Zhang Junzhe, Yang Kaini, Bu Junfeng, Yan Jiayan, Hu Xiaoqiang, Liu Ke, Gao Si, Tang Shuibin, Gao Lili, Chen Wei
Department of Biliary-Pancreatic Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai, China.
Front Oncol. 2022 Oct 13;12:1035871. doi: 10.3389/fonc.2022.1035871. eCollection 2022.
Recent studies have reported that IGF2BP3 is linked to the pathogenesis of various malignancies. Since IGF2BP3 is associated with poor outcomes of gallbladder carcinoma (GBC), we aimed to explore the association between its N-methyladenosine (m6A) RNA methylation and GBC progression.
Bioinformatic analysis of GSE136982, GSE104165, and RNA-seq was performed. and gain- and loss-of-function assays were done. qPCR, Western blotting, and IHC were conducted in cells or in collected clinical tissue samples. RNA immunoprecipitation, RNA stability measurement, methylated RNA immunoprecipitation, and dual-luciferase reporter assays were performed in this study.
The expression of IGF2BP3 was higher in GBC tissues than in peritumoral tissues. Functions such as cell proliferation and migration, both and , were inhibited by downregulation of IGF2BP3. The analysis of RNA-seq indicated that KLK5 was a downstream target of IGF2BP3. The expression of KLK5 was measured in GBC cells and tumor samples. It was found to be positively correlated with IGF2BP3 level. Upon IGF2BP3 depletion, ectopic expression of KLK5 could rescue cell function in part. Mechanistically, we found that IGF2BP3 directly binds to KLK5 mRNA and regulates its stability in an m6A-dependent manner. As a result, inhibition of KLK5 decreased the expression of PAR2, and deregulated phospho-Akt. Using bioinformatic prediction combined with miRNA microarray analysis, we identified that let-7g-5p is an inhibitor of IGF2BP3, and let-7g-5p expression was negatively correlated with IGF2BP3. Overexpression of let-7g-5p affected the aggressive phenotype of GBC cells by deregulating IGF2BP3, and inhibiting the KLK5/PAR2/AKT axis.
Our data showed that IGF2BP3 is associated with the aggressive phenotype of GBC. Mechanistically, IGF2BP3 activated the PAR2/AKT axis by stabilizing KLK5 mRNA in an m6A-dependent manner. The loss of let-7g-5p enhanced the expression of IGF2BP3 and improved GBC progression. Thus, IGF2BP3 plays a crucial role in GBC, and the let-7g-5p/IGF2BP3/KLK5/PAR2 axis may be a therapeutic target for GBC.
近期研究报道称，IGF2BP3与多种恶性肿瘤的发病机制相关。由于IGF2BP3与胆囊癌（GBC）的不良预后有关，我们旨在探讨其N-甲基腺苷（m6A）RNA甲基化与GBC进展之间的关联。
对GSE136982、GSE104165进行生物信息学分析以及RNA测序。进行了功能获得和功能缺失实验。在细胞或收集的临床组织样本中进行了qPCR、蛋白质免疫印迹和免疫组化实验。本研究还进行了RNA免疫沉淀、RNA稳定性测量、甲基化RNA免疫沉淀和双荧光素酶报告基因实验。
IGF2BP3在GBC组织中的表达高于瘤旁组织。IGF2BP3的下调抑制了细胞增殖和迁移等功能。RNA测序分析表明KLK5是IGF2BP3的下游靶点。在GBC细胞和肿瘤样本中检测了KLK5的表达。发现其与IGF2BP3水平呈正相关。在IGF2BP3缺失后，KLK5的异位表达可部分挽救细胞功能。从机制上讲，我们发现IGF2BP3直接与KLK5 mRNA结合，并以m6A依赖的方式调节其稳定性。因此，抑制KLK5可降低PAR2的表达，并使磷酸化Akt失调。通过生物信息学预测结合miRNA微阵列分析，我们确定let-7g-5p是IGF2BP3的抑制剂，且let-7g-5p的表达与IGF2BP3呈负相关。let-7g-5p的过表达通过调节IGF2BP3并抑制KLK5/PAR2/AKT轴来影响GBC细胞的侵袭性表型。
我们的数据表明IGF2BP3与GBC的侵袭性表型有关。从机制上讲，IGF2BP3通过以m6A依赖的方式稳定KLK5 mRNA来激活PAR2/AKT轴。let-7g-5p的缺失增强了IGF2BP3的表达并促进了GBC进展。因此，IGF2BP3在GBC中起关键作用，let-7g-5p/IGF2BP3/KLK5/PAR2轴可能是GBC的治疗靶点。
