Viral IL-10 regulates nasopharyngeal carcinoma cell progression via the JAK1/STAT3 signalling pathway

doi:10.21203/rs.3.rs-5246904/v1

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Viral IL-10 regulates nasopharyngeal carcinoma cell progression via the JAK1/STAT3 signalling pathway

https://doi.org/10.21203/rs.3.rs-5246904/v1

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Purpose Nasopharyngeal carcinoma is a common clinical malignant tumour in the nasopharynx. The secretion of vIL-10 by EB virus can promote the development of nasopharyngeal carcinoma. The purpose of this study was to provide a theoretical basis for potential targets and mechanisms of vIL-10 in nasopharyngeal carcinoma.

Methods A total of 100 ng/mLvIL-10 was applied to the nasopharyngeal carcinoma CNE-2 cell line for 48 h,and then whole transcriptome sequencing analysis was performed to identifypotential targets and signalling pathways of vIL-10 innasopharyngeal carcinoma. The effects of vIL-10 on the proliferation, migration, invasion and apoptosis of human nasopharyngeal carcinoma cells were determined by ELISA, Ki67 immunofluorescence, colony formation, Transwell migration/invasion, Hoechst 33258 staining, flow cytometry and other experiments, and the potential effects of vIL-10 on nasopharyngeal carcinoma cells were verified at the cellular level. Western blotting was performed to measure the changes in key factors of the JAK1-STAT3 signalling pathway in nasopharyngeal carcinoma cells after vIL-10 treatment.

Results The whole transcriptome gene sequencing results showed that vIL-10 could effectively activate the JAK-STAT signalling pathway and upregulatethe expression of JAK1 and STAT3. Moreover, vIL-10 inhibited the apoptosis of nasopharyngeal carcinoma cells, enhanced their migration and invasion capabilities, and further promoted the proliferation of nasopharyngeal carcinoma cells.

Conclusion VIL-10 regulates the JAK1-STAT3 signalling pathway, promotes the proliferation of NPC cells, enhances their migration and invasion capabilities, inhibits tumour cell apoptosis, and participates in the progression of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma

Virus interleukin-10

JAK1-STAT3 signal pathway

BCRF-1

Nasopharyngeal carcinoma (NPC) is a common malignant tumour in the clinic that occurs frequently in the anterior parietal wall of the nasopharynx and pharyngeal recess. The incidence of NPC is geographically different. Most new cases of NPC are in East Asia and Southeast Asia, with low incidence in the West. Due to the hidden anatomical position of the nasopharynx, patients with early NPC do not have characteristic symptoms, so the diagnosis is often delayed. Most patients are in the advanced stage of NPC when they are diagnosed [1]. Clinically, some patients have a cervical mass as the initial symptom, and most patients with NPC have cervical lymph node metastasis [2]. The 5-year survival rate of some patients with advanced NPC is only 10% [3]. The global geographical distribution of NPC is uneven. The age-standardized rate (ARS) in China is 3.0/100,000, and that in the white population is 0.4/100,000 [4]. Current research shows that NPC is caused by Epstein–Barr virus (EBV) infection, environmental factors, family heredity and other factors.

EBV is a human lymphocyte-addicted gamma-DNA herpesvirus. Current research shows that EBV infection has a significant serological relationship with NPC, among which undifferentiated NPC is the most closely related to EBV infection [1.5]. EBV can enhance tumorigenicity by regulating and controlling many immunosuppressive factors, such as IL-10 and PD1, and form an immunosuppressive microenvironment to help tumour cells accomplish immune escape [6]. Viral interleukin-10 is a secreted protein produced by EBV that was first discovered by Moore and Hsu. It is a protein encoded by the BCRF-1 gene in EBV and has 84% homology with human IL-10 [7]. VIL-10 has a relative molecular mass of 17 kD and consists of two regions composed of homodimers. Each monomer contains 6 helical structure regions (A, B, C, D, E, and F), in which 4 helical structures participate in forming one region and the remaining 2 helical structures form another region, with a total of 145 amino acids. IL-10 is mainly a cytokine produced by Th2 cells and mononuclear macrophages that can inhibit the cellular immune response through various mechanisms [8], thus promoting the immune escape of tumour cells [9], and can also play a role in immune regulation, promoting the differentiation and proliferation of B cells and mast cells [10]. VIL-10 can exert the immunosuppressive effect of IL-10 but lacks the immunoregulatory effect of IL-10 [11]. VIL-10 exhibits immunosuppressive properties related to cell ligands, such as inhibition of the Th1 polarization reaction, but does not have immunostimulatory properties related to IL-10, such as activation of dendritic cells, NK cells and some T cells [12]. These results suggest that vIL-10 plays an important role in the development of NPC.

