Rockville, MD, USA), according to the manufacturer’s instructions. The U6 small RNA served as the internal control.

RNA fluorescence in situ hybridization

In accordance with the methodology described in reference,²⁴ cells were fixed in 4% paraformaldehyde for 15 minutes to preserve cellular morphology and structural integrity. Following fixation, the cells were permeabilized with 0.5% Triton X-100 for 15 minutes at 4°C to facilitate probe access to the intracellular environment.

Digoxigenin-labeled probes specific for NAALADL2-AS2, along with appropriate control probes, were then prepared and incubated with the cells at 55°C for 4 hours to allow for hybridization. After hybridization, the cells underwent three washes with a saline solution to remove unbound probes and minimize background signal.

Subsequently, the cells were incubated with an NAALADL2-AS2 antibody (cat no. NAALADL2-AS2-20-RE, Empire Genomics, Williamsville, NY, USA) to detect the presence of the lncRNA, followed by additional washes to eliminate any unbound antibody. For nuclear counterstaining, 4’,6’-diamidino-2-phenylindole (DAPI) was employed to visualize the cell nuclei and enhance the overall contrast of the fluorescent images.

Fluorescent images were captured using a fluorescence microscope, allowing for the assessment of both hybridization specificity and efficiency. The verification of hybridization efficiency was conducted utilizing a rigorous experimental design that included both positive and negative controls. Control samples comprised non-DLBCL tissues to ensure the accuracy and reliability of the results obtained. The specific sequence of NAALADL2-AS2 is provided in Supplementary Table S3 for detailed reference.

DLBCL tissue microarray

The DLBCL tissue chip (cat no. HLymB085PT01) was purchased from Shanghai Xinchao Biotechnology Co., Ltd. (China). It included 15 cases of tonsillitis lymphoid tissue (1 case was missing in the experiment, leaving 14 cases), 13 cases of diffuse large B-cell lymphoma tissue (GCB type) (4 cases were missing in the experiment, leaving 9 cases), and 57 cases of diffuse large B-cell lymphoma tissue (non-GCB type) (12 cases were missing in the experiment, leaving 45 cases). All patients had not received radiotherapy, chemotherapy, or another anti-tumor treatment before surgery and had been pathologically diagnosed with diffuse large B-cell lymphoma. The patients’ ages ranged from 19 to 92 years old.

ATP-TCA experiments

DLBCL cells, transfected with NAALADL2-AS2 siRNA, the control alone, or the siRNA and the control in combination, were treated with different concentrations of doxorubicin and/or rituximab (0, 0.01, 0.1, 1, 10 times the peak plasma concentration). Eight groups were set up for drug sensitivity testing, namely: (1) doxorubicin (0, 0.01, 0.1, 0.3, 1, 3, 10, 30, 100 µM) + si-NC; (2) doxorubicin (0, 0.01, 0.1, 0.3, 1, 3, 10, 30, 100 µM) + si-NAALADL2-AS2; (3) rituximab (0, 0.01, 0.1, 0.3, 1, 3, 10, 30 µg/ml) + si-NC; (4) rituximab (0, 0.01, 0.1, 0.3, 1, 3, 10, 30 µg/ml) + si-NAALADL2-AS2; (5) 2 × IC₅₀ doxorubicin + rituximab (0, 0.01, 0.1, 0.3, 1, 3, 10, 30 µg/ml) + si-NC; (6) 2 × IC₅₀ doxorubicin + rituximab (0, 0.01, 0.1, 0.3, 1, 3, 10, 30 µg/ml) + si-NAALADL2-AS2; (7) 2 × IC₅₀ rituximab + doxorubicin (0, 0.01, 0.1, 0.3, 1, 3, 10, 30, 100 µM) + si-NC; and (8) 2 × IC₅₀ rituximab + doxorubicin (0, 0.01, 0.1, 0.3, 1, 3, 10, 30, 100 µM) + si-NAALADL2-AS2. Tumor cells were co-cultured with chemotherapeutic drugs in 96-well plates, and a blank control group was included. The concentrations of doxorubicin and rituximab were prepared using a 10-fold gradient dilution from 100 µM to 0.01 µM. After 24 h of culture, ATP was extracted, and luminescence was measured using a chemiluminescence analyzer. The inhibitory rate of each drug concentration was determined, and the tumor cell growth inhibition curves were plotted. IC₅₀ values were calculated to evaluate the effectiveness of the drugs in killing tumor cells.

