The preprocessed level 3 RNA-seq data and corresponding clinical information of lymphoma patients were collected from TCGA database (http://cancergenome.nih.gov/). Data from TCGA were downloaded to integrate the expression of SPAG6 and survival data. Survival analysis was performed with the survival R package and using Kaplan-Meier analysis (log-rank test) (12).

All the cell lines were purchased from Nanjing Kaiji Bio-tech Co., Ltd. The human normal peripheral-blood B-lymphocyte (IM-9) and four BL cell lines (CA46, NAMALWA, Daudi and Raji) were cultured in RPMI-1640 medium (HyClone; Cytiva) supplemented with 10% FBS (HyClone; Cytiva) and 1% penicillin and streptomycin (Beyotime Institute of Biotechnology). All cells were maintained at 37°C in 5% CO₂ during all experiments.

SPAG6 shRNA and scrambled control-shRNA were purchased from Shanghai GenePharma Company. For shRNA transfection, 293T cells were grown to 30–50% confluence in 6-well culture plates and transfected with scrambled shRNA or SPAG6 shRNA using Lipofectamine™ LTX reagent with PLUS™ reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h. Then, the supernatant from 293T cells was harvested and added to Daudi and Raji cells for 72 h, followed by selection with 1 µg/ml puromycin for 1 month. Plasmids (pcDNA3.1-SPAG6 and pcDNA3.1) were purchased from Obio Technology Co., Ltd. For plasmid transfection, CA46 and NAMALWA cells (70% confluence) were plated in 6-well culture plates. Plasmids were purified and transfected with pcDNA3.1-SPAG6 or pcDNA3.1 for 72 h using Lipofectamine™ LTX reagent with PLUS™ reagent (Invitrogen; Thermo Fisher Scientific, Inc.), followed by selection with 1 µg/ml puromycin for 1 month.

Cell proliferation was tested with the Cell Counting Kit-8 (CCK-8) Assay (Dojindo Molecular Technologies, Inc.). Briefly, cells (2,000–5,000) were seeded in 96-well plates. After incubation for 24, 48, 72 and 96 h, 10 µl CCK-8 reagent was added into each well of the 96-well plates. After incubation with the CCK-8 solution for 1 h, the final optical density (OD) values at 450 nm were calculated.

Daudi and Raji cells (2×10⁵ cells/well) were cultured in 6-well culture plates. After incubation, the cells were washed with PBS and centrifuged at 500 × g for 5 min at 4°C. Then, the cell suspension was re-suspended with binding buffer, followed by the addition of 5 µl Annexin V and 5 µl propidium iodide (PI) (Invitrogen; Thermo Fisher Scientific, Inc.) for 15 min in the dark. Cells subsequently were counted by a flow cytometer (BD Biosciences Inc.).

Cells were harvested and total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA (1 µg) was reverse-transcribed at 37°C for 15 min and 85°C for 5 sec using the PrimeScript Reverse Transcriptase system (Takara Biotechnology Co., Ltd.). qPCR amplification was performed using SYBR Green Master Mix (Takara Biotechnology Co., Ltd.) under the following conditions: 94°C for 1 min, 30 cycles at 94°C for 30 sec, 50°C for 30 sec, 72°C for 45 sec, 72°C for 5 min. The relative expression of SPAG6 was calculated using the 2^−ΔΔCq method. Primers for PCR included: SPAG6, 5′-GACGTGGTCCCAACAATTCAA-3′ (forward) and 5′-ACAGCTTCTGCTAGGTCATCAT-3′ (reverse); GAPDH, 5′-TGACTTCAACAGCGACACCCA-3′ (forward) and 5′-ACCCTGTTGCTGTAGCCAAA-3 (reverse).

Cells were harvested and lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology) with protease inhibitors (Thermo Fisher Scientific, Inc.). The protein concentration was determined by a BCA kit (Thermo Fisher Scientific, Inc.). For western blotting, samples were electrophoresed on 8–12% acrylamide gradient Tris-Tricine Ready Gels and transferred to PVDF membranes (EMD Millipore). Then, the PVDF membranes were blocked with 5% milk for 1 h at room temperature and incubated with the primary antibodies anti-SPAG6 (Abcam, ab155653, 1:1,000 dilution), anti-β-actin (Sigma-Aldrich; Merck KGaA, A5441, 1:5,000 dilution), anti-Bcl-2 [Cell Signaling Technology, Inc. (CST), cat. no. 3498, 1:1,000 dilution], anti-Bax (CST, cat. no. 5023, 1:1,000 dilution), anti-caspase-8 (CST, cat. no. 4790, 1:1,000 dilution), anti-cleaved-caspase-8 (CST, cat. no. 9748, 1:1,000 dilution), anti-poly(ADP-ribose) polymerase (PARP; CST, cat. no. 9542, 1:1,000 dilution), anti-cleaved-PARP (CST, cat. no. 52873, 1:1,000 dilution), anti-caspase-3 (CST, cat. no. 14220, 1:1,000 dilution), anti-cleaved-caspase-3 (CST, cat. no. 9664, 1:1,000 dilution), anti- phosphatase and tensin homolog (PTEN; CST, cat. no. 9188, 1:1,000 dilution), anti-AKT (CST, cat. no. 4685, 1:1,000 dilution) and anti-p-AKT (CST, cat. no. 4060, 1:1,000 dilution) overnight at 4°C, followed by incubation with secondary antibodies (CST, cat. no. 7074, 1:5,000 dilution) at room temperature for 2 h.

