基于网络互作研究雷丸蛋白pPeOp调控Cdc42/JAK/STAT3信号通路抑胃癌作用机制

Gastric cancer is one of the most common clinical malignant tumor, which be treated by surgery auxiliary with chemotherapy and radiotherapy. However, the efficacy of such treatment is not ideal as expectable. Hence, it`s very important to develop multi-target and multi-pathway anti-cancer drugs to significantly improve the efficacy and survival rate of patients. In our preliminary study, the results suggested that pPeOp protein from Omphalia lapidescen has significantly down-regulate the JAK/STAT3 signaling pathway, meanwhile down-regulate the expression and activity of Cdc42 protein. Since Cdc42 is a key regulatory factor in Cdc42 phosphorylation, we speculate that the anti-tumor effect of pPeOp should be related with Cdc42/JAK/STAT3 signal pathway. Therefore, the present study intends to establish Cdc42 and STAT3 gene silencing cell models by lentiviral transfection, and further to establish the naked mice model with the transfected gastric cancer cells. Meanwhile, making the wild type cells as the control group, we try to detect the variation of Cdc42/JAK/STAT3 signaling pathway in gastric cancer cells and tumor body after the pPe0p protein treatment. Furthermore, we tend to analyze the mRNA interaction, miRNA interaction and protein interaction by network pharmacology and bioinformatics, and to analyze the effect of pPeOp protein on Cdc42/JAK/STAT3 network. Then, the key regulatory nodes of Cdc42/JAK/STAT3 signaling pathway would be identified and validated. Finally, we propose to clarify the anti-gastric cancer target network of pPe0p protein with in vitro and in vivo experimental data.

胃癌是临床常见恶性肿瘤，多以手术切除辅以放、化疗治疗，但疗效不显著，若开发多靶点、多途径抗肿瘤药物，将显著提高疗效。本项目前期研究表明，抗肿瘤中药雷丸所含pPeOp蛋白负调控JAK/STAT3通路抗胃癌时，可显著抑制Cdc42的表达与活性。鉴于Cdc42是STAT3磷酸化的另一重要调控因子，我们推测pPeOp蛋白的抗胃癌作用可能与Cdc42/JAK/STAT3通路的网络调控有关。因此，本申请项目拟采用慢病毒转染胃癌细胞，构建Cdc42和STAT3沉默细胞并制备荷瘤裸鼠模型。IP和ChIP-qPCR分析pPeOp蛋白干预后，Cdc42和STAT3与胞内蛋白及靶基因互作，网络药理学方法分析mRNA、miRNA和蛋白互作效应，确定pPeOp蛋白对Cdc42/JAK/STAT3通路的网络调控作用，明确关键调控靶点，阐明pPeOp蛋白的抗胃癌作用与Cdc42/JAK/STAT3通路网络调控的相关性。

胃癌是我国高发的恶性肿瘤之一，目前胃癌治疗面临预后差、术后复发率高的瓶颈。本项目前期发现抗肿瘤中药雷丸所含pPeOp蛋白能显著抑制STAT3/Cdc42的转录，而Cdc42/Rac1是STAT3磷酸化的重要调控因子，pPeOp蛋白的抗胃癌作用可能与Cdc42/Rac1调控STAT3磷酸化有关。本项目采用慢病毒转染胃癌细胞HGC-27和MC-4，构建了Cdc42敲低和STAT3敲低稳转株并制备荷瘤裸鼠模型。细胞表征结果表明，敲低Cdc42或STAT3能够显著增强pPeOp抑制胃癌细胞的活性、增殖和迁移能力，促进细胞凋亡，阻滞细胞周期于G1期。深入研究表明，pPeOp蛋白抑制了促进STAT3磷酸化相关蛋白JAK1-3、Cdc42等的表达，促进了抑制STAT3磷酸化相关蛋白SOCS1和SOCS3的表达，导致以p-STAT3为核心的蛋白网络被显著负调控。裸鼠荷瘤实验表明，pPeOp蛋白对肿瘤生长具有显著抑制作用，敲低STAT3或Cdc42对抑瘤效果有一定增强作用。病理学结果证实，pPeOp蛋白的干预可显著降低瘤体组织中肿瘤细胞的核分裂相数量及显著增加凋亡细胞数量，同时显著抑制Cdc42、Rac1和STAT3的表达或磷酸化。综上所述，雷丸蛋白pPeOp通过负调控Cdc42/JAK/STAT3信号通路抑制STAT3磷酸化是pPeOp发挥抗胃癌作用的重要途径之一。