BrdU cell proliferation assay

DLBCL cells, transfected with NAALADL2-AS2 siRNA, the control alone, or the siRNA and the control in combination, were treated with different concentrations of doxorubicin and/or rituximab (0, 0.01, 0.1, 1, 10 times the peak plasma concentration). The groups were as follows: (1) 2 × IC₅₀ doxorubicin + si-NC; (2) 2 × IC₅₀ doxorubicin + si-NAALADL2-AS2; (3) 2 × IC₅₀ rituximab + si-NC; (4) 2 × IC₅₀ rituximab + si-NAALADL2-AS2; (5) 2 × IC₅₀ doxorubicin + 2 × IC₅₀ rituximab + si-NC; and (6) 2 × IC₅₀ doxorubicin + 2 × IC₅₀ rituximab + si-NAALADL2-AS2. After 24 h of culture, the cells were collected. In brief, 1 × 10⁷ cells were labeled in vitro with 10 µM BrdU at 37°C for 45 min. Cells were fixed, and 5 µL of anti-BrdU fluorescent antibody was added to each sample, followed by incubation at room temperature for 25 min in the dark. Cell proliferation was assessed using flow cytometry (FACSAria II, Becton Dickinson, Franklin Lakes, NJ, USA).

Apoptosis assay

The groups of drugs and drug treatments were the same as those described for the BrdU assay. After 24 h of culture, the cells were collected. In brief, 5 μL of Annexin V-fluorescein isothiocyanate was added, followed by incubation at room temperature for 15 min in the dark. Approximately 5 min before measurement, 5 μL of propidium iodide and 200 μL of 1× binding buffer were added. Cell apoptosis was assessed using flow cytometry.

Establishment of tumor-bearing mouse model

Nude mice were purchased from GemPharmatech Co., Ltd. (Nanjing, China). DLBCL cell lines, namely U2932 (ABC type) and OCI-Ly19 (GCB type), which exhibit high expression levels of NAALADL2-AS2, MYC, and BCL-2, were separately inoculated into athymic BALB/c nude mice. Nude mice were subjected to whole-body irradiation at a dose of 600cGy 1 day prior to inoculation. In brief, 1 × 10⁷ DLBCL cells were subcutaneously inoculated into the right flank of mice, leading to the establishment of the DLBCL tumor-bearing mouse model within 14–21 days.

The construction of the lentiviral vector for inhibiting NAALADL2-AS2 expression was performed as follows: the NAALADL2-AS2 siRNA fragment was inserted downstream of the CMV promoter to construct the NAALADL2-AS2 siRNA lentiviral vector plasmids (Tween, pRRL-CMVPGK-GFP-WPRE), and these plasmids were transfected into packaging cells (293T cells). A packaging cell strain capable of producing high-titer virus was screened and confirmed using the enzyme digestion method. The lentivirus solution (containing 1 × 10⁷ viral particles carrying NAALADL2-AS2-siRNA) was subcutaneously injected near the tumor on the right side of the chest in the tumor-bearing mice, and the lentivirus without NAALADL2-AS2 siRNA served as the control.

The following groups were set up: (1) sh-NC; (2) sh-NAALADL2-AS2; (3) doxorubicin (3 mg/kg) + sh-NC; (4) doxorubicin (3 mg/kg) + sh-NAALADL2-AS2; (5) rituximab (10 mg/kg) + sh-NC; and (6) rituximab (10 mg/kg) + sh-NAALADL2-AS2. Tumor size changes in the tumor-bearing mice were observed and recorded before and after injection. Two weeks after the injection of the lentiviral solution, the tumor-bearing mice were euthanized, and relevant tissue samples were collected. The volume and weight of the transplanted tumors were measured, and the tumor inhibition rate was calculated.