Current research indicates that the JAK1/3 signalling pathway is involved in the pathogenesis of inflammatory diseases, and the JAK1/2 signalling pathway is involved in the development of various malignant tumours, including leukaemia, lymphoma and myeloproliferative tumours [13]. In NPC cells, the JAK2/STAT3 signalling pathway can activate its downstream target gene, promote tumour neovascularization and help tumour cells metastasize. Activation of STAT3 can promote the progression and metastasis of NPC and is clinically related to advanced NPC (stage III or IV). The increase in activated STAT3 expression levels is closely related to the poor prognosis of NPC patients[14].

Furthermore, in prophase work, CCK8 assays showed that vIL-10 could promote the proliferation of NPC cells. However, the specific mechanism of vIL-10 in NPC is unclear. Therefore, the purpose of this study was to explore the mechanism by which vIL-10 promotes the development of NPC and reveal its potential promoting effect in vitro .

Preparation of vIL-10 protein solution

BSA (Solarbio, Beijing, China) powder (0.05 g) was added to 50 mL of 1× PBS to prepare a 0.1% BSA solution. A total of 1000 µg of 0.1% BSA solution was added to 100 µg of vIL-10 (R&D Systems, Inc., Minneapolis, MN, USA) powder so that the final concentration of vIL-10 was 100 µg/mL. Every 50 µL of prepared vIL-10 liquid was packed into one sterile EP tube and stored in a -20℃ refrigerator.

Cell lines and cell cultures

The NPC cell line CNE-1 is a highly differentiated squamous carcinoma cell of the human nasopharynx with negative EBV produced by Shanghai Enzyme Research Biotechnology Co., Ltd. The NPC cell line CNE-2 is an EBV-negative human nasopharyngeal poorly differentiated squamous carcinoma cell line produced by Shanghai Enzyme Research Biotechnology Co., Ltd. The NPC cell line C666-1 is an EBV-positive human NPC cell line produced by Zhejiang Meisen Cell Technology Co., Ltd. All three cell lines were cultured in RPMI-1640 (Gibco, Carlsbad, CA, USA) medium supplemented with 10% foetal bovine serum (Biological Industries, Israel) in an incubator at 5% CO₂ and 37°C.

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. Total RNA was extracted, and a total amount of ≥ 800 ng, was used to construct a library using the library construction kit of the nebnextultratnallibrary prep kit from Illumina. The data at the molecular level were enriched and analysed through the KEGG signalling pathway, and finally, the statistical enrichment of differentially expressed genes in the KEGG pathway was analysed by using cluster profiler software.

Western blot analysis

After treating CNE1, CNE2 and C666-1 cells with 100 ng/mL vIL-10 for 24 h and 48 h, the cells were incubated in RIPA lysis buffer to obtain total cell protein. The protein concentration was determined using a BCA kit (Biyun, Shanghai, China). The protein samples were separated by SDS‒PAGE, transferred to a nitrocellulose membrane (PVDF membrane, Millipore), and then blocked with fresh 5% skim milk (Coolaber, Beijing, China) at room temperature for 2 h. The membrane was then mixed with primary antibodies against GAPDH (5619; Proteintech, China), IL-10R1 (Ab225820; Abcam, Cambridge, MA, USA), p-JAK1 (74129S; CST), JAK1 (66466; Proteintech, China), p-STAT3 (9145S; CST), and STAT3 (10253; Proteintech, China) and incubated overnight at 4°C. After washing the membrane with Tris-buffered saline and Tween-20 (TBST), the HRP-coupled secondary antibody was incubated at room temperature for 2 h. After washing, the proteins bands on the membrane were detected using an enhanced chemiluminescence detection reagent (Millipore). The expression levels of GAPDH, IL-10R1, p-JAK1, JAK1, p-STAT3 and STAT3 were normalized with ImageJ software (BIO-RAD, Harkles, CA).

