The Role Of APOM In Sunitinib Resistance In Clear Cell Renal Cell Carcinoma

ObjectiveRenal cell carcinoma(RCC)is one of the top ten most common malignancies in humans,with its incidence and mortality rates increasing annually.Clear cell RCC(ccRCC)is the most common pathological type of RCC and is often associated with poor prognosis.In addition,ccRCC frequently exhibits innate or acquired resistance to molecular targeted drugs,leading to reduced treatment efficacy.However,the mechanisms underlying the occurrence and development of ccRCC and its potential resistance to sunitinib are not yet clear.Apolipoprotein M(APOM),encoded by the APOM gene,is a novel lipid-related plasma protein of the apolipoprotein family.Aberrant expression of this gene is closely related to the occurrence and development of various tumors.The purpose of this study is to explore the potential mechanisms of APOM in the occurrence and development of ccRCC and its resistance to sunitinib.MethodsFirstly,we conducted bioinformatics analysis of the APOM gene.To this end,we used the GEPIA2 online database to analyze the expression of APOM gene in various tumor and normal tissues,and the ULCAN database to analyze the differential expression of APOM in ccRCC.We used the Kaplan-Meier Plotter database to analyze the relationship between APOM expression levels and overall survival(OS)and progression-free survival(PFS)of ccRCC patients.We constructed a protein-protein interaction(PPI)network of the target gene to identify the core genes closely related to the target gene,and then performed GO and KEGG pathway analysis.In addition,based on the expression analysis of APOM,we identified differentially expressed genes using the mRNA expression matrix in the TCGA database,and conducted GO and KEGG pathway analysis on these genes.Secondly,we collected tumor tissue and adjacent normal kidney tissue samples from 40 ccRCC patients.We used qRT-PCR and Western Blot techniques to analyze the expression of the target gene in clinical tissue samples.We also used qRT-PCR and Western Blot techniques to detect the expression of APOM mRNA and protein in ccRCC cells(786-O,A498,ACHN and Caki-1)and renal tubular epithelial cells(293T and HK-2).Finally,we established cell lines with high expression of the target gene in 786-O and A498 cell lines using lentivirus transfection technology.We validated the successful modeling by detecting the mRNA and protein expression levels of the target gene using qRT-PCR and Western Blot techniques.We used CCK-8 assay and colony formation assay to detect the effects of the target gene on the proliferation and drug resistance of ccRCC cell lines 786-O and A498.We used cell scratch assay and Transwell assay to detect the effects of the target gene on the migration and invasion ability of ccRCC cell lines 786-O and A498.Furthermore,we conducted in vivo experiments in nude mice to analyze the subcutaneous tumor formation results and explore the effects of APOM on the tumorigenicity and drug resistance of ccRCC cell line 786-O.We analyzed the results of the two pathway analyses and referred to relevant literature to explore the signaling pathways that the target gene may be involved in.ResultsThrough analysis of online databases,significant differences were found in the expression of the APOM gene in different tumors.In ccRCC tissue,both mRNA and protein expression levels of APOM were significantly decreased(P<0.05).Low expression of APOM was associated with poor OS in patients(P<0.05),while it was not associated with PFS(P>0.05).Molecular functions of APOM and related signaling pathways were predicted by GO analysis and KEGG pathway analysis.Validation of mRNA and protein levels of APOM in tumor tissue was conducted through qRT-PCR and Western blot experiments,which revealed that APOM expression was significantly lower than in adjacent normal tissue(P<0.05).In ccRCC cells,both mRNA and protein levels of APOM were significantly lower than in renal tubular epithelial cells(P<0.05).The effect of APOM on ccRCC was verified through in vitro and in vivo experiments using cell and animal models,respectively.High expression of APOM in ccRCC cell lines inhibited cell proliferation,migration,invasion,and drug resistance in vitro,as well as tumor formation and drug resistance in vivo.Conversely,low expression of APOM in ccRCC cell lines promoted cell proliferation,migration,invasion,and drug resistance.Combining two different pathway analysis methods and reviewing relevant literature,it was found that APOM may be associated with the PPAR signaling pathway.ConclusionThe above study indicates that based on online databases and clinical specimens,APOM is significantly downregulated in ccRCC,and low expression of APOM is associated with poor prognosis in patients.In cell experiments,high expression of APOM can inhibit the proliferation,migration,invasion,and sunitinib resistance of ccRCC cells.Conversely,low expression of APOM promotes the proliferation,migration,invasion,and sunitinib resistance of ccRCC cells.In animal experiments,high expression of APOM also exhibits a significant inhibitory effect on ccRCC tumorigenicity and sunitinib resistance.The specific molecular mechanisms underlying these findings require further investigation,and APOM may serve as a biomarker for evaluating tumor prognosis.