The Role Of Lipid Rafts And DCC Signaling In The Regulation Of Netrin-1 In Cell Polarity In Hepatocellular Carcinoma Cells

Backgrounds and aimsCell polarity plays a key role in the biological process of cells and the loss of polarity is a hallmark of cancer. Previous research suggests that cancer cells obtain the ability of invasion principally through the EMT (epithelial-mesenchymal transition). The disruption of cell polarity is the initial stage of EMT, and the exact mechanism of which remains unclear. Netrin-1 is a diffusible laminin-like chemotactic protein which has been shown to act as guidance cues for developing axons and migrating neuroblasts and it also plays an important part in the regulation of the apoptosis and invasion of tumor cells. The aim of the project study is to investigate the role of Netrin-1 and the lipid rafts-dependence receptor DCC signaling in the regulation of cell polarity in hepatocellular carcinoma cells.MethodsWe established the physical hypoxia model of hepatocellular carcinoma cells. Cell images were captured by inverted microscope. Immunofluorescence staining and confocal laser microscopy were employed to detect the change of cell polarity and the influence of shRNA-Netrin-1 on the cell polarity under hypoxic conditions. The Netrin-1 plasid was stably transfected into heptocellular carcinoma cell HepG2. Immunofluorescence staining and confocal laser microscopy were employed to detect the the colocalization of Netrin-1's receptor DCC and lipid rafts at the cell membranes, the disruption of cell polarity and actin cytoskeleton rearrangement of the cells. Western blot was used to measure the change of expression of specific polarity proteins such as Cdc42 and Rac-1. The influence of Netrin-1 on cell polarity and the change of E-cadherin were detected after the transfected cells were treated with methyl-β-cyclodextrin (MCD).ResultsAfter being cultured under hypoxia conditions, hepatocellular carcinoma cells, which transformed from epithelia to mesenchymal and lost cell contact. Hypoxic promoted the disruption of cell polarity. The shRNA of Netrin-1 could inhibit the disruption of cell polarity. The HepG2 cells transfected with Netrin-1 showed that the loss of cell polarity, the rearrangement of actin cytoskeleton and the increased expression of cell polarity proteins such as Cdc42 and Rac-1. We also found that the Netrin-1 receptor DCC and lipid rafts colocalized at the cell membranes. The disruption of cell polarity could be inhibited after the cells transfected with Netrin-1 being treated with methyl-β-cyclodextrin (MCD) and the sign of epithelial E- cadherin underwent substantial changes.ConclusionsHypoxic conditions and Netrin-1 can promote the disruption of cell polarity of hepatocellular cells, which occurred because of the Netrin-1 and the lipid rafts-dependence receptor DCC signaling. It reveals the role of cell polarity in the early stage of metastasis in hepatocellular carcinoma, which is important for us to continue to investigate the intervention targets in the metastasis of heptocellular carcinoma.