Regulation of lymphoma in vitro by CLP36 through the PI3K/AKT/CREB signaling pathway

Bioinformatics

The CLP36 expression pattern in lymphoma and the association between CLP36 expression and the survival of lymphoma patients were predicted based on relevant data from the cancer genome atlas program (https://www.cancer.gov/ccg/research/genome-sequencing/tcga) (Shahrajabian & Sun, 2023). The correlation between CLP36 and PI3K/AKT/CREB was also determined.

Cell culture and intervention

Human lymphoma cell lines, including B-cell lymphoma cell lines WSU-DLCL2 (ACC 575), SU-DHL-8 (ACC 573), OCI-LY19 (ACC 528), SU-DHL-2 (ACC 902), and Burkitt lymphoma cell lines BJAB (ACC 757) and RA 1 (ACC 603) were all ordered from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) and cultured as follows. For B-cell lymphoma cell lines, 90% Roswell Park Memorial Institute-1640 medium (90023; Solarbio LifeSciences, Beijing, China) or α-minimal essential medium (M0644; Millipore Sigma, Darmstadt, Germany) with the supplementation of 10% fetal bovine serum (12103C; Millipore Sigma, Darmstadt, Germany) was applied for the culture. Meanwhile, 80% Roswell Park Memorial Institute-1640 medium and 20% heat-inactivated fetal bovine serum (12106C; Millipore Sigma, Darmstadt, Germany) were used for the culture of Burkitt lymphoma cells. All cells were incubated at 37 °C with 5% CO₂.

For the intervention assays, the small interfering RNAs (siRNAs) against CLP36 (si-CLP36#1, target sequence: 5′-TGGGGAAAATACTAGCAATATGA-3′; si-CLP36#2, target sequence: 5′-GTCATCCATACAAGATGAATTTA-3′) and the corresponding negative control with scramble target sequences (5′-ATTAGTGCAGATACATCTTACAA-3′) were all synthesized by GenePharma (Shanghai, China) and transfected into BJAB and RA 1 cells using Invitrogen™ Lipofectamine 2000 transfection reagent (11668027; ThermoFisher Scientific, Waltham, MA, USA) for 48 h, as recommended by the protocols of the producer.

Cell counting kit-8 assay

Cell viability of CLP36-silencing lymphoma cells was gauged with a commercial CCK-8 assay kit (CK04; Dojindo, Kumamoto, Japan). In detail, lymphoma cells BJAB and RA 1 were seeded in a 96-well plate at the density of 2 × 10³ cells/well and cultured for 12, 24 and 36 h. Then 10 μL CCK-8 solution was added to the cells for another 4-h culture. IMark™ microplate absorbance reader (1681130; Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to measure the OD value at 450 nm (Ruan et al., 2024).

Cell proliferation assays

For colony formation assay, following the transfection, BJAB and RA 1 cells were grown in a 6-well plate at 1 × 10³ cells/well and cultured at 37 °C for 14 days. Hereafter, these cells were fixed in methanol (M813896; Macklin, Shanghai, China) for 15 min and dyed using crystalline violet solution (C776042; Macklin, Shanghai, China) for 30 minu. The colonies formed were observed and photographed and the number was hereafter quantified.

For the second part, BJAB and RA 1 cells were seeded in 96-well plates at the density of 3 × 10³ cells/well for overnight culture, followed by the incubation with 50 μM 5-ethynyl-2′-deoxyuridine (EdU, E796036; Macklin, Shanghai, China) reagent at 37 °C for 1 h. Further, DAPI solution (C0065; Solarbio LifeSciences, Beijing, China) at 0.1 μg/mL was adopted for nuclei dyeing for 20 min at room temperature and the results were finally observed in a fluorescence microscope (DM6B; Leica Microsystems, Wetzlar, Germany) (Wang et al., 2022).

Cell apoptotic assay

FITC Annexin V/PI Apoptosis Detection Kit (CA1020; Solarbio LifeSciences, Beijing, China) was applied in cell apoptotic assay. Following the transfection, the treated BJAB and RA 1 cells (1 × 10⁶ cells in total) were suspended in the binding buffer and centrifuged to remove the supernatant. Then, these cells were sequentially added with the working solutions of both FITC Annexin V and PI (5 μL for each) and cultured for 10 min. Ultimately, the results were analyzed in a flow cytometer (FACSVERSE; BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo 10 (FlowJo, LLC., Ashland, OR, USA) was utilized for data analysis.

Quantitative PCR

The total RNA from cells were extracted using Invitrogen™ TriZol assay kit (15596026; Thermo Fisher Scientific, Waltham, MA, USA) and then reverse-transcribed into cDNA using first-strand cDNA synthesis kit (11119ES60; Yeason, Shanghai, China). Hereafter, the PCR was initiated in a PCR thermocycler (CFX96; Bio-Rad Laboratories, Inc., Hercules, CA, USA) using the Hieff^® qPCR Green Master Mix (11204ES03; Yeason, Shanghai, China) at 95 °C for 1 min and 40 cycles of 95 °C for 10 s, 60 °C for 20 s and 72 °C for 20 s. The relative mRNA levels of genes of interest were finally calculated with the method 2^−ΔΔCt with GAPDH as the housekeeping control (Livak & Schmittgen, 2001; Sindhuja, Amuthalakshmi & Nalini, 2022). The primers used are shown in Table 1.

