| Objective:The incidence of Laryngeal squamous cell carcinoma(LSCC)and hypopharyngeal squamous cell carcinoma(HSCC)is significantly higher than other regions in our country,especially in the northeast.Their poor prognosis seriously affects the quality of patients'life.The therapeutic methods of LSCC(HSCC)mainly including surgery,radiotherapy and radiotherapy have been improved.However,the 5-year survival rate of patients has not improved.Therefore,it has always been a hot topic for researchers to explore the mechanism of tumor development and find new therapeutic methods to optimize the available treatment strategies.The study found that the occurrence of tumor is the result of immune escape,which provides us with a new idea that we can obtain promising results by activating the immune response in vivo.The development of the tumor is closely related to organism immune microenvironment,in which the infiltration of lymphocytes plays an important role in the anti-tumor process.Programmed death-1(PD-1)and programmed death ligands-1(PD-L1)are important immunological checkpoints,which can inhibit the activation of T cells and participate in the immune escape of tumors.Apurinic/apyrimidinic endonuclease 1(APE1)is a functional protein with redox function,which can regulate various transcription factors and the expression level of different proteins.Multiple researches reported that APE1 and PD-L1 participated tumor infiltraion,metastasis and prognosis.In the first part of this study,we firstly examined the number of CD3~+T lymphocytes in the tissues of LSCC(HSCC)at random high magnification,so as to understand the infiltration of T lymphocytes in LSCC(HSCC).And then,we detected the expression of PD-1/PD-L1 at protein level and their clinical significance.The second part was to detect the expression of PD-L1 and APE1 at mRNA level in LSCC and analyze their correlation,and to preliminary explore the interaction mechanism of APE1 and PD-L1 in Hep-2 cells by siRNA transfection technique.Methods:We collect the tumor samples from 70 patients with laryngeal and laryngopharynx squamous cell carcinoma(treated by surgery in the Department of Otorhinolaryngology,affiliated to Shengjing Hospital of China Medical University,from2013 to 2015 in the first part of the study(60 of them were LSCC and 10 of them were HSCC),35 adjacent carcinoma tissues(1 cm over the margin of the tumor)of which were randomly selected as control group.The the number of cells with positive expression of PD-1,PD-L1 and CD3 in laryngeal carcinoma and hypopharyngeal carcinoma tissues were detected by immunohistochemical method.The number of apoptotic cells was detected by TUNEL staining.And we analyzed the relationship between the result of them with clinical parameters.In the second part,we extrated the total RNA of LSCC and adjacent tissues and detected the expression of PD-L1 and APE1 at mRNA level in LSCC and adjacent carcinoma tissues through real-time quantitative PCR.The correlation between the two mRNA expression were analyzed.We designed and synthesized siRNA to silence the expression of APE1 in Hep-2 cells by liposome lipo2000 transfection technique.We also interved Hep-2 cells with LPS(NF-?B agonist)and PDTC(NF-?B inhibitor).Western blot was used to detect the protein expression of APE1?PD-L1?NF-?B?pNF-?B.The changes of cell cycle and apoptosis after transfection were analyzed by flow cytometry.Results:1.Immunohistochemical results showed that PD-1 protein was mainly expressed in tumor infiltrating T lymphocytes.PD-L1 protein was mainly expressed in tumor cells,while only a few was expressed in tumor infiltrating lymphocytes.Both of the two proteins were located in cytomembrane.In addition,a small amount of PD-L1was expressed in the cytoplasm of the tumor cells.The expression level of both the protein in LSCC and HSCC was significantly higher than that in adjacent carcinoma tissues(P<0.05).The expression of PD-L1 was related to the location of the tumor(P<0.05)without relationship to the age,sex,smoking history,LN metastasis and TNM stage of the patients(P>0.05).The expression level of PD-L1 in glottic and subglottic carcinoma was significantly higher than that of supraglottic carcinoma(P<0.05).At the same time,the PD-L1 expression in supraglottic and subglottic carcinoma was significantly higher than that of HSCC.PD-1 expression has relationship to LN metastasis and TNM stage(P<0.05).There was no correlation between the expression of PD-1 and the age,sex,smoking history,the location of tumor and even pathological differentiation(P>0.05).The infiltration degree of PD-1+T cells in LSCC(HSCC)with lymph node metastasis was significantly higher compared with that in tissues without lymph node metastasis(P<0.01).?-?phase of LSCC(HSCC)tissues'PD-1+T cell infiltration degree was significantly higher than that of?-?phase(P<0.05).There was a positively correlation betwenn the protein expression of PD-1and PD-L1 in human laryngeal carcinoma(r~2=0.29,P<0.01).CD3~+represented the infiltration degree of mature T lymphocytes in LSCC(HSCC).But the level of infiltration was not statistically significant with patients'age,sex,smoking history,tumor location,and degree of differentiation(P>0.05).Tunel results was aimed to show the relationship between the number of apoptotic cells and TNM stage,lymph node metastasis,pathological classification.There was no statistically significant difference between the two groups by t test(P=0.172,P=0.084,P=0.155).2.The mRNA expression of APE1,PD-L1 in Laryngeal cancer tissues was significantly higher than that in adjacent mucosa tissues(P<0.05).There was a significant positive correlation between the APE1 and PD-L1(r=0.37,P<0.01).Hep-2 cells transfected by APE1-siRNA significantly inhibited the protein expression of APE1,NF-?B,pNF-?B,PD-L1(P<0.05),however cell apoptosis rate was increased.In addition,in a certain range concentration of LPS and PDTC intervention,PD-L1 and NF-?B protein expression was gradually increased,while the protein expression was decreased treated by PDTC in a certain range concentration(P<0.05).We treated the cell with LPS after silenced APE1 intervention.NF-?B and PD-L1 protein expression was significantly decreased compared with both the control group and LPS group(P<0.05).Conslusions:1.The infiltration degree of PD-1~+T cells in human LSCC(HSCC)is closely related to the occurrence and development of the carcinoma.There was no clear correlation between the infiltration degree of mature T lymphocyte(CD3~+)infiltration and the development of LSCC(HSCC).2.The protein expression of PD-L1 in glottic and subglottic carcinoma was significantly higher compared with supraglottic carcinoma and hypopharyngeal squamous cell carcinoma.It prompts that there may exist a relatively active tumor immune microenvironment in the glottic area and the subglottic area in LSCC tissues,and we could get a better prognosis using immunotherapy such as immune checkpoint inhibitors in glottic caicinoma and the subglottic carcinoma,compared with supraglottic carcinom and HSCC.3.The results of the study on Hep-2 cells showed the APE1 could regulate the expression of PD-L1 through NF-?B signaling pathway.The combined detection of PD-1/PD-L1 and APE1 expression has a predictive value in the treatment of immunocheckpoint inhibitors in LSCC. |
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