The pathway by which vIL-10 promotes the development of NPC cells was explored through transcriptome sequencing.
The EBV-negative NPC cell line CNE-2 was treated with 100 ng/mL vIL-10 for 48 h, and then the transcriptome was sequenced. The experimental results of whole transcriptome gene sequencing of CNE-2 NPC cells before and after vIL-10 action revealed 5,899 differentially expressed genes (P < 0.05), 3,136 upregulated genes and 2,763 downregulated genes, among which JAK1 and STAT3 were upregulated genes (Fig. 1(A and B)). The repeatability within the sample group was relatively good (Fig. 1(C)).
A: Genes shown in the heatmap were significantly upregulated (red) and downregulated (blue). After vIL-10 acts on the NPC cell line CNE-2, differentially expressed genes were detected in the cancer cells.
B: Volcano map showing that compared with the control group, mRNA transcription-related genes in the vIL-10 treatment group were significantly upregulated (red), downregulated (blue) and had no difference (black). A total of 5899 differentially expressed genes were found (P < 0.05). Among them, there were 3136 upregulated genes and 2763 downregulated genes, among which JAK1 and STAT3 were upregulated genes.
C: Specific principal component analysis (PCA): The scattered points corresponding to the samples are clustered together within the group, and the repeatability within the group was good.
VIL-10 promotes the expression of IL-10R1 in NPC cells.
VIL-10 is a factor secreted by EBV-positive NPC cells, and we explored the difference in the secretion of vIL-10 between EBV-positive NPC cells and EBV-negative NPC cells. ELISA was conducted, and the cell culture medium of NPC cell lines EBV(+)C666-1 and EBV(-)CNE-2 were collected for the experiment. The results showed that the concentration of vIL-10 secreted by the NPC cell line C666-1 was 26.13 pg/mL, which was significantly higher than that secreted by CNE-2 cells (3.29 pg/mL) (P < 0.0001), suggesting that EBV-positive NPC cells could secrete VIL-10 spontaneously (Fig. 2(A)). The expression level of IL-10R1 protein was detected after the NPC cell CNE-1 was treated with 100 ng/mL vIL-10 for 24 h and 48 h. The experimental results showed that the expression of IL-10R1 protein in NPC cell CNE-1 could be upregulated with the prolonged treatment time of vIL-10 (Fig. 2(B)). After CNE-1, CNE-2 and C666-1 NPC cells were treated with 100 ng/mL vIL-10 for 24 h, the fluorescence expression level of IL-10R1 in the cells was detected by an upright fluorescence microscope. Experimental results showed that vIL-10 could effectively promote the expression of IL-10R1 in NPC cells, and IL-10R1 was mainly expressed in the cytoplasm of NPC cells (Fig. 2(C, D, and E)).
A: Secretion of vIL-10 by CNE-2 and C666-1 NPC cell lines detected by ELISA. B: Western blot analysis to detect the level of IL-10R1 protein in each group after a 100 ng/mL vIL-10 treatment of CNE-10 NPC cells for 10 h, 24 h and 48 h. C-E: CNE-1 (C), CNE-2 (D) and C666-1 cells (E) were incubated with 100 ng/mL vIL-10 for 0 h, 24 h or 48 h, and the fluorescence expression level of IL-10R1 in the cells was detected by immunofluorescence. The data show an average SD for three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
VIL-10 promotes the proliferation of NPC cells.
Ki67 staining is mainly used to label cells in the proliferation state. Cells in the G0 phase do not express Ki67, so it is often used to measure the growth index of tumour cells. After treating CNE-1, CNE-2 and C666-1 NPC cells with 100 ng/mL vIL-10 for 24 h and 48 h, the fluorescence expression of the Ki67 factor was detected. The results showed that the immunofluorescence intensity of Ki67 in the experimental group was significantly different from that in the control group at 24 h and 48 h, and the immunofluorescence intensity of Ki67 was directly proportional to the action time of vIL-10 (Fig. 3(A, B, and C)).
A-C: Ki67 staining experiment results revealing the expression of Ki67 factor fluorescence in each group after three NPC strains were exposed to 100 ng/mL vIL-10 for 0 h, 24 h and 48 h. The data show the average SD of the three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Cell colony formation experiments to detect cell proliferation ability.