ELISA

The level of vIL-10 secreted by NPC cells was detected by ELISA (Jiangsu Enzyme Immunoassay, China). Nasopharyngeal carcinoma CNE-2 and C666-1 cell cultures were collected in sterile centrifuge tubes and centrifuged at room temperature for 25 min at 3000 rpm, and the centrifuged supernatant was collected. Standard wells, 3 blank wells and 3 sample wells were set. The enzyme-labelled reagent was added, incubated, and adjusted to zero according to the absorbance of blank wells, and then the absorbance of each well was measured in turn. Attention was given to the absorbance measurement as soon as possible after the termination of the reaction, which was within 15 min to avoid inaccurate measurement. A standard curve was drawn according to the standard substance, and the vIL-10 concentration of each well was obtained from the absorbance of each well.

Immunofluorescence experiment

CNE-1, CNE-2 and C666-1 cells were placed in a 24-well plate with a 15 mm diameter cell cover slip, and the cells were cultured overnight to control the cell density to approximately 50%. The 24-well plate was removed from the cells after 100 ng/mL vIL-10 was applied to CNE-1, CNE-2, and C666-1 NPC cells for 24 h and 48 h. The culture medium was discarded, and 0.5 mL of fixative (Bi Yun Tian, China) was added to each well, and the cells were fixed at room temperature for 20 min. The fixative was discarded, and the cells were washed for 5 min with wash buffer (Bi Yun Tian, China) 3 times. Then, 0.5 mL of sealing solution (Bi Yun Tian, China) was added to each well, blocked for 60 min, and gently shaken using a shaker. The sections were incubated with primary antibody (IL-10R1 antibody Abcam; Ki67 antibody), secondary antibody (Elabscience, China) and DAPI (Solibao, China) and sealed. IL-10R1 and Ki67 immunofluorescence was photographed under an OLYMPUS microscope.

Cell colony formation experiment

CNE-1, CNE-2 and C666-1 NPC cells were seeded into 6-well plates at a concentration of 1000 cells per well to form colonies, with 3 wells per group. After 12 days of culture, the cells were fixed with methanol for 10 min, stained with 0.10% crystal violet (Solabel, China) solution, and then stained with clones containing more than 50 cells. The colony formation rate was observed under an OLYMPUS microscope.

Transwell cell migration and invasion experiment

For the invasion experiment, Matrigel (ABW, China) matrix gel was spread in a small Transwell chamber (Selection, China) to simulate the state of the extracellular matrix. The experimental group received 600 L of 100 N g/mL VIL-10 in 30% FBS RPMI-1640 complete medium in the lower chamber, while the blank group received 600 L of RPMI-1640 complete medium containing 30% FBS. Two hundred microlitres of CNE-1, CNE-2 and C666-1 NPC cell suspensions (5×104 cells) were added to the upper chamber. Attention was given to avoid bubble generation between the upper and lower chambers, which would affect the experimental results. There were 3 wells per group. The 24-well plate for the Transwell Migration Test was cultured under the original conditions for 24 h, and the 24-well plate for the Transwell Invasion Test was cultured under the original conditions for 36 h.

Hoechst 33258 staining was used to detect apoptosis

The cells were inoculated on a cell climbing plate at a cell density of 3×105 cells/mL and treated with 100 ng/mL vIL-10 for 24 h and 48 h. Then, the culture medium was removed, and 1 mL of 4% paraformaldehyde (Sorebo, China) was added to each well and fixed at room temperature for 30 min. The paraformaldehyde was discarded, the cells were rinsed 3 times with 1×PBS, and then the cells in each well were stained with 1 mL of Hoechst 33258 (MedChemExpress, USA) staining solution for 15 min. Afterward, the cells were rinsed 3 times with PBS for 3 min each. After sealing, the film was observed and photographed under a fluorescence microscope.

Flow cytometry

Apoptosis was detected by a flow cytometry kit (Biyun, China). CNE1, CNE2 and C666-1 NPC cells were placed in a 6-well plate and cultured normally in a constant temperature incubator. The cells were treated with 100 ng/mL vIL-10 for 24 h and 48 h. After digestion and centrifugation, 100,000 resuspended cells were taken according to the cell count and centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Then, 1× binding buffer (175 µL) was used to resuspend the cells. Non-dyed tubes, Annexin V-FITC single-dyed, PI single-dyed, NC, 24 h, and 48 h tubes were set. For Annexin V-FITC labelling, 5 µL of Annexin V-FITC was added and mixed well while avoiding light exposure. For PI labelling, 10 µL of PI staining solution was added while avoiding light exposure. The samples were placed on ice and incubated for 15 min. Before the operation, 400 µL of 1× binding buffer was added to each flow tube and mixed evenly. Apoptosis was detected by flow cytometry (BD, USA).

