1. Cell migration assays illustrated an increased migratory capacity of HepG2 OCLN cells in comparison to control. The migratory capacity of both HepG2 naïve and OCLN over expressing cells were the same after 2 days. This is due to the transitory lentiviral expression only lasting up to two days after the cell migration began. OCLN has been seen as a molecule that would slow migration however in other cell types OCLN regulates polarised migration by recruiting aPCK-Par3/PATJ to the leading edge (Dan Du 2010). Further research is going to analyse the proteins recruited by OCLN during over expression and if the are located in the correct area of the cell. Quantitative RT-PCR in OCLN over-expressing cells shows a 2-fold increase in E-cadherin mRNA levels and also a 3.4 fold increase in Vimentin the mesenchymal protein. This shows changes in the migratory capacity within HepG2 cells which complements the migration assay data. Treatment of transformed cells with the de-polarizing cytokine Oncostatin M resulted in a 2.4 fold increase in E-cadherin compared to a 1.2 fold increase in naive HepG2 cells, showing a possible utilization of increased OCLN in a compensatory capacity during depolarization events to maintain cellular polarity. Vimentin mRNA levels increase with OCLN overexpression as the migration data suggests. Vimentin mRNA levels also increase when challenged with Onc M but not when Onc M challenged OCLN overexpressing cells showing OCLN is also activating a compensatory mechanism which further research will identify. Concomitant changes in protein localisation of TJ proteins OCLN and adhesion junction protein E-cadherin in these cells were observed using fluorescent confocal microscopy. It was found even though both OCLN and E-cadherin proteins are overexpressed but the location has changed which complements the PCR and migration data. These data illustrate the role of OCLN in liver cell physiology linked to maintenance of cell polarity. Chiba, H., Osanai, M., Murata, M., Kojima, T., and Sawada, N. (2008) 'Transmembrane Proteins of Tight Junctions'. Biochimica Et Biophysica Acta (BBA)- Biomembranes 1778 (3), 588-600 Du, D., Xu, F., Yu, L., Zhang, C., Lu, X., Yuan, H., Huang, Q., Zhang, F., Bao, H., and Jia, L. (2010) 'The Tight Junction Protein, Occludin, Regulates the Directional Migration of Epithelial Cells'. Developmental Cell 18 (1), 52-63 Endendijk (2011) Endendijk 3D Design Visualisation Realisation [online] available from [04/03 2014] Ibidi Culture-Insert Family [online] available from [04/03 2014] Lee, N. P. and Luk, J. M. (2010) 'Hepatic Tight Junctions: From Viral Entry to Cancer Metastasis'. World Journal of Gastroenterology: WJG 16 (3), 289 Martin, T. A. and Jiang, W. G. (2009) 'Loss of Tight Junction Barrier Function and its Role in Cancer Metastasis'. Biochimica Et Biophysica Acta (BBA)- Biomembranes 1788 (4), 872-891 St Michaels Hospital (2014) Flow Cytometry and Sorting Intracellular Staining [online] available from [04/03 2014] Hepatocytes account for 60-70% of cells in an adult liver and due to the vascular architecture of the organ it is essential for hepatocytes to be highly polarised (Lee and Luck 2010). Tight junctions (TJs) allow hepatocytes to form intracellular bound sheets lining the organ cavities vital for efficient liver function. TJs are dynamic complexes that are comprised of three main proteins occludin (OCLN), claudins (CLDN) and junction adhesion molecules (JAMs) along with scaffolding and signalling proteins (Chiba 2008). A change in TJ protein expression or localisation causes reduced contact with neighbouring cells therefore loss of contact inhibition and stimulation of epithelial to mesenchymal transition in cancer (Martin et al. 2009). Human oncostatin M (Onc M) is a cytokine normally found in foetal or wounded liver where cell migration is needed however it is found in hepatocellular carcinoma. The role of Onc M during cancer is to depolarise cells promoting a remodelling of the extracellular matrix and epithelial to mesenchymal