Four-week-old male severe combined immunodeficiency (SCID) mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. SPAG6 stable knockdown or control Raji cell lines (5×10⁶ cells in 50 µl DMEM) were subcutaneously injected into the left axilla of 15 SCID mice. When the tumors reached 100 mm³, the mice were randomized into three groups: i) nc/DMSO group, intraperitoneal injection of DMSO (50 µl daily) (n=5); ii) shSPAG6/DMSO group, intraperitoneal injection of DMSO (50 µl daily) (n=5); and iii) shSPAG6/SF1670 group, intraperitoneal injection of SF1670 (10 µmol/kg diluted in 50 µl DMSO; n=5) (13). The length (a) and width (b) of the tumors were calculated every 3 days and the volume (V) was calculated using the formula V=1/2ab². After 3 weeks of treatment, the mice were euthanized by CO₂ asphyxiation at a flow rate of 25% volume/min of gas displacement in a chamber for 5 min in December of 2019. Death of all mice were confirmed before removing them from the chamber. Then the tumors were harvested and weighed. During the entire animal experiment, we performed this experiment following the Ethical Guidelines of Animal Experiment version 1.0 of the Affiliated Huaian No. 1 People's Hospital of Nanjing Medical University released on October 28th, 2019.

The expression of proliferation- associated antigens Ki67 and proliferating cell nuclear antigen (PCNA) in tissues was measured by immunohistochemistry assay. After conventional paraffin embedding, sectioning, dewaxing and hydration, the tumor tissues were treated with antigen repair buffer (pH 6.0). Following treatment with 0.3% H₂O₂ for 20 min, the tumor tissue sections were blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) and incubated with primary antibodies (anti-Ki-67, Abcam, ab15580, 1:200 dilution; anti-PCNA, Dako, M0879, 1:50 dilution) overnight at 4°C. On the following day, the tissue sections were washed with PBS three times and incubated with secondary antibodies at 37°C for 1 h, followed by treatment with DAB chromogen.

TUNEL staining was performed to assess the tumor cell apoptosis. For the TUNEL assay, tissues were fixed with 4% formalin for 24 h at 4°C and then embedded in paraffin; TUNEL staining was carried out using a TUNEL kit (Roche Diagnostics). Specifically, the tumor tissue sections were deparaffinized in xylene and hydrated through graded alcohols. The tissue slides were blocked with PBST solution (PBS with 0.1% Triton X-100) for 20 min. After washing with PBS three times, the slides were incubated with TUNEL reaction mixture (5 µl of enzyme solution and 45 µl of label solution) for 2 h in the dark at 37°C. Then, the tissue sections were washed and incubated with DAPI (1:1,000 dilution, Beyotime Institute of Biotechnology) for 20 min in the dark.

GraphPad Prism 7.0 (GraphPad Software, Inc.) was used to create all graphs and perform statistical analyses. The two-tailed Student's t-test and one-way ANOVA were used to performed statistical analyses. P<0.05 was considered to indicate statistically significant differences.

In order to examine the effects of SPAG6 and its clinical relevance in BL patients, the association between the levels of SPAG6 and the survival of BL patients was investigated in the TCGA database (12). The result indicated that higher expression of SPAG6 is associated with worse prognosis of lymphoma patients (Fig. 1A), indicating that SPAG6 expression is closely linked to the development and progression of lymphoma.

BL, a germinal center B-cell-derived tumor, is the most frequently occurring non-Hodgkin lymphoma (NHL), accounting for 40% of childhood NHLs (14,15). Despite the fact that a high-dose combination of chemotherapy is an effective treatment strategy for BL, a poor prognosis is unavoidable. Furthermore, the exact mechanism underlying the development and progression of BL remains unclear. Hence, BL was selected as the study focus to assess the role of SPAG6. The expression of SPAG6 was analyzed in human normal peripheral blood B-lymphocytes (IM-9) and BL cell lines (CA46, NAMALWA, Daudi and Raji). The RT-PCR result revealed that the mRNA levels of SPAG6 in CA46, NAMALWA, Daudi and Raji cells were 1.80±0.26; 2.88±0.31; 6.17±0.65 and 7.70±1.14 times higher, respectively, compared with that noted in the IM-9 cells (P<0.05; Fig. 1B). Moreover, the protein levels of SPAG6 were significantly increased in the BL cell lines compared with this level in the IM-9 cells (P<0.05) as detected by western blot analysis (Fig. 1C and D).

