| Background and Objective Heptocellular carcinoma(HCC)ranks the fifth most comman malignancy and the second leading cause of cancer-related death wordwide.Recoverence and metastasis are responsible for more than 90% of cancer-related mortality.Microscopic intrahepatic metastase after surgery are thought to be the main reason for the frequent recurrence in HCC.Epithelial-mesenchymal transition(EMT)is a key step in cancer invasion and micro-metastasis,and create a satisfactory microenvironment for cancer stem cells(CSCs).CSCs account for cancer recurrence,metastasis and chemotherapy resistance in many malignancies.To clarify the mechanisms of microscopic intrahepatic metastase and biology of CSCs may provide new approaches for exploring noval theraputic strategies and drug targets.In this study,we propose to analyze clinical significance of c PLA2α and its influence on prognosis by using HCC clinical tissues,and explore the role and mechanisms of c PLA2α in the EMT of HCC by using HCC cell lines and athymic BALB/C nude mice.We will further reveal the role of c PLA2α in regulating the transition between epithelial typed HCC CSCs and mesenchymal typed HCC CSCs.Methods 1)Immunohistochemical staining and Western Blot of fresh frozen tissue were used to analysis clinical significance of c PLA2α and its influence on prognosis.2)Lentivirus mediated sh RNA delivery systems were used to knockdown and overexpress c PLA2α in Hep G2 cells and HLE cells.3)Invasion assay,chemotaxis assay,migration assay,cell adhesion assay and F-actin polymerization assay were used to examine the effect of c PLA2α different levels on chemotaxis and migration capability of Hep G2 cells.4)Athymic BALB/C nude mice was used to evaluate the effect of c PLA2α different levels on metastasis of HCC cells in vivo. 5)Cell morphology observasion,western blooting and immonofluresence were used to investigate the role of c PLA2α in regulating the EMT of HCC induced by EGF and TGFβ.6)Western blotting was used to explore the mechanism of c PLA2α in regulating EMT and metastasis of HCC.7)Spheroid formation assay was used to analysis the role of c PLA2α in influencing the sphere morphology.8)Western Blot,RT-PCR were used to analysis the effect of c PLA2α on stem cell markers(nanog,oct4,sox2)and EMT markers(E-cadherin,N-cadherin and vimentin).9)Spheroid formation assay,colony formation assay and differentiation assay were used to analysis the impact of c PLA2α on cancer stem cell-like properties including self-renew,proliferation,invasion and differentiation.10)Tet-on inducible systems were used to inveatigate the role of c PLA2α in mediating inversible EMT of cancer stem cell in hepatocellular carcinoma.11)Tet-on inducible systems were used to inveatigate the role of c PLA2α in mediating the transition between two different HCC CSCs through PKCζ.Results 1)c PLA2α was higher in HCC tumor tissues and especially high in tumor edges,microscopic invasive foci and tumor thrombus,and was associated with poorer prognosis.2)c PLA2α facilitated the chemotaxis and invasive capabilities of HCC cells.3)c PLA2α was associated with serious distant metastasis in vivo.4)Knockdown of c PLA2α rendered cells stayed in cobblestone shape with an increase in E-cadherin,and decreases in N-cadherin and vimentin,while overexpression of c PLA2α promoted the formation of spindle-shaped cells with a decrease in Ecadherin,and increase in N-cadherin and vimentin.5)Stimulated by EGF,HCC cells acquired a mesenchymal typed morphology with a decrease in E-cadherin,and increase in N-cadherin and vimentin.The effects were inhibited by c PLA2α deletion and enhanced by c PLA2α overexpression.6)EGF induced the phosphorylation of Akt 473 and ERK1/2,and the increase of EMT related transcription factors.The effects of EGF were inhibited by c PLA2α deletion and enhanced by c PLA2α overexpression.LY294002,inhibitor of PI3 K,blocked the effect of EGF.Arachidonic acid(AA)and LPI,key products of c PLA2α enzymatic reaction,rescued the effect of EGF.7)Stimulated by TGFβ,HCC cells acquired a mesenchymal typed morphology with a decrease in E-cadherin,and increase in N-cadherin and vimentin.The effects were inhibited by c PLA2α deletion and enhanced by c PLA2α overexpression.8)The phosphorylation of smad2 and ERK1/2,and increase of EMT related transcription factors induced by TGFβ was bolcked by c PLA2α deletion and LY294002,and recued by AA.9)c PLA2α knockdown cells formed epithelial-typed tight spheres in serum free medium with a high expression level of E-cadherin,while cells with c PLA2α overexpression formed mesenchymal-typed loose grape shaped spheres with high expression levels of N-cadherin and vimentin.10)Epithelial-typed spheres with lower c PLA2α expression showed stronger properties of proliferation and mesenchymal-typed spheres with higher c PLA2α expression showed stronger properties of invasion.11)c PLA2α mediated the inversible EMT of cancer stem cell in hepatocellular carcinoma by regulating the phosphorylation of PKCζ.Conclusions c PLA2α was higher in HCC tumor tissues and especially high in tumor edges,microscopic invasive foci and tumor thrombus,and was associated with poorer prognosis.c PLA2α mediated the chemotaxis and invasion of HCC cells in vitro and in vivo.c PLA2α regutaed the migration of HCC cells by promoting EMT through PI3 K signaling pathway induced by EGF.c PLA2α played an important role in TGFβ induced EMT through smad signaling pathway,which is depend on the activation of PI3 K signaling pathway.There were two different HCC CSCs,epithelial-typed CSCs and mesenchymal-typed CSCs.c PLA2α influenced the stemness-associated properties and mediated the the inversible EMT of cancer stem cell in hepatocellular carcinoma by regulating the phosphorylation of PKCζ. |
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