Immunohistochemistry

Tissue samples were collected, fixed with 4% paraformaldehyde for 3 h, dehydrated, and embedded in paraffin. Following dewaxing, rehydration, heat-mediated antigen retrieval, and nonspecific site blocking with BlockAid solution (Thermo Fisher Scientific), the sections were incubated with MYC (cat no. ab32072, Abcam), BCL-2 (cat no. GB113375, Service-Bio), and KI67 (cat no. GB111499, Service-Bio) antibodies overnight at 4°C. On the following day, the sections were incubated with a goat anti-rabbit secondary antibody (cat no. G1213, Service-Bio) for 10 min at room temperature. Sections were counterstained with hematoxylin, mounted after dehydration, and photographed using a light microscope (Leica, BOND-III, Wetzlar, Germany). Perform semi-quantitative analysis of immunohistochemical staining intensity using ImageJ software (version 1.54j, USA).

Detection of apoptosis by TUNEL assay

Tissue samples were collected, dewaxed, and hydrated. Cells were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline and incubated with TUNEL reaction solution at 37°C for 1 h. Following the DAB reaction, the cells were photographed using a fluorescence microscope. Perform semi-quantitative analysis of TUNEL fluorescence intensity using ImageJ software (version 1.54j, USA).

Luciferase reporter assay

The target fragment was inserted into the pGL3-basic vector (Promega, Beijing, China). miR-NC/miR-34a/miR-125a was co-transfected with the empty vector or the vector-NAALADL2-AS2 WT/Mut plasmids. After 24 h of transfection, luciferase activity was detected using the Promega Dual-Luciferase system kit (Promega). The sequences are provided in Supplementary Table S4.

Statistical analysis

According to the methodology provided by the University of Düsseldorf, this study utilized G*Power to perform power calculations,²⁵ with the specific power (1-β error probability) presented following the relevant results. Statistical analyses were carried out using IBM SPSS version 22.0 (Chicago, IL, USA), while graphical representations were generated using GraphPad Prism version 8.0 (San Diego, CA, USA). Differences between two groups were assessed using Student’s t-test, and one-way ANOVA followed by Tukey’s post hoc test was employed for comparisons involving more than two groups. Two-tailed tests were utilized for analyzing differences between groups, and p-values of less than 0.05 were considered statistically significant.

Results

Localization and expression of NAALADL2-AS2 in DLBCL tissues

The results of the RNA fluorescence in situ hybridization assay revealed the expression of NAALADL2-AS2 in both the cytoplasm and nucleus of DLBCL cells (U-2932 and OCI-Ly19) (Figure 1a). By comparing the expression of NAALADL2-AS2 in DLBCL tissues and control samples using tissue microarray analysis, we found that the average density value of NAALADL2-AS2 was significantly higher in DLBCL tissues than in control samples (p < .05), suggesting NAALADL2-AS2 exhibits high expression in DLBCL tissues (Figure 1b,c). The actual P-values and corresponding statistical significance markers for all figures in this study are listed in Supplementary Table S5.

Figure 1.

Localization and expression of NAALADL2-AS2 in DLBCL cells and tissues (a, left) fluorescent in situ hybridization (FISH) assay was used to detect the localization of NAALADL2-AS2 in U2932 and OCI-Ly19 cells. (a, right) localization of NAALADL2-AS2 in cell lines at different magnifications (4X, 40X). (b) Representative fluorescent images of NAALADL2-AS2 expression in DLBCL tissues and tonsillitis lymphoid tissues (normal) as detected by FISH, in which blue corresponds to DAPI and red corresponds to NAALADL2-AS2. (c) Statistical analysis of the average optical density values of DLBCL tissues and tonsillitis lymphoid tissues. Statistical significance between the two groups was analyzed using Student’s t-test. Data were expressed as mean ± standard deviation.*, P < 0.05.

