Dissecting the invasive potential of esophageal cancer by siRNA library screening.

Abstract

86 Background: The low survival for esophageal cancer is in part attributed to its high invasive potential and distant metastasis. In cancer, abnormal cell migration is an essential component of metastasis, and it is reasonable to consider that the conversion between different forms of morphology permits tumor cells to metastasise. Discovering regulators of esophageal cancer morphogenesis may aid in the development of novel targeted therapies that limit metastatic potential. Methods: GOhTRT cells were seeded and treated with siRNA (Human Druggable Genome, Dharmacon) by reverse transfection. Cells were fixed, immunostained for DNA, tubulin and actin and imaged with automated microscope. Cell images were processed using the InCell Analyzer software and the R statistical software systems CellHTS2 and RNAither. The effect of RNAi knockdown on cell viability and cell motility were assessed using MTT cell proliferation assay and scratch wound assay. Results: 127 gene candidates greatly exhibited effects on F-actin and α-tubulin area staining. This list was refined to six high quality candidates (RRM2, ITGB8, GPS1, SPRY1, NOL1 and MYO9B). 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. There was no significant difference in cell viability absorbance values for siGPS1 (0.9037 ± 0.06575; p =0.1905) and siSPRY1 (0.9088 ± 0.09849; p =0.2985), which suggests that the increased rate of wound closure previously seen is by virtue of migratory signalling as oppose to an increase in cell 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). Conclusions: In summary, this screen successfully identified high confidence hits associated with cytoskeletal remodelling, 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 patients.