【作者】 李磊；

【导师】 苏玲； 刘相国；

【作者基本信息】 山东大学 ， 细胞生物学， 2020， 硕士

【摘要】 PI3Ks-AKT信号通路在细胞信号转导过程中有极为重要的功能,在众多肿瘤中都保持较高的激活状态。PI3Ks是一种可将PIP2催化为PIP3的酶,PIP3再招募PDK1和PDK2对AKT进行磷酸化。PI3Ks信号参与细胞增殖,凋亡和分化等众多细胞功能,与肿瘤的产生和恶化密切相关。而肿瘤发生的过程中,PI3Ks-AKT途径经常会发生异常调节进而异常调节细胞的代谢,增殖以及凋亡等功能。PIK3CA是I型PI3Ks的一个重要的催化亚基,具有五个域:Adaptor-binding domain(ABD),Ras-binding domain(RBD),C2 domain,螺旋结构域(he1ica1),激酶结构域(Kinase)。当PIK3CA基因在细胞内表达出错时,会导致Akt的活化发生异常,从而导致细胞发生错误的行为。但是,目前有关于PIK3CA如何影响肺癌的发展以及PIK3CA的降解方式的研究还并不深入。我们首先在H1299和H1792中转染RNF19B的质粒,发现RNF19B能够在肺癌细胞中下调PIK3CA的蛋白水平,并且是在转染14 h后发现PIK3CA开始降解。之后我们用E64D和MG132处理细胞,来进一步探究PIK3CA是如何降解的。实验结果显示,用MG132处理细胞后可以恢复由于过表达RNF19B而下调的PIK3CA的蛋白水平,表明RNF19B影响了 PIK3CA通过蛋白酶体途径的降解。通过免疫共沉淀实验和免疫荧光实验,发现RNF19B与PIK3CA具有相互作用。接下来我们克隆出了不同分段的RNF19B domain,探究RNF19B通过其哪个domain发挥作用。实验结果显示,PIK3CA主要与RNF19B的IBR domain结合。接下来我们又鉴定了 RNF19B对于PIK3CA的泛素化水平的影响,western b1ot结果显示,过表达RNF19B促进了 PIK3CA的多聚泛素化水平,敲低RNF19B抑制了 PIK3CA的多聚泛素化水平。然后我们将位于PIK3CA的C2 domain以及Kinase domain的几个赖氨酸突变为精氨酸,当PIK3CA这两个区域发生突变后,突变后PIK3CA的泛素化在过表达RNF19B后也不能增加,表明这两个区域在PIK3CA泛素化过程中有很重要的作用。之后我们将泛素分子的7个赖氨酸突变为精氨酸,发现第11和48位点的Lys残基是介导PIK3CA泛素化的重要位点。通过饥饿实验,发现过表达RNF19B 10 min后,AKT(S473)的磷酸化水平下降。之后通过EdU实验和平板克隆实验,证明RNF19B能够抑制肺癌细胞的增殖及其克隆的形成。由于AKT也可以通过多种方式影响凋亡,我们用两种化疗药物Doxorubicin和Pemetrexed处理两种肺癌细胞,实验结果证明RNF19B能够促进肺癌细胞凋亡。之后,我们证明了 RNF19B促进肺癌细胞凋亡是通过PIK3CA。更多还原

【Abstract】 The PI3Ks-AKT signaling pathway plays an important role in the process of cell signal transduction.It is an important signaling pathway for tumor cells with immortalization ability,and it maintains a high activation state in many tumors.PI3Ks is an enzyme that catalyzes PIP2 to PIP3.PIP3 recruits PDK1 and PDK2 to phosphorylate AKT.The functions of the PI3Ks-AKT pathway include controlling intracellular vesicle transport,cell movement,and nutrient metabolism.In the process of tumorigenesis,the PI3Ks-AKT pathway often undergoes abnormal regulation and abnormally regulates cell metabolism,proliferation and apoptosis.PIK3CA is an important catalytic subunit of type I PI3Ks,which has five domains:ABD,RBD,C2,helical,Kinase.When PIK3CA functions abnormally,Akt will be activated abnormally,which will cause abnormal behavior of cells.however,there are few studies on how PIK3CA affects the development of lung cancer and the degradation of PIK3CA.We transfected the plasmid of RNF19B,and found that RNF19B can down-regulate the level of exogenous PIK3CA protein in lung cancer cells,and found that PIK3CA began to degrade after 14 h of transfection.We then treated the cells with the lysosomal pathway inhibitor E64D and the proteasome pathway inhibitor MG132,respectively.The experimental results show that treatment of cells with MG 132 can restore the protein level of PIK3CA down-regulated due to overexpression of RNF19B,indicating that RNF19B affects the degradation of PIK3CA by the proteasome pathway.Next,we found that RNF19B interacts with PIK3CA through co-immunoprecipitation experiments.Immunofluorescence experiments also prove this.We cloned RNF19B domains in different segments and explored which domain RNF19B plays an important role.Next,we identified the effect of RNF19B on the ubiquitination level of PIK3CA.The ressults of co-immunoprecipitation experiments showed that overexpression of RNF19B promoted the polyubiquitination level of PIK3CA.Then we knocked down RNF19B and found that knockdown of RNF19B inhibited the polyubiquitination level of PIK3CA.Then we mutated the two lysines located in the C2 domain and the Kinase domain of PIK3CA to arginine.When the two regions of PIK3CA were mutated,the ubiquitination of PIK3CA after mutation could not be increased,indicating that these two regions play a very important role.Later,we mutated 7 lysines of ubiquitin molecules to arginine,and we found that K11 and K48 sites are important sites that mediate PIK3CA ubiquitination.We found through starvation experiments that 10 minutes after RNF19B overexpression,the phosphorylation level of AKT(S473)decreased significantly.Later,we used plate cloning experiments and EdU cell proliferation detection methods to detect whether RNF19B has an effect on tumor cell proliferation.The experimental results show that RNF19B can inhibit the formation and proliferation of lung cancer cells.Since AKT can also affect apoptosis in a variety of ways,we treated two lung cancer cells with two chemotherapy drugs,doxorubicin and pemetrexed.The experimental results show that RNF19B can promote the apoptosis of lung cancer cells due to chemotherapy drugs.Later,we also demonstrated that RNF19B promotes lung cancer cell apoptosis through PIK3CA.更多还原