miR-23a/27a/24-2分子在宫颈癌中的异常表达及参与NF-kB信号通路的反馈调节机制研究

miRNAs are a class of small regulatory non-coding RNAs. It is involved in various biological processes including cancers. In our previous work, we found that miR-23a and miR-429 were significantly up-regulated upon silencing NF-κB1 expressions. MiR-429 played tumor suppressor roles in cervical cancer cells by inhibiting cell proliferation and promoting apoptosis. Meanwhile, miR-429 can directly bind to the 3’UTR of IKKβ, inhibit its expression and negatively regulate NF-κB signaling pathway. The other preliminary results showed that miR-27a was significantly up-regulated in cervical cancers compared with the matched normal cervical tissues and miR-23a can directly target the 3’UTR of IKKα and active the NF-κB signaling pathway. ..Based on these findings, we are planning to further evaluate their downstream functional roles and the upstream regulatory mechanisms in cervical cancer cells. As for the downstream functional aspects, we will focus on the phenotype changes and the regulatory mechanisms. We will systematically investigate the phenotype effects of miRNAs on cell proliferation, apoptosis, autophagy, vascular mimicry formation, migration and invasion, etc. In addition, we will analyze the malignancy effects of the targets of miRNAs and perform rescue experiments for functional targets verification. As for the upstream regulatory aspects, we will first construct a series of promoter reporter vectors, determine their promoter activities and map the core promoter regions. Subsequently, we will focus on NF-κB by searching for the key factors which can potentially be regulated by NF-κB and bind to the promoter regions of the miRNAs. Moreover, we will also focus on the HPV E6/E7 oncoproteins, which were reported to regulate the expressions of miR-23a/27a, by exploring the molecular mechanisms involved in the biogenesis of miR-23a/27a and their molecular mechanisms at the transcriptional level. This will help us further understanding the mechanisms of miRNA dysregulations at transcriptional level...This project will enrich our understandings for the molecular mechanisms of miRNAs in tumor developments, for the crosstalk and feedback loop between miRNAs and NF-κB signaling pathways, as well as the regulatory networks between miRNAs and the HPV viral oncoproteins. Meanwhile, this project will also provide us some valuable clues for the transcriptional regulations of miRNA genes, especially for miRNA cluster members.

前期研究发现miR-23a和miR-429受NF-κB的调节，在宫颈癌细胞中miR-429可直接靶定IKKβ并抑制其表达，对NF-κB形成负反馈调节；miR-23a/27a在宫颈癌组织中高表达，miR-23a可直接靶定IKKα，激活NF-κB信号通路。在此基础上，我们拟在宫颈癌细胞中进行miR-23a/27a等的功能表型研究，发挥功能的具体机制研究及其上游的表达调控研究；在表达调控研究中，将围绕NF-κB这一关键分子，寻找受其调节并与miRNA启动子结合的关键分子，从转录水平上研究其失调表达机制；同时也围绕HPV癌蛋白 E6/E7对miRNA表达的影响，从miRNA的生物合成过程以及转录水平探讨其相关的分子机制，进一步完善对miRNA的失调表达的机制研究。本研究将揭示miRNA与NF-κB信号通路的反馈调节机制及病毒癌基因对miRNA的作用机制，为发现新的宫颈癌标志分子提供理论与实验依据。

本项目在前期研究的基础上，以mir-23a/27a/24-2基因簇为例，进一步对其在宫颈癌细胞中的功能及异常表达机制进行深入探讨。前期已证实，该基因簇中miRNA分子的表达受NF-kB的调节。本项目中，我们围绕着NF-kB信号通路，主要以miR-27a-3p为依托进行项目研究。.本项目的主要研究内容包括miRNA的下游功能作用机制和上游表达调控机制两部分。下游功能机制研究中，我们利用生物信息学分析和实验相结合的方法，通过寻找miR-27a-3p下游的靶基因，将其与NF-kB信号通路建立直接联系。我们筛选并证实了TAB3为miR-27a-3p分子的下游直接靶基因，miR-27a-3p通过与TAB3的3’UTR直接结合，促进其mRNA转录及蛋白翻译；TAB3本身在宫颈癌细胞中发挥促癌基因的效应，功能挽救实验证实miR-27a-3p可通过对TAB3的上调表达发挥促癌效应；上游表达调控机制中，我们主要围绕着转录因子NF-kB展开，发现并证实了miR-27a-3p分子上游序列中与NF-kB/P65直接结合的一个位点，转录因子NF-kB通过与此位点的直接结合可促进miRNA簇的基因转录，进而增强整个基因簇中各成熟miRNA分子的表达；此外，初步研究还显示高危型HPV癌蛋白E6/E7的表达也直接或间接影响着宫颈癌细胞中miR-27a-3p的异常表达。.我们的研究围绕着miR-27a-3p、TAB3和NF-kB信号通路进行，提出了NF-kB/miR-27a-3p/TAB3/NF-kB这一闭合的正反馈调节环路，不仅有助于我们理解miR-27a-3p分子在宫颈癌细胞中发挥癌基因效应的分子机制，更重要的是从不同角度（包括基因转录、miRNA生成过程以及致癌病毒癌蛋白的影响等）阐述了肿瘤细胞中miRNA分子异常表达的机制，为未来开发具有临床意义的新型生物标志物提供了理论与实践依据。