Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of human colorectal cancer HCT116 and HT29 cells by the distinct conditions of trypsin digestion

ABSTRACT: In the process of cell culture in vitro, we usually find that the cells show signs of aging, which is that the passed cells are unable to stick to the wall. Experience tells us that the time it takes for trypsin to digest the adherent cells is important. If the digestion time is too short, enough cells cannot be obtained for passage, while if the digestion time is too long, a large number of senescent cells will also be digested. The substances released by the death and decomposition of these senescent cells will affect the function of those active cells, resulting in the failure of the recovery of the whole daughter cell line. This project starts from the question of the global difference among cells undergoing the multiple rounds of mitosis in one culture dish. RNA sequencing was conducted to investigate the transcriptome signatures of cells with different sensitivity response to trypsin.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Yihang Shen

PROVIDER: E-MTAB-14651 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Gliadin triggers T-cell mediated immunity in celiac disease, and has cytotoxic effects on enterocytes mediated through obscure mechanisms. In addition, gliadin transport mechanisms, potential cell surface receptors and gliadin-activated downstream signaling pathways are not completely understood. In order to screen for novel downstream gliadin target genes we performed a systematic whole genome expression study on intestinal epithelial cells. Undifferentiated Caco-2 cells were exposed to pepsin- and trypsin- digested gliadin (PT-G), a blank pepsin-trypsin control (PT) and to a synthetic peptide corresponding to gliadin p31-43 peptide for six hours. RNA from four different experiments was used for hybridization on Agilent one color human whole genome DNA microarray chips. The microarray data were analyzed using the Bioconductor package LIMMA. Genes with nominal p < 0.01 were considered statistically significant. Compared to the untreated cells 1705, 1755 and 211 probes were affected by PT-G, PT and p31-43 respectively. 46 probes were significantly different between PT and PT-G treated cells. Among the p31-43 peptide affected probes, 10 and 21 probes were affected by PT-G and PT respectively. Only PT-G affected genes could be validated by quantitative real-time polymerase chain reaction. All the genes were, nonetheless, also affected to a comparable level by PT treated negative controls. In conclusion, we could not replicate previously reported direct effects of gliadin peptides on enterocytes. The PT-G affected genes in the microarray analysis were validated by qRT-PCR, however these genes were also affected by PT treated negative controls suggesting that certain epitopes derived from pepsin and trypsin may also affect epithelial cell gene transcription. Our study demonstrates novel non-enzymatic effects of pepsin and trypsin on cells and calls for proper controls in pepsin and trypsin digested gliadin experiments. It is conceivable that gliadin effects on enterocytes are secondary mediated through oxidative stress, NFkB activation and IL-15 up-regulation. In total, 16 samples were analyzed of which 4 were control (MED) samples, 4 samples of p31-43 treatment, 4 samples of PT treatmetn and 4 samples of PT-G treatment

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RNA-seq of human colorectal cancer HCT116 and HT29 cells by the distinct conditions of trypsin digestion

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