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Wnt signaling inhibition in human colon cancer cell lines (SW480 and SW620) drives changes in gene programs for cell adhesion, mobility, and cell junction

ABSTRACT: Overactive Wnt signaling has been found to be a significant driver in colon cancer carcinogenesis targeting many cellular processes such as cel proliferation, migration, and metabolism. The purpose of this study was to use high throughput transcriptional profiling (RNA-seq) to identify Wnt signaling gene targets in human colon cancer cells. Transcriptional profiles of human colon cancer cell lines (SW480 and SW620) with overactive Wnt signaling were compared to their matching Wnt low genetically modified stable cell (SW480-dnLEF1 and SW620-dnLEF1) through high throughput RNA-seq with four biological replicates. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat2 followed by HTseq and DESEQ2. Using an optimized data analysis workflow, we mapped about 30-40 million sequence reads per sample to the human genome (build hg38). RNAseq analysis of dnLEF1 expression in SW480 and SW620 cells cultured in vitro showed changes in gene programs for cell adhesion, mobility, cell junction, and extracellular matrix.

ORGANISM(S): Homo sapiens

PROVIDER: GSE131948 | GEO | 2020/05/31

REPOSITORIES: GEO

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Project description:Protein extraction and proteolytic digestion were performed using a Filter-Assisted Sample Preparation (FASP) protocol. In case of phosphopeptide enrichment immobilized Fe(III) affinity chromatography (Fe-IMAC) was performed. The peptides were fractionated using an HPLC system fitted with an SCX column. 10 sample datasets were used: 1. CRC_N: Normal tissue samples were obtained from 20 colorectal cancer patients, no quantification.Phosphopeptide enrichment was performed. 2. CRC_T: Human colorectal cancer tissue samples were obtained from 20 patients, no quantification. Phosphopeptide enrichment was performed. 3. CRC_iTRAQ: Human colorectal cancer tissue samples were obtained from 24 patients, iTRAQ quantification. 4. CRC_phospho_iTRAQ:Human colorectal cancer tissue samples were obtained from 24 patients, iTRAQ quantification.Phosphopeptide enrichment was performed. 5. HCT116_iTRAQ: Human colon cancer cell HCT116, iTRAQ quantification. 6. HCT116_phospho_iTRAQ: Human colon cancer cell HCT116, phosphopeptide enrichment, iTRAQ quantification. 7. SW_SILAC_HL: Human colon cancer cell SW480 and SW620, SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW480; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW620; heavy). 8. SW_SILAC_LH: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW620; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW480; heavy). 9. SW_phospho_SILAC_HL: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW480; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW620; heavy). Phosphopeptide enrichment was performed. 10. SW_phospho_SILAC_LH: SW480 and SW620 were grown in SILAC media, containing l-arginine (Arg0) and l-lysine (Lys0) (SW620; light), or 13C615N4-l-arginine (Arg10) and 13C6-l-Lysine (Lys6) (SW480; heavy). Phosphopeptide enrichment was performed. For creation of xml and mgf files, Proteome Discoverer 1.3 and Mascot v2.3 software were used.

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Wnt signaling inhibition in human colon cancer cell lines (SW480 and SW620) drives changes in gene programs for cell adhesion, mobility, and cell junction

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