NAALADL2-AS2 upregulates BCL-2 protein expression by inhibiting miR-34a and miR-125a

Using miRcode bioinformatics analysis, the potential miRNA binding sites for MYC and BCL-2 in the NAALADL2-AS2 sequence were identified. The TarBase v7.0 miRNA – target gene interaction database indicated that miR-24, miR-34a, miR-125a/b, and miR-181a/b/c/d could inhibit MYC and BCL-2 by targeting them. To determine the functional impact of these findings, siRNA was employed to disrupt NAADL2-AS2 expression in U-2932 and OCI-Ly19 cells. qPCR results demonstrated an upregulation of miR-34a and miR-125a and a downregulation of miR-181a in U2932 cells, whereas OCI-Ly19 cells showed an upregulation of miR-34a and miR-125a/b (Figure 2a,b). Western blotting results supported these findings, showing a significant decrease in MYC and BCL-2 protein expression in both U-2932 and OCI-Ly19 cells (Figure 2c,d). The observed upregulation of BCL-2 mediated by NAALADL2-AS2 underscores the critical role of this lncRNA in modulating the apoptotic threshold in DLBCL cells, thereby contributing to mechanisms of drug resistance.

Figure 2.

Effects of NAALADL2-AS2 interference on the expression of miRnas, MYC, and BCL-2 (a, b) expression changes of miRNAs (miR-24, 34a, 181a/b/c/d, 125a/b) in U2932 cells (a) and OCI-Ly19 cells (b) after transfection of NAALADL2-AS2 siRNA as detected by qPCR. (c, d) expression changes of MYC and BCL-2 protein in U2932 cells and OCI-Ly19 cells after transfection of NAALADL2-AS2 siRNA as detected by western blotting. The control groups in these experiments consisted of cells treated with non-targeting siRNA. Statistical significance between the two groups was analyzed using Student’s t-test. Data were obtained from at least three independent experiments and expressed as mean ± standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

U2932 and OCI-Ly19 cells were subjected to lentiviral transfection to overexpress miRNAs (miR-24, 34a, 181a/b/c/d, 125a/b). The levels of NAALADL2-AS2, MYC mRNA, and BCL-2 mRNA were examined by qPCR, and the levels of MYC and BCL-2 proteins were detected by western blotting. Upon overexpression of miR-34a and miR-125a, NAALADL2-AS2 and BCL-2 mRNA levels (Figure 3a,b) and the BCL-2 protein level were significantly downregulated (Figure 3d,e). However, the overexpression of these miRNAs did not lead to changes in MYC mRNA and protein expression (Figure 3c, d–f). To determine whether NAALADL2-AS2 directly binds to miR-34a or miR-125a, we predicted the binding sequence of NAALADL2-AS2 for miR-34a and miR-125a using a bioinformatics approach. NAALADL2-AS2, containing either the wild-type or mutant binding sequence, was cloned into the luciferase reporter plasmid and co-transfected into 293T cells with miR-34a/miR-125a mimic or empty vector. The empty vector group overexpressing miR-34a/miR-125a and the mutant NAALADL2-AS2 group exhibited no significant change in the luciferase activity, whereas the wild-type NAALADL2-AS2 group displayed a significant decrease in the luciferase activity (Figure 3g,h), indicating a direct binding effect between NAALADL2-AS2 and miR-34a/miR-125a. These results reveal that NAALADL2-AS2 upregulates the expression of BCL-2 protein by inhibiting miR-34a and miR-125a. However, the regulation of MYC protein by NAALADL2-AS2 was not mediated through miRNA. Instead, other post-transcriptional regulatory pathways may be involved.

Figure 3.

NAALADL2-AS2 upregulates BCL-2 protein expression by inhibiting miR-34a and miR-125a (a–f) miR-24, 34a, 181a/b/c/d, and 125a/b were overexpressed in U2932 and OCI-Ly19 cells by lentivirus infection. Untransfected cells and those transfected with miR-nc served as the controls. Expression changes of NAALADL2-AS2 (a), MYC (b), and BCL-2 (c) as detected by qPCR. Expression changes of MYC (d, e) and BCL-2 (d, f) as detected by western blotting. (g, h) luciferase activity in 293T cells transfected with the luciferase reporter gene containing the miR-34a sequence (g), miR-125a sequence (h), wild-type or mutant NAALADL2-AS2 binding site, or blank vector. The control groups in these experiments consisted of cells treated with non-targeting siRNA. Statistical significance between the two groups was analyzed using Student’s t-test. Data were obtained from at least three independent experiments and expressed as mean ± standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

In vitro interference with NAALADL2-AS2 expression enhances the sensitivity of DLBCL cells to doxorubicin and rituximab

