Abstract
Aim: To quantitatively assess complex cancer cell phenotypes after gene inhibition by RNAi interference (RNAi), and assess the effect of these distinct morphologies on cell viability and cell motility using semi-automated image-based high-throughput screening. Method: GOhTRT cells were seeded and treated with the siRNA Human Druggable Genome Library (Dharmacon) by reverse transfection. Cells immunostained for DNA, tubulin and actin were imaged with the InCell Analyzer 1000 and processed using the InCell Analyzer software, CellHTS2 and RNAither. Statistical z-score analysis was performed on the combined A-T metric (F-actin area-α-tubulin area). The effect of RNAi knockdown on cell viability and cell motility were assessed using MTT cell proliferation assay and scratch wound assay. Results:127 high confidence hits (Z-factor>2) was refined to six genes (RRM2, ITGB8, GPS1, SPRY1, NOL1, MYO9B) on the basis of distinct morphologies, reproducible metrics, and functional pathway analysis. In siRRM2 cells nuclear displacement (ND) and A-T area was 1.598±0.076; p <0.0001 and 285.70±35.48; p<0.05, respectively. siGPS1 cells had an ND and A-T area of 0.845±0.036; p =0.0369 and 266.201±25.629; p =0.0326, respectively. Silencing of GPS1, MYO9B and SPRY1 increased the rate of migration in a scratch wound assay, with 86.98%±3.097%, 75.78%±5.454% and 72.97%±5.463% (p =0.0022) respectively. No significant difference in cell viability existed for siGPS1 (0.9037 ± 0.06575; p = 0.1905) and siSPRY1 (0.9088 ± 0.09849; p =0.2985), suggesting that wound closure is by virtue of migratory signalling and not proliferation. Cell viability was decreased considerably in siRRM2 cells (0.2492±0.02798; p <0.0001) and siMYO9B (0.4048±0.04663; p <0.0001) in comparison to siNT cells (1.046±0.07712). Discussion:In summary, this approach successfully identified genes regulating oesophageal cancer cell cytoskeletal remodelling and metastasis using in-vitro assays, some of which are already associated with metastasis in literature and database searches. Further mechanistic studies and gene pathway analysis of candidate genes will provide novel therapeutic targets which can be utilised to block the spread of cancer in oesophageal adenocarcinoma patients.
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