Statistical analysis

Statistical analysis was performed via Student’s t test using SPSS version 23.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 6.0 (GraphPad, La Jolla, CA, USA). The results are described as the mean ± SD. Differences were considered statistically significant at P < 0.05.

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)).

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.

NPC is a malignant tumour of the nasopharynx mucosa epithelium that mostly occurs in the top of the nasopharynx, followed by the lateral wall and pharyngeal recess. According to the summary released by the International Agency for Research on Cancer (IARC), in 2018, a total of 129,000 new cases of NPC occurred in 185 countries around the world, accounting for 0.7% of the total number of cancers diagnosed in these countries in that year. The number of deaths due to NPC was approximately 73,000, accounting for 0.8% of the total number of deaths due to cancers [15]. The incidence has geographical distribution differences; more than 70% of new cases are in East Asia or Southeast Asia. Moreover, the prevalence of EBV infection in the affected population indicates that NPC is caused by EBV infection, genetic susceptibility, environmental factors and other factors. Current studies indicate that there are three pathological types of NPC: keratinized squamous cell carcinoma, non-keratinized squamous cell carcinoma and basal-like squamous cell carcinoma. Non-keratinized NPC can be divided into differentiated tumours and undifferentiated tumours. Keratotic subtypes account for less than 20% of global cases and are relatively rare in endemic areas such as southern China. Non-keratinized subtypes constitute the majority of cases (> 95%) in epidemic areas and are associated with EBV infection in patients [16.17.18]. At present, EBV infection, host genetics, environmental factors and other factors are considered to be involved in the occurrence and development of NPC. The HLA gene located in the MHC region of chromosome 6p21 has been widely considered the major risk site leading to the development of NPC [19.20].

vIL-10 is a viral product similar to IL-10 encoded by the BCRF1 gene in EBV. vIL-10 is produced in the late stage of the EBV dissolution cycle. When the virus protein particles are translated and synthesized, the released virus particles can reinfect B cells, and the accompanying release of vIL-10 can inhibit the activity of host T cells and NK cells, thus promoting the infection of EBV on B cells. vIL-10 can reduce the recognition and attack of cytotoxic T lymphocytes on cancer cells, mainly by inhibiting the activation of NF-κB, nuclear transfer and binding ability with DNA in NPC, thus inhibiting the transcription of NF-κB-targeted genes, affecting the activation of cytotoxic CD8 + T cells, exerting an immunosuppressive effect and reducing antigen presentation [21]. HIL-10 produced in the incubation period of EBV-positive Burkitt lymphoma (BL) can function as an autocrine growth factor. However, the production of hIL-10 has the characteristics of both immunostimulation and immunosuppression and can carry out immune regulation. When the cells enter the virus lysis phase, vIL-10 has a unique immunosuppressive function, which can create conditions for virus propagation [22]. To identify potential targets of vIL-10 acting on NPC, single-cell transcriptome sequencing was used in this study. Through KEGG signalling pathway enrichment analysis, JAK-STAT was found to be the main signalling pathway, and JAK1 and STAT3 were upregulated after vIL-10 treatment.

Current research indicates that the JAK1/2 signalling pathway is involved in the development of various malignant tumours, including leukaemia, lymphoma and myeloproliferative tumours[23]. In NPC cells, the JAK2/STAT3 signalling pathway can promote tumour neovascularization by activating its downstream target gene, thus promoting tumour cell metastasis. STAT3, as a carcinogenic gene, participates in many cellular processes, including invasion, metastasis, angiogenesis, proliferation and immune escape. The increase in STAT3 activation plays a role in the progression and metastasis of NPC and is clinically associated with advanced NPC (stage III or IV). The increased expression of activated STAT3 is closely related to the poor prognosis of NPC patients[24].

