Abstract
BackgroundTransgelin, an actin-binding protein, is associated with cytoskeleton remodeling. Findings from our previous studies demonstrated that transgelin was up-regulated in node-positive colorectal cancer (CRC) versus node-negative disease. Over-expression of TAGLN affected the expression of 256 downstream transcripts and increased the metastatic potential of colon cancer cells in vitro and in vivo. This study aims to explore the mechanisms through which transgelin participates in the metastasis of colon cancer cells.MethodsImmunofluorescence and immunoblotting analysis were used to determine the cellular localization of endogenous and exogenous transgelin in colon cancer cells. Co-immunoprecipitation and subsequently high-performance liquid chromatography/tandem mass spectrometry were performed to identify the proteins that were potentially interacting with transgelin. The 256 downstream transcripts regulated by transgelin were analyzed with bioinformatics methods to discriminate the specific key genes and signaling pathways. The Gene-Cloud of Biotechnology Information (GCBI) tools were used to predict the potential transcription factors (TFs) for the key genes. The predicted TFs corresponded to the proteins identified to interact with transgelin. The interaction between transgelin and the TFs was verified by co-immunoprecipitation and immunofluorescence.ResultsTransgelin was found to localize in both the cytoplasm and nucleus of the colon cancer cells. Approximately 297 proteins were identified to interact with transgelin. The overexpression of TAGLN led to the differential expression of 184 downstream genes. Network topology analysis discriminated seven key genes, including CALM1, MYO1F, NCKIPSD, PLK4, RAC1, WAS and WIPF1, which are mostly involved in the Rho signaling pathway. Poly (ADP-ribose) polymerase-1 (PARP1) was predicted as the unique TF for the key genes and concurrently corresponded to the DNA-binding proteins potentially interacting with transgelin. The interaction between PARP1 and transgelin in human RKO colon cancer cells was further validated by immunoprecipitation and immunofluorescence assays.ConclusionsOur results suggest that transgelin binds to PARP1 and regulates the expression of downstream key genes, which are mainly involved in the Rho signaling pathway, and thus participates in the metastasis of colon cancer.
Highlights
Transgelin, an actin-binding protein, is associated with cytoskeleton remodeling
Localization of transgelin in human colon cancer cell lines The expression of transgelin in colon cancer cell lines (HCT116, SW480, RKO and LOVO) was detected by immunofluorescence and immunoblotting assays
Identification of proteins potentially interacting with transgelin in the RKO cells To explore the proteins that were potentially interacting with transgelin, we performed immunoprecipitation in the RKO-TAGLN-FLAG cells and the RKO-CTRLFLAG cells using an anti-flag monoclonal antibody
Summary
Transgelin, an actin-binding protein, is associated with cytoskeleton remodeling. Findings from our previous studies demonstrated that transgelin was up-regulated in node-positive colorectal cancer (CRC) ver‐ sus node-negative disease. This study aims to explore the mechanisms through which transgelin participates in the metastasis of colon cancer cells. Colorectal cancer (CRC) is a frequent malignant tumor in the gastrointestinal tract worldwide. Mechanisms of CRC tumorigenesis and metastasis have been extensively studied, high. Lew et al Cancer Cell Int (2020) 20:366 mortality rates are still reported, especially in patients with advanced disease [1]. The spread of cancer cells from the primary tumor to other areas of the body, is a complex process associated with remodeling of the cytoskeleton. Abnormal expression of related genes or effector proteins may lead to the activation of various signaling pathways, promoting tumor metastasis [2,3,4,5]
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