局部注射胆固醇包裹let-7a miRNA模拟物下调人3种Ras表达抑制裸鼠皮下肝癌移植瘤的生长和转移  

Local Injections of Cholesterol-conjugated let-7a Mimics Inhibit Tumor Growth and Metastasis of Hepatocellular Carcinoma in a Subcutaneous Xenograft Nude Mouse Model by Targeting K-Ras,H-Ras,and N-Ras

作　　者：刘明洋[1] 陈杰[1] 关健[1] 

机构地区：[1]中国医学科学院北京协和医学院北京协和医院,北京100730

出　　处：《协和医学杂志》2015年第2期133-139,共7页Medical Journal of Peking Union Medical College Hospital

摘　　要：目的研究胆固醇包裹let-7a模拟物(mimics)是否能通过下调人3种Ras抑制肝癌发展,寻找肝癌的潜在治疗策略。方法采用MTT增殖分析、PI单染和Annexin V/FITC双染方法检测let-7a mimics在体外对肝癌细胞的作用。使用裸鼠皮下移植瘤模型和通过肿瘤局部注射方法观察let-7a mimics对体内肝癌的作用。采用实时定量PCR和Western blot方法检测let-7a及其靶点Ras的表达。结果与阴性对照组相比,let-7a mimics转染的肝癌细胞let-7a水平升高,细胞增殖缓慢(P均<0.05);let-7a mimics阻滞更多肝癌细胞停留在G0-G1期且促进肝癌细胞凋亡(P均<0.05)。经let-7a mimics治疗的裸鼠体内肿瘤体积较阴性对照组减小,是阴性对照组的54.97%(P=0.039);局部浸润和肝脏转移较阴性对照组明显减轻。肝癌细胞和移植瘤组织内let-7a上调的同时,人K-Ras、H-Rras和N-Ras mRNA和蛋白的表达降低(P均<0.05)。结论 let-7a mimics下调人3种Ras mRNA和蛋白的表达,影响细胞周期,进而抑制细胞增殖和促进细胞凋亡。瘤周多点注射胆固醇包裹的let-7a mimics能抑制移植瘤的生长、浸润和转移。提示let-7阻断Ras可成为肝癌治疗的研究策略。Objective To investigate the potential antitumor effects of cholesterol-conjugated let-7a mim- ics （let--Ta mimics） on hepatocellular carcinoma （HCC） by down-regulating all 3 human Ras, with the aim to explore alternative therapeutic strategy for HCC. Methods Effects of let-Ta mimics on HCC cells in vitro were detected using MTT-based cell proliferation assays in combination with propidium iodide staining and annexin-V/ FITC double staining. The antitumor effects in vivo of let-Ta mimics on HCC growth were analyzed in subcutane- ous xenograft nude mice with intra-tumoral injections. Expressions of let-Ta and its target human Ras were exam- ined with real-time PCR and Western blot. Results let-Ta mimics-transfected HCC cells showed increased let-Ta levels and slower proliferation compared with the negative controls （both P 〈 0.05 ）. let-？a mimics induced more cell-cycle arrest at the G0-G1 phase and promoted apoptosis of HCC cells （both P 〈 0.05）. In addition, signifi- cant reductions in tumor size, local invasion and metastasis to liver were observed in let-？a mimics-treated nude mice compared to negative controls. The tumor size after let-Ta treatment was 54.97% that of negative controls （P =0. 039）. Furthermore, the up-regulation of let-7a coincided with the reduction of K-Ras, Iq-Rras, and N-Ras mRNA and protein expressions in HCC cells and xenograft tumor （ all P 〈 0. 05 ）. Conclusions let-7a mimics could affect cell cycle, inhibit cell proliferation and promote cell apoptosis by downregulation of mRNA and protein expressions of all the 3 human Ras. Local injections of cholesterol-conjugated let-7a mimics could ef- fectively suppress the growth, invasion and metastasis of xenograft tumor. These findings suggest that Ras-targe- ting let-7 mimics could be a potential therapeutic option for ttCC.

关 键 词：肝癌 let-7a K-RAS H-RAS N-RAS 细胞周期 局部注射治疗 

分 类 号：Q74[生物学—分子生物学]