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Reg3A Overexpression Facilitates Hepatic Metastasis by Altering Cell Adhesion in LoVo Colon Cancer Cells.

DNA and Cell Biology 2024 April 27
Reg3A is upregulated in various cancers and considered a potential target for antitumor treatments. However, the effect of Reg3A in metastasis has been elusive. This study aims to disclose the role of Reg3A overexpression in hepatic metastasis of LoVo colon cancer cells. A stable cell line of LoVo cells overexpressing Reg3A (LoVo-luc-Reg3A), labeled with luc reporter gene, was constructed. Cell proliferation, apoptosis, migration, and invasion were determined using MTT, EdU, Hoechst's staining, flow cytometry, and transwell assays, respectively. Hepatic metastasis of LoVo-luc-Reg3A cells was investigated in BALB/c nude mice. Living bioluminescence imaging, histological examination, and mRNA sequencing (mRNA-seq) were performed to assess the metastatic efficiency and gene expression alteration. Reg3A content was determined by Western blotting and Enzyme-Linked Immunosorbent Assay. Cell attachment capacity was determined in the Matrigel culture. Reg3A overexpression did not promote LoVo cell proliferation or apoptosis, but facilitated cell migration and invasion. In the hepatic metastasis model, Reg3A overexpression increased the number of metastatic colonies. The result of mRNA-seq suggested 349 differentially expressed genes (DEGs) by Reg3A upregulation, many of which were related to colon adenocarcinoma tumorigenesis compared to normal colon tissue. Gene ontology enrichment assay indicated that the DEGs are mainly associated with cell adhesion, leukocyte regulation, extracellular matrix (ECM) remodeling, integrin binding, and STAT protein binding. Reg3A overexpression led to an enrichment of Reg3A protein in local tumor tissue of liver metastasis and ECM/intracellular space in ex vivo cultured cells. However, Reg3A concentration in serum and culture medium was relatively low. Reg3A overexpression also resulted in an increased number of cells that attach to Matrigel, which was attenuated by treatments of siRNA-Reg3A and single-chain variable fragment against Reg3A. Endogenous Reg3A overexpression facilitates hepatic metastasis of LoVo colon cancer cells. The prometastatic effect could be contributed by Reg3A enrichment in ECM, which alters the cell adhesion behavior.

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