Expression Of IL-35 In Oral Squamous Cell Carcinoma And Its Effect In Tumor Microenvironment

Part Ⅰ Expression and clinical significance of IL-35 in OSCCObjectiveTo detect the expression of Interleukin-35（IL-35）subunits IL12A（IL-12 alpha）and EBI3（Epstein – Barr virus induced gene 3）in oral squamous cell carcinoma and their relationship with clinicopathological features and prognosis of patients of oral squamous cell carcinoma.Materials and Methods1、 186 cases of oral squamous cell carcinoma received and diagnosed in the Department of pathology of the Affiliated Stomatological Hospital of Wuhan University from 2008 to 2012 were collected for immunohistochemical detection.2、 The stained sections were scanned into digital pathological images.Four parts of IL12 A and EBI3 stained sections were selected for analysis by using Aprio Image Scope software,and the results were recorded and sorted out.3、 Using TCGA database,the transcriptome and clinical information of all patients with head and neck squamous cell carcinoma were downloaded,and a total of 494 cases were sorted out for follow-up analysis.4、 Chi square test was used for the obtained data,Kaplan-Meier method was used for survival analysis,Spearman analysis was used for the correlation between the expression of IL12 A and EBI3 in oral squamous cell carcinoma,P < 0.05 was considered statistically significant.5、 qRT-PCR and Western Blot were used to detect the expression levels of IL12 A and EBI3 in HIOEC、Cal27、HSC3、SCC25、SCC23 and EBI3 knockdown efficiency.6、 HSC3-sh EBI3 cell lines were constructed by lentiviral packaging system,and the knockdown efficiency of IL12 A and EBI3 genes were examined.7、 CCK-8 experiment was used to evaluated the effects of EBI3 knockdown on the proliferation of OSCC cell line.Result1、 IL12 A and EBI3 were widely expressed in tumor tissues and cells,and the expression was significantly higher than that in normal epithelium（P < 0.001）.In the normal epithelium close to the tumor,some cells expressed IL12 A and EBI3,and the expressions were enhanced in the abnormal epithelium.The expression partner of IL12 A and EBI3 in lymph node metastases was similar to that in primary lesions,and there was no significant difference in the expression level.2、 The expression of IL12 A and EBI3 in Cal27,HSC3,SCC25 and SCC23 cell lines was significantly higher than that in HIOEC,IL12 A increased by 5.87 and 6.49 times in HSC3 and SCC23 cell lines,respectively;EBI3 increased in Cal27,HSC3,SCC25 and SCC23 cell lines by 1.24,1.58,1.30 and 1.61 respectively.3、 The expression of IL12 A and EBI3 in oral squamous cell carcinoma was positively correlated（P < 0.01,r = 0.45）.4、 The 5-year survival rate was lower in the high expression group of IL12 A and EBI3（P < 0.05）.TCGA data analysis showed that the ten-year disease-free interval was shorter in the high expression group of IL12 A and EBI3（P < 0.05）.5、 The expression of IL12 A and EBI3 in oral squamous cell carcinoma was significantly different in pathological grade（P < 0.05）;There was no significant difference in gender,age,tumor diameter,lymph node metastasis,distant metastasis and clinical stage.6、 In HSC3 EBI3 knockdown cell line,IL12 A decreased by 87% and 58%,and EBI3 decreased by 82% and 71%,respectively.The results of CCK-8 experiment showed that the proliferative activity of HSC3-EBI3-sh1 /sh2 knockdown group decreased by 37% and 35% compared with the control group（P < 0.001）.ConclusionThe expression of IL-35 subunits IL12 A and EBI3 increased and correlated in oral squamous cell carcinoma.The expression of IL12 A and EBI3 was positively correlated with the pathological grade of patients,and were poor prognostic factors for oral squamous cell carcinoma.Part Ⅱ The effect of IL-35 on tumor microenvironment of OSCCObjectivePrevious experimental results showed that the high expression of IL12 A and EBI3 were related to the poor prognosis of oral squamous cell carcinoma.By studying the effects of IL12 A and EBI3 on the tumor microenvironment of oral squamous cell carcinoma,to explore the possible mechanism of IL-35 affecting the prognosis of oral squamous cell carcinoma.Materials and Methods1.Use timer2 0 database to calculate the effects of IL12 A and EBI3 expression on the infiltration of T cells,B cells,macrophages,dendritic cells and neutrophils.2.The correlation between IL12 A and EBI3 and PD1 / PD-L1 was analyzed by TIMER database.3.CD4 + T cells were stained and analyzed;Neutrophils were observed and counted in digital sections.4.SPSS 21.0 statistical software was used to analyze the data,the chi square analysis was used for counting data,and Kaplan-Meier method was used for survival analysis.Spearman analysis was used for correlation analysis.P < 0.05 was considered to be statistically significant.Result1.TIMER2.0 database data show that high expression of IL12 A will reduce the proportion of CD8 + T cells and B cells,and increase the proportion of CD4 + T cells and neutrophils.The high expression of EBI3 will reduce the proportion of CD8 + T cells and neutrophils,and increase the proportion of CD4 + T cells,DC cells and neutrophils.2.The correlation coefficients between IL12 A and PD1 / PD-L1 were 0.438 and 0.236,respectively,P < 0.01;The correlation coefficients between ebi3 and PD1 /PD-L1 were 0.701 and 0.361,respectively,P < 0.01.3.Immunohistochemical analysis of 186 cases of oral squamous cell carcinoma in the early stage of the research group showed that EBI3 was significantly positively correlated with the degree of neutrophil infiltration（P < 0.0001,r = 0.311）;There was a weak positive correlation between IL12 A and CD4 + T cell infiltration（P = 0.0316,r = 0.254）.4.According to the IHC expression of IL12 A and EBI3 and the degree of infiltration of neutrophils（N）and CD4 + T cells in tumor tissues,samples were divided into high expression or high infiltration group（+）and low expression or low infiltration group（-）.The different groups of EBI3 and neutrophils were combined and classified,and then the prognosis was analyzed.The results showed that the group with high neutrophil infiltration and low EBI3 expression had the best prognosis,and there was no significant difference among the other three groups.The prognosis of different groups of IL12 A and CD4 + T was analyzed after combination and classification.The data of tissue microarray showed that the prognosis of CD4 + T cell high infiltration and IL12 A high expression group（CD4 + T + IL12 A +）was the worst（P < 0.0001）.ConclusionIL12A and EBI3,two subunits of IL-35,affect the infiltration of immune cells in oral squamous cell carcinoma and the prognosis of oral squamous cell carcinoma.