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目的通过RNA干扰(RNAi)技术,观察肿瘤转移相关基因1(MTA1)对裸鼠喉鳞癌移植瘤生长和转移能力的影响。方法设计MTA1的短发夹RNA(shRNA)慢病毒干扰载体,进行包装与转染Hep-2细胞。将无关shRNA对照组和MTA1shRNA感染的Hep-2细胞接种于裸鼠爪垫,每组5只。复制人喉癌移植瘤淋巴转移模型,9周后处死裸鼠,取下肿瘤组织及腹股沟淋巴结,进行HE染色形态学观察,反转录PCR检测MTA1、β联蛋白(β-catenin)、基质金属蛋白酶9(MMP-9)、细胞周期蛋白D1(cyclin D1)mRNA的水平,Western blot法检测MTA1、β-catenin、MMP-9、cyclin D1蛋白水平。结果慢病毒MTA1干扰载体筛选成功;2组人喉癌裸鼠爪垫移植瘤100%成瘤;MTA1 shRNA转染细胞裸鼠肿瘤体积在同一时间点均明显小于无关shRNA对照组;MTA1 shRNA转染细胞5只裸鼠爪垫肿瘤未发现淋巴结转移;无关shRNA对照组5只裸鼠腹股沟淋巴结切片均可见喉癌细胞;与无关shRNA对照组相比,MTA1 shRNA转染细胞MTA1、β-catenin、MMP-9、cyclin D1的mRNA和蛋白水平明显降低。结论抑制MTA1基因能够减缓裸鼠喉鳞癌的生长并降低其转移能力。
Objective To observe the effect of tumor metastasis-associated gene 1 (MTA1) on the growth and metastasis of laryngeal squamous cell carcinoma in nude mice by RNA interference (RNAi) technique. Methods Short hairpin RNA (shRNA) lentiviral vector of MTA1 was designed and transfected into Hep-2 cells. Hep-2 cells infected with MTA1 shRNA and non-related shRNA control group were inoculated into nude mouse paw pads with 5 mice in each group. The nude mice were sacrificed after 9 weeks and the tumor tissues and inguinal lymph nodes were removed for HE staining. Morphological changes of MTA1, β-catenin, The levels of MMP-9 and cyclin D1 mRNA were detected by Western blot. The protein levels of MTA1, β-catenin, MMP-9 and cyclin D1 were detected by Western blot. Results The lentivirus MTA1 interference vector was successfully screened. Two groups of human laryngeal carcinoma xenografts in nude mice were 100% tumorigenic. The tumor volume of MTA1 shRNA transfected nude mice was significantly smaller than that of non-related shRNA control group at the same time point. MTA1 shRNA transfection MTA1 shRNA transfected cells transfected with MTA1, β-catenin, MMPs were not found in 5 nude mouse paw tumor. No significant correlation was found between MTA1 shRNA transfected MTA1 shRNA and control group -9, cyclin D1 mRNA and protein levels were significantly lower. Conclusion Inhibition of MTA1 gene can reduce the growth of nude mice with laryngeal squamous cell carcinoma and reduce its metastatic capacity.