摘要
目的探讨CrkⅡ表达水平对喉癌细胞Hep-2增殖、迁移及侵袭的影响。方法采用免疫组化法检测CrkⅡ在108例喉癌患者喉癌组织及癌旁正常组织中的表达情况,分析不同临床特征喉癌患者喉癌组织中CrkⅡ蛋白的阳性表达情况。构建并验证CrkⅡRNA干扰质粒,将培养后的Hep-2细胞分为空白对照组(未转染质粒)、阴性对照组(转染阴性对照质粒)和实验组(转染CrkⅡ干扰质粒),细胞转染后行实时聚合酶链反应(PCR)扩增。采用蛋白质印迹法(Western blot)检测转染后Hep-2细胞中CrkⅡ的表达水平,噻唑蓝(MTT)法检测细胞的增殖情况,Transwell实验检测细胞的侵袭和迁移能力,细胞划痕实验检测细胞的迁移情况。结果喉癌组织中CrkⅡ蛋白的阳性表达率明显高于癌旁正常组织(P﹤0.01)。临床分期为Ⅲ~Ⅳ期、组织分化程度为中低分化、有淋巴结转移喉癌患者喉癌组织中CrkⅡ蛋白的阳性表达率均高于临床分期为Ⅰ~Ⅱ期、组织分化程度为高分化、无淋巴结转移的喉癌患者(P﹤0.05)。实验组喉癌细胞Hep-2中CrkⅡmRNA和蛋白的相对表达量均低于阴性对照组、空白对照组(P﹤0.05)。实验组穿过基质膜至下室的细胞数目低于阴性对照组和空白对照组(P﹤0.05)。转染48 h时,实验组细胞的迁移率低于阴性对照组、空白对照组(P﹤0.05)。细胞培养24、48、72 h时,实验组Hep-2细胞的光密度(OD)值均低于空白对照组和阴性对照组(P﹤0.05)。结论喉癌细胞Hep-2中CrkⅡ的表达下调可降低细胞的增殖、迁移和侵袭能力,CrkⅡ有可能成为喉癌临床治疗的新靶点。
Objective To investigate the effect of CrkⅡ expression on proliferation, migration and invasion of laryngeal carcinoma Hep-2 cells. Method The expression of CrkⅡ in the laryngeal carcinoma tissues and adjacent normal tissues collected from 108 cases with laryngeal carcinoma were detected immunohistochemically, and the positive expression of CrkⅡ protein in laryngeal carcinoma patients with differing clinical characteristics were analyzed. CrkⅡ RNA interference plasmid was constructed and validated, thereafter the cultured Hep-2 cells were divided into three groups:blank control group(no transfected plasmid), negative control group(transfected with negative control plasmid), and study group(transfected with CrkⅡ interference plasmid). Cells were amplified by real-time polymerase chain reaction(PCR), and Western blot were used to detect the expression of CrkⅡ in Hep-2 cells after transfection. Methylthiazolyl tetrazolium(MTT) assay, Transwell chamber assay and cell scratch assay were employed to detect cell proliferation, migration and invasion, respectively. Result The positive expression rate of CrkⅡ protein were significantly higher in laryngeal carcinoma tissues compared to adjacent normal tissues(P<0.01). Significant increases of CrkⅡ protein expression were noted in the laryngeal carcinoma tissues with clinical stage Ⅲ-Ⅳ, histological differentiation of low or moderate degree,and positive lymph node metastasis versus those with clinical stage Ⅰ-Ⅱ, high degree of histological differentiation, and negative lymph node metastasis(P<0.05). The relative expression of Crk Ⅱ mRNA and protein in Hep-2 cells of study group were lower than those in negative control group and blank control group(P<0.05). Significantly less cells invading membrane to the lower compartmentin Transwell chamber were observed in study group compared with negative control group or blank control group(P<0.05). In 48 h after transfection, the study group cells had lower migration rate versus negative control and blank control group, respectively(P<0.05). The OD values in study group Hep-2 cells were lower after 24, 48 and 72 hours of cell culture in comparison with those in negative control and blank control group(P<0.05).Conclusion The expression of CrkⅡ in laryngeal carcinoma Hep-2 cell are down-regulated, thereby decreasing the ability of cell proliferation, migration and invasion. CrkⅡ may become a new target in the treatment of laryngeal carcinoma.
作者
刘东东
周李芳
杨华晖
LIU Dongdong;ZHOU Lifang;YANG Huahui(Department of Otorhinolaryngology-Head and Neck Surgery,Jingzhou Hospital of Traditional Chinese Medicine,Jingzhou 448002,Hubei,China)
出处
《癌症进展》
2020年第5期500-504,共5页
Oncology Progress
关键词
喉癌
HEP-2细胞
CrkⅡ
增殖
迁移
侵袭
laryngeal carcinoma
Hep-2 cell
Crk II
proliferation
migration
invasion
作者简介
通信作者:杨华晖,邮箱:csnb8877@sina.com。