U2932 and OCI-Ly19 cells were exposed to different concentrations of doxorubicin and rituximab, either individually or in combination, in NAALADL2-AS2 siRNA and control groups. The ATP-TCA drug sensitivity test was used to assess changes in cell growth before and after transfection and to observe alterations in drug sensitivity. In the experimental group (si-NAALADL2-AS2), the cytotoxic effects of both doxorubicin and rituximab were heightened compared to the control group (si-NC group) (Figure 4a,b). When the cells in the experimental group were concomitantly treated with doxorubicin and rituximab, the cytotoxic effect was further augmented compared to the control group (Figure 4c,d). These results indicate that interfering with the expression of NAALADL2-AS2 has the potential to heighten the sensitivity of DLBCL cells to both doxorubicin and rituximab.

Figure 4.

Proliferation, apoptosis, and sensitivity of DLBCL cells to doxorubicin and rituximab after in vitro interference of NAALADL2-AS2 expression (a, b) DLBCL cells transfected with NAALADL2-AS2 siRNA and the control group were treated with different concentrations of doxorubicin (a) and rituximab (b) alone. Changes in drug sensitivity were detected by ATP-TCA assay. (c) DLBCL cells transfected with NAALADL2-AS2 siRNA and the control group were treated with different concentrations of doxorubicin and 2×IC₅₀ rituximab. Changes in drug sensitivity were detected by ATP-TCA assay. (d) DLBCL cells transfected with NAALADL2-AS2 siRNA and the control group were treated with different concentrations of rituximab and 2×IC₅₀ doxorubicin. Changes in drug sensitivity were detected by ATP-TCA assay. (e, f) DLBCL cells transfected with NAALADL2-AS2 siRNA and the control group were treated with 2×IC₅₀ doxorubicin and 2×IC₅₀ rituximab alone or in combination. Cell proliferation was detected by BrdU/DNA assay. (g, h) DLBCL cells transfected with NAALADL2-AS2 siRNA and the control group were treated with 2×IC₅₀ doxorubicin and 2×IC₅₀ rituximab alone or in combination. Cell apoptosis was detected by annexin V-FITC/PI assay. In all experiments described above, the cells were collected after 24 h of drug treatment. The control groups in these experiments consisted of cells treated with non-targeting siRNA. Statistical significance between the two groups was analyzed using Student’s t-test. Data were obtained from at least three independent experiments and expressed as mean ± standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.

(Continued).

Cell proliferation in experimental and control groups was assessed using flow cytometry and BrdU/DNA double staining, whereas cell apoptosis was detected using the Annexin V-fluorescein isothiocyanate/propidium iodide kit. In U-2932 cells, when doxorubicin was used alone or in combination with rituximab, cell proliferation in the experimental group (si-NAALADL2-AS2) was reduced compared to that in the control group. However, there was no significant change in cell proliferation between the two groups when rituximab was used alone (Figure 4e). For OCI-Ly19 cells, the use of both drugs resulted in reduced cell proliferation in the experimental group (si-NAALADL2-AS2) compared to that in the control group. When treated with doxorubicin or rituximab alone, there was no significant change in cell proliferation between the two groups (Figure 4f). In U2932 cells, the experimental group treated with si-NAALADL2-AS2 exhibited a significant increase in apoptosis efficiency compared to the control group (si-NC). Specifically, with doxorubicin treatment, the apoptosis efficiency increased by 48.45% (power = 0.99), while with rituximab treatment, the increase was 43.84% (power = 0.99). Furthermore, the combination treatment group demonstrated a notable 25.16% (power = 0.99) enhancement in apoptosis efficiency (Figure 4g). In OCI-Ly19 cells, the experimental group showed a 45.59% (power = 0.98) increase in apoptosis efficiency with doxorubicin treatment and a 53.34% (power = 0.99) increase with rituximab treatment. Notably, the combination treatment group exhibited a substantial enhancement of 70.47% (power = 0.99) in apoptosis efficiency compared to the control group (Figure 4h). In terms of apoptosis, both U2932 and OCI-Ly19 cells exhibited increased cell death in the experimental group compared to the control group, whether the two drugs were used alone or in combination (Figure 4g,h). In summary, the findings suggest that targeting NAALADL2-AS2 resulted in a greater proportion of apoptosis in diffuse large B-cell lymphoma cells following treatment with chemotherapy agents, as compared to the control group. These results demonstrate that interference with NAALADL2-AS2 effectively enhances chemosensitivity. Numerous studies have confirmed that lncRNAs play a critical role in the anti-apoptotic signaling pathways associated with doxorubicin resistance in various cancers. Research on NAALADL2-AS2 enriches our understanding of effective strategies to overcome doxorubicin resistance in DLBCL patients.²⁶