As shown in Fig. 1B-D, the expression of SPAG6 was the highest in the Daudi and Raji cells. To ascertain whether SPAG6 expression is associated with the proliferative activity of BL cells, SPAG6 expression was first knocked down in Daudi and Raji cells using shRNAs. The results revealed that the expression of SPAG6 was significantly reduced in the knockdown groups compared with the control groups in both Daudi and Raji cells, as shown by RT-PCR and western blot assays (Fig. 2A and B). Furthermore, a proliferation assay was performed to determine whether SPAG6 depletion could affect the proliferation ability of Daudi and Raji cells. As shown in Fig. 2C, cell viability was markedly decreased at 72 h in the shSPAG6 groups compared with the control groups, as shown by the CCK-8 assay.

Based on the expression of SPAG6 in BL cell lines, CA46 and NAMALWA cells were selected to construct cell lines stably overexpressing SPAG6 (Fig. 1B-D). Using plasmid transfection, CA46 and NAMALWA cell lines that stably overexpressed SPAG6 were generated and the transfection efficiency testing indicated that the levels of SPAG6 were significantly higher in the SPAG6 overexpression groups compared with those in the control groups, as shown by RT-PCR and western blot assays (Fig. 2D and E). By contrast, the CCK-8 assay revealed that overexpression of SPAG6 significantly enhanced cell proliferation from 48 h (Fig. 2F). These results suggest that the expression of SPAG6 is correlated with the proliferation of BL cells.

Abnormal apoptosis, including resistance to apoptosis, is considered to be an important mechanism underlying tumorigenesis (16). Previous studies have found that, in most types of lymphoma, apoptosis is significantly reduced and abnormal or reduced apoptosis is the main cause behind the occurrence and development of lymphoma (17). Therefore, promoting the apoptosis of lymphoma cells is one of the strategies employed for treating lymphoma. We next sought to investigate whether the expression of SPAG6 is also associated with reduced apoptosis of BL cells. The cells were stained with Annexin V-PI solution to evaluate the apoptosis rate by flow cytometry. Specifically, the percentage of apoptotic cells in the blank control and SPAG6-knockdown groups of Daudi cells was 4.62±0.28 and 12.32±2.34%, respectively (P<0.01) (Fig. 3A). An increased apoptosis rate was also observed in the SPAG6-silenced group compared with the non-transfected group of Raji cells (P<0.01; Fig. 3B).

c-MYC is an established indicator of BL (18), and it also plays an important role in numerous biological processes, including cell growth and proliferation, cell cycle progression and apoptosis (19). The expression of c-MYC was reduced after the knockdown of SPAG6 in Daudi and Raji cells, as shown by western blot analysis (Fig. 3C). In addition, the western blot results demonstrated that the expression of Bcl-2 (a well-known anti-apoptotic protein) in the SPAG6-knockdown group of Daudi and Raji cells was significantly downregulated, while the expression of Bax (a well-known pro-apoptotic protein) was markedly enhanced (Fig. 3C). Moreover, the protein levels of cleaved-caspase-8, cleaved-caspase-3 and cleaved-PARP were obviously increased in the SPAG6-knockdown groups compared with the control groups in Daudi and Raji cells (Fig. 3C). In summary, the aforementioned data indicate that SPAG6 expression is correlated with the apoptosis of BL cells.

The PTEN/PI3K/AKT signaling pathway plays a key role in the development of various cancers and the regulation of essential tumor cell functions (20). In particular, PTEN, a tumor suppressor, can negatively regulate the PI3K/AKT signaling pathway (21). To confirm whether the effects of SPAG6 on the viability of BL cells were mediated via the modulation of the PI3K/AKT pathway, AKT, p-AKT and PTEN were tested in SPAG6-depleted or SPAG6-overexpressed cells by western blotting. The experimental results uncovered that the level of PTEN protein was significantly increased, while p-AKT protein was markedly reduced in the SPAG6-knockdown group (shSPAG6) compared with the blank control group (nc-shSPAG6) in Daudi and Raji cells, as shown by western blot analysis (Fig. 4A). Conversely, the expression of PTEN was obviously decreased and the protein levels of p-AKT was significantly increased in the SPAG6-overexpression group (pcDNA-SPAG6) compared with the control group (pcDNA) in CA46 and NAMALWA cells (Fig. 4E). We further explored whether the PI3K/PTEN/AKT pathway is implicated in the SPAG6-mediated enhancement of cell proliferation. For that purpose, PTEN was knocked down by siRNAs in Daudi and Raji cells (Fig. 4B). Interestingly, silencing of SPAG6 suppressed the proliferation of Daudi and Raji cells, whereas PTEN knockdown (siPTEN) rescued the tumor-inhibiting effect of SPAG6 depletion (Fig. 4C). Similarly, SF1670, a specific PTEN inhibitor, also reversed the anti-proliferative effect exerted by SPAG6 knockdown (Fig. 4D).

