AIM To establish a permanent piwi like RNA-mediated genesilencing 1(PIWIL1) gene knockout in AGP01 gastric cancer cell line using CRISPR-Cas9 system and analyze phenotypic modifications as well as gene expression alte...AIM To establish a permanent piwi like RNA-mediated genesilencing 1(PIWIL1) gene knockout in AGP01 gastric cancer cell line using CRISPR-Cas9 system and analyze phenotypic modifications as well as gene expression alterations.METHODS CRISPR-Cas9 system used was purchased from Dharmacon GE Life Sciences(Lafayette, CO, United States) and permanent knockout was performed according to manufacturer's recommendations. Woundhealing assay was performed to investigate the effect of PIWIL1 knockout on migration capability of cells and Boyden chamber invasion assay was performed to investigate the effect on invasion capability. For the gene expression analysis, a one-color microarray-based gene expression analysis kit(Agilent Technologies, Santa Clara, CA, United States) was used according to the protocol provided by the manufacturer. RESULTS PIWIL1 gene knockout caused a significant decrease in AGP01 migration capacity as well as a significant decrease in cell invasiveness. Moreover, functional analysis based on grouping of all differentially expressed m RNAs identified a total of 35 genes(5 up-regulated and 30 down-regulated) encoding proteins involved in cellular invasion and migration. According to current literature, 9 of these 35 genes(DOCK2, ZNF503, PDE4 D, ABL1, ABL2, LPAR1, SMAD2, WASF3 and DACH1) are possibly related to the mechanisms used by PIWIL1 to promote carcinogenic effects related to migration and invasion, since their functions are consistent with the changes observed(being up-or down-regulated after knockout). CONCLUSION Taken together, these data reinforce the idea that PIWIL1 plays a crucial role in the signaling pathway of gastric cancer, regulating several genes involved in migration and invasion processes; therefore, its use as a therapeutic target may generate promising results in the treatment of gastric cancer.展开更多
基金Supported by Fundacao Amazonia de Amparo a Estudos e Pesquisa(FAPESPA),No.174/2014
文摘AIM To establish a permanent piwi like RNA-mediated genesilencing 1(PIWIL1) gene knockout in AGP01 gastric cancer cell line using CRISPR-Cas9 system and analyze phenotypic modifications as well as gene expression alterations.METHODS CRISPR-Cas9 system used was purchased from Dharmacon GE Life Sciences(Lafayette, CO, United States) and permanent knockout was performed according to manufacturer's recommendations. Woundhealing assay was performed to investigate the effect of PIWIL1 knockout on migration capability of cells and Boyden chamber invasion assay was performed to investigate the effect on invasion capability. For the gene expression analysis, a one-color microarray-based gene expression analysis kit(Agilent Technologies, Santa Clara, CA, United States) was used according to the protocol provided by the manufacturer. RESULTS PIWIL1 gene knockout caused a significant decrease in AGP01 migration capacity as well as a significant decrease in cell invasiveness. Moreover, functional analysis based on grouping of all differentially expressed m RNAs identified a total of 35 genes(5 up-regulated and 30 down-regulated) encoding proteins involved in cellular invasion and migration. According to current literature, 9 of these 35 genes(DOCK2, ZNF503, PDE4 D, ABL1, ABL2, LPAR1, SMAD2, WASF3 and DACH1) are possibly related to the mechanisms used by PIWIL1 to promote carcinogenic effects related to migration and invasion, since their functions are consistent with the changes observed(being up-or down-regulated after knockout). CONCLUSION Taken together, these data reinforce the idea that PIWIL1 plays a crucial role in the signaling pathway of gastric cancer, regulating several genes involved in migration and invasion processes; therefore, its use as a therapeutic target may generate promising results in the treatment of gastric cancer.