Immunoblotting

In the present study, in order to further determine the expression of CLP36, as well as to assess the activation status of the signaling pathway, we performed assays using the western blotting assay. Cell lysates were prepared with the RIPA lysis buffer (20115ES60; Yeason, Shanghai, China) and the protein concentration was tested via BCA method. Following the separation in SDS-PAGE separation gel, the protein samples (30 μg) were transferred to the PVDF membrane which was hereafter blocked with 5% defat milk for 1 h at room temperature. PI3 Kinase p85 Antibody (#4292, 1:1000; Cell Signaling Technology, Danvers, MA, USA), Phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) Antibody (#4228, 1:1000; Cell Signaling Technology, Danvers, MA, USA), Akt Antibody (#9272, 1:1000; Cell Signaling Technology, Danvers, MA, USA), Phospho-Akt (Ser473) Antibody (#9271, 1:1000; Cell Signaling Technology, Danvers, MA, USA), CREB (48H2) Rabbit mAb (#9197, 1:1000; Cell Signaling Technology, Danvers, MA, USA), Phospho-CREB (Ser133) (87G3) Rabbit mAb (#9198, 1:1000; Cell Signaling Technology, Danvers, MA, USA) and GAPDH (14C10) Rabbit mAb (#2118, 1:1000; Cell Signaling Technology, Danvers, MA, USA) were applied for the overnight culture at 4 °C, followed by the further reaction with Anti-rabbit IgG, HRP-linked Antibody (#7074, 1:1000; Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. The visualization on the membrane was completed with the ECL visualization reagent (36208ES60; Yeason, Shanghai, China) and the densitometry on the protein band was determined with ImageJ 5.0 (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical analysis

All statistical analysis of this study was performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA) and all data of independent triplicate were expressed in the form of mean ± standard deviation. Differences among groups were compared by student’s t-test. Statistically significant was defined when a P-value was lower than the threshold of 0.05.

CLP36 expression analysis in lymphoma and determination on the association with the prognosis

In the beginning, with the purpose of exploring the specific effects of CLP36 in lymphoma, we searched and downloaded the data of CLP36 expression pattern in lymphoma from The Cancer Genome Atlas program. Relevant data have suggested that CLP36 (shown as PDLIM1 in the figures) was highly expressed in lymphoma (Fig. 1A, P < 0.05), and such high CLP36 expression in lymphoma was related to an unfavorable overall survival of lymphoma patients (Fig. 1B, logrank P = 0.02).

Effects of CLP36 silencing on the survival and proliferation of lymphoma cells

Then, the level of CLP36 in lymphoma cells was quantified accordingly, where a relatively higher expression was seen in BJAB and RA 1 cells as compared to that in WSU-DLCL2 cells (Figs. 2A–2C, P < 0.01). These two cells were hence applied in subsequent assays. Then, the level of CLP36 was forcibly knockdown in BJAB and RA 1 cells using the relevant siRNAs, and the successful transfection was witnessed, as indicated by the reduced level of CLP36 in these cells (Figs. 2D and 2E, P < 0.0001). The CCK-8 assay was applied to determine the viability of BJAB and RA 1 cells, as seen in the reduced OD value at 12, 24 and 36 h (Figs. 2F and 2G, P < 0.01).

The proliferation of CLP36-silenced lymphoma cells BJAB and RA 1 was evaluated based on colony formation assay (Figs. 3A and 3B) and EdU staining (Figs. 3C and 3D). Accordingly, it was visible that CLP36 silencing led to the reduced number of colonies formed (Figs. 3A and 3B, P < 0.01) and the number of EdU-positive cells (Figs. 3C and 3D, P < 0.05). Further, the levels of metastasis-related proteins cadherin 2 (CDH2) and vimentin (VIM) were calculated to evaluate the metastasis potential of lymphoma cells, and it was evident that the knockdown of CLP36 led to the diminished levels of these two proteins in lymphoma cells (Figs. 3E and 3F, P < 0.05).

Effects of CLP36 knockdown on the apoptosis of lymphoma cells

The effects of CLP36 silencing on the apoptosis of lymphoma cells were further explored, revealing the elevated apoptosis rate in lymphoma cells BJAB and RA 1 following the knockdown of CLP36 (Figs. 4A–4D, P < 0.01). Such pro-apoptotic effects of CLP36 silencing on lymphoma cells BJAB and RA-1 were further confirmed by qPCR, which was applied to calculate the levels of relevant proteins Bax and caspase-3 (CASP3). Specifically, the silencing of CLP36 caused the elevation of Bax and CASP3 expressions in lymphoma cells BJAB and RA 1 (Figs. 4E and 4F, P < 0.05).

Effects of CLP36 silencing on PI3K/AKT/CREB pathway in lymphoma cells

Finally, we determined the downstream pathway underlying the effects of CLP36 silencing on lymphoma cells. The function of PI3K/AKT signaling pathway caught our attention, and the correlation between CLP36 and PI3K/AKT/CREB was determined firstly. Positive correlations were seen in CLP36 (shown as PDLIM1) and PI3K (Fig. 5A, P = 0.00066, R = 0.48), AKT (Fig. 5B, P = 0.04, R = 0.3) and CREB (Fig. 5C, P = 0.014, R = 0.36). Additionally, the phosphorylation level of PI3K, AKT and CREB was witnessed to be sharply diminished in lymphoma cells after the silencing of CLP36 (Figs. 5D and 5E, P < 0.05).