信号传导及转录激活子3通路参与卷烟烟气凝集物诱导永生化人支气管上皮细胞的恶性转化

STAT3 signaling pathway participates in malignant transformation of Immortalized human bronchial epithelial cells induced by cigarette smoke condensate

目的检测肺鳞癌组织、鳞状细胞不典型增生组织和癌旁组织中磷酸化信号传导及转录激活子3(p STAT3)蛋白的表达,比较其表达与吸烟的关系;探讨STAT3通路在烟草诱导细胞恶性转化过程中的作用。方法免疫组化法检测288例肺鳞癌组织、108例鳞状细胞不典型增生组织、112例癌旁组织中p STAT3蛋白的表达,比较其表达与吸烟的相关性。构建卷烟烟气凝集物(CSC)诱导第10,20,30,40,50,60及70代细胞(P10,P20,P30,P40,P50,P60及P70)永生化人支气管上皮细胞(BEP2D)恶性转化模型。从血清抗性及锚着独立性等方面对CSC诱导各代BEP2D细胞的恶性转化特征进行鉴定。Western蛋白印迹法检测CSC诱导各代BEP2D细胞中p STAT3的表达。MTT法和流式细胞术分别检测JSI-124 0.25~10μmol·L^(-1)对P70细胞存活及凋亡的影响。JSI-124处理CSC诱导P70 24 h后检测存活蛋白表达的变化。结果 p STAT3蛋白在肺鳞癌组织中表达高于鳞状细胞不典型增生和癌旁组织(P<0.05);p STAT3在目前吸烟者中的表达均高于曾经吸烟者和从不吸烟者(P<0.05),且随着吸烟指数增加两者表达水平逐渐升高(P<0.01)。随着转化代数的增高,细胞血清抗性增加,锚着独立性增强,P30细胞以后尤为明显。p STAT3在正常对照组和乙醇对照组中弱表达,且两者之间无显著性差异;CSC诱导的各组BEP2D细胞中,p STAT3的表达均显著高于正常对照组和乙醇对照组(P<0.05),并随细胞代数的增高而增高。JSI-124抑制P70细胞增殖、促进P70细胞凋亡呈浓度及时间依赖性。JSI-124 1μmol·L^(-1)作用P70细胞24 h后,存活蛋白表达显著降低(P<0.05)。结论 STAT3信号通过调控存活蛋白表达抑制细胞凋亡,参与烟草诱导永生化人支气管上皮细胞的恶性转化。

OBJECTIVE To detect the expression of p STAT3 protein in lung specimens of squamous cell carcinoma,squamous dysplasia and normal bronchial epithelial tissues,analyze the relationship between its expression and smoking,and to explore the role of STAT3 signaling in the process of lung cancer induced by smoking. METHODS p STAT3 protein expression was assessed by immunohistochemistry in 288 samples of lung specimens of squamous cell carcinoma,108 samples of squamous dysplasia and 112 samples of normal bronchial epithelial tissues. The relationship between its expression and smoking was analyzed. Immortalized human bronchial epithelial cells induced by CSC were divided to 9 groups. Malignant transformation was assessed by resistance to serum-induced terminal differentiation and anchorage-independence growth. p STAT3 protein expression was detected by Western blotting. The proliferation and apoptosis of P70 treated with JSI-124 0.25-10 μmol·L^（-1）were explored by MTT assay and flow cytometry,respectively. Survivin expression in P70 treated with JSI-124 was detected by reverse transcription PCR and Western blotting,respectively. RESULTS Expression of p STAT3 protein showed a significant upward trend from normal bronchial epithelial tissues were compared to squamous dysplasia and lung specimens of squamous cell carcinoma. p STAT3 expression wa closely relate to the smoking exposure of patients. With the increase in transformation generation,the resistance to serum-induced terminal differentiation and anchorage-independence growth were enhanced,especially after P30. p STAT3 protein expression in normal untreated control and alcohol-treated control groups had no significant difference. p STAT3 protein expression in BEP2 D induced by CSC was higher than that in the two control groups（P〈0.05）and their expression increased gradually with the increase in transformation generation. JSI-124 inhibited proliferation and promoted apoptosis in a concentrationand time-dependent manner. The expression of survivin m RNA and protein in P70 treated with JSI-1241 μmol·L^（-1）was significantly higher than that in DMSO group（P〈0.05）. CONCLUSION STAT3 signaling may participate in the malignant transformation of human bronchial epithelial cells induced by tobacco by inhibiting apoptosis of cells through regulation of survivin.