SPANXN2 functions a cell migration inhibitor in testicular germ cell tumor cells

Human testicular samples

The adjacent normal testicular tissue and TGCTs tissue samples used in this study were obtained from the Affiliated Cancer Hospital of Central South University (Changsha, China). Five adjacent normal tissue samples had been removed during para-testicular tumor surgery and the TGCT tissue samples were obtained from 11 testicular seminomas and three non-seminomas. Fresh tissues were collected and frozen in liquid nitrogen for storage at −180 °C. All the tissues were confirmed by histopathological examination. The patients provided written informed consent to tissue sample collection, which was performed with the authorization of the Ethics Committee of Central South University (Approve No.: LLSB-2017-002).

Quantitative RT-PCR

The total RNA was extracted using TRIzol Reagent (Invitrogen, USA). The amount and purity of each RNA sample were quantified by Agilent2100 (Agilent, Wilmington, DE, USA). The cDNA was synthesized from 1 µg RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche, USA). The real-time PCR system (LightCycler480, Roche, USA) was used to measure the relative expression level of the SPANXN2 gene and the β-actin was used as the housekeeping gene for normalization. Amplification was performed with the following thermo-cycling conditions: initial denaturation at 95 °C for 5 min, followed by 45 cycles of 95 °C for 10s and 60 °C for 10 s, and a final extension at 72 °C for 10 s. The LightCycler480 software was used to analyze the threshold cycle (CT) values and the 2^−ΔΔCT method was used to evaluated relative gene expression. The gene-specific primers used were as follows:

SPANXN2 forward: 5′-GTGTATTACTACAGGAAGCATACG-3′;

reverse: 5′-CTCCTCCTCTTGGACTGGATT-3′

β − actin forward: 5′-TCACCAACTGGGACGACATG-3′;

reverse: 5′-GTCACCGGAGTCCATCACGAT-3′

Cell culture

The human TGCT cell line NCCIT was bought from the American Type Culture Collection (ATCC, VA, USA), and the human TGCT cell line TCAM-2 was obtained from Dr. Yuxin Tang (Peng et al., 2019; Gan et al., 2016). NCCIT cells were cultured in RPMI-1640 medium (GIBCO, USA), and TCAM-2 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO, USA). All cells were cultured in medium containing 10% fetal bovine serum (FBS, GIBCO, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (GIBCO) and were incubated at 37 °C under 5% CO₂.

Cell transfection

The sequence of SPANXN2 was cloned into the CMV-MCS-DsRed2-SV40-Neomycin-GV147 vector. Cells were cultured as described above and divided into negative control (NC) and test (SPANXN2) groups and transfected with the GV147 empty vector (NC) and the GV147 vectors expressing SPANXN2, respectively. Briefly, cells were seeded in a 6-well culture plate (5 ×10⁵ cells/well) and transfected at 70% confluence. Cells were transfected with 2.5 µg of the GV147 empty vector and 2.5 µg of the GV147 vectors expressing SPANXN2 using DNA Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. Cells were harvested 36 h or 48 h after transfection for use in the following experiments.

Plate colony formation assay

Cells were seeded in a 6-well plate (300 cells/well) and incubated at 37 °C under 5% CO₂ for 12–14 days when most single-cell colonies consist of >50 cells. The cells were washed three times with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 30 min and then stained for 15 min with 0.5% crystal violet. After staining, colonies containing >50 cells were observed under a microscope and the colony number (a single colony with >30 cells) was counted using Adobe Photoshop CC 2018.

MTT assay

The proliferation of TGCT cells was evaluated by MTT assay. The cells were seeded into 96-well plates at 5 ×10³ cells/ml (200 µl/well). After 6 h, 20 µl MTT solution (5 mg/ml) (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, MTT) reagent (Sigma Chemicals, St Louis, MO, USA) was added to the 96-well plates. After incubation for 6 h at 37 °C, the supernatant was discarded from each well and 200 µl DMSO was added to each well. The absorbance at 492 nm was measured using a Nanodrop 1000 spectrophotometer (Thermo Fisher, USA). This process was repeated every 24 h for 1, 2, 3, and 4 days.

Flow cytometric cell cycle analysis

At 48 h after transfection, cells were first enzymatically dissociated into single cells by 0.05% trypsin and then fixed with 75% cold ethanol in PBS overnight at 4 °C. Second, the cells were washed with PBS three times. Finally, the cells were double-stained with a propidium iodide solution (PI, 50 µg/mL) and treated with RNase (1 mg/mL) at 37 °C for 30 min in the dark. Subsequently, samples were analyzed using an Accuri C6 flow cytometer (BD) with the Accuri software. For each sample, data from approximately 20,000 cells were acquired in list mode using logarithmic scales. FlowJo7.6.2 was used to analyze the cell cycle distribution according to the guideline.

Wound healing assay

Cells were seed in a 6-well culture plate (5 ×10⁵ cells/well) and the next day, transfected at 70% confluence. At 48 h after transfection, we drew three straight lines with an average interval in the layer of cells with a 100 µl of the tip. Cells were then washed three times with PBS to remove the scraped suspended cells. Cells were then washed cultured in medium containing 2% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37 °C under 5% CO₂. The wound healing areas were recorded by observation under a microscope at 0 h, 24 h and 48 h, and Image-Pro Plus 6.0 software was used to analyze the width of wound healing.

