Expression analysis of autophagy protein and mTOR protein in oral squamous cell carcinoma
张斐 1高华 2翟兴华 3苏明 4阴旭斌3
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作者信息
1. 100070,首都医科大学附属北京天坛医院口腔科;321200,浙江省金华市武义县口腔医院
2. 100070,首都医科大学北京市神经外科研究所
3. 100070,首都医科大学附属北京天坛医院口腔科
4. 100050,首都医科大学附属北京口腔医院
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摘要
目的 比较口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)患者肿瘤及其临近组织的自噬相关蛋白和哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)蛋白表达水平,并探讨自噬蛋白与mTOR通路蛋白表达的相关性.方法 26例肿瘤标本来源于首都医科大学附属北京口腔医院确诊为OSCC并行病灶切除手术的患者,将标本组织分为3组,其中肿瘤组为肿瘤中心区域组织,正常组为距肿瘤组织边缘2 cm的正常组织,两者之间的组织为交界组.采用免疫荧光实验和蛋白免疫印迹实验检测各组样本的自噬相关蛋白微管蛋白1轻链3(microtubule associated protein 1 light chain 3,LC3)Ⅱ和p62的表达水平,采用免疫组织化学染色方法和蛋白免疫印迹实验检测mTOR通路相关蛋白的表达水平,并通过Spermann相关性分析方法探讨自噬相关蛋白与mTOR通路相关蛋白表达水平的相关性.结果 免疫荧光实验结果显示,肿瘤组和交界组LC3-Ⅱ和p62的表达水平均高于正常组.蛋白免疫印迹实验结果亦表明,肿瘤组和交界组中LC3-Ⅱ和p62的表达水平均高于正常组(P<0.001),而肿瘤组与交界组LC3-Ⅱ和p62的表达水平之间的差异并无统计学意义(P>0.05).免疫组织化学结果显示,交界组和肿瘤组的mTOR、4E-BP1的表达及其磷酸化4E-BP1水平均高于正常组.蛋白免疫印迹实验结果表明,交界组mTOR和4E-BP1及其磷酸化水平最高,肿瘤组次之,正常组最低(P<0.01);而p70S6K及其磷酸化水平在各组间比较,差异无统计学意义(P>0.05).Spermann相关性分析结果显示,LC3-Ⅱ与p62的表达水平在肿瘤组和交界组中均呈正相关(P<0.05),mTOR与LC3-Ⅱ和p62的表达水平在肿瘤组和交界组中均呈正相关(P<0.05).结论 OSCC患者的肿瘤中心区域和肿瘤与正常组织交界区域的mTOR通路活跃而自噬流过程受损,靶向抑制mTOR通路为OSCC患者的临床诊疗提供新的建议.
Abstract
Objective To compare the expression levels of autophagy-related proteins and mTOR pathway proteins in tumors and their adjacent tissues of patients with oral squamous cell carcinoma (OSCC) and to explore the correlation between autophagy proteins and mTOR pathway protein expression. Methods Twenty-six specimens were collected from patients diagnosed with OSCC and underwent resection of the lesions in Beijing Stomatology Hospital,The specimen tissues were divided into three groups,in which the tumor group(T) was the tissues in the central area of the tumor,the normal group(N) was the normal tissues 2 cm away from the edge of the tumor lesion,and the junction tissues (margin group M) were the tissues in between the two groups. Immunofluorescence assay and protein immunoblotting assay were used to detect the expression levels of autophagy-related proteins LC3-Ⅱ and p62 in each group of samples,Immunohistochemical staining method and protein immunoblotting assay were used to detect the expression levels of mTOR pathway-related proteins,and to explore the correlation between the expression levels of autophagy-related proteins and mTOR pathway-related proteins by the Spermann' s correlation analysis method. Results The findings of immunofluorescence experiments showed that the expression levels of LC3-Ⅱ and p62 were higher in both the tumor group and the junction group than in the normal group. Significantly,the expression levels of LC3-Ⅱ and p62 were higher than those of the normal group in both the junction group and the tumor group (both P<0.001),and the differences between the tumor group and the junction group were not statistically significant (P>0.05). Immunohistochemistry results showed that the expression of mTOR and 4E-BP1 and their levels of phosphorylated 4E-BP1 were higher in the tumor and margin groups than in the normal group. The results of protein immunoblotting experiments also showed that the levels of mTOR and 4E-BP1 and their phosphorylation were the highest in the margin group,followed by the tumor group,and the lowest in the normal group (all P<0.01);whereas p70S6K and its phosphorylation levels did not differ statistically between groups (P>0.05). Spermann correlation analysis showed that the expression levels of LC3-Ⅱ and p62 were positively correlated in both tumor and margin groups (both P<0.05),and mTOR was positively correlated with the expression levels of LC3-Ⅱ and p62 in both tumor and margin groups (both P<0.05). Conclusions OSCC patients have active mTOR pathway and impaired autophagic flow process in the central region of the tumor and the tumor-normal tissue junction region,and targeted inhibition of the mTOR pathway provides a new proposal for the clinical management of OSCC patients.
关键词
口腔肿瘤/癌,鳞状细胞/自噬相关蛋白类/哺乳动物雷帕霉素靶蛋白/信号通路/机制
Key words
Mouth Neoplasms/Carcinoma,Squamous Cell/Autophagy-Related Proteins/Mammalian target of rapamycin/Signal pathway/Mechanisms
维普
万方数据