协和医学杂志

2015, v.6(02) 133-139

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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

刘明洋;陈杰;关健;
LIU Ming-yang;CHEN Jie;GUAN Jian;Peking Union Medical College Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College;

摘要(Abstract):

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 mimics( let-7a 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-7a 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-7a mimics on HCC growth were analyzed in subcutaneous xenograft nude mice with intra-tumoral injections. Expressions of let-7a and its target human Ras were examined with real-time PCR and Western blot. Results let-7a mimics-transfected HCC cells showed increased let-7a levels and slower proliferation compared with the negative controls( both P < 0. 05). let-7a mimics induced more cell-cycle arrest at the G0-G1 phase and promoted apoptosis of HCC cells( both P < 0. 05). In addition,significant reductions in tumor size,local invasion and metastasis to liver were observed in let-7a mimics-treated nude mice compared to negative controls. The tumor size after let-7a 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,H-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 effectively suppress the growth,invasion and metastasis of xenograft tumor. These findings suggest that Ras-targeting let-7 mimics could be a potential therapeutic option for HCC.

关键词(KeyWords): 肝癌;let-7a;K-Ras;H-Ras;N-Ras;细胞周期;局部注射治疗
hepatocellular carcinoma;let-7a;K-Ras;H-Ras;N-Ras;cell cycle;local injection treatment

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作者(Author): 刘明洋;陈杰;关健;
LIU Ming-yang;CHEN Jie;GUAN Jian;Peking Union Medical College Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College;

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