Ma Ming-Zhe, Li Chun-Xiao, Zhang Yan, Weng Ming-Zhe, Zhang Ming-di, Qin Yi-Yu, Gong Wei, Quan Zhi-Wei
Department of General Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, 1665 Kongjiang Road, Shanghai 200092, People's Republic of China.
Mol Cancer. 2014 Jun 23;13:156. doi: 10.1186/1476-4598-13-156.
Protein coding genes account for only about 2% of the human genome, whereas the vast majority of transcripts are non-coding RNAs including long non-coding RNAs. A growing volume of literature has proposed that lncRNAs are important players in cancer. HOTAIR was previously shown to be an oncogene and negative prognostic factor in a variety of cancers. However, the factors that contribute to its upregulation and the interaction between HOTAIR and miRNAs are largely unknown.
A computational screen of HOTAIR promoter was conducted to search for transcription-factor-binding sites. HOTAIR promoter activities were examined by luciferase reporter assay. The function of the c-Myc binding site in the HOTAIR promoter region was tested by a promoter assay with nucleotide substitutions in the putative E-box. The association of c-Myc with the HOTAIR promoter in vivo was confirmed by chromatin immunoprecipitation assay and Electrophoretic mobility shift assay. A search for miRNAs with complementary base paring with HOTAIR was performed utilizing online software program. Gain and loss of function approaches were employed to investigate the expression changes of HOTAIR or miRNA-130a. The expression levels of HOTAIR, c-Myc and miRNA-130a were examined in 65 matched pairs of gallbladder cancer tissues. The effects of HOTAIR and miRNA-130a on gallbladder cancer cell invasion and proliferation was tested using in vitro cell invasion and flow cytometric assays.
We demonstrate that HOTAIR is a direct target of c-Myc through interaction with putative c-Myc target response element (RE) in the upstream region of HOTAIR in gallbladder cancer cells. A positive correlation between c-Myc and HOTAIR mRNA levels was observed in gallbladder cancer tissues. We predicted that HOTAIR harbors a miRNA-130a binding site. Our data showed that this binding site is vital for the regulation of miRNA-130a by HOTAIR. Moreover, a negative correlation between HOTAIR and miRNA-130a was observed in gallbladder cancer tissues. Finally, we demonstrate that the oncogenic activity of HOTAIR is in part through its negative regulation of miRNA-130a.
Together, these results suggest that HOTAIR is a c-Myc-activated driver of malignancy, which acts in part through repression of miRNA-130a.
蛋白质编码基因仅占人类基因组的约2%，而绝大多数转录本是非编码RNA，包括长链非编码RNA。越来越多的文献表明，长链非编码RNA在癌症中起着重要作用。HOTAIR先前被证明是多种癌症中的致癌基因和不良预后因素。然而，导致其上调的因素以及HOTAIR与微小RNA之间的相互作用在很大程度上尚不清楚。
对HOTAIR启动子进行计算机筛选以寻找转录因子结合位点。通过荧光素酶报告基因检测法检测HOTAIR启动子活性。通过在假定的E盒中进行核苷酸替换的启动子检测来测试HOTAIR启动子区域中c-Myc结合位点的功能。通过染色质免疫沉淀测定法和电泳迁移率变动测定法证实c-Myc与体内HOTAIR启动子的关联。利用在线软件程序搜索与HOTAIR具有互补碱基配对的微小RNA。采用功能获得和功能缺失方法研究HOTAIR或微小RNA-130a的表达变化。检测了65对配对的胆囊癌组织中HOTAIR、c-Myc和微小RNA-130a的表达水平。使用体外细胞侵袭和流式细胞术检测法测试了HOTAIR和微小RNA-130a对胆囊癌细胞侵袭和增殖的影响。
我们证明，在胆囊癌细胞中，HOTAIR通过与HOTAIR上游区域假定的c-Myc靶反应元件（RE）相互作用，是c-Myc的直接靶标。在胆囊癌组织中观察到c-Myc与HOTAIR mRNA水平呈正相关。我们预测HOTAIR含有一个微小RNA-130a结合位点。我们的数据表明，该结合位点对于HOTAIR对微小RNA-130a的调控至关重要。此外，在胆囊癌组织中观察到HOTAIR与微小RNA-130a呈负相关。最后，我们证明HOTAIR的致癌活性部分是通过其对微小RNA-130a的负调控实现的。
总之，这些结果表明，HOTAIR是一种由c-Myc激活的恶性肿瘤驱动因子，其部分作用是通过抑制微小RNA-130a实现的。
