SPARC Promotes Epithelial-Mesenchymal Transition Of Hepatocellular Carcinoma Cells And Acquisition Of Cancer Stem Cell Phenotypes

Background: Hepatocellular carcinoma(HCC)is a common malignant tumor,and its mortality ranks second in tumor death worldwide.The important factors of death are high metastatic rate and high invasiveness of tumor.There are multiple therapeutic modalities available for tumor treatment,including resection,radiofrequency ablation,transcatheter arterial chemoembolization and immunotherapy.But the overall prognosis of patients is still poor.Therefore,it is vitally important to identify mechanisms of HCC development and metastasis.And it is also an effective way to reduce mortality from liver cancer.The epithelial-mesenchymal transition(EMT)is the transformation process of epithelium to mesenchyme.Epithelial cells lose their typical intercellular connections,remodel the cytoskeleton,and become fibroblasts during EMT,leading to cell apoptosis resistance and enhance movement ability.EMT plays an important role in the invasion,proliferation and migration of tumor cells.Inhibition of EMT can effectively inhibit the metastasis of tumor cells.Therefore,research elucidating the mechanism of EMT development in HCC can provide an effective solution for inhibiting the metastasis of liver cancer.Cancer stem cells(CSCs)are a class of cells that possess cell immortalization capacity,differentiation potential and tumorigenesis.These CSC populations are associated with metastasis,tumor relapse and resistance to conventional anti?cancer therapies.Although the presence of CSCs has been found in a variety of solid tumors,there are still many unknowns in the specific mechanism of tumorigenesis and development.Therefore,a better understanding of the biology of CSCs will uncover new strategies to diagnose tumors earlier,treat them more efficaciously,and prevent them from relapses.SPARC(secreted protein acidic and rich in cysteine),also called Osteonectin,is an extracellular protein that plays important roles in cancer cell migration,proliferation,angiogenesis,matrix cell adhesion,and tissue remodeling.It has been reported that SPARC plays a role in promoting cancer process in highly metastatic tumors,and the expression of SPARC is also closely related to poor prognosis.Although many studies have evaluated the importance of SPARC expression,its role in the development of liver cancer remains unclear.Therefore,it is significant to explore the regulation of SPARC on the development of liver cancer,and it can provide an effective way and better prognosis for the treatment of HCC.Part ? Regulatory effect of SPARC on acquisition of cancer stem cell phenotypes Objective:1.To compare the expression difference of SPARC in liver cancer stem cells and liver cancer cells.2.To investigate the regulation of overexpression of SPARC on the phenotype of liver cancer stem cells.Methods:1.Firstly,MHCC-97 H and SMMC-7721 liver cancer cells were cultured in a serum-free stem cell culture medium.The expression of SPARC in enriched cell spheres and non-stem hepatoma cells was detected by qPCR and Western blot.2.The SPARC/SPARC-NC stably overexpressing cell line was constructed by lentiviral vector.The transfection efficiency was detected by qPCR and Western blot.We used qPCR,Western blot and flow cytometry to detect the expression of CD133 and Oct4.Then the capacity of sphere-forming in two groups was compared.Results:1.After the cells were cultured in serum-free stem cell medium for 2 weeks,we observed the formation of cell spheres.And the expression of SPARC in the spheres was higher than that of adherent cells.2.Compared with the control group,overexpressing SPARC increased the expression of CD133 and Oct4.The percentage of CD133+ in SPARC overexpressing cells was significantly higher than that in the control group.The forming ability of SPARC overexpressing was observed significantly increased under a fluorescence microscope.Conclusion: SPARC promotes the acquisition of liver cancer stem cell phenotypePart ?Regulatory effect of SPARC on liver cancer EMT Objective:To determine the role of SPARC overexpression in liver cancer EMT.Methods:1.Use lentiviral vector to construct SPARC stably overexpressing MHCC-97 H,SMMC-7721 and Huh-7 cell lines.Use qPCR and Western blot to detect the expression level of EMT markers E-cadherin,N-cadherin and vimentin.2.Use the Transwell chamber migration assay to test the migratory ability of overexpressing SPARC liver cancer cells.3.Perform a wound-healing experiment to determine the wound healing ability.Results:1.Compared with the control group,the expression level of E-cadherin was decreased,and the expression levels of N-cadherin and vimentin were increased in the SPARC overexpression group.2.Lentiviral overexpression of SPARC in MHCC-97 H and SMMC-7721 cells increased their migration ability compared to the NC group.3.The wound-healing capacity was significantly enhanced in the SPARC-LV group in MHCC-97 H and Huh-7.Conclusion: SPARC promotes EMT in vitro.Part ?The role of SPARC in tumor growth Objective:To determine the effect of SPARC on tumor growth.Methods:1.Cells were resuspended in complete culture medium and injected into the skin of nude mice.Observing the speed and size of subcutaneous tumor formation in nude mice on time.2.Extracting the subcutaneous tumor tissues of nude mice,then using qPCR and Western blot to detect the expressions of E-cadherin,N-cadherin and vimentin in tumor tissues.Results:1.SPARC overexpression group exhibited enhanced growth and larger tumor size than the control group.2.Compared with the control group,the expression level of E-cadherin was decreased,and the expression levels of N-cadherin and vimentin were increased in SPARC overexpressing tumor tissues.Conclusion: SPARC promotes tumor growth in vivo.