In vivo interference of NAALADL2-AS2 expression upregulates miR-34a and miR-125a expression and downregulates BCL-2 expression, increasing the sensitivity of DLBCL cells to doxorubicin and rituximab

To study the impact of interfering NAALADL2-AS2 expression on the sensitivity of DLBCL cells to doxorubicin and rituximab, a tumor-bearing mouse model was established. Compared to the control groups (sh-NC group, doxorubicin + sh-NC group, rituximab + sh-NC group), the tumor volume and weight in the experimental groups (sh-NAALADL2-AS2 group, doxorubicin + sh-NAALADL2-AS2 group, rituximab + sh-NAALADL2-AS2 group) were significantly reduced (Figure 5a–c). Specifically, the doxorubicin + sh-NAALADL2-AS2 group and the rituximab + sh-NAALADL2-AS2 group demonstrated reductions of 221.49% (power = 0.98) and 332.65% (power = 0.98) in tumor weight, respectively, compared to their respective treatment controls. Additionally, there were decreases of 42.60% (power = 0.96) and 50.37% (power = 0.97) in tumor volume for the combination treatment groups (Figure 5a–c). Immunohistochemical staining revealed decreased expression levels of Ki67, MYC, and BCL-2 in the experimental groups compared to the control groups (Figure 5d–e). The TUNEL method was used to detect the apoptosis of tumor cells. Compared to the control group, the experimental group showed an increased apoptosis rate (Figure 5f–g). Subsequently, qPCR analysis was employed to observe changes in the expression levels of NAALADL2-AS2, MYC, BCL-2, miR-34a, and miR-125a in the various treatment groups. We further investigate the changes of these RNAs in the tumor microenvironment. Compared to the control groups, the experimental groups showed decreased expression of NAALADL2-AS2, MYC, and BCL-2 (Figure 5h), but increased expression of miR-34a and miR-125a (Figure 5i). Taken together, the results indicate that interference of NAALADL2-AS2 expression in vivo inhibits tumor growth, upregulates the expression of miR-34a and miR-125a, downregulates the mRNA and protein expression of MYC and BCL-2, and increases the sensitivity of DLBCL cells to doxorubicin and rituximab. Recent studies suggest that lncRNAs play a critical role in drug resistance across various cancer therapies by reestablishing essential signaling pathway.²⁷ The potential of NAALADL2-AS2 interference as an adjuvant therapy in conjunction with conventional chemotherapy agents such as doxorubicin is suggested by its ability to significantly reduce tumor volume.

Figure 5.

Sensitivity of DLBCL cells to doxorubicin and rituximab after in vivo interference of NAALADL2-AS2 expression (a–c) subcutaneous tumor size (a), tumor volume (b), and tumor weight (c) of nude mice in the different treatment groups. (d-e) immunohistochemistry detection of representative images (d) and statistical results (e) of KI67, MYC, and BCL-2 protein expression in tumor tissues from different treatment groups. (f-g) TUNEL assay for detecting representative images (f) and statistical results (g) of apoptosis in DLBCL cells from different treatment groups. (h, i) expression of NAALADL2-AS2, miR-34a, miR-125a, MYC, and BCL-2 mRNA in DLBCL tissues of the different treatment groups as detected by qPCR. The control groups in these experiments consisted of cells treated with non-targeting siRNA. Statistical significance between the two groups was analyzed using Student’s t-test. Data were obtained from at least three independent experiments and expressed as mean ± standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Discussion