To explore the effect of vIL-10 on the proliferation of CNE-1, CNE-2 and C666-1 cells, colony formation experiments were used. The results showed that the number of cell colonies formed by CNE-1, CNE-2 and C666-1 cells treated with 100 ng/mL vIL-10 for 24 h and 48 h was significantly different from that of the control group (Fig. 4(A and B)).
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Effect of vIL-10 on the proliferation of NPC cells investigated through a cell colony formation experiment, with the average SD (n = 3), *P < 0.05, and *** P < 0.001.
VIL-10 can promote the migration and invasion of NPC cells.
To explore the effect of vIL-10 on the migration and invasion ability of NPC cells, a Transwell experiment was performed. The results showed that the number of cells passing through the Transwell microcells in the experimental group was significantly higher than that in the control group after the treatment of different NPC cells with vIL-10 for 24 h. This finding suggests that vIL-10 can significantly promote the vertical migration of NPC cells (Fig. 5(A)).
Transwell invasion experiments are commonly used to explore tumour invasion ability. Compared with the migration experiment, the difference is that the Transwell chamber is coated with Matrigel to simulate the state of the extracellular matrix to detect cell invasion ability. The experimental results showed that after the CNE-1, CNE-2 and C666-1 NPC cells were treated with 100 ng/mL vIL-10 for 36 h, the invasion ability of different NPC cell lines was significantly enhanced, and the number of NPC cells passing through the Transwell matrix gel and small cell micropores was significantly different from that of the control group (Fig. 5(B)).
A .B: Transwell assay results showing the effect of vIL-10 on the migration and invasion abilities of CNE-1, CNE-2 and C666-1 cells. The data are expressed as the mean ± SD (n = 3), **P < 0.01, and ***P < 0.001.
VIL-10 inhibits the apoptosis of NPC cells.
Hoechst 33258 dye is commonly used for apoptosis detection, and the nuclei of apoptotic cells are stained bright blue. After CNE-1, CNE-2 and C666-1 NPC cells were treated with 100 ng/mL vIL-10 for 24 h and 48 h, Hoechst 33258 staining was performed. The bright blue nuclei of the experimental group were significantly different from those of the control group (Fig. 6(A)), which indicated that VIL-10 could inhibit the apoptosis of NPC cells. Flow cytometry is a common method to study apoptosis. Therefore, to explore the effect of vIL-10 on the apoptosis of NPC cells, Annexin V-FITC/PI double staining was performed to detect the apoptosis rate. The results indicated that the percentage of apoptotic CNE-1, CNE-2 and C666-1 NPC cells decreased with prolonged vIL-10 treatment time. The percentage of apoptotic cells in the experimental group was significantly different from that in the control group (Fig. 6(B)), suggesting that vIL-10 may inhibit the apoptosis of NPC cells.
A: Hoechst 33258 staining results showing the effect of vIL-10 on the apoptosis of NPC cells.
B: Flow cytometry results to determine apoptosis. Compared with the control group (NC), the percentage of apoptosis in the experimental group (24 h and 48 h) was significantly different. Note: average SD (n = 3), *p < 0.05,**P < 0.01,***P < 0.001, and ****P < 0.0001.
VIL-10 regulates the JAK-STAT signalling pathway.
Through KEGG pathway analysis, the differentially expressed genes were found to be mainly enriched in biological processes such as herpes simplex virus infection 1, cytokine interaction, MAPK signal pathway, human T-cell leukaemia virus type 1 infection, NOD-like receptor signal pathway, JAK-STAT signal pathway, non-alcoholic fatty liver, apoptosis, autophagy, TNF signal pathway, NF-κB signal pathway, nicotinate and nicotinamide metabolism, glycine, serine, threonine metabolism, iron death, etc. Furthermore, vIL-10 could effectively activate the JAK1-STAT3 signalling pathway, and the cancer-related signalling pathway also involves the JAK1-STAT3 signalling pathway. Therefore, we speculated that vIL-10 could promote the development of NPC through the JAK1-STAT3 signalling pathway.
A: KEGG signalling pathway enrichment analysis results. The colour change from red to purple indicated that the P value changed from small to large. The redder the colour, the more significant the enrichment of the core targets. The size of the bubbles represents the ratio of genes. B: Level of p-JAK1 protein in the control group (NC) and experimental group (24 h and 48 h) after vIL-10 treatment of C666-1 cells was detected by Western blotting. C: Levels of p-STAT3 protein expression in the control group (NC) and experimental group (24 h and 48 h) after the three NPC cell lines treated with vIL-10 were measured by Western blotting.