By establishing a cell model, we explored the effects of vIL-10 on the proliferation, migration, invasion and apoptosis of NPC cells and the related pathways. vIL-10 is a secreted protein produced by EBV. By collecting the culture supernatant of the EBV-positive NPC cell line C666-1 and the EBV-negative NPC cell line CNE-2, the secretion of vIL-10 was detected by ELISA. The results showed that the level of vIL-10 secreted by C666-1 cells was significantly higher than that secreted by CNE-2 cells, suggesting that EBV-positive NPC cells could secrete vIL-10 spontaneously. Therefore, for patients with EBV-positive NPC, vIL-10 may play a role in promoting the onset of NPC in these patients.

The mobility and invasiveness of tumour cells as cancer progresses mainly depend on complicated biochemical and biological changes in the tumour cells and related matrix. However, the mechanism of tumour invasion and metastasis is still poorly understood. In this study, a Transwell migration experiment was conducted to explore the migration ability of NPC cells. The results showed that vIL-10 enhanced the migration and invasion of NPC cells after it was applied to NPC cells. This result suggests that when patients with NPC have EBV infection, their tumour migration and invasion ability may be stronger due to the secretion of vIL-10 by EBV.

In conclusion, this study preliminarily found that vIL-10 may be oncogenic in the development of NPC and affect its cell biological function, which may be closely related to the activation of the JAK1/STAT3 signalling pathway, providing a theoretical basis for the pathogenesis of EBV-positive NPC patients. However, as vIL-10 is a factor derived from EBV, it is difficult to construct NPC cell lines that knock down or overexpress vIL-10. Animal experiments investigating NPC cells in which the vIL-10 gene is knocked down or overexpressed have not been carried out, and in vivo experiments have not been conducted. This study mainly explored the possible mechanism of vIL-10 in the development of NPC at the cellular level.

Our research shows that vIL-10 can promote the proliferation, migration, and invasion, and inhibit the apoptosis of CNE1, CNE2 and C666-1 NPC cells; the above may be due to activating the JAK1/STAT3 signalling pathway. These findings provide a theoretical basis for the study of the mechanism of vIL-10 as an oncogene in the development of NPC.

Conflict of interest The authors declare that they have no confict of interest.

Informed consent

For this type of study, formal consent is not required.

Author Contribution

Author contributions Conception and design: QQ,XY,XL;Collection and assembly of data: YR, HN;Data analysis and interpretation: QQ,XY;Manuscript writing: All authors;Final approval of manuscript: All authors.

Acknowledgment

This research was funded by the National Natural Science Foundation of China (No.81960489 and No.82260485).Yunnan provincial health commission medical reserve talent training program (H-201634),Yunnan Ten Thousand People Plan“Young Top Talents”Project (YNWR-QNBJ-2019-056),General project of Yunnan basic research plan(202001AT070056), Science Research Fund Project of Yunnan Education Department in2019༈2019J1287༉.

Data availability

The authors will provide data included in the present research upon request.

DATA AVAILABILITY STATEMENT

Data used in this study are available from the corresponding author upon reasonable request.

