Ye Zhen-long, Huang Yao, Li Lin-fang, Zhu Hai-li, Gao Hai-xia, Liu Hui, Lv Sai-qun, Xu Zeng-hui, Zheng Luo-ning, Liu Tao, Zhang Jing-lei, Jin Hua-jun, Qian Qi-jun
Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
Xinyuan Institute of Medicine and Biotechnology College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
Acta Pharmacol Sin. 2015 Oct;36(10):1237-45. doi: 10.1038/aps.2015.18. Epub 2015 May 4.
Argonaute2 (AGO2) protein is the active part of RNA-induced silencing complex, cleaving the target mRNA strand complementary to their bound siRNA. An increasing number of miRNAs has been identified as essential to angiogenesis of hepatocellular carcinoma (HCC). In this study we investigated how AGO2 affected HCC angiogenesis.
Human HCC cell lines HepG2, Hep3B, Huh7, SMMC-7721, Bel-7404, MHCC97-H and LM-3, and human umbilical vein endothelial cells (HUVEC) were tested. The expression of AGO2 in HCC cells was knocked down with siRNA and restored using recombinant adenovirus expressing Ago2. The levels of relevant mRNAs and proteins were examined using RT-PCR, Western blot and EILSA. Nude mice were implanted with Huh7 or SMMC-7721 cells, and tumor volumes were measured. After the mice were euthanized, the xenograft tumors were used for immunohistological analysis.
In 6 HCC cell lines, AGO2 protein expression was significantly correlated with VEGF expression (r=+0.79), and with VEGF secretion (r=+0.852). Knockdown of Ago2 in Huh7 cells and SMMC-7721 cells substantially decreased VEGF expression, whereas the restoration of AGO2 reversed both VEGF expression and secretion. Furthermore, knockdown of Ago2 significantly up-regulated the expression of PTEN (a tumor suppressor involved in the inhibition of HCC angiogenesis), and vice versa. Moreover, the specific PTEN inhibitor bisperoxovanadate (7, 14, 28 nmol/L) dose-dependently restored the expression of VEGF and the capacity of HCC cells to induce HUVECs to form capillary tubule structures. In the xenograft nude mice, knockdown of Ago2 markedly suppressed the tumor growth and decreased PTEN expression and CD31-positive microvascular in the xenograft tumors.
A direct relationship exists between the miRNA processing machinery AGO2 and HCC angiogenesis that is mediated by the AGO2/PTEN/VEGF signaling pathway. The results suggest the high value of Ago2 knockdown in anti-angiogenesis therapy for HCC.
Argonaute2（AGO2）蛋白是RNA诱导沉默复合体的活性部分，可切割与其结合的小干扰RNA（siRNA）互补的靶mRNA链。越来越多的微小RNA（miRNA）已被确定对肝细胞癌（HCC）的血管生成至关重要。在本研究中，我们调查了AGO2如何影响HCC血管生成。
对人HCC细胞系HepG2、Hep3B、Huh7、SMMC-7721、Bel-7404、MHCC97-H和LM-3以及人脐静脉内皮细胞（HUVEC）进行检测。用siRNA敲低HCC细胞中AGO2的表达，并使用表达Ago2的重组腺病毒进行恢复。采用逆转录聚合酶链反应（RT-PCR）、蛋白质印迹法和酶联免疫吸附测定（ELISA）检测相关mRNA和蛋白质水平。将Huh7或SMMC-7721细胞接种到裸鼠体内，测量肿瘤体积。小鼠安乐死后，将异种移植瘤用于免疫组织学分析。
在6种HCC细胞系中，AGO2蛋白表达与血管内皮生长因子（VEGF）表达显著相关（r = +0.79），与VEGF分泌显著相关（r = +0.852）。敲低Huh7细胞和SMMC-7721细胞中的Ago2可显著降低VEGF表达，而恢复AGO2则可逆转VEGF表达和分泌。此外，敲低Ago2可显著上调抑癌基因PTEN（参与抑制HCC血管生成）的表达，反之亦然。此外，特异性PTEN抑制剂双过氧钒酸盐（7、14、28 nmol/L）剂量依赖性地恢复VEGF表达以及HCC细胞诱导HUVEC形成毛细血管小管结构的能力。在异种移植裸鼠中，敲低Ago2可显著抑制肿瘤生长，并降低异种移植瘤中PTEN表达和CD31阳性微血管数量。
miRNA加工机制AGO2与HCC血管生成之间存在直接关系，该关系由AGO2/PTEN/VEGF信号通路介导。结果表明敲低Ago2在HCC抗血管生成治疗中具有很高的价值。
