慢病毒介导的c-met RNA干扰对人乳头状甲状腺癌K1细胞生物学行为的影响

Effects of lentivirus-mediated c-met RNAi on biological behaviors of human papillary thyroid cancer K1 cells

目的:探讨c-Met在乳头状甲状腺癌(PTC)中的表达;构建针对人c-met基因的RNA干扰(RNAi)慢病毒载体,并观察其对K1细胞c-met基因的沉默效应及对细胞生物学行为的影响。方法:免疫组化方法检测35例乳头状甲状腺癌及25例良性甲状腺疾病手术切除标本中c-Met的表达;构建人c-met基因的RNAi慢病毒载体,应用荧光定量RT-PCR与Western blotting检测其对c-met的干扰效率,应用克隆形成实验、流式细胞术、划痕实验和Transwell实验检测其对细胞克隆形成、周期、迁移、侵袭能力的影响,应用裸鼠模型评估RNA干扰后细胞成瘤能力的影响。结果:c-Met在PTC中的表达明显高于良性甲状腺组织;成功构建了c-met RNAi慢病毒载体,RT-PCR及Western blotting实验显示其对乳头状甲状腺癌K1细胞c-met mRNA及蛋白表达的抑制均较明显;克隆形成实验显示c-met RNAi慢病毒载体能够抑制K1细胞的克隆形成能力;流式细胞术检测显示c-met RNAi慢病毒载体能够减少S细胞比列;划痕实验及Transwell实验显示c-met RNAi慢病毒载体能够抑制细胞迁移和侵袭;裸鼠成瘤实验显示RNA干扰后细胞的成瘤能力降低。结论:c-met RNAi慢病毒载体能够抑制K1细胞的克隆形成能力、细胞周期进程、迁移、侵袭及成瘤能力。

AIM： To evaluate the expression of c - Met in papillary thyroid cancer （PTC） by constructing lentiviral vectors for RNA interference （RNAi） of c - met gene and detecting its silencing effect on the biological behaviors of human papillary thyroid cancer cell line K1 cells. METHODS ： Immunohistochemical assay was performed to detect the expression of c - Met protein in 35 cases of PTC and 25 cases of benign thyroid disease. Lentiviral vector for RNA of c - met gene was constructed and the silencing effect was detected by RT - PCR and Western blotting. The colony - forming ability, cell cycle, migration and invasion of KI cells were measured by colony - forming assay, flow cytometry, wound - healing observation and Transwell experiment, respectively. In vivo tumorigenicity assay was performed to analyze in vivo proliferation of K1 cells in a xenograft model. RESULTS ： The expression of c - Met in PTC was significantly higher than that in benign thyroid tissues. Lentiviral RNAi vectors targeting c - met gene were successfully constructed, and they＂ effi- ciently inhibited the expression of c - met at mRNA and protein levels. Transfection of c - met lentiviral RNAi vectors inhibited the colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells. CONCLUSION： Lentivirus - mediated c - met RNAi efficiently inhibits colony formation, cell cycle progression, migration, invasion and tumorigenicity of K1 cells.