DLBCL and normal B lymphocyte (normal) samples were collected from 30 patients with DLBCL and 30 healthy control volunteers, respectively, who were admitted to The Affiliated Hospital of Qingdao University (Qingdao, China) from September 2018 to May 2019 (mean age, 65.6 years; age range, 45-78-years; male to female ratio, 1.3:1). Patients were selected if they met the following inclusion criteria: i) Confirmed histopathological diagnosis of DLBCL cases; ii) had not received chemoradiotherapy or other forms of treatment before surgery; and iii) voluntarily provided samples with complete information.

The specimens were immediately frozen in liquid nitrogen and then transferred to a -80˚C freezer for preservation until use. The present study was approved by The Ethics Committee of The Affiliated Hospital of Qingdao University (approval no. bc2020012). All participants provided written informed consent.

GM12878 cells, OCI-Ly7 cells, OCI-Ly10 cells and SUDHL-4 cells were obtained from American Type Culture Collection. GM12878 cells and SUDHL-4 cells were grown in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.); OCI-Ly10 cells were grown in Iscove's modified Dulbecco's medium (IMDM; Gibco; Thermo Fisher Scientific, Inc.); OCI-Ly7 cells were grown in IMDM with 0.3 g/ml glutamine. All media included 10% fetal bovine serum (HyClone; Cytiva), and all cells were cultured at 37˚C in a 5% CO₂ incubator.

Small interfering RNA (si-RNA) targeting CACNA1G-AS1 siRNA (si-CACNA1G-AS1) and its non-targeting sequence negative control (si-NC), miRNA mimic negative control (miR control), miR-3160-5p mimic, miR-4739 mimic, miR-3160-5p antisense oligonucleotides (ASO) and ASO-NC were purchased from Suzhou GenePharma Co., Ltd. pGL3/CACNA1G-AS1-wild-type (wt; seed sequence, 5'-CGAAAGA-3') and pGL3/CACNA1G-AS1-mutant (mut; seed sequence, 5'-CTCCTAA-3') constructs were synthesized by Suzhou GenePharma Co., Ltd. DLBCL cells, OCI-Ly10 cells and SUDHL-4 cells were seeded into six-well plates at a density of 3x10⁵ cells/well and were transfected with the respective oligonucleotides using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 48 h of transfection at 37˚C, the cells were used in follow-up experiments.

TRIzol^® reagent (Thermo Fisher Scientific, Inc.) was utilized to extract total RNA from sample DLBCL tissues or cells, and the purity of RNAs was identified. Then, total RNA was reverse transcribed into cDNA according to the instructions of the Bestar^® qPCR RT kit (DBI Bioscience). The target genes were amplified based on the instructions of the SYBR^® Green qPCR kit (Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95˚C for 8 min; denaturation at 95˚C for 25 sec, annealing at 60˚C for 30 sec; extension at 72˚C for 30 sec; and final extension at 72˚C for 10 min. The 2^-ΔΔCq method was applied to calculate relative gene expression (33). Primer sequences are displayed in Table I.

DLBCL cells from each group were harvested, and the concentration was adjusted to 5x10⁴ cells/ml. After seeding cells in a 96-well plate, 20 µl of MTT solution (5 g/l; Beijing Solarbio Science & Technology Co., Ltd.) was added at 0, 24, 48 and 72 h. After 4 h at 37˚C, the product was dissolved in 200 µl of dimethyl sulfoxide and the absorbance at 490 nm was measured using a microplate reader.

Transfected OCI-Ly10 and SUDHL-4 cells (0.5x10³ cells/well) in a six-well plate were incubated for 14 days in an incubator at 37˚C on soft agar. Then, the cells were fixed using methanol at room temperature for 15 min and stained using crystal violet at room temperature for 20 min (Sigma-Aldrich; Merck KGaA). The number of colonies containing >50 cells was counted by a light microscope (Nikon Corporation; magnification, x100).

Transfected OCI-Ly10 and SUDHL-4 cells were collected, and the concentration was adjusted to 2x10⁶ cells/ml. After centrifugation, the cell pellet was resuspended in 100 µl of PBS binding buffer (BioVision, Inc.). Subsequently, 5 µl of propidium iodide and 5 µl of Annexin V-FITC were added for 20 min in the dark according to the instructions of the FITC Annexin V/PI Apoptosis Detection kit I (Guangzhou RiboBio Co., Ltd.). Flow cytometry (CytoFLEX; Beckman Coulter, Inc.) analysis was performed using FlowJo 10 software (FlowJo LLC).

