干扰PAG1介导肿瘤相关巨噬细胞自噬状态提高放射抗拒喉鳞癌的放射敏感性研究  

Interfering with PAG1-mediated tumor-associated macrophage autophagy to improve radiosensitivity of radioresistance to laryngeal squamous cell carcinoma

作　　者：张鄂[1] 黄畅 费永光 岑瑞祥[3] 陈沛[1] ZHANG E;HUANG Chang;FEI Yongguang;CEN Ruixiang;CHEN Pei(Department of Otolaryngology Head and Neck Surgery,Wuhan First Hospital,Wuhan 430022,China)

机构地区：[1]武汉市第一医院耳鼻咽喉头颈外科,武汉430022 [2]赤壁市人民医院耳鼻咽喉头颈外科,赤壁437300 [3]鄂东医疗集团黄石市中心医院耳鼻咽喉头颈外科,黄石435000

出　　处：《中国免疫学杂志》2023年第1期131-138,共8页Chinese Journal of Immunology

基　　金：湖北省卫生健康委员会联合基金项目(WJ2019H459)。

摘　　要：目的:探究鞘糖脂微结构域1相关磷酸化蛋白(PAG1)在喉鳞癌(LSCC)组织中的表达,以及干扰肿瘤相关巨噬细胞PAG1表达对放射抗拒喉鳞癌细胞敏感性的影响。方法:收集武汉市第一医院收治的40例LSCC肿瘤组织及邻近癌旁组织,免疫组织化学染色和RT-qPCR检测PAG1表达;将人单核白血病细胞株THP-1诱导为M2型肿瘤相关巨噬细胞,通过脂质体介导法将PAG1-siRNA转染至M2型肿瘤相关巨噬细胞,RT-qPCR和蛋白质印迹法检测转染效果,ELISA法检测巨噬细胞分泌相关细胞因子IL-6、IL-12、IL-4及IL-10含量;建立肿瘤相关巨噬细胞与放射抗拒人喉鳞癌细胞Hep-2max的共培养体系,培养4 d后收集Hep-2max细胞,经8 Gy X射线照射24 h后,MTT法检测细胞活性,流式细胞术和TUNEL染色检测细胞凋亡,免疫荧光染色和蛋白质印迹法检测细胞自噬水平。结果:喉鳞癌肿瘤组织中PAG1蛋白和mRNA表达水平显著高于癌旁组织(P<0.01);THP-1细胞诱导后呈不规则多边形,且胞体有尾足伸出,CD86表达减少,CD206表达明显增加(P<0.01),提示成功诱导肿瘤相关巨噬细胞;在PAG1-siRNA转染肿瘤相关巨噬细胞后,细胞中PAG1 mRNA和蛋白相对表达量较对照组均显著下降(P<0.01),培养液上清中IL-6和IL-12水平升高,IL-4和IL-10水平降低(P<0.01);诱导后的肿瘤相关巨噬细胞转染PAG1-siRNA与Hep-2max共培养,再经过8 Gy X射线照射,细胞活性降低(P<0.05),细胞凋亡率升高(P<0.01),TUNEL阳性率升高(P<0.01),同时,LC3蛋白绿色荧光强度明显减弱,细胞中Beclin-1蛋白表达水平下降(P<0.01),P62蛋白表达水平升高(P<0.01),LC3-Ⅱ/LC3-Ⅰ下调(P<0.01)。结论:干扰肿瘤相关巨噬细胞中PAG1表达可改变放射抗拒人喉鳞癌细胞自噬状态,增强放射抗拒人喉鳞癌细胞的放射敏感性,诱导放疗后细胞凋亡发生。Objective:To investigate expression of phosphoprotein associated with glycosphingolipid microdomains 1(PAG1)in laryngeal squamous cell carcinoma(LSCC)tissue,and influence of interference with PAG1 expression in tumor-associated macrophages on sensitivity of radioresistance to laryngeal squamous cell carcinoma cells.Methods:Forty cases of LSCC tumor tissues and adjacent tissues in Wuhan First Hospital were collected,expression of PAG1 was detected by immunohistochemical staining and RTqPCR;induced human monocytic leukemia cell line THP-1 into M2 tumor-associated macrophages,transfection of PAG1-siRNA into M2 tumor-associated macrophages by liposome-mediated method,RT-qPCR and Western blot method to detect transfection effect,ELISA to detect levels macrophages related cytokines IL-6,IL-12,IL-4 and IL-10;a co-culture system of tumor-associated macrophages and radiation-resistant human laryngeal squamous cell carcinoma Hep-2max was established.After 4 days of culture,Hep-2max cells were collected,and after 8 Gy X-ray irradiation for 24 h,MTT method was used to detect cell viability,flow cytometry and TUNEL staining were used to detect cell apoptosis,immunofluorescence staining and Western blot were used to detect level of autophagy.Results:Expression levels of PAG1 protein and mRNA in tumor tissue of laryngeal squamous cell carcinoma were significantly higher than that in adjacent tissues(P<0.01);after THP-1 cells were induced,they showed irregular polygons,and the cell bodies had tailfoot protruding.Expression of CD86 was reduced,while expression of CD206 was significantly increased(P<0.01),indicating that tumor-associated macrophages were successfully induced;after PAG1-siRNA transfection of tumor-associated macrophages,relative expression of PAG1 mRNA and protein in cells were significantly lower than those in control group(P<0.01),levels of IL-6 and IL-12in supernatant of culture medium were increased,while levels of IL-4 and IL-10 were decreased(P<0.01);the induced tumor-associated macrophages were transfected w

关 键 词：鞘糖脂微结构域1相关磷酸化蛋白 喉鳞癌 自噬 凋亡 放射敏感性 

分 类 号：R739.65[医药卫生—肿瘤]