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RNA sequencing to study transcriptomic changes in DLD-1 (colorectal adenocarcinoma) cells exposed to soft polyacrylamide matrices (~2 kPa and ~55 kPa) for short time scale of 90 minutes

ABSTRACT: Aim: To examine transcriptional changes in DLD-1 cells exposed to softer matrices (2 kPa and 55 kPa) and identify the chromosomes that are enriched with maximmally deregulated genes Methods: DLD-1 cells (otherwise growing on stiff tissue culture plastic substrates) were exposed to softer matrices for 90 minutes and to collagen coated glass coverslips (served as control) served as control) Results: RNA sequencing revealed nearly equivalent transcriptional deregulation in cells on both the polyacrylamide matrices (783 genes up and 872 genes down on 2 kPa, 649 genes up and 783 genes down on 55 kPa) when compared to cells on glass. Additionally, GO classification revealed that unique sets of transcriptionally deregulated genes (log fold≥2) belonged to pathways associated with transcription regulation, chromatin organization, cell cycle and DNA damage/repair Results: We identified chromosomes 1, 2, 3, 6, 7, 10, 12, 14, 17 and 19 to be maximally enriched with the deregulated genes on softer matrices (log fold≥2), while chromosomes 13, 18 and 21 showed minimal enrichment of deregulated genes. We also examined the spatial organization of chromosome 1, 18 and 19 territories in cells on softer matrices (using 3D-FISH) and observed that these chromosomes were mislocalized away from their conserved nuclear locations Conclusions: Our study reports the transcriptomic changes in DLD-1 cells upon lowering of extracellular substrate stiffnes and its impact on the spatial positioning of chromosome territories

ORGANISM(S): Homo sapiens

PROVIDER: GSE108907 | GEO | 2018/04/05

REPOSITORIES: GEO

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Project description:Purpose: When we infect MDCK epithelial cells we low dosage of Listeria monocytogenes, we observe that at late times (24 h) post-infection the infected cells pertaining in infection foci get collectively extruded out of the epithelial monolayer forming a 3D mound. The formation of the infection mound results from the competition between two populations: the uninfected cells ("surrounders") that eventually squeeze and extrude the infected cells ("mounders") in the infected wells. The extracellular matrix where the host cells adhere appears to impact the formation of infection mounds with more mounding on glassr as opposed to soft 3 kPa substrata, as evidenced by confocal 3D microscopy. Here we built poalyacrylamide hydrogels of varing stiffness (3 kPa soft or 70 kPa stiff) and coated them with collagen I rat tail. We also placed host cells on glass collagen I-coated surfaces. We then infected for 24 h (or not) the host cells with Listeria monocytogenes to infer how matrix stiffness-dependent gene expression of MDCK cells in monolayers changes in uninfected versus infected wells, through RNA-Sequencing. Our goal was to understand how matrix stiffness impacts transcriptional regulation of host cells and whether that could modulate the collective extrusion of infected epithelial cells triggered by surrounding uninfected cells. Methods and Results: By analyzing the differentially expressed genes between all conditions we find a significant number of up- or down-regulated genes between all groups, other than between cells residing on soft versus stiff polyacrylamide hydrogels that behave similarly in terms of gene expression. Through pathway enrichement analysis, we find that cells residing on polyacrylamide hydrogels as opposed to cells residing on glass show higher upregulation of a number of innate immune signaling related pathways (e.g. MAPK, TNF) even when infection is not involved. When comparing infected versus not wells, the relative differences in gene expression for cells residing on glass are higher than for cells residing on hydrogels. However certain specific innate immune signaling pathways that regulate mound formation (https://www.sciencedirect.com/science/article/abs/pii/S1534580721000708), show higher upregulation in infection for cells on hydrogels as opposed to glass (e.g. IL-17, TNF, NF-κΒ; evidenced by pathway enrichement analysis). Finally, through pathway enrichement analysis we find that the relative change based on enrichement score when comparing uninfected versus infected settings for cells residing on glass versus on polyacrylamide hydrogels is lower for the former (i.e. for cells residing on glass) for NF-κB, TNF and IL17. However, with regards to the MAPK pathway the enrichment score of cells from infected versus uninfected wells, is much higher for cells residing on glass as opposed to polyacrylamide hydrogles. Conclusions: Infection of MDCK epithelial cells with Listeria monocytogenes significantly alters the transcriptome of the infected host cells when those are examined 24 h post infection and compared to uninfected cells on all different substrata. Compared to cells residing on glass substrates cells residing on polyacrylamide hydrogels (irrespective of whether the stiffness is low i.e. 3 kPa or high i.e. 70 kPa) show significant differences both when uninfected settings and when infected ones are compared.

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RNA sequencing to study transcriptomic changes in DLD-1 (colorectal adenocarcinoma) cells exposed to soft polyacrylamide matrices (~2 kPa and ~55 kPa) for short time scale of 90 minutes

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