Transwell migration assay

Transwell Cell Culture Inserts (8 µm pore size, FALCON, USA) were used to measure cell migration capacity in 24-well plates. In brief, TCAM-2 and NCCIT cells (2 ×10⁴ cells in 200 µl of 2% FBS medium) were added to the upper chamber, and 800 µl 15% FBS medium was added to the lower chamber. After incubation at 37 °C under 5% CO₂ for 48 h, the cells on the upper surface were wiped with cotton swabs, while the cells that migrated through the filter pores were fixed in 4% paraformaldehyde for 30 min. After staining with 0.5% (w/v) crystal violet (Sigma) in PBS (GIBCO) for 15 min, cells were observed and photographed under an inverted microscope. Cells were counted in five randomly selected fields of view.

Western Blot analysis

Cells were collected and lysed with RIPA lysis buffer (Co Win Biosciences, China) according to the manufacturer’s instructions, and proteins were harvested. Protein quantification was performed using a BCA Protein Quantification Kit (Thermo, USA), and samples were adjusted to the same concentration. After boiling in 6 × Loading Buffer for 5 min, the proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membrane was incubated overnight at 4 °C with the appropriate primary detection antibodies: GAPDH (Sigma, 1:1,000), α-Tubulin (Sigma, 1:600), Snail (Cell Signaling Technology, 1:1,000), and Vimentin (Cell Signaling Technology, 1:1,000); AKT (ProteinTech, 1:1,000) and p-AKT (ProteinTech, 1:1,000). After washing three times with TBST for 5 min, the membrane was incubated with secondary antibodies (Cell Signaling Technology, 1:1,000) for 1 h at 37 °C. After washing three times with TBST for 15 min each time, the protein bands were visualized by fluorography using an enhanced chemiluminescence system (Millipore, USA); the expression levels of α-Tubulin and GAPDH were used as internal references.

Statistical analysis

All assays were repeated three times and all samples were analyzed in triplicate. The data were expressed as the mean ± SD or mean ± SEM of at least three independent experiments. Student’s t-test or a two-way ANOVA was used to determine the significance of differences between groups, and P < 0.05 was deemed statistically significant. Curve fitting analyses were performed with GraphPad Prism Software5.0 (GraphPad Software, USA).

Validation of SPANXN2 expression in TGCTs

To verify the reliability of differential SPANXN2 expression, we analyzed its expression in TGCTs in the GEPIA database (Tang et al., 2017) (http://gepia.cancer-pku.cn/index.html), and the results showed that SPANXN2 was significantly downregulated in TGCT tissues (Fig. 1A). Subsequent qRT-PCR analysis of TGCT samples and normal samples also confirmed the differential expression of SPANXN2 (Fig. 1B). These results revealed that TGCT tissues express a lower level of SPANXN2 relative to those detected in normal tissues.

Transfection of TGCT cell lines

As shown in Figs. 2A, 2B, most cells (>75%) showed red fluorescence that confirmed transfection with plasmids targeting SPANXN2, or an empty-plasmid control. Quantitative RT-PCR was used to detect the relative expression of SPANXN2 in these two transfected TGCT cell lines (TCAM-2, NCCIT), using β − actin as a reference (Figs. 2C and 2D). The results confirmed the relative overexpression of SPANXN2 in theSPANXN2 group compared with that in the NC group, laying the foundation for the study of its function.

SPANXN2 upregulation inhibited colony formation ability with no effect on cell proliferation

The effect of SPANXN2 on the ability of TGCT cells to form colonies was investigated in colony formation assays. The colony formation ability of TGCT cells overexpressing SPANXN2 was significantly decreased compared with that of the cells in the NC group (P < 0.05, Figs. 3A and 3B).

To investigate the effect ofSPANXN2 overexpression on TGCT cell proliferation. NCCIT and TCAM-2 cells were transfected with the SPANXN2 plasmid and their negative control plasmid. The MTT assay results revealed no significant difference in the cell proliferation rates of the NC andSPANXN2 groups (P >  0.05, Figs. 3C and 3D). Furthermore, flow cytometric analysis carried out 48 h after transfection showed that there were no significant differences in the cell cycle distribution of TGCT cells overexpressing SPANXN2 compared with the cells in the NC group (P > 0.05, Figs. 4A–4F). In addition, no marked cell apoptosis was observed at 24 h and 48 h after transfection with the SPANXN2 plasmid. These results indicated that SPANXN2 overexpression inhibited the colony formation ability of TGCT cells, but has no significant effect on the cell cycle distribution and apoptosis.

SPANXN2 negatively regulates TGCT cell migration

The effect of SPANXN2 overexpression on the migration of TGCTs cells was investigated in wound healing and Transwell assays. As shown in Fig. 5, the wound healing gap in the NC group was significantly smaller than that in theSPANXN2 group. At 48 h after transfection, there were significantly fewer migrated cells in theSPANXN2 group compared with that in the NC group (P < 0.05, Figs. 6A and 6B). The results indicated that SPANXN2 overexpression inhibited TGCT cell migration.

SPANXN2 influences the expression of EMT- and AKT-related proteins

EMT describes the transition of epithelial cells into mesenchymal cells, some molecules of which will be changed during the process of metastasis of many tumors. To explore how SPANXN2 inhibited TGCT cell migration, the expression levels of EMT-related proteins were detected by western blot analysis. These results revealed that the expression levels of the EMT related proteins, Vimentin, and Snail were decreased (Fig. 6C), suggesting that SPANXN2 regulates the EMT-related proteins. Similarly, the AKT-related molecules were also detected. AKT and p-AKT protein expression levels were lower after SPANXN2 overexpression (Fig. 6D). These results showed that SPANXN2 overexpression regulated EMT- and AKT-related proteins in TGCT cells.