Studies On The Apoptosis Of HepG2 Cells Through GPC3 Receptor Stress In Wnt/?-catein Pathway

With the improvement of living standards and changes in diet of our nationals,liver cancer has become a frequent malignant tumor.More than 85% of the cases are primary hepatocellular carcinoma(HCC),and its cases in China account for more than 50% of the world's cases,which has a serious impact on people's physical and mental health and quality of life.So far,how to effectively prevent and treat HCC is still a very important scientific issue concerned by the whole society.Therefore,it is of great significance and value to carry out relevant research that can achieve the purpose of early diagnosis and treatment of HCC.As one of the model cells of HCC,the regulation and of HepG2 cell has become the basis for many researchers to carry out the prevention and treatment of HCC.In this paper,the research group used the innovative synthesis of multi-branched sea-urchin like gold nanostars(MSGNS)-Anti-Glypican 3 antibody(Anti-GPC3 antibody)nano-polymer(MSGNS-aGPC3),to explore the effect of HepG2 under the continuous activation of the membrane surface receptor GPC3,and explain the mechanism of HepG2 cell apoptosis based on the GPC3 receptor pathway.The main research contents are as follows:(1)We used a modified seed-mediated growth method to synthesize multi-branched gold nanoparticles with cell membrane adsorption.The morphology and characteristics of MSGNS nanomaterials at different synthesis stages were characterized by transmission electron microscopy,ultraviolet light,and nanoparticle size,and the growth mechanism of MSGNS was analyzed.Modification of MSGNS with SH-PEG improved the stability and biocompatibility of MSGNS.The difference in particle size,dispersion and Zeta potential verified the successful modification of MSGNS-PEG.CCK-8 was used to test the cytotoxicity of MSGNS-PEG and it was found that MSGNS-PEG has low cytotoxicity.Compared with the traditional seed growing method,it had a shorter period and easier steps.The synthesized multi-branched gold nanoparticles would not have surfactant,which was beneficial to the later modification and application of materials,and provided a basis for subsequent experiments after coupling Anti-GPC3.(2)To study the adsorption of MSGNS-a GPC3 nano-polymer on HepG2 cell membrane and its effect on HepG2 cell apoptosis.MSGNS-PEG and Anti-GPC3 antibody were incubated to form MSGNS-aGPC3.MSGNS-aGPC3 and MSGNS-PEG were co-cultured with HepG2 cells respectively.The results showed that,compared with MSGNS-PEG,MSGNS-aGPC3 could be efficiently adsorbed on the cell membrane for a long time to achieve the effect of continuously stimulating the receptor GPC3 on the surface of the cell membrane.Using in situ fluorescence detection method,it was found that after MSGNS-aGPC3 was co-cultured with HepG2 cells for 72 hours,HepG2 cells were apoptotic.We used western blotting to measure the changes of ?-catenin,Cyclin-D1,and GPC3.The results showed that under the stimulation of GPC3 receptor,the expression of ?-catenin,Cyclin-D1,and GPC3 protein decreased significantly compared with the control group.The above results indicated that Anti-GPC3 antibody conjugated to MSGNS maintained biological activity and induced apoptosis by down-regulating the expression of ?-catenin,Cyclin-D1,and GPC3 on the Wnt/?-catenin pathway of Hep G2 cells.(3)In order to analyze the mechanism of Wnt/?-catenin pathway change induced by GPC3 receptor stress in HepG2 cells,WAY262611 and KYA1797 K were used to up-regulate and down-regulate the expression of Wnt/?-catenin pathway respectively.Western blotting,adenosine triphosphate(ATP)detection,and reactive oxygen species(ROS)detection were used to explore the protein changes and energy differences of HepG cell under the continuous activation of GPC3 under the condition that Wnt/?-catenin pathway was activated or blocked.The results showed that when Wnt/?-catenin was blocked,?-catenin expression in HepG2 cells decreased significantly,the expression of GPC3 was almost unchanged,and the ATP content was greatly reduced,and the cell reactive oxygen content also decreased greatly.It is speculated that when the Wnt/?-catenin pathway is blocked,the GPC3 receptor stress signal may no longer be expressed.Using flow cytometry and CCK-8 kit to detect the apoptosis of HepG2 cells,and there was no significant difference in apoptosis rate between test group and control group.This result indicated that GPC3 receptor stimulation after blocking Wnt/?-catenin pathway could not achieve the effect of apoptosis of liver cancer cells.