Study On The Mechanism Of PDGF-D-mediated EMT In Gemcitabine Resistance Of Hepatoma Cells

Background and objective:Hepatocellular carcinoma (HCC) is one of the most common malignant tumor of the clinical.HCC threats our nation’s health with highly malignant, difficultly early detection and poorly prognosis. Systemic chemotherapy is an important treatment for advanced HCC, but the clinical benefit of traditional cytotoxic brought is limited. The new cytotoxic drugs such as oxaliplatin (OXA), gemcitabine (GEM) and others show some prospects, but these drugs will still encounter acquired resistance and lead to treatment failure.Epithelial-to-mesenchymal transition(EMT) is the process of epithelial cells into mesenchymal cells which acquire the abilities of migration and invasion. EMT is closely related to tumor drug resistance, Platelet-derived growth factor-D(PDGF-D) plays a key role in the induction of EMT.We used a previously established in vitro cell model of GEM-resistant(GR) HCC cells (HepG2GR and SMMC-7721GR),we found that GR cells have occurred EMT morphological changes and PDGF-D is highly expressed in them. Our research uses the resistant cells model to study whether the occurrence of acquired resistance phenotypes of GR cells is resulted from the PDGF-D-mediated EMT or not. And the molecular mechanism of how the acquired resistance of GEM can be revealed that will give theories and treatment clues to overcome the secondary resistance.Methods:1. We developed HCC GR (HepG2GR and SMMC-7721GR)cells using the method of increasing concentration gradient. The level of resistance was measured by MTT.2. The morphological changes of HCC GR cells were observed.The biological activity of GR cells were assessed by attachment and detachment assays and migration and invasion assays respectively.The EMT markers(E-cadheri、Snail、Slug Vimentin) were detected by Real-time PCR (RT-PCR) and Western blot.3. PDGF-D and its receptor PDGFRβ were detected by RT-PCR and Western blot. 4. Using siRNA interference technology to silence the expression of PDGF-D transiently in GR cells. To investigate the change of biological activity and EMT markers in GR cells after the gene knockout of PDGF-D.Results:1.We observed that parent cells showed loss of cell polarity and increased formation of pseudopodia, leading to elongated, irregular fibroblastoid morphology.The ability of migration and invasion is enhanced and the capacity of attachment and detachment is increased associated with the EMT phenotype. This observation was supported by expression analysis of classic EMT markers,we found that the expression of E-cadherin was significantly reduced in HCC GR cells. On the contrary, the expression of mesenchymal markers including Vimentin, Snail and Slug was elevated in HCC GR cells.2.The expression of PDGF-D and PDGFRβ were elevated in HCC GR cells.3. Knockdown of PDGF-D in HCC GR cells resulted in changes from fibroblast-like shapes to cobblestone appearance with little pseudopodia,decline in cell migration and invasion,a significant increased in the expression of epithelial markers and decreased the levels of mesenchymal markers including Vimentin, Snail and Slug.Conclusion:1.GEM resistance cells of HCC have EMT phenotype.2.PDGF-D plays an important role in GR-induced EMT in HCC, inhibition of PDGF-D led to the reversal of EMT.