IL-33在鼻咽癌中的作用及其潜在的分子机制

Role of IL-33 in nasopharyngeal carcinoma and its potential molecular mechanisms

目的探讨白细胞介素-33(IL-33)促进鼻咽癌恶化的作用,并研究其潜在的分子机制。方法选取2015年7月至2018年1月该院收治的78例鼻咽癌患者,同期收治的30例鼻咽黏膜慢性炎症患者作为对照。采用ELISA法检测鼻咽癌和鼻咽黏膜慢性炎症患者的血清IL-33水平。采用Western blot和免疫组化(IHC)分析鼻咽癌患者肿瘤组织及癌旁正常组织中IL-33的表达水平。成球实验观察重组IL-33蛋白对人鼻咽癌细胞株5-8F与CNE-2肿瘤干性的影响。Real-time PCR与Western blot检测重组IL-33蛋白对CNE-2细胞肿瘤干性基因NANOG、NOTCH3与OCT3/4的影响。Western blot检测重组IL-33蛋白对CNE-2细胞ERK、MAPK、NF-κB及c-Jun信号通路的影响。构建IL-33-BALB/c转基因小鼠,建立鼻咽癌小鼠模型,统计分析小鼠的肿瘤生长及生存曲线。Western blot检测小鼠肿瘤组织NANOG、NOTCH3、OCT3/4及磷酸化c-Jun的表达水平。流式细胞术检测小鼠肿瘤组织中CD68+F4/80+巨噬细胞数量。结果鼻咽癌患者的血清IL-33水平明显高于鼻咽黏膜慢性炎症患者(P<0.05)。鼻咽癌患者肿瘤组织IL-33水平明显高于癌旁正常组织(P<0.05)。重组IL-33蛋白能明显增加5-8F与CNE-2细胞的成球数量与大小(P<0.05)。重组IL-33能明显增加CNE-2细胞NANOG、NOTCH3与OCT3/4的mRNA及蛋白水平(P<0.05)。重组IL-33能明显增加c-Jun的磷酸化水平(P<0.05),使用JNK抑制剂能明显抑制重组IL-33对鼻咽癌细胞NANOG、NOTCH3与OCT3/4的作用(P<0.05)。IL-33转基因小鼠的肿瘤生长曲线与生存曲线均明显差于对照小鼠(P<0.05)。IL-33转基因小鼠肿瘤组织NANOG、NOTCH3、OCT3/4及磷酸化c-Jun水平均明显高于对照小鼠(P<0.05)。IL-33转基因小鼠肿瘤组织中CD68+F4/80+巨噬细胞数量明显高于对照小鼠(P<0.05)。结论鼻咽癌患者血清及肿瘤组织中IL-33水平均明显升高,IL-33可能通过调控巨噬细胞介导肿瘤干性从而促进鼻咽癌的恶化。

Objective To investigate the role of IL-33 in promoting nasopharyngeal carcinoma(NPC)deterioration and to explore its potential molecular mechanisms.Methods A total of 78 cases of NPC treated in this hospital from July 2015 to January 2018 were included.Contemporaneous other 30 cases of chronic inflammation of nasopharyngeal mucosa(CIONM)treated in this hospital were selected as the control.The serum levels of IL-33 in NPC and CIONM patients were determined by adopting ELISA.The expression levels of IL-33 in tumor and paracancerous normal tissues were determined by Western blot and immunohistochemical staining(IHC).The effect of recombinant IL-33 protein on the tumor stemness of human nasopharyngeal carcinoma cell lines 5-8F and CNE-2 was detected by the sphere formation assay.The effects of recombinant IL-33 on tumor stemness genes NANOG,NOTCH3 and OCT3/4 in CNE-2 cells were detected by the real time PCR and Western blot.Western blot was used to examine the effect of recombinant IL-33 on ERK,MAPK,NF-κB and c-Jun signal pathways.The IL-33-BALB/c transgenic mice were constructed,and the nasopharyngeal carcinoma mice models were established.The tumor growth and survival curves in mice were statistically analyzed.The expression levels of NANOG,NOTCH3,OCT3/4 and phosphorylated c-Jun in tumor tissues were detected by Western blot.The number of CD68+F4/80+macrophages in tumor tissues were detected by the flow cytometry.Results The level of serum IL-33 in NPC patients was significant higher than that in CIONM patients(P<0.05).The expression level of IL-33 in tumor tissues of the NPC patients was significant higher than that in paracarcinoma tissues(P<0.05).Recombinant IL-33 protein significantly increased the number and size of spheres in 5-8F and CNE-2 cells(P<0.05).Recombinant IL-33 increased the mRNA and protein level of NANOG,NOTCH3 and OCT3/4 in CNE-2 cells significantly(P<0.05).Recombinant IL-33 significantly increased the phosphorylation level of c-Jun(P<0.05),and using the JNK inhibitor significantly inhibited the effect of recombinant IL-33 on NANOG,NOTCH3 and OCT3/4(P<0.05).The tumor growth and survival curves of IL-33 transgenic mice were significantly worse than those of control mice(P<0.05).The level of NANOG,NOTCH3,OCT3/4 and phosphorylated c-Jun in tumor tissues of IL-33 transgenic mice were all significantly higher than those in the control mice(P<0.05).The number of CD68+F4/80+macrophages in tumor tissues of IL-33 transgenic mice was significant higher than that of the control mice(P<0.05).Conclusion The IL-33 levels in serum and tumor tissue of the patients with NPC increase significantly,and IL-33 may promote nasopharyngeal carcinoma deterioration via regulating tumor stemness mediated by macrophage.