1] Department of Immunology and Key Laboratory of Molecular Medicine of Ministry of Education, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China [2] Biotherapy Research Center of Fudan University, Shanghai, People's Republic of China [3] Department of Hematology/Oncology, The Second Hospital of Anhui Medical University, Hefei, Anhui, People's Republic of China.
1] Department of Immunology and Key Laboratory of Molecular Medicine of Ministry of Education, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China [2] Biotherapy Research Center of Fudan University, Shanghai, People's Republic of China.
Br J Cancer. 2014 Jan 21;110(2):353-62. doi: 10.1038/bjc.2013.728. Epub 2013 Nov 26.
MicroRNA-7 (miR-7) has been reported to be a tumour suppressor gene. However, whether it has a role in the growth of non-small-cell lung cancer (NSCLC) and what is its target involved in the tumour growth is still under investigation.
NSCLC tissue sample, NSCLC cell lines and tissue microarray were investigated in this study. Total RNA, miRNA and protein were used for RT-PCR and western blot analysis. Immunohistochemistry was performed in tissues microarray. Cell culture and intervention experiments were performed in vitro and in vivo. Bioinformatics prediction, western blot and luciferase assay were identified the target of miR-7.
In this study, we found that the expression of miR-7 was significantly downregulated not only in NSCLC cell lines, but also in human NSCLC tissues compared with the matched adjacent tissues. Restoration of its expression through miR-7 mimics in A549 and H1299 NSCLC cells inhibited cell proliferation, colony formation, and cell-cycle progression in vitro. More importantly, the tumorigenicity in nude mice was reduced after administration of miR-7 in vivo. In advance, through bioinformatic analysis, luciferase assay and western blot, we identified a novel target of miR-7, PA28gamma (a proteasome activator) to be enrolled in the regulation with tumour. PA28gamma mRNA and protein levels are markedly upregulated in NSCLC cell lines and tumour samples, exhibiting a strong inverse relation with that of miR-7. In addition, knockdown of PA28gamma induced similar effects as overexpression of miR-7 in NSCLC cells. Furthermore, miR-7 overexpression or silencing of PA28gamma reduced the cyclinD1 expression at mRNA and protein level in NSCLC cell lines.
All these findings strongly imply that the overexpression of PA28gamma resulted from miR-7 downexpression in NSCLC has an important role in promoting cancer cell progress and consequently results in NSCLC growth. Thus, strategies targeting PA28gamma and/or miR-7 may become promising molecular therapies in NSCLC treatment.
MicroRNA-7（miR-7）已被报道为一种肿瘤抑制基因。然而，它是否在非小细胞肺癌（NSCLC）的生长中发挥作用，以及其在肿瘤生长中涉及的靶标是什么，仍在研究中。
本研究调查了 NSCLC 组织样本、NSCLC 细胞系和组织微阵列。使用 RT-PCR 和 Western blot 分析进行总 RNA、miRNA 和蛋白质分析。在组织微阵列中进行免疫组织化学分析。在体外和体内进行细胞培养和干预实验。通过生物信息学预测、Western blot 和荧光素酶测定鉴定 miR-7 的靶标。
在这项研究中，我们发现 miR-7 的表达不仅在 NSCLC 细胞系中，而且在与匹配的相邻组织相比的人 NSCLC 组织中都明显下调。通过 miR-7 模拟物在 A549 和 H1299 NSCLC 细胞中恢复其表达，可抑制体外细胞增殖、集落形成和细胞周期进程。更重要的是，体内给予 miR-7 后可降低裸鼠的肿瘤生成能力。在前期，通过生物信息学分析、荧光素酶测定和 Western blot，我们确定了 miR-7 的一个新靶标，即 PA28gamma（一种蛋白酶体激活剂），参与了肿瘤的调控。PA28gamma mRNA 和蛋白水平在 NSCLC 细胞系和肿瘤样本中明显上调，与 miR-7 的水平呈强烈的负相关。此外，在 NSCLC 细胞中敲低 PA28gamma 可诱导与 miR-7 过表达相似的作用。此外，miR-7 过表达或 PA28gamma 沉默可降低 NSCLC 细胞系中环蛋白 D1 的 mRNA 和蛋白水平。
所有这些发现都强烈表明，NSCLC 中 miR-7 下调导致的 PA28gamma 过表达在促进癌细胞进展中起着重要作用，进而导致 NSCLC 生长。因此，针对 PA28gamma 和/或 miR-7 的策略可能成为 NSCLC 治疗的有前途的分子治疗方法。
