miRNA-150靶向p27基因转录后调控的分子机制

Although p27Kip1(p27) is characterized as a tumor suppressor, inactivating point mutations with loss of heterozygosity are rarely observed in human cancer. Therefore, the low levels of p27 protein observed in many aggressive types of cancer are likely to be mediated by other mechanisms. The abundance of p27 protein is largely controlled through a variety of post-transcriptional regulatory mechanisms. MicroRNAs (miRNAs) are a class of small non-coding RNAs that function to control gene expression through association with the 3'-UnTranslated Region (3'-UTR) of protein coding genes and subsequent induction of translation inhibition. As p27Kip1 is mostly controlled at the post-transcriptional level and miRNAs are potent regulators of gene expression, we hypothesized a role for miRNAs in the contribution of cancer progression through the suppression of p27 expression. However, the exact mechanism of post-transcriptional regulation of p27 is still unclear. Here, we used bioinformatics tools to predict and identify the potential miRNAs binding sites located at the 3'-UTR of p27, a cell cycle inhibitor and tumor suppressor. Our previous results indicated that miR-150 act as a potent regulators of p27. Using miRNA inhibitors, we demonstrate that certain cancer cell lines require high activity of miR-150 to maintain low p27 levels and continuous proliferation. To verify and quantify the effect of miR-150 on p27 post-transcriptional regulation, we subcloned the p27-3′UTR downstream of luciferase. Analysis of transiently transfected cells revealed that the miR-150 expressing vector significantly suppressed p27-3'UTR activity. To further substantiate the specificity of p27-3'UTR-mediated suppression by miR-150. We concluded that miR-150 is a potential regulator of the 3'UTR of p27. Further, using luciferase reporter assays, we have mutated and deleted these predicted sites in order to examine their requirement for miR-150 function to completely refractory to the miR-150 suppressive effect. Flow cytometry,RT-PCR and cell cycle profile analysis to verify miR-150 as potent suppressors of p27 expression. Current study will suggest that it might be possible to use antagomiR-150 as a target of cancer therapy.

肿瘤细胞中p27基因转录后调控机制并不完全清楚。课题组利用生物信息学方法筛选p27基因3'-非编码序列(3'-UTR)中可能结合的microRNA分子，试验初步证实miRNA-150能够靶向p27基因，并介导其功能改变。为进一步阐明miRNA-150在p27基因转录后调控的作用机制，本课题拟运用荧光素酶报告基因试验、位点突变、荧光实时定量RT-PCR、原位杂交、基因共转染等实验技术，深入研究miRNA-150靶向调控p27基因的确切机制以及对乳腺癌细胞生物学特性的影响。以上研究为进一步阐明肿瘤细胞中p27基因功能的分子调控机制提供实验依据，并为逆转乳腺癌细胞生物学特性提供潜在干预靶点。

p27是细胞周期调控机制中最关键的抑制因子，几乎可以抑制所有的CDK和cyclin复合物的激酶活性，是调控细胞周期并抑制细胞分裂的一个重要因子，在细胞增殖、分化以及凋亡等过程中起着相当重要的作用。miRNA通过与靶基因mRNA 3′-UTR区结合，从而降解mRNA或抑制其翻译对靶标基因的表达水平进行调控进而抑靶基因的表达，在基因的转录后调控中起着关键的的作用。为进一步阐明miRNA在p27基因转录后调控中的确切机制，我们在前期研究p27基因启动子区和外显子区的基础上，通过生物信息学技术预测并验证了p27 3'-UTR中可能结合的miRNA分子——miR-150。我们还发现miR-1470通过靶向调控c-jun参与拉帕替尼对p27表达调控。我们的研究还发现p73基因不同启动子区的甲基化状态在乳腺癌细胞增殖和凋亡中具有相反的生物学功能。Nrf-2与p73基因不同启动子区P1及P2的结合受到甲基化状态的影响。乳腺癌组织中高甲基化状态可使Nrf-2与P1结合减少，而与P2结合增加，从而导致TAp73表达减少而ΔNp73表达增加，首次证实Nrf-2能够与Tp73/ΔNp73结合并影响其转录。此外，我们还发现了三阴性乳腺癌细胞株和非三阴性细胞株中差异的一系列的miRNAs。课题组研究miRNA在p27基因转录后调控中的作用机制，为阐明 p27功能异常的分子基础提供理论依据，进而逆转肿瘤细胞生物学特性提供潜在的干预靶点。