IGF2BP3 Upregulates The PPARD Pathway To Promote Bile Duct Cancer Cell Proliferation

BackgroundCholangiocarcinoma(CCA)is a rare and highly malignant liver tumor with a poor prognosis.In the last 20 years,the incidence of CCA has been increasing globally.However,the 5-year survival rate is less than 10%.Most patients have no specific presentation in the early stages,but are already in advanced stage or have distant metastasis at the time of diagnosis.Treatment options for CCA are still suboptimal,and early surgical resection remains the only possible treatment.Unfortunately,most patients with CCA are not sensitive to chemotherapy or radiation therapy.As a result,the search for new molecular markers and the exploration of their pathogenesis are imperative in the study of CCA to predict early-stage CCA.Insulin-like growth factor-2messenger RNA-binding protein 3(IGF2BP3)is a family of insulin-like growth factor 2m RNA-binding proteins,including IGF2BP1,IGF2BP2 and IGF2BP3.IGF2BP3 is clearly involved in ribonucleic acid(RNA)transport and stabilization,cell growth and migration during early embryonic development.Recent studies have shown that IGF2BP3 is importantly associated with many aggressive and advanced cancers and isspecifically expressed in many malignancies,including colorectal,gastric,bladder,pancreatic and renal cancers.In addition,previous literature has shown that IGF2BP3 is highly expressed in CCA tissues and is associated with poor clinical outcomes.However,these studies only reported the relationship between IGF2BP3 and CCA clinicopathology,while there are few basic studies on IGF2BP3 at the molecular level in CCA.Therefore,the present study attempted to explore the roleand molecular mechanism of action of IGF2BP3 in CCA.MethodsIn this study,we investigated the expression of the target gene IGF2BP3 in CCA by analyzing data from the biomarker database The Cancer Genome Atlas(TGGA)and Gene Expression Omnibus(GEO)databases.Survival prognostic information of GSE107943 patients was downloaded through the official website of GEO to evaluate the prognostic value of IGF2BP3 in CCA.To further investigate the sensitivity and specificity of IGF2BP3 gene in the diagnosis of CCA,sequencing data of GSE107943 were extracted and ROC curve analysis of IGF2BP3 was performed.We detected the protein expression level of IGF2BP3 in four CCA tissues by immunoblot analysis.The si RNA-IGF2BP3 transfected beads were constructed for cell proliferation test and colony formation test to demonstrate the role of IGF2BP3 in promoting the proliferation of CCA cells.Next,single gene GSEA analysis and KEGG enrichment analysis were performed to clarify the possible potential effector pathways of IGF2BP3 gene in the development of CCA,and human lentiviral vectors overexpressing IGF2BP3 were constructed for blocking assays to verify the specific effector mechanism of IGF2BP3 regulating PPARD pathway.Finally,immunohistochemical analysis of 20 pairs of CCA specimens was performed to clarify the correlation between IGF2BP3 and PPARD pathway.ResultsWe found that IGF2BP3 m RNA expression was significantly higher in CCA tissues than in the corresponding paracancerous tissues,as also demonstrated by immunoblot analysis.high expression of IGF2BP3 correlated with poor prognosis.ROC curve analysis of IGF2BP3 showed an AUC value of 0.901 between tumor and normal tissues,indicating that IGF2BP3 has reasonable diagnostic ability.The results of cell proliferation test and colony formation test showed that transfection of si RNA-IGF2BP3 in Hucct-1 and RBE cells significantly inhibited the proliferation of CCA cells(p < 0.05).GSEA analysis and KEGG enrichment analysis showed that IGF2BP3 expression is associated with the peroxisome proliferator-activated receptor(PPAR)signaling pathway and found that si-IGF2BP3 silencing in Hucct-1 cells,PPARD expression was decreased,and the same result was observed in RBE cells,indicating that IGF2BP3 expression is consistent with PPARβ/δ(PPARD)in Hucct-1 cell lines.By immunohistochemical analysis,we found that CCA tissues with high IGF2BP3 expression also had high PPARD protein,and CCA tissues with low IGF2BP3 expression had low PPARD.We also found that CCA cell proliferation after the addition of the PPARD inhibitor GSK3787,even after overexpression of IGF2BP3 in the Hucct-1 cell line or RBE cell line was significantly inhibited.In addition,colony formation in Hucct-1 cell line and RBE cell line showed the same results.ConclusionThis study confirmed that IGF2BP3,an oncogene,can regulate the proliferation of CCA cells,and that the PPARD pathway is a molecular mechanism by which IGF2BP3 functions.This provides a new theoretical basis for future exploration of the proliferation mechanism of CCA and increases the possibility that it may become a new prognostic marker and therapeutic target for CCA.