Total protein was extracted from transfected DLBCL cells using RIPA lysis buffer (cat no. P0013B, Beyotime Institute of Biotechnology). The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc.). After quantification, the protein (40 µg/lane) in each group was separated by 10% SDS-PAGE and transferred to PVDF membranes (cat. no. 3010040001; Roche Applied Science). After blocking in 5% non-fat milk at room temperature for 2 h, the membranes were incubated with the corresponding primary antibodies at 4˚C overnight, followed by a donkey anti-rabbit IgG HRP-conjugated secondary antibody (1:1,000, cat. no. ab6802; Abcam) at room temperature for 1 h. Then, the results were visualized using an ECL system (Thermo Fisher Scientific, Inc.) and semi-quantitatively analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.). The following primary antibodies were used: Proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. ab18197), Bcl-2 (1:1,000; cat. no. ab194583) and Actin (1:2,000; cat. no. ab8227) (all Abcam).

A total of 10 male BALB/c nude mice (20±2 g, 4 weeks old) BALB/c nude mice were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. All animals were housed in individual air laminar flow chamber with a humidity of 55-60% at 28˚C on a 12 h light/dark cycle, with access to SPF food and water ad libitum. The health and behavior of mice were monitored each day in whole study. The mice were divided into the si-NC group (n=5) and si-CACNA1G-AS1 group (n=5). All animal care and experiments were performed according to the guidelines of the National Institutes of Health and approved by The Animal Ethics Committee of The Affiliated Hospital of Qingdao University (approval no. bc20191015-52). OCI-Ly10 cells (200 µl; 1.5x10⁶ cells/ml) transfected with si-NC or si-CACNA1G-AS1 were subcutaneously injected into the right dorsal of the mice. The tumor volume was monitored for 21 days. The weights for the mice were measured every 3 days with the electronic balance and tumor volumes were measured every 3 days with the vernier caliper. After that, the mice were sacrificed by cervical dislocation under sodium pentobarbital anesthesia (50 mg/kg; intraperitoneal injection). Sacrifice was confirmed when the heart and spontaneous breathing stopped and nerve reflexes disappeared. There were no mice mortalities before sacrifice. If mice tumor grew >10% of the original body weight of the animal, with an average tumor diameter of >20-mm in mice, or when the tumor metastasized or rapidly grew to ulceration, causing infection or necrosis, the mice should be euthanized for humane endpoints.

Wt and mut CACNA1G-AS1 plasmids (pGL3-CACNA1G-AS1-wt and pGL3-CACNA1G-AS1-mut, respectively) were purchased from Hanbio Biotechnology Co., Ltd. OCI-Ly10 and SUDHL-4 cells (1x10⁴ cells/well) were co-transfected with either pGL3-CACNA1G-AS1-WT plasmid (0.5 µg) or pGL3-CACNA1G-AS1-Mut plasmid (0.5 µg) and miR-3160-5p mimic (50 nM, forward 5'-GGCUUUCUAGUCUCAGCUCUCC-3', reverse 5'-AGAGCUGAGACUAGAAAGCCGC-3') or miR control (50 nM, 5'-CGAUCGCAUCAGCAUCGAUUGC-3') using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 24 h, the fluorescence activity was determined using a Dual-Luciferase Assay System (Promega Corporation), and Renilla luciferase activity was set as a control.

The secondary structure of single stranded RNA was predicted using the RNAalifold web server (http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi). The binding sites and the secondary structures were predicted by RegRNA2 (http://regrna2.mbc.nctu.edu.tw/index.html).

Data were analysed and visualized using SPSS 23.0 (IBM Corp.) and Graphpad Prism 5.0 (GraphPad Software, Inc.). The non-parametric Mann-Whitney U test was used to compare gene expression of lncRNAs between patients with DLBCL and normal controls. A paired t-test was performed to compare the apoptosis rate between two groups. Statistical differences among three groups were tested with one-way analysis of variance test. The Tukey's honest significant difference post hoc test was used for multiple comparisons. A two-tailed P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR analysis revealed that CACNA1G-AS1 expression was significantly elevated in DLBCL tissues compared with normal tissues (P<0.05; Fig. 1A). Simultaneously, CACNA1G-AS1 was revealed to be significantly upregulated in III-IV stage DLBCL tissues compared with I-II stage DLBCL tissues (P<0.05; Fig. 1B). Additionally, the expression levels of CACNA1G-AS1 were significantly increased in OCI-Ly7, OCI-Ly10 and SUDHL-4 cells compared with B cells (P<0.05; Fig. 1C). Thus, these findings confirmed that CACNA1G-AS1 was highly expressed in DLBCL.

