Effect Of Cancer-associated Fibroblast Activation On The Proliferation Of Hepatocellular Carcinoma Cells

Background and objective: Hepatocellular Carcinoma(HCC)represent majority of primary malignancy of liver disease with high incidence rate and lethality.Tumor microenvironment(TME)is closely related to HCC proliferation and autophagy.On one hand,microenvironmental autophagy can promote the proliferation of HCC by releasing amino acids such as glutamine;On the other hand,nonautonomous autophagy of HCC cells mediated by TME factors can promote HCC growth by providing necessary oxidative substrate.Considering cancer cells are more dependent on autophagy than stromal cells,we focus on the role of nonautonomous autophagy in HCC.Studies have found that autophagy plays different roles in different stages of HCC.It can inhibit the occurrence of HCC in the early stage.While in the advanced stage,nonautonomous autophagy can promote the proliferation and growth of HCC.Cancer associated fibroblasts(CAF),which are closely related to the proliferation,growth and prognosis of HCC,are one of the important cell components in TME.However,it is unclear whether the proliferative effect of CAF on hepatoma cells is related to nonautonomous autophagy.Our pre-experimental studies have found that CAF can promote the proliferation and growth of hepatoma cells in vivo and in vitro,and its role in promoting proliferation and growth is related to autophagy of hepatoma cells.The purpose of this study is to explore what active factors in activated CAF may promote the proliferation and growth of HCC as well as to explore the possible mechanism of its regulation of HCC autophagy,so as to provide new ideas and directions for the treatment of targeting tumor microenvironment CAF and autophagy of HCC cells.Methods: 1.human primary CAF and PAF were isolated and identified by primary cell culture technique.To explore the effect and possible mechanism of activated CAF on the proliferation of hepatoma cells,primary CAF conditioned medium(CM)was used to stimulate hepatoma cells compared with CM of PAF,human hepatic stellate cell line LX-2 and human synovial fibroblast cell line HFL.Cell counting kit 8(CCK8)experiment was used to compare and analyze the effects of activated CAF CM on the proliferation and viability of hepatoma cells;Taking the nude mice tumorigenic group inoculated with Hep G2 cells alone as the control,the tumorigenic experiment of nude mice inoculated with CAF and Hep G2 cells and transcriptome sequencing analysis was conducted to explore the possible mechanism of CAF promoting HCC growth in vivo.In our pre-experiment,we found that hepatoma cell autophagy can mediate the regulation of CAF on hepatoma cell proliferation.After inhibiting hepatoma cell autophagy by using autophagy blocker,Western blot,real-time polymerase chain reaction(real-time PCR),confocal and transmission electron microscopy and other experimental methods were used to analyze whether CAF CM could promote the proliferation of hepatoma cells by inducing autophagy.2.In order to explore whether the regulation of activated CAF on hepatoma cell autophagy depends on the interaction between secretory growth differentiation factor 15(GDF15)and hepatoma cell membrane receptor GFRAL,we found that GDF15 was the most significant gene between CAF and PAF through literature and bioinformatics analysis as well as RNA sequencing analysis between primary CAF and PAF;After knocking down the expression of GDF15 in CAF by lentivirus transfection,we analyzed whether the promoting effect of CAF on autophagy and proliferation of hepatoma cells partially dependent on GDF15 paracrine by using mouse tumorigenesis,Western blot,real time PCR and immunohistochemical staining;Bioinformatics and transcriptome sequencing were used to test the