APOC2 Affects The Wnt Pathway By Regulating The Stability Of β-catenin In Lung Adenocarcinoma

Objective:Globally,China has one of the higher incidences of lung cancer in the world.And the incidence of lung adenocarcinoma(LUAD)is growing in recent years.Although the overall 5-year survival rate of lung cancer is no more than 20%,the overall 5-year survival rate for early-stage LUAD is approximately 50%.Thus,the thoracic surgeons are placing the early detection and treatment of LUAD at the top of their priority list.Among early-stage LUAD,our interest was piqued because they display pathological features such as pure ground-glass opacity(GGO)and solid nodules.Thus,our department has undertaken a series of studies on earlystage LUAD,to understand its pathogenesis,develop novel diagnosis and treatment strategies,and enrich the diagnosis and treatment biomarkers for LUAD.In this study,clinical samples were obtained from patients with early-stage LUAD that underwent surgery in our hospital and sent to RNAseq.The APOC2 gene was screened from the sequencing results for follow-up research.We constructed APOC2 overexpression and knockdown cell lines and performed transcriptome sequencing,bioinformatics analysis,CO-IP experiments,and mass spectrometry analysis to reveal the molecular mechanisms of APOC2 on LUAD.Our findings may provide new light on the diagnosis and treatment of LUAD.Materials and Methods:1.Transcriptome sequencing was used to analyze the differentially expressed genes(DEGs).The DEGs function analysis was performed by GO pathways enrichment analysis,and the core genes were screened by Cyto Hubba for study.2.The expression of APOC2 was preliminarily explored by the TCGA pan-cancer database,TCGA-LUAD,and GEO lung adenocarcinoma database.3.Kaplan-Meier survival curve for prognosis difference between low and high expression APOC2 in TCGA pan-cancer.Logical regression was used to verify whether APOC2 was an independent prognostic indicator of lung adenocarcinoma in TCGA-LUAD and GEO databases.4.GSEA enrichment analysis was used to explore the activation or inhibition of signaling pathways caused by APOC2 expression changes.5.m RNA and protein of clinical samples and lung cancer cell lines were extracted and their APOC2 expression levels were detected by RTPCR,immunohistochemistry,and WB.6.APOC2 overexpressed and knockdown cell lines were established to explore the effects of APOC2 on the biological behavior of lung adenocarcinoma by CCK-8 assay experiment,Transwell experiment,plate cloning assay experiment,and lipid staining assay.7.The downstream target genes of APOC2 were identified by RNAseq,CO-IP,and mass spectrometry.8.The specific localization of APOC2 and its target gene was explored by cytoplasmic and nuclear isolation experiments,immunohistochemistry,and immunofluorescence co-localization of transplanted tumor models.9.APOC2 mutants and truncated downstream target genes were constructed to explore the domain of interaction between APOC2 and downstream target genes.10.To explore the effects of APOC2 on the degradation patterns and stability of downstream target genes by adding inhibitors of different molecular degradation pathways.Results:1.The transcriptome results suggest that the Wnt signaling pathway,lipid metabolism-related signaling pathway,and immune-related signaling pathway play an important role in the occurrence and development of early lung adenocarcinoma(P<0.05,FDR q<0.25).2.In the TCGA-LUAD and GEO databases of lung adenocarcinoma,the m RNA expression level of APOC2 in tumor tissues was lower than that in normal tissues,especially in patients with AJCC stage IA(P<0.05).APOC2 was differentially expressed in lung adenocarcinoma and normal lung tissues,and m RNA and protein levels were lower in lung adenocarcinoma tissues(P<0.05).3.APOC2 expression is an independent prognostic indicator of lung adenocarcinoma(HR<1;P<0.05).The level of APOC2 expression is correlated with the prognosis of lung adenocarcinoma patients,and the prognosis of patients with high APOC2 expression is better than that of patients with low expression(P<0.05).4.GSEA analysis indicated that increased APOC2 expression inhibited RNA degradation,Wnt signaling pathway,spliceosome,cell cycle,and other signaling pathways.Activation of cytokine receptor interactions,chemokine signaling pathways,lysosomes,NK cell-mediated cytotoxicity,antigen processing and presentation,JAK-Stat signaling pathway,and other signaling pathways(P<0.05,FDR q<0.25).5.Overexpression of APOC2 significantly inhibited the proliferation ability,invasion ability,and migration ability of LUAD(P<0.05).APOC2 knockdown significantly promoted the proliferation ability,invasion ability,and migration ability of LUAD(P<0.05).APOC2 overexpression could significantly promote intracellular lipid accumulation in lung adenocarcinoma cells(P<0.05).Knockdown APOC2 significantly inhibited intracellular lipid accumulation in lung adenocarcinoma cells(P<0.05).6.The co-immunoprecipitation experiment confirmed that APOC2 interacts with β-catenin.Further IP experiments using mutants and truncations of APOC2 and β-catenin provided further evidence that APOC2 and β-catenin directly interact.The sites of action are APOC2(The amino acid sequence: 35～63)and β-catenin(The amino acid sequence:233～297).7.APOC2 interacts with β-catenin proteins mainly in the cytoplasm.Increased APOC2 expression promoted β-catenin degradation in the cytoplasm,inhibited β-catenin entry into the nucleus,and inhibited the Wnt/β-catenin signaling pathway and EMT signaling pathway activation.Knockdown APOC2 increased the protein expression of β-catenin in the cytoplasm and nucleus and activated the Wnt/β-catenin signaling pathway and EMT signaling pathway.8.APOC2 overexpression may initiate ubiquitination of β-catenin and decrease the stability of β-catenin protein while knocking down APOC2 increased the stability of the β-catenin protein.Conclusion:1.APOC2 tended to be a prognostic biomarker for lung adenocarcinoma patients,and its expression is significantly associated with survival.2.APOC2 could significantly inhibit the proliferation ability,invasion ability,migration ability,and tumor formation ability of LUAD.3.APOC2 and β-catenin can interact directly,and their interaction site is APOC2(The amino acid sequence: 35～63)and β-catenin The amino acid sequence: 233～297).4.APOC2 affects the stability of β-catenin to inhibit the Wnt/β-catenin signaling pathway and EMT signaling pathway.