The effect of aquaporin-5 knockdown on HT29 colon cancer cell proliferation and migration Y Tomita1, 2, H Dorward1, J Wrin1, RJ Vary1, ML De Ieso2, AJ Yool2, TJ Price2, 3, JE Hardingham1,2. 1. Molecular Oncology, The Basil Hetzel Institute, The Queen Elizabeth Hospital Adelaide, SA, Australia; 2. University of Adelaide, Adelaide, SA, Australia; 3. Medical Oncology, The Queen Elizabeth Hospital, Adelaide, SA, Australia. Introduction Colorectal Cancer (CRC) CRC is a highly prevalent malignancy, accounting for 10 and 9% of malignancy cases in males and females worldwide (WHO. 2012). Systemic therapy has become the standard of care in patients at high risk of recurrence after primary surgery and those with metastatic disease, however, its potential toxicities and emergence of resistance remain problematic. Aquaporin 5 (AQP5) AQP5 is a hydrophobic integral transmembrane protein, which has a dual water and ion transporter function (Ribatti et al. 2014 & Wang et al. 2015). Expression of AQP5 is induced at early stages of CRC development (Moon et al. 2003) and it is implicated in enhanced cell proliferation and migration, features pathognomonic to tumours (Woo et al. 2008 & Jung et al. 2011). AQP5 expression in CRC correlates with histological differentiation and tumour-node-metastasis (TNM) staging (Wang et al. 2012) The aim of the project was to investigate the functional significance of AQP5 in HT29 colon cancer cell line. Specifically the objectives were; To perform RNA interference (RNAi) using small interfering RNA (siRNA) to down-regulate the expression of AQP5 To perform functional assays to assess if down-regulation of AQP5 has an effect on proliferation and/or migration Cell culture HT29 cells represents primary colon adenocarcinoma and are known to harbour multiple mutation including APC, Braf, PIK3CA, SMAD4, TP53 (Trust Sanger Institute). siRNA transfection HT29 cells were transfected with siGENOME SMARTPool AQP5 siRNA (GE Healthcare) using a reverse transfection protocol. Transfected cells were incubated for 48 hours before down-stream assays. Assessment of AQP5 expression Expression of AQP5 was examined in AQP5 siRNA transfected (AQP5 siRNA), mock transfected (MT) and untreated (UT) HT29 cells by performing quantitative Polymerase Chain Reaction (qPCR) and western blot. Circular wound healing migration assay Transfected HT29 cells in a 96-well plate were treated with mitomycin C to halt proliferation and incubated for 12 hours to achieve 100% confluency. The cell monolayer was wounded with a pipette tip and a fresh mitomycin C containing media was added. After 60-minute incubation, serial images of the scratch were captured at 0, 20 and 30 hours. Proliferation Assay Proliferation assay was performed on transfected HT29 cells in a 96-well plate, using CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). Down-regulation of AQP5 Expression from AQP5 siRNA Transfection Figure 1: AQP5 mRNA expression after AQP5 siRNA transfection N=3, **p<0.01, ***p<0.001 Error bar represents +/- standard error of mean. AQP5 expression in siRNA transfected cells was down-regulated by 59% and 55% compared to MT and UT cells. A B Figure 2: Western blot for AQP5 A Chemiluminescent blot (A) showed discrete bands at MW 212, 56, and 28 KD corresponding to AQP5 tetramers, dimers and monomers across all the transfection types. Volume quantification and normalisation to total protein of the 3 bands combined showed AQP5 protein expression in siRNA transfected cells was down-regulated by 24% and 34% compared to MT and UT cells (B) . Migration Assay Figure 4: Circular wound healing migration assay N=9, **p<0.01 Error bar represents +/- standard error of mean. Closure of circular wound for siRNA transfected cells was delayed by 19% compared to MT and 18% compared to UT at both 20 and 30 hours. Proliferation Assay Figure 5: Proliferation assay N=5, ***p<0.001 Proliferation in siRNA transfected cells was reduced by 16 and 14% compared to MT and UT at 90 minutes. Up-regulation of AQP1 Expression from AQP5 siRNA Transfection Figure 6: AQP1 mRNA expression after AQP5 siRNA transfection N=3, **p<0.01 Error bar represents +/- standard error of mean. AQP1 expression in AQP5 siRNA transfected cells was up-regulated by 77 and 133% compared to MT and UT cells. AQP1 has been implicated in tumour angiogenesis and migration (Papadopoulos and Saadoun. 2014). AQP5 siRNA transfection of HT29 cells resulted in successful knockdown of AQP5 expression at both mRNA and protein level. Reduction in migratory and proliferative properties of transfected HT29 cells supports the role of AQP5 in tumour growth and invasion of CRC, however, the magnitude of functional alteration observed was relatively small. Lack of sufficient AQP5 knockdown may explain this and alternative silencing methods such as CRISPR is being investigated. Compensatory up-regulation of AQP1 expression was likely to have enhanced migration of the AQP5 down-regulated cells. As HT29 cells harbour multiple mutated cancer genes, a counteractive act of the intricate molecular signalling mechanisms underlying the CRC tumorigenicity is considered. Future study examining the major oncogenic signalling pathways and AQP1 during AQP5 down-regulation is suggested. ** ** Results ** *** Aims and Objectives Conclusion *** *** Methods References WHO., 2012. GLOBOCAN 2012: Estimated Cancer Incidence, Mortality and Prevalence World Wide in 2012 [Online]. Jung, H. J. et al., 2011. Aquaporin-5: a marker protein for proliferation and migration of human breast cancer cells. PLoS One, 6, e28492. Ribatti, D. et al., 2014. Aquaporins in cancer. Biochim Biophys Acta, 1840, 1550-3. Wang, W. et al., 2012. Expression of AQP5 and AQP8 in human colorectal carcinoma and their clinical significance. World J Surg Oncol, 10, 242. Wang, J. et al., 2015. Aquaporins as diagnostic and therapeutic targets in cancer: How far we are? J Transl Med, 13, 96. 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