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MiRNA-mediated expression switch of cell adhesion genes driven by microcirculation in chip

ABSTRACT: Changes in cell adhesion molecule (CAM) expression and miRNAs regulating them are known to be involved in malignant progression in colon cancer. We investigated expression profiles of CAM genes and non-coding RNAs in CaCo2 colon cancer cells in static culture and under dynamic flow conditions perfused in microfluidic chip emulating physiological microenvironment. We incubated monolayers of CaCo2 cells in Transwell® units either under static conditions or under flow in a microfluidic chip. We identified 7 up-regulated CAM genes (CD44, CDH7, CEACAM5, CEACAM6, CYR61, L1CAM and VCAN), 7 down-regulated genes (COL12A1, FGA, FGB, FGG, GJA1, ITGA5 and LAMA1) and 69 miRNAs targeting them under the influence of microcirculation. The revealed network comprised CAM genes known to interact with each other and 13 miRNAs simultaneously regulating more than one of them.

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE81867 | GEO | 2016/05/26

SECONDARY ACCESSION(S): PRJNA322822

REPOSITORIES: GEO

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Project description:Blood flow promotes emergence of definitive hematopoietic stem cells (HSCs) in the developing embryo, yet the signals generated by hemodynamic forces that influence hematopoietic potential remain poorly defined. In transplantation assays of hematopoietic reconstitution, we find that fluid shear stress endows long-term multilineage engraftment potential upon early hematopoietic tissues at E9.5 not previously described to harbor HSCs. Effects on hematopoiesis appear to be mediated in part by prostaglandin E2 (PGE2) and the cyclic AMP-protein kinase A (cAMP-PKA) signaling axis. Studies of Ncx1 cardiac mutants corroborate that blood flow is required for sufficient COX2 levels and phosphorylation of CREB. Further implicating PGE2 in mediating the effects of shear stress, we find that E10.5 and E11.5 AGM treated transiently with the synthetic analog dmPGE2 engraft more robustly and contribute to greater lymphoid reconstitution. These data provide a mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential. - AGM from C57BL/6J embryos at 10.5 days gestation (E10.5) were isolated by microdissection from uteri of pregnant dams, following by gentle dissociation by Accutase with agitation at room temperature for 20 minutes. Single cell suspension was seeded on microfluidic IBIDI VI^0.4 6-channel slides (0.8 to 1x10^7 cells per channel) and permitted to attach for 8 hours. Fluid movement was then applied to each channel using a Harvard Apparatus PHD ULTRA programmable syringe pump for manangement of M5300 Myelocult medium. Cells were exposed either to static/low flow (0.0001 dyne/cm^2) or wall shear stress (WSS) of 5 dyne/cm^2 for 6 hours or 36 hours. In addition, some cells were treated with 10 uM indomethacin (indo) to inhibit COX2 activity and PGE2 synthesis. Upon collection of cells with RLT lysis buffer (QIAGEN RNeasy kit), six channels of identical treatment were pooled to comprise a single sample. 24 samples total are included in this study. 12 samples were collected after 6 hours and 12 after 36 hours. In detail, samples included at 6 hours: 3 static, 3 static with indo, 3 WSS, 3 WSS with indo; and at 36 hours: 3 static, 3 static with indo, 3 WSS, 3 WSS with indo. Sample labels begin with the timepoint collected and end with the replicate number, i.e., 06WSS1 for the 6 hour collection of the first replicate of the WSS sample.

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MiRNA-mediated expression switch of cell adhesion genes driven by microcirculation in chip

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