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Abstract

The study aimed to analyze the gene expression patterns of LS174T colorectal cancer cells as they proliferate within the liver of nude rats over defined time intervals. LS174T cells were initially introduced into the rat mesenteric vein, facilitating subsequent growth within the liver. To streamline the experimental process and ensure precise tumor detection at specific stages (days 3, 6, 9, 14, and 21), LS174T cells were transfected with a luciferase marker gene for bioluminescence imaging. For efficient re-isolation of tumor cells, rat liver perfusion was done, followed by fluorescent activated cell sorting (FACS) and subsequent reculturing for further analysis. Total RNA extracted from re-isolated LS174T cells was subjected to chip array analysis, comparing re-isolated cells to those originally cultured before implantation into nude rats. Additionally, miRNA profiles were obtained from re-isolated cells at corresponding time points. Data analysis employed two approaches: analysis using the R programming language and Ingenuity Pathways Analysis (IPA). The R analysis focused on identifying gene categories associated with metastatic progression and important miRNAs related to tumor advancement. IPA was utilized to identify key signaling pathways and functional annotations associated with LS174T progression in the liver, comparing chip array data sets from a rat colorectal cancer cell line (CC531) and rat/human pancreatic cancer cells (ASML and Suit2-007). Analysis using the R-program revealed expression patterns of 21,448 genes at different stages of LS174T growth in the liver. Gene categories associated with tumor progression were identified, including those decreasing over time, upregulated during early stages (Early up), increasing over time, and exhibiting late-stage upregulation (Late up). Selected genes increasing over time were confirmed via qPCR and western blot, including MGST1, FTL, and RAB15. Furthermore, miRNA analysis identified different categories, with some exhibiting similar expression patterns to genes, such as miR-200b-3p and miR-141-3p. Furthermore, among the miRNAs categorized as 'early down,' notable members include miR-141-3p, miR-200c-3p, and miR-17-5p. Intriguingly, miRNA-1260a, exhibiting downregulation during the late stages (days 14 & 21), displayed a negative correlation with MGST1, FTL, and RAB15, all of which demonstrated an increase over time. IPA analysis identified 20 signaling pathways and functional annotations based on significant Z-scores, including the S100 family pathway, tumor microenvironment pathway, and colorectal cancer pathway. Additionally, common functions relevant to metastasis, such as cell survival, migration, proliferation, and angiogenesis, were identified. Overall, this study provides valuable insights for future research, particularly in identifying target genes for colorectal cancer therapy.