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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
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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 目的 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。 Abstract: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. -
Key words:
- hepatocellular carcinoma /
- let-7a /
- K-Ras /
- H-Ras /
- N-Ras /
- cell cycle /
- local injection treatment
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图 1 let-7a mimics对两种肝癌细胞的增殖、细胞周期和凋亡的影响
A.肝癌细胞MTT生长曲线分析；B.let-7a mimics对肝癌细胞周期的影响
let-7a: let-7a mimics治疗组；NC-Ctrl:阴性对照组
图 2 let-7a mimics对裸鼠皮下肝癌移植瘤的影响
A.裸鼠皮下移植瘤生长曲线；B.局部注射给药4周，移植瘤实验结束后移植瘤大小及其侵及皮肤溃疡情况；C.移植瘤组织病理学，蓝色箭头示坏死区，黄色箭头示细胞分裂象(HE染色)
let-7a、NC-Ctrl:同图 1 -
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