P-0193 Transgelin Promotes The Invasiveness of Human Colon Cancer Cells in Vitro

Abstract

ABSTRACT Introduction Transgelin was the top-ranked marker of metastatic potential identified in the comparison of node-positive colorectal cancer (CRC) versus node-negative CRC in our previous study1. Transgelin is localized in the nucleus of cultured CRC cells and microRNA-mediated knockdown of TAGLN (the gene encoding transgelin) expression modulates the expression of genes involved in the epithelial-to-mesenchymal transition1. This study was initiated to investigate the hallmarks of metastasis in human colon cancer cell line RKO with stable expression of TAGLN in vitro. We also aimed to identify the downstream targets of transgelin in RKO cells. Methods The plasmid containing human TAGLN was generated using pcDNA6.2/EmGFP-Bsd/V5-DEST vector. Lipofectamine-mediated transfection was performed and stable transfectants were selected. The stable expression of TAGLN in RKO cells was validated by real-time RT PCR, western blotting and immunofluorescence. The Transwell invasion assay, clonogenic survival assay, cell proliferation assay and flow cytometry were performed in the RKO cells with stable expression of TAGLN and the control vector. DNA microarray was used to detect the mRNA expression of the downstream targets of transgelin in the RKO cells. Results Real-time RT PCR, western blotting and immunofluorescence indicated that the expression of TAGLN increased by 5-fold in the RKO cells transfected with pcDNA6.2/EmGFP-Bsd/V5-TAGLN plasmid as compared to those transfected with the control vector (P63:4650.01). Overexpression of TAGLN increased the invasion by 180% in RKO cells (P63:4650.01), and increased the clonogenic survival by approximately 129% (P63:4650.01). However, overexpression of TAGLN had minimal impact on cell proliferation and apoptosis of RKO cells (P>0.05). Preliminary analysis of the expression microarray of the downstream targets of transgelin in RKO cells indicated that many cancer-related pathways were involved, notably the integrin-PI3K/Akt pathway, Ras-Rac-JNK pathway and the ezrin-radixin-moesin (ERM) proteins. Conclusion Transgelin was able to promote the invasiveness of human colon cancer cell line RKO in vitro and enhance the clonogenic survival. Transgelin could be a determinant of metastatic potential in human colon cancer cells, which effects through many cancer-related pathways.