Zhou Ling, Guo Hui, Liao Quan, Zou Jianping, Le Yi, Fang Ziling, Xiong Jianping, Huang Shanshan, Deng Jun, Xiang Xiaojun
Department of Oncology, The First Affiliated Hospital of Nanchang University, Nanchang, China.
Jiangxi Key Laboratory for Individualized Cancer Therapy, Nanchang, China.
J Cell Mol Med. 2023 Oct;27(20):3090-3106. doi: 10.1111/jcmm.17880. Epub 2023 Aug 9.
Malignant cell growth and chemoresistance, the main obstacles in treating gastrointestinal cancer (GIC), rely on the Hippo and p53 signalling pathways. However, the upstream regulatory mechanisms of these pathways remain complex and poorly understood.
Immunohistochemistry (IHC), western blot and RT-qPCR were used to analyse the expression of RNF146, miR-3133 and key components of Hippo and p53 pathway. CCK-8, colony formation, drug sensitivity assays and murine xenograft models were used to investigate the effect of RNF146 and miR-3133 in GIC. Further exploration of the upstream regulatory mechanism was performed using bioinformatics analysis, dual-luciferase reporter gene, immunoprecipitation assays and bisulfite sequencing PCR (BSP).
Clinical samples, in vitro and in vivo experiments demonstrated that RNF146 exerts oncogenic effects in GIC by regulating the Hippo pathway. Bioinformatics analysis identified a novel miRNA, miR-3133, as an upstream regulatory factor of RNF146. fluorescence in situ hybridization and RT-qPCR assays revealed that miR-3133 was less expressed in gastrointestinal tumour tissues and was associated with adverse pathological features. Functional assays and animal models showed that miR-3133 promoted the proliferation and chemotherapy sensitivity of GIC cells. miR-3133 affected YAP1 protein expression by targeting RNF146, AGK and CUL4A, thus activating the Hippo pathway. miR-3133 inhibited p53 protein degradation and extended p53's half-life by targeting USP15, SPIN1. BSP experiments confirmed that miR-3133 promoter methylation is an important reason for its low expression.
miR-3133 inhibits GIC progression by activating the Hippo and p53 signalling pathways via multi-targets, including RNF146, thereby providing prognostic factors and valuable potential therapeutic targets for GIC.
恶性细胞生长和化疗耐药是治疗胃肠道癌（GIC）的主要障碍，这依赖于Hippo和p53信号通路。然而，这些通路的上游调控机制仍然复杂且了解甚少。
采用免疫组织化学（IHC）、蛋白质印迹法和逆转录定量聚合酶链反应（RT-qPCR）分析RNF146、miR-3133以及Hippo和p53通路关键成分的表达。采用细胞计数试剂盒-8（CCK-8）、集落形成、药敏试验和小鼠异种移植模型研究RNF146和miR-3133在GIC中的作用。利用生物信息学分析、双荧光素酶报告基因、免疫沉淀试验和亚硫酸氢盐测序PCR（BSP）进一步探索上游调控机制。
临床样本、体外和体内实验表明，RNF146通过调节Hippo通路在GIC中发挥致癌作用。生物信息学分析鉴定出一种新型微小RNA（miRNA），即miR-3133，作为RNF146的上游调控因子。荧光原位杂交和RT-qPCR分析显示，miR-3133在胃肠道肿瘤组织中表达较低，且与不良病理特征相关。功能试验和动物模型表明，miR-3133促进GIC细胞的增殖和化疗敏感性。miR-3133通过靶向RNF146、AGK和CUL4A影响Yes相关蛋白1（YAP1）蛋白表达，从而激活Hippo通路。miR-3133通过靶向泛素特异性蛋白酶15（USP15）、SPIN1抑制p53蛋白降解并延长p53的半衰期。BSP实验证实miR-3133启动子甲基化是其低表达的重要原因。
miR-3133通过多靶点激活Hippo和p53信号通路抑制GIC进展，这些靶点包括RNF146，从而为GIC提供预后因素和有价值的潜在治疗靶点。