Non-coding RNAs, particularly long lncRNAs, have gained significant attention due to their crucial roles in both physiological and pathological processes. Among these, several lncRNAs have been found to be dysregulated in tumor tissues, making them potential markers for distinguishing between benign and malignant tumors, predicting prognosis, and offering promising targets for tumor therapy. Increasing evidence suggests that many lncRNAs play vital roles in tumor initiation, progression, invasion, and metastasis in DLBCL. For instance, Zhao et al. found that SMAD5-AS1, an lncRNA, inhibited the expression of miR-135b-5p through a competitive sponging effect, thereby weakening the binding of the 3’ end of the APC gene to miR-135b-5p, and consequently indirectly increasing APC expression and suppressing tumor cell proliferation.²⁸ Huang et al. confirmed that LINC00857 promotes proliferation and lymphoma development in DLBCL by regulating the miR-370-3p/CBX3 axis.²⁹ Wang et al. reported elevated expression of MALAT1 in DLBCL cells and tissues. This lncRNA was found to act as a competitive sponge, suppressing miR-195, which induced PD-L1 expression and promoted immune escape.³⁰ Similarly, SNHG14 induced PD-L1 signaling by competitively inhibiting miR-5590-3p to achieve a pro-apoptotic effect in CD8⁺ T cells.³¹ In these studies, lncRNAs, functioning as ceRNAs, competitively bind to miRNA response elements, thereby weakening the inhibitory effect of miRNAs on target genes. This, in turn, indirectly regulates the expression of target genes and influences tumor progression. In our study, we used high-throughput lncRNA microarray analysis to identify NAALADL2-AS2, which showed the most significant upregulation in expression in DLBCL, and verified its expression level in DLBCL cells.²¹ We found that NAALADL2-AS2 was expressed in the cytoplasm and nucleus of DLBCL cells and that its expression was significantly increased in DLBCL tissues compared to normal tissues. NAALADL2-AS2 is located on human chromosome 3q26.3. Studies have shown that NAALADL2-AS2 is highly expressed in prostate cancer tissues, and NAALADL2-AS2, which is regulated by androgens, promotes the survival of prostate cancer cells.³² These results are consistent with recent research indicating that lncRNAs such as MALAT1 and HOTAIR regulate apoptotic pathways in B-cell malignancies, further corroborating the role of NAALADL2-AS2 as a critical modulator of chemoresistance. However, the precise mechanism of NAALADL2-AS2 in DLBCL remains unclear.

Abnormal expression of lncRNAs is linked to the occurrence, progression, and therapeutic resistance of various cancers. A growing body of research is investigating the potential of lncRNAs as therapeutic targets in cancer treatment, exploring both monotherapy and combination strategies to overcome resistance.^33–35 Our results indicate that NAALADL2-AS2 acts as a ceRNA to repress the expression of miR-34a and miR-125a, thereby indirectly upregulating the expression of BCL-2. However, the regulation of MYC by NAALADL2-AS2 may not be realized through the lncRNA – miRNA – target gene module. Studies have demonstrated that miR-34a functions as a tumor suppressor in various malignancies. For example, its overexpression has been shown to inhibit malignant biological behaviors, such as cancer cell proliferation, migration, and invasion, which have been confirmed in solid tumors, including bladder cancer, ovarian cancer, prostate cancer, and liver cancer.^36–39 In hematological malignancies, such as acute myeloid leukemia and chronic lymphocytic leukemia, decreased miR-34a expression may be associated with disease pathogenesis. Asmar et al. reported the role of the miR-34 family in regulating cell cycle dynamics and apoptosis, with its loss of expression associated with poor treatment outcomes in DLBCL patients.⁴⁰ Another study has demonstrated that miR-34a overexpression may heighten the sensitivity of DLBCL cells to doxorubicin by downregulating the expression of its target FOXP1.⁴¹ The tumor-suppressive function of miR-34 mainly involves its direct regulation of genes related to cell proliferation or apoptosis, as well as its indirect participation in tumor development by mediating cell cycle arrest, weakening immune resistance, inhibiting tumor activity, and affecting various signaling pathways.