Next, PTEN overexpression was achieved using plasmids in CA46 and NAMALWA cells (Fig. 4F). SPAG6 overexpression promoted the proliferation of CA46 and NAMALWA cells, whereas PTEN overexpression rescued the SPAG6 overexpression-mediated tumor-promoting effect (Fig. 4G). Furthermore, stably overexpressing SPAG6 cell lines were treated using the PI3K-specific inhibitor LY294002, and reversal of the proliferative effect of SPAG6 overexpression on CA46 and NAMALWA cells was observed by inhibiting the PI3K pathway (Fig. 4H). Overall, these data indicate that SPAG6 may promote the proliferation of BL cells via the PTEN/PI3K/AKT pathway.

It was verified that SPAG6 induced growth promotion in human BL cells by activating the PTEN/PI3K/Akt signaling pathway. We further examined whether SPAG6 promotes the AKT pathways associated with apoptosis. Raji cells had highest expression of SPAG6 in the BL cell lines (Fig. 1C and D), so this cell line was chosen for the subsequent apoptosis experiments. Annexin V-PI staining was performed to detect the apoptosis rate in four groups of Raji cells (nc-shRNA, shSPAG6, shSPAG6/siPTEN and shSPAG6/SF1670) by flow cytometry assay. It was demonstrated that the apoptotic rates in the four groups were 4.91±0.98, 11.34±2.31, 6.22±1.81 and 5.63±0.73, respectively (Fig. 5A). SPAG6 knockdown enhanced the apoptosis of Raji cells, while PTEN knockdown by siRNA or inhibitor reversed the apoptosis activation caused by SPAG6 depletion. The apoptosis-related protein levels detected by western blotting confirmed these results. As shown in Fig. 5B, the protein levels of cleaved-caspase-3 and cleaved-PARP were obviously increase in the SPAG6-knockdown group compared with the control group, while the increased protein levels induced by SPAG6 knockdown were partly reversed after silencing of PTEN. These results indicated that SPAG6 may inhibit the apoptosis of BL cells though the PTEN/PI3K/AKT pathway.

As mentioned above, SPAG6 was shown to promote cell proliferation and inhibit cell apoptosis via the PTEN/PI3K/AKT pathway in BL cells in vitro (Fig. 6). To observe whether SPAG6 exerts functional effects on BL progression in vivo, a Raji BL cell xenograft mouse model was established. The mice were divided into three groups: nc-shRNA/DMSO group (injection with control cells and treatment with DMSO); shSPAG6/DMSO group (injection with SPAG6 stable knockdown cells and treatment with DMSO); and shSPAG6/SF1670 group (injection with SPAG6 stable knockdown cells and treatment with SF1670). It was confirmed that the tumor size and weight in the tumor tissues from the SPAG6-knockdown (shSPAG6/DMSO) group were smaller compared with those in the control tissues (nc-shRNA/DMSO group) (Fig. 7A-C). Consistently, the expression of the proliferation-related proteins Ki67 and PCNA in BL tumor tissues was reduced following SAPG6 knockdown compared with the blank group, as demonstrated by immunohistochemistry (Fig. 7D). Furthermore, TUNEL assay revealed that the apoptotic cell numbers were obviously increased in the shSPAG6/DMSO group (17.64±1.76) compared with the nc-shRNA/DMSO group (7.27±2.77; P<0.05; Fig. 7E). Interestingly, silencing of PTEN using the inhibitor SF1670 (shSPAG6/SF1670 group) reversed the tumor growth induced by SPAG6 depletion compared with the shSPAG6/DMSO group (Fig. 7A-C). Moreover, there was no difference in the expression of Ki67 or PCNA between the shSPAG6/SF1670 and the nc-shRNA/DMSO groups, whereas the expression of Ki67 and PCNA was higher in the shSPAG6/SF1670 group compared with the shSPAG6/DMSO group (Fig. 7D). In addition, PTEN knockdown in the shSPAG6/SF1670 group (10.06±2.96) partly rescued the apoptotic ratio mediated by SPAG6 depletion compared with the shSPAG6/DMSO group (17.64±1.76), as shown by the TUNEL assay (Fig. 7E). Collectively, these results indicate that SPAG6 promotes tumor growth via the PTEN/PI3K/AKT pathway in vivo.