transition. The investigation into over-expression of OCLN was performed using transient Lentiviral expression systems resulting in a 30-40% increase in OCLN protein expression in liver HepG2 cells. Migration assays, qPCR and confocal microscopy give a picture of how hepatocytes act during cancer metastasis and when challenged with Onc M. Introduction References Experimental Procedures and Results Discussion Migration AssayqPCR AnalysisConfocal Microscopy Figure 2: Naïve HepG2 vs Occuldin overexpressing HepG2 cell migration Figure 4: Naïve HepG2 vs HepG2 OCLN overexpressing cells E-cadherin mRNA expression. Figure 5. Naïve HepG2 vs HepG2 OCLN overexpressing cells Vimentin mRNA expression 1.Sterilise cell culture inserts and place in a cell culture well. 2.Pipette 3.5x10 4 cells in 70µl of media into each well of the cell culture insert and incubate for 24 hours. 3.Remove cell culture inserts. 4.Propagate in 500µl of fresh media. 5.Track c ell migration over 72 hours. (Ibidi 2011) 1. 2. 3. 4. Cell culture insert Before PCR: Place PCR master mix, primers and cDNA into a PCR plate. 1: Initial denaturation of cDNA 95 o c for 3 minutes. 2: Denaturation of cDNA at 95 o c for 25 seconds – Annealing of primers to amplicon at 60 o c for 30 seconds – Extension of amplicon 72 o c for 30 seconds for 40 cycles. Amplicon detection occurs after each cycle. 3: Final extension 10 minutes at 72 o C. Detection cycle of a target in relation to detection cycle of a control give fold difference in expression. Step 1: Incubate for 5 minutes in ice cold methanol then in PBS/BSA (0.5%) with 0.1% saponin for 30 minutes. 1 o antibody 2 o antibody Step 3: Wash cells 3x in PBS then incubate cells in PBS/BSA (0.5%) with 0.1% saponin with 2 o antibody with fluorescent tag for 1 hour. Step 4: Assess fluorescence and location of targets using a confocal microscope Step 2: Wash cells 3x in PBS then incubate cells in PBS/BSA (0.5%) with 0.1% saponin with 1 o antibody for 1 hour. Figure 1; Cell culture inserts in a cell culture well Migration Assay Data.qPCR Analysis Data. Figure 4 shows HepG2 cells challenged with Onc M produces 1.2 fold more E-cadherin mRNA compared to a naïve cells. OCLN overexpressing HepG2 cells result in a 2 fold increase in mRNA E-cadherin expression. OCLN overexpressing cells that were also challenged with Onc M shows the largest difference in mRNA expression with a 2.4 fold increase of E-cadherin. Figure 5 shows HepG2 cells challenged with Onc M produce 6.6 fold more Vimentin mRNA compared to a naïve cells. OCLN overexpressing HepG2 cells result in a 3.4 fold increase in mRNA Vimentin expression. OCLN overexpressing cells that were also challenged with Onc M shows only a 3.6 increase. Figure 3: Migration of HepG2 naïve cells vs OCLN overexpressing cells over 72 hours OCLN Naïve 0 Hours 72 Hours Leading edge of migrating OCLN overexpressing cells After 72 hours OCLN overexpressing cells have a higher migratory capacity compared to naïve cells. Time course of migration. OCLN over expressing cells have a higher migratory capacity compared to naïve cells for up to 2 days migrating 10% further. The motility decreases between days two and three inline with the motility of naïve HepG2 cells. (St Michaels Hospital 2014) (Endendijk n.d.) Occludin localisation at the apical lumen Confocal Microscopy Data. Figure 6: Confocal microscopy cell preparation Figure 7: OCLN over expression localisation The localisation of OCLN through fluorescently tagged protein via the lentiviral system. OCLN localises to the edges of cells as expected. Figure 8: E-Cadherin protein levels in OCLN vs naïve. Overexpressed green OCLN tagged with a red antibody is very pronounced however naïve cells OCLN protein is localised to specific areas. Figure 8: E-Cadherin protein levels in OCLN vs naïve. E-cadherin protein expression is amplified in OCLN overexpressing cells compared to naïve cells. OCLN overexpressed OCLN cells + OCLN ABNaïve cells + OCLN AB OCLN cells + E-cadherin AB Naïve cells + E-cadherin AB HepG2 OCLN