Chen YP, Chan ATC, Le QT, et al. Nasopharyngeal carcinoma [J]. Lancet, 2019,394(10192):64-80.
Lee HM, Okuda KS, González FE, et al. Current Perspectives on Nasopharyngeal Carcinoma［J］． Adv Exp Med Biol，2019，1164:11-34．
Gong L, Kwong DL, Dai W, et al.Comprehensive single-cell sequencing reveals the stromal dynamics and tumor-specific characteristics in the microenvironment of nasopharyngeal carcinoma[J]. Nat Commun, 2021, 12(1): 1540. DOI: 10.1038/s41467-021-21795-z.
Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018; 68: 394–424.
Tsao SW, Tsang CM, Lo KW. Epstein-Barr virus infection and nasopharyngeal carcinoma. Philos Trans R Soc Lond B Biol Sci. 2017;372(1732):20160270. doi:10.1098/rstb.2016.0270
Fang W, Zhang J, Hong S, et al.EBV-driven LMP1 and IFN-γ up-regulate PD-L1 in nasopharyngeal carcinoma: Implications for oncotargeted therapy. ［J］． Oncotarget，2014，5 ( 23 ) :12189－12202
Moore KW, Vieira P, Fiorentino DF, et al. Homology of cytokine synthesis inhibitory factor (IL-10) to the Epstein-Barr virus gene BCRFI [published correction appears in Science 1990 Oct 26;250(4980):494]. Science. 1990;248(4960):1230-1234.
Chan LL, Cheung BK, Li JC, et al.A role for STAT3 and cathepsin S in IL-10 down-regulation of IFN-gamma-induced MHC class II molecule on primary human blood macrophages, J Leukoc Biol, 2010, 88(2):303-31.
García-Rocha R, Moreno-Lafont M, Mora-García ML, et al. Mesenchymal stromal cells derived from cervical cancer tumors induce TGF-β1 expression and IL-10 expression and secretion in the cervical cancer cells, resulting in protection from cytotoxic T cell activity, Cytokine,2015,76(2):382-390.
Li YF, Yang PZ, Li HF, et al. Functional polymorphisms in the IL-10 gene with susceptibility to esophageal, nasopharyngeal, and oral cancers. Cancer Biomark. 2016 ,16(4):641-651.
Yoon SI, Jones BC, Logsdon NJ, et al. Epstein-Barr virus IL-10 engages IL-10R1 by a two-step mechanism leading to altered signaling properties. J Biol Chem. 2012 ,287(32):26586-26595.
Alexandra Lucas, Grant McFadden.Secreted Immunomodulatory Viral Proteins as Novel Biotherapeutics. J Immunol 2004;173;4765-4774.
Roskoski R Jr. Janus kinase (JAK) inhibitors in the treatment of inflammatory and neoplastic diseases. Pharmacol Res. 2016 Sep;111:784-803.
Si Y, Xu J, Meng L, et al. Role of STAT3 in the pathogenesis of nasopharyngeal carcinoma and its significance in anticancer therapy. Front Oncol. 2022 Oct 13;12:1021179.
Ferlay J, Colombet M, Soerjomataram I, et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. [J].Int J Cancer. 2019;144(8):1941-1953.
Wang HY, Chang YL, To KF, et al. A new prognostic histopathologic classification of nasopharyngeal carcinoma. Chin J Cancer 2016; 35: 41.
Pathmanathan R, Prasad U, Chandrika G, et al. Undifferentiated, nonkeratinizing, and squamous cell carcinoma of the nasopharynx. Variants of Epstein-Barr virus-infected neoplasia. Am J Pathol 1995; 146: 1355–67.
Young LS, Dawson CW. Epstein-Barr virus and nasopharyngeal carcinoma. Chin J Cancer 2014; 33: 581–90.
Chua mLK, Wee JTS, Hui EP, et al. Nasopharyngeal carcinoma. Lancet 2016; 387: 1012–24.
Bei JX, Zuo XY, Liu WS, et al. Genetic susceptibility to the endemic form of NPC. Chin Clin Oncol 2016; 5: 15.
Ren YX, Yang J, Sun R M, et al. Viral IL-10 down-regμLates the" MHC-I antigen pro cessing operon" through the NF-кB signaling pathway in nasopharyngeal carcinoma cells [J].Cytotechnology,2016,68(6):2625-2636.
Ressing ME, Horst D, Griffin BD, et al. Epstein-Barr virus evasion of CD8(+) and CD4(+) T cell immunity via concerted actions of mμLtiple gene products. Semin Cancer Biol. 2008 ,18(6):397-408.
Roskoski Robert.Janus kinase (JAK) inhibitors in the treatment of inflammatory and neoplastic diseases.PHARMACOLOGICALRESEARCH.2016;111：784-803.doi:10.1016/j.phrs.2016.07.038
Si Yishimei, Xu Jinjing, Meng Linghan, et al.Role of STAT3 in the pathogenesis of nasopharyngeal carcinoma and its significance in anticancer therapy.Frontiers in oncology.2022;12：1021179.doi:10.3389/fonc.2022.1021179

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Viral IL-10 regulates nasopharyngeal carcinoma cell progression via the JAK1/STAT3 signalling pathway

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Abstract

Figures

Introduction

Materials and methods

Preparation of vIL-10 protein solution

Cell lines and cell cultures

Transcriptome sequencing

Western blot analysis

ELISA

Immunofluorescence experiment

Cell colony formation experiment

Transwell cell migration and invasion experiment

Hoechst 33258 staining was used to detect apoptosis

Flow cytometry

Statistical analysis

Results

Discussion

Conclusion

Declarations

Author Contribution

Acknowledgment

Data availability

DATA AVAILABILITY STATEMENT

References

Additional Declarations

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