Because CACNA1G-AS1 is highly expressed in DLBCL, it was hypothesized that CACNA1G-AS1-knockdown may prevent DLBCL malignancy. First, CACNA1G-AS1 was silenced using siRNA in OCI-Ly10 and SUDHL-4 cells, and RT-qPCR data demonstrated that CACNA1G-AS1 was significantly downregulated in the si-CACNA1G-AS1 group compared with the si-NC group (P<0.05; Fig. 2A). Next, the cytotoxicity of OCI-Ly10 and SUDHL-4 cells were significantly decreased in the si-CACNA1G-AS1 group compared with the si-NC group (P<0.05; Fig. 2B). Similarly, CACNA1G-AS1-knockdown significantly reduced the colony number of OCI-Ly10 and SUDHL-4 cells compared with the si-NC (P<0.05; Fig. 2C). In addition, CACNA1G-AS1-knockdown significantly increased the apoptosis rates in OCI-Ly10 and SUDHL-4 cells compared with the si-NC groups (P<0.05; Fig. 2D). Overall, CACNA1G-AS1-silencing enhanced cytotoxicity and promoted apoptosis in DLBCL cells.

To identify the potential mechanism of CACNA1G-AS1, its secondary structure was analyzed using the RNAalifold web server (Fig. 3A). Bioinformatics analysis revealed that there were binding sites between miR-3160-5p and -4739 and CACNA1G-AS1; the binding sites in the secondary structures of miR-3160-5p and miR-4739 are also presented (Fig. 3B). To screen the most promising miRs, the changes in the luciferase intensity of CACNA1G-AS1 after miR-3160-5p or -4739-overexpression were monitored. Firstly, transfection success was verified (Fig. 3C). Subsequently, the data confirmed that the luciferase intensity was significantly reduced in the miR-3160-5p group compared with the miRNA control group, indicating that CACNA1G-AS1 could interact with miR-3160-5p (P<0.05; Fig. 3C). However, luciferase intensity in the miR-4739 group had no obvious changes. Furthermore, the interaction between CACNA1G-AS1 and miR-3160-5p was verified by a luciferase reporter assay using CACNA1G-AS1-wt and CACNA1G-AS1-mut. The results indicated that miR-3160-5p significantly reduced the CACNA1G-AS1-wt luciferase intensity in OCI-Ly10 and SUDHL-4 transfected cells (P<0.05; Fig. 3D and E); however, this alteration disappeared in CACNA1G-AS1-mut-transfected cells (P>0.05). The transfection success of miR-3160-5p ASO was verified, as presented in Fig. 3F, and miR-3160-5p ASO decreased the CACNA1G-AS1-wt luciferase intensity in transfected cells. Overall, this indicated that CACNA1G-AS1 interacted with miR-3160-5p as a competitive endogenous RNA in DLBCL.

Subsequently, the influence of miR-3160-5p on the cytotoxicity and apoptosis of DLBCL cells was further analyzed. First, miR-3160-5p overexpression was confirmed in OCI-Ly10 and SUDHL-4 cells due to the high level of miR-3160-5p expression in the miR-3160-5p transfection group (P<0.05; Fig. 3C). Secondly, the MTT results revealed that the cytotoxicity of OCI-Ly10 and SUDHL-4 cells was significantly lower in the miR-3160-5p groups compared with the control groups (P<0.05; Fig. 4A). Furthermore, colony formation results revealed that miR-3160-5p also significantly decreased the colony number of transfected OCI-Ly10 and SUDHL-4 cells (P<0.05; Fig. 4B). Next, the apoptosis rates in OCI-Ly10 and SUDHL-4 cells were demonstrated to be significantly elevated in the miR-3160-5p groups compared with the control groups (P<0.05; Fig. 4C). As Fig. 4D depicted, CACNA1G-AS1-silencing reduced the colony formation rate of OCI-Ly10 and SUDHL-4 cells in the si-CACNA1G-AS1 + anti-control group compared with the si-NC + anti-control group; which was then significantly increased by miR-3160-5p overexpression (P<0.05). Compared with si-NC + anti-control, CACNA1G-AS1 depletion markedly elevated the apoptosis rate in both OCI-Ly10 and SUDHL-4 cells; whereas miR-3160-5p overexpression exhibited the opposite impact, evidenced by the significant descrease in apoptosis rate in si-CACNA1G-AS1 + Anti-3160-5p (P<0.05; Fig. 4E). Overall, these data confirmed that CACNA1G-AS1-knockdown enhanced cytotoxicity and induced apoptosis in DLBCL cells via miR-3160-5p.

The impact of CACNA1G-AS1 on tumor growth and apoptosis in vivo was further identified. SUDHL-4 cells transfected with si-NC or si-CACNA1G-AS1 were subcutaneously injected into nude mice, and the tumor volumes were monitored weekly. The data indicated that silencing CACNA1G-AS1 resulted in a significant inhibition of tumor growth (P<0.05; Fig. 5A). The level of CACNA1G-AS1 was also significantly lowered in the si-CACNA1G-AS1 group compared with the si-NC group (P<0.05, Fig. 5B). In addition, the western blotting data revealed that the protein expression levels of PCNA and Bcl-2 were also significantly attenuated in the si-CACNA1G-AS1 groups compared with the si-NC groups (P<0.05; Fig. 5C and D). Overall, this revealed that CACNA1G-AS1-knockdown also prevented DLBCL progression in vivo.