title: Identification of hepatic microvascular adhesion-related genes of human colon cancer cells using random homozygous gene perturbation.
abstract: Random homozygous gene perturbation (RHGP), in combination with liver sinusoidal endothelial cell (LSEC) adhesion screening of clonal colon cancer cells with perturbed genes, was used to identify genes contributing to the hepatic microvascular adhesion of colon cancer cells. Plasmid vector encoding transactivator and gene search vector were transfected into HT-29 human colorectal cancer cells to create a HT-29 RHGP cell library; the adhesion of these library cells to primary cultured mouse LSEC significantly decreased in the presence of RSL1 ligand (inducer), indicating that most of the genes contributing to HT-29 adhesion to LSEC were altered. Next, HT-29 RHGP cell library fractions with upregulated or silenced LSEC adhesion-related genes were isolated. Around 160 clones having altered expression in LSEC adhesion-related genes were obtained, and nine relevant protein-coding genes were identified. Some were proadhesive genes detected because of their overexpression in adherent HT-29 cells (DGCR8 and EFEMP1 genes) and their silenced status in nonadherent HT-29 cells (DGKE, DPY19L1, KIAA0753, PVR and USP11 genes). Others were antiadhesive genes detected because of their overexpression in nonadherent HT-29 cells (ITPKC gene) and their silenced status in adherent HT-29 cells (PPP6R2 gene). Silencing of PVR, DGCR8 and EFEMP1 genes decreased adhesion to LSEC and hepatic microvascular retention of HT-29 cells. The results conclude that RHGP was a valuable strategy for the discovery of mechanisms regulating microvascular adhesion of circulating colon cancer cells before hepatic metastasis formation. Identified genes may contribute to understand the metastatic process of colon cancer and to discovering molecular targets for hepatic metastasis therapeutics.
authors: Department of Cellular Biology and Histology, Basque Country University School of Medicine and Dentistry, Leioa, Bizkaia, Spain.
authors: Joana
authors: J
authors: Márquez
authors: Manu
authors: M
authors: Kohli
authors: Beatriz
authors: B
authors: Arteta
authors: Shaojing
authors: S
authors: Chang
authors: Wu-Bo
authors: WB
authors: Li
authors: Michael
authors: M
authors: Goldblatt
authors: Fernando
authors: F
authors: Vidal-Vanaclocha
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D002448:Cell Adhesion / Q000235:genetics*; D002478:Cells, Cultured; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D019073:HT29 Cells; D006801:Humans; D008099:Liver / Q000098:blood supply* / Q000378:metabolism / Q000473:pathology; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D014408:Biomarkers, Tumor; D012333:RNA, Messenger
keywords: HT29; RHGP; cancer cell adhesion-related genes; colon cancer