Recent research has revealed the abnormal expression of miR-125 in various hematological tumors, where it has important roles in disease development and progression, albeit with varying roles. In acute lymphoblastic leukemia and acute myeloid leukemia, miR-125 functions as an oncogene, whereas it operates as a tumor suppressor in chronic lymphocytic leukemia and multiple myeloma.^42,43 In the context of DLBCL, a study has indicated that high miR-125b expression correlates with disease recurrence and poor prognosis. Its dysregulation may even precede clinical manifestations and diagnostic findings, making it a potentially valuable target for assessing therapeutic effects.⁴⁴ Furthermore, miR-125 has been implicated in the activation of NF-κB signaling by targeting tumor necrosis factor-alpha-induced pathway protein 3 (TNFAIP3, A20), thereby impacting the development and progression of DLBCL.⁴⁵ We confirmed that NAALADL2-AS2 functions as a miRNA sponge, specifically for miR-34a and miR-125a in DLBCL, and this interaction upregulates BCL-2 expression, which mediates the apoptosis of DLBCL cells.

DLBCL, an overly aggressive and lethal type of non-Hodgkin’s lymphoma, presents a significant clinical challenge. While the use of the R-CHOP regimen in the past 20 years has improved the therapeutic effect, as many as 40% of patients remain refractory to treatment or experience relapse after achieving complete remission, and the prognosis of these patients is poor. Once resistance to R-CHOP occurs, even if the drug dose or intensity is increased or a new therapy is added to the R-CHOP backbone, there is little effect on prolonging overall survival. Therefore, an in-depth understanding of the mechanism of drug resistance in DLBCL is crucial for developing new and effective therapeutic strategies. Multiple mechanisms mediate drug resistance in DLBCL, including genetic and/or epigenetic modifications and infiltration of immune cells and stromal cells to protect DLBCL cells from apoptosis.⁴⁶ Given that a significant number of DLBCL patients develop resistance to R-CHOP, targeting NAALADL2-AS2 could serve as a novel therapeutic approach to improve sensitivity to current chemotherapeutic regimens. In this study, we verified by in vitro and in vivo experiments that interference of NAALADL2-AS2 expression can upregulate miR-34a and miR-125a, thereby downregulating BCL-2 mRNA and protein expression, promoting DLBCL cell apoptosis, increasing sensitivity to doxorubicin and rituximab, and reversing drug resistance. Our results explain the mechanism of DLBCL drug resistance from the perspective of epigenetic modifications and provide new targets for the development of DLBCL therapeutic drugs.

Conclusions

In conclusion, our study found a significant upregulation of NAALADL2-AS2 in DLBCL tissues. We demonstrated that NAALADL2-AS2 functions as a ceRNA, decreasing the expression of miR-34a and miR-125a and indirectly increasing the expression of the target gene BCL-2. Moreover, interference of NAALADL2-AS2 expression promoted the apoptosis of DLBCL cells and enhanced the sensitivity of these cells to doxorubicin and rituximab, effectively reversing drug resistance. The recognition of NAALADL2-AS2 as a critical regulator of apoptosis and drug resistance positions it as a promising target for novel therapies in treatment-resistant DLBCL. Taken together, our findings suggest that NAALADL2-AS2/miR-34a and miR-125a/BCL-2 networks hold promise as potential therapeutic targets for DLBCL treatment. To fully evaluate the therapeutic potential of NAALADL2-AS2 inhibition, future studies should investigate its combined effects with other targeted therapies.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Funding Statement

This work was financially supported by the National Natural Science Foundation of China [grant no. 81770212] and Changzhou Science and Technology Project [grant no. CJ20220094, CJ20210096].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Xiaoli Xu, Danxia Zhu, Jingting Jiang, and Changping Wu conceived and designed the experiments, analyzed the data, and prepared the figures and tables. Data collection was performed by Juan Liu, Cheng Fang, and Xu Deng. The first draft of the manuscript was written by Xiaoli Xu, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All animal studies were approved by the Animal Use and Care Committee of the Third Affiliated Hospital of Soochow University (Changzhou, China; August 8, 2017/protocol no. 038) and performed in accordance with ARRIVE guidelines (https://arriveguidelines.org). All experiments with human tissues were approved by the Ethics Committee of the Third Affiliated Hospital of Soochow University (Changzhou, China; August 8, 2017/protocol no. 038). All procedures were conducted in accordance with the Declaration of Helsinki, as well as with relevant guidelines and regulations. The tissues from all human subjects were obtained with written informed consent.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15384047.2024.2432690