| Background Interleukin 35(IL-35) is a newly found cytokine of IL-12 cytokine family. As IL-35 plays important roles in auto-immune diseases, inflammation diseases and some tumors, it could be used as a potent therapy target. In the previous study we found that compared with normal tissues and cell lines, pancreatic tissue and cells had high IL-35 expression. We designed this experiment to further clarify the IL-35 expression pattern in pancreatic cancer tissues and cells; to explore the function of tumor_derived IL-35 in metastasis and progression process of pancreatic cancer and the underlying regulation mechanism from the clinical level, cell level and animal model level.Method 1. Immunohistochemistry staining analysis in consecutive sections of paraffin-fixed pancreatic tissue was carried out to detect the two subunits of IL-35(p35 and EBI3) and the two IL-35 receptor subunits(IL12rβ2 and gp130). The expression pattern of IL-35 and its receptor and their correlation was analyzed is SPSS software. The clinical data including the follow-up information was collected.The relationship between the expression levels of IL-35 and its receptor with clinicopathological parameters was analyzed.2. Western blot, RT-PCR and ELISA assays were used to detect the expression levels of IL-35 and its receptor in five pancreatic cancer cell lines, Treg cells(as a positive control) and normal pancreatic ductal cell line. Immunofluorescence was used to detect the cellular localization of IL-35 and its receptor. Co-immunoprecipitation(COIP) was used to explore the bingding mode of the two subunits of IL-35 in pancreatic cancer cells.3. The IL-35-overexpressing and IL-35-sh RNA plasmid was constructed. Lentiviral stable transfection system was used to establish stably overexpressing or downexpressing IL-35 cell lines. Western blot, RT-PCR and ELISA assays was used to verify the IL-35 expression levels in the stable cell lines. EDU proliferation assay was employed to confirm the biological function of IL-35 secreted by the stable cell lines.4. Umbilical vein endothelial cells(HUVEC) adhesion assays and transendothelial migration(TEM) assays was used to study the role of IL-35 in promoting metastasis of pancreatic tumor cells in vitro.5. Western blot, RT-PCR and immunofluorescence assay was applied to detect the JAK-STAT pathway activation process. The transcriptome sequencing was performed to screen the target genes by which IL-35 promotes tumor metastasis. Then Western blot and RT-PCR experiments was done to confirm the screening results. Blocking tests and bottleneck experiments was used to further identify the targets of IL-35 in promoting tumor metastasis.6. Orthotopic pancreatic tumor model in NOD-SCID mouse was used to validate the role of IL-35 in promoting pancreatic cancer cell metastasis in vivo.The results 1. Through the immunohistochemical staining in the 123 cases of pancreatic tissue we found that in a variety of pancreatic histological type the expression of two subunits of IL-35(p35 and EBI3) was positively expressed, while the corresponding normal pancreatic tissues do not express or weakly expressed IL-35. The correlation analysis was performed and found that the distribution of p35 and EIB3 was significantly correlated(r = 0.950; p <0.0001). Both high p35 and EBI3 expression was defined as high IL-35 expression, otherwise were low IL-35 expression. As a result, high expression of IL-35 accounted for 48.8%(60 cases). We further explore the relationship between IL-35 expression and clinicopathological parameters and found that high levels of IL-35 were associated with advanced TNM stage, positive lymph node involvement and positive vascular invasion(P values were 0.01, 0.027 and 0.007, respectively). We found that higher levels of IL-35 expression in patients had poorer overall survival(median: 13.70 ± 0.722 months vs 18.5 ± 1.50 months) and poorer relapse-free survival(median: 11.39 ± 0.54 months vs 16.87 ± 2.00 months); Univariate and multivariate analysis in Cox risk model was performed and found that IL-35 expression levels were independent predictors of overall survival and relapse-free survival.The two subunits of IL-35 receptor gp130 and IL12rβ2 were positively expressed in pancreatic cancer, whereas the expression levels of the two is not always consistent. There was a significant correlation between IL-35 ligand and receptor expression levels(r = 0.384, P <0.0001). When grouped the cases according to IL-35 ligand and receptor level into four groups, we found that patients in IL-35(high) IL-35 receptor(+) group had the poorest overall survival and relapse-free survival.2. RT-PCR, Western blot, immunofluorescence and ELISA assays were performed and found IL-35 was positively expressed in all the five pancreatic cancer cell lines(PANC-1, MIA Pa Ca-2, CFPAC-1, As PC-1 and Bx PC-3). COIP experiments was carried out in MIA Pa Ca-2 and CFPAC-1 cells and confirmed that the two subunits of IL-35 bind directly.3. Two IL-35-overexpressing pancreatic cancer cell lines(PANC-1 and Bx PC-3) and two IL-35-downexpressing pancreatic cancer cell lines(MIA Pa Ca-2 and CFPAC-1) were successfully constructed. The IL-35 expression levels of these cell lines were detected by Western blot, RT-PCR and ELISA assays. The biological function of IL-35 in the constructed IL-35-overexpressing cells was comfirmed by EDU proliferation assays in vitro experiments.4. In vitro functional assays demonstrated that overexpression of IL-35 enhanced the HUVEC adhesion and TEM capacity, while downexpression of IL-35 reduced the HUVEC adhesion and TEM capacity.5. After IL-35 stimulation JAK-STAT signaling pathway was activated in pancreatic cancer cells, with elevated phosphorylation level of STAT1 and STAT4. And the accumulation of p-STAT1 and p-STAT4 in the nucleus was noticed in the immunofluorescence assays. After transcriptome sequencing we found that after IL-35 stimulation the expression level of intercellular adhesion molecule 1(ICAM1) were significantly upregulated, which was further verified by Western blot experiments. Bottleneck experiments was performed and found that when the ICAM1 was knockdown the increased HUVEC adhesion and TEM capabilities of the IL-35-overexpressing stable cells no longer existed. Preliminarily treating HUVEC with ICAM1 neutralizing antibody could partially blocked the incresed adhesion capability to HUVEC by IL-35 overexpression.6. Compared with control cells, IL-35-over-expressed Pan02 cells can form significantly more metastases in mice omentum, peritoneum and live. Pan02 cells with IL-35 overexpression had much less metastasizes. The bottleneck experiments in vivo found that knockdown of ICAM1 expression in Pan02 cells with over-expressed IL-35 would reduce the number of metastases significantly.Conclusions 1. IL-35 and its receptor are positively expressed in pancreatic cancer tissues. IL-35 high expression levels associated with poor prognosis.2. IL-35 protein and its receptor are positively expressed in five pancreatic cancer cell lines.3. IL-35 overexpression will activate JAK-STAT signaling pathway, upregulated ICAM1, thereby enhance the ability of HUVEC adhesion and TEM in pancreatic cancer cells. By promoting tumor cell adhesion to endothelium, IL-35 can enhance the metastasis ability of pancreatic cancer cells. |
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