Introduction The CXC-chemokine CXCL4 is released from alpha granules of activated platelets and is involved in platelet aggregation. This protein is a chemo-attractant for numerous other cell types and also functions as an inhibitor of hematopoiesis, T-cell function, tumor-induced angiogenesis and modulation of host inflammatory response. Due to its multiple functions, CXCL4 is a potential clinical anti-tumor target. We examined the effects of the CXCL4 gene on biological properties of a colorectal cancer cell line by in vitro functional tests. RNA interference (RNAi) methodology by means of gene specific stealth siRNA technology was used to control the expressional level of the CXCL4 gene. Aim of study * To determine the effect of CXCL4 knockdown by RNAi methodology on biological properties of CC531 cells in vitro. * To evaluate the role of CXCL4 gene in colorectal cancer and later on its metastasis in animal rat liver model. Materials and Methods * Rat colorectal CC531 cancer cells were inplanted into the rat liver. After selected time intervals, CC531 cancer cells were re-isolated and their expression profile was studied by mRNA micro-array analysis. In vitro cultured CC531 cells were used as control in this study. * Transient knockdown of the CXCL4 gene was achieved in CC531 cells by using Lipofectamine 2000 transfection reagent along with 100nM gene specific stealth siRNA. * After 24, 48 and 72 h of treatment with siRNA, the extent of knockdown of CXCL4 was determined at mRNA level by RT-PCR and qPCR. * Functional effects of CXCL4 knockdown on CC531 cells were investigated by proliferation, colony formation, migration and scratch assays. Results In vivo Expression of CXCL4 Implanted colorectal CC531 cancer cells exhibited 3-4 times lower expression in the rat liver for an initial period of 3 weeks and came back to normal level after week 4. Knock-down of CXCL4 Exposure to siRNA species reduced the expression of CXCL4 at mRNA level after 24-72h with maximum reduction of 70% after 24h. MTT Assay Following the post-transfection period, we found a moderate decline in cell proliferation by 20, 26 and 8% for 24, 48 and 72h, respectively, in knockdown CC531 cells. Colony Formation Assay We observed reduced ability of knockdown CC531 cells to produce small and large colonies. Migration Assay Significant reduction in directional migration was observed in the knockdown CC531 cells especially after 48 and 72hr. Scratch Assay Photomicrographs, obtained after 12 and 24 hours of scrape wounding showed a significant reduction of knockdown CC531 cells to cover the scratched area as compare to control CC531 cells. Conclusion Higher expression of CXCL4 contributes to enhanced cell proliferation, migration, colonization and wound healing abilities of colorectal CC531 cancer cells in vitro. Based on these preliminary results, we will also study the effect of CXCL4 in a liver cancer metastasis model to evaluate an anticipated correlation with colorectal cancer progression.


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