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Next generation sequencing comparing effects of FFSS, FAK inhibitor and FAK gene deletion. [FFSS]

ABSTRACT: Next generation sequencing comparing effects of FFSS, FAK inhibitor and FAK gene deletion. [FFSS]

PROVIDER: PRJNA603271 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10969104.fastq.gz	Fastqsanger.gz
	SRR10969105.fastq.gz	Fastqsanger.gz
	SRR10969106.fastq.gz	Fastqsanger.gz
	SRR10969107.fastq.gz	Fastqsanger.gz
	SRR10969108.fastq.gz	Fastqsanger.gz

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Project description:Purpose: Focal adhesion kinase (FAK), hyaluronan (HA), and hyaluronan synthase-3 (HAS3) have been implicated in cancer growth and progression. FAK inhibition with the small molecule inhibitor Y15 decreases colon cancer cell growth in vitro and in vivo. HAS3 inhibition in colon cancer cells decreases FAK expression and activation, and exogenous HA increases FAK activation. We sought to determine the genes affected by HAS and FAK inhibition and hypothesized that dual inhibition would synergistically inhibit viability. Methods: We treated SW620 colon cancer cells with Y15 to inhibit FAK. We used two strategies to inhibit HAS: (1) cells were transfected with siRNA (HAS3 inhibited); a scrambled sequence was used as a control (HAS3 scrambled), and (2) cells were treated with the HAS inhibitor 4-methylumbelliferone (4-MU). To determine the effect on viability, MTT assays were performed on transfected cells treated with Y15, and wild type cells treated with Y15 alone, 4-MU alone or Y15+4-MU. Treated and untreated cells were submitted to the gene microarray facility for expression profiling. RT-PCR was done to confirm the results. Results: HAS and FAK inhibition affected cell viability. Y15 and 4-MU decreased viability in a dose-dependent manner; viability was further inhibited by treatment with Y15+4-MU in combination (p<0.05). HAS-inhibited cells treated with as little as 2 M of Y15 showed significantly decreased viability compared to HAS scrambled cells treated with the same dose (p<0.05), suggesting synergistic inhibition of viability with dual FAK/HAS inhibition. Microarray analysis showed more than 2-fold up- or down-regulation of 121 genes by HAS inhibition, and 696 genes by FAK inhibition (p<0.05). Of 29 genes that were common to both groups, 9 were down-regulated (CBS, DHRS3, EEPD1, ESPN, FAM46C, GRTP1, IL20RA, INHBE, SCNN1A) and 4 were up-regulated (ANXA1, MALL, RGS2, SNAI2). RT-PCR confirmed these findings. Among the genes affected by FAK or HAS3 inhibition were FOX genes (apoptosis, cell cycle regulation), ANXA1 (apoptosis, proliferation), IL8 (cell cycle regulation, adhesion, proliferation), RGS2 (cell cycle regulation), CEACAM6 (adhesion), SNAI2 (transcription regulation), and SFRP5 (apoptosis). Several genes were specific to either FAK or HAS3 inhibition and several were common to both. Gene expression profiles of samples isolated from human colorectal cancer cells (SW620). A comparison of gene expression between untreated cells and cells treated with 4mcM of Y15. A second comparison between cells transfected with siRNA to HAS3 (HAS3-silenced) and cells transfected with a scrambled control sequence (sc). Two replicates each.

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Next generation sequencing comparing effects of FFSS, FAK inhibitor and FAK gene deletion. [FFSS]

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