靶向敲低肿瘤转移相关基因1可减缓裸鼠喉鳞癌移植瘤生长并降低其转移能力

MTA1-specific shRNA inhibits the growth and metastasis of mouse xenograft of laryngeal squamous cell carcinoma

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 metastasis-associated gene 1（MTA1） on the growth and metastasis of laryngeal squamous cell carcinoma in nude mice using RNA interference technology（ RNAi）. Methods Lentiviral vector of short hairpin RNA（ shRNA） targeting MTA1 was constructed and packaged to transfect Hep-2 cells. Hep-2 cells transfected with scramble shRNA and MTA1 shRNA were injected into the paw pad of nude mice（ n = 5 per group）. Nine weeks after modeling of the lymphatic metastasis of laryngeal carcinoma,the mice were sacrificed,and tumor tissues and inguinal lymph node were harvested to be subjected to HE staining,reverse transcription PCR and Western blotting. Results The gene screening showed the lentiviral vector of MTA1 shRNA was constructed successfully,and those tumor cells were transplanted and grew well in all mice. The size of tumor in the mice of MTA1 shRNA tansfected group was obviously smaller compared to the scramble shRNA transfected group at the same time point. No inguinal lymph node metastasis was found in the mice of MTA1 shRNA group. In contrast,the tumor cells were seen in the inguinal lymph nodes of the scramble shRNA infected mice. Reverse transcription PCR and Western blotting showed that the mRNA and protein levels of MTA1,β-catenin,matrix metalloproteinase-9（ MMP-9）,cyclin D1 were obviously reduced in MTA1 shRNA infected mice compared with the scramble shRNA infected mice. Conclusion The inhibition of MTA1 gene could depress the growth and metastasis of laryngeal squamous cell carcinoma in nude mice.