基于PI3K/AKT/mTOR信号通路对GDNF促胶质瘤细胞增殖的miRNAs调控网络研究

Glial cell line-derived neurotrophic factor (GDNF) can promote glioma cells proliferation, but its mechanism is not yet clear. We have previously found dramatic changes in the expression of a few specific miRNAs and mRNAs in glioma cells by microarray. Particularly, the expression of phosphatase and tensin homolog (PTEN) is decreased significantly. We hypothesize that GDNF may promote glioma cell proliferation by means of a specialized miRNAs network which down-regulates PTEN and activates the PI3K/AKT/mTOR signalling pathway. We have already cloned the PTEN gene from U251 glioma cell line. In order to prove the above hypothesis, we plan to use TALENs technique to construct a PTEN glioma reporter cell line. In addition, we plan to construct miRNAs sponge (such as miR-26 sponge) and miRNA over-expression vectors, which will then be delivered into the PTEN glioma reporter cell line by high-efficiency lentivirus transduction system. Whether the miRNAs target PTEN will be determined by whether the reporter cell line fluoresces or not. Moreover, we plan to confirm that our miRNAs of interest and PTEN participate in this regulatory network by measuring their expression as detected by ST-RT PCR, qPCR and other techniques. Lastly, we will compare differences in the phosphorylation levels of downstream protein kinases AKT and mTOR and their effects on the PI3K/AKT/mTOR signalling pathway in those cells by western blot, and cell proliferation as detected by MTT assay. Based on the above studies, we can prove that GDNF regulates the PI3K/AKT/mTOR signalling pathway through this miRNAs network. Our study not only lays a foundation for the mechanism of glioma cell proliferation, but also provides new ideas for the development of advanced pharmaceutical treatments for glioma.

胶质细胞系源性神经营养因子(GDNF)能够促进胶质瘤细胞增殖，但机理不明，芯片检测到大量miRNAs和mRNAs发生变化，其中磷酸酶张力蛋白类似物（PTEN）mRNA显著下降，推测GDNF可能通过miRNAs网络下调PTEN激活PI3K/AKT/mTOR信号通路促进胶质瘤细胞的增殖。为此，分离了PTEN基因，拟利用TALENs技术构建胶质瘤PTEN报告细胞系，用非对称酶技术快速构建miRNAs海绵和其超表达载体，将其分别导入到报告细胞系。通过荧光变化分析每个miRNAs与PTEN基因的调控关系。利用ST-RT和qPCR等技术监测miRNAs与PTEN基因表达的变化，确定miRNAs调控网络。并比较PI3K/AKT通路的主要蛋白激酶磷酸化水平，结合细胞增殖实验，探明GDNF通过miRNAs网络对该信号通路的调控，为深入研究胶质瘤细胞增殖机制奠定基础，也为研发预防和治疗胶质瘤的药物提供新思路。

由于胶质瘤细胞GDNF异常表达造成细胞周围浓度升高，推测在胶质瘤.细胞中普遍高表达的GDNF或许对胶质瘤的发生发展具有促进作用，本研究通过外源性添加GDNF至胶质瘤细胞系培养基中，来研究其对细胞增殖作用的影响并初步探求其基本机制。由于本项目对GDNF用量很大，而商品化的GDNF比较昂贵，所以我们自己用大肠杆菌BL2表达并纯化了与商品化GDNF具有相似活性的蛋白。本项目microRNA 芯片与基因转录组表达谱芯片的联合应用，通过生物信息学技术、基因克隆等方法，对GDNF作用下的胶质瘤细胞U251的mRNA和miRNA检测，发现大量miRNAs发生表达变化，本项目分离到5种上调基因hsa-miR-26、hsa-miR-21、hsa-miR-519、hsa-miR-10、hsa-miR-17，5种下调基因hsa-miR-15b、hsa-miR-128、hsa-miR-320、hsa-miR-342、hsa-miR-222和靶基因PTEN，并采用自主研发的RNA海绵构建体系构建具有eGFP报告基因的海绵和超表达载体，并借助经典高效的慢病毒系统进行介导，利用先进的TALENs技术构建瘤细胞的PTEN报告细胞系，进而通过荧光的改变很容易确定hsa-miR-26等miRNAs与PTEN的靶向性关系，通过上调miRNAs基因hsa-miR-26、hsa-miR-21的海绵和下调基因hsa-miR-15b的过表达载体对GDNF作用下增高或下调的miRNAs的恢复作用，从而部分抑制GDNF的促细胞增殖作用。通过qPCR和Western Bot，检测AKT和mTOR的磷酸化水平，从而初步确定GDNF通过某种信号机制上调has-miR-26调控miRNAs并通过PI3/AKT/mTOR信号通路促进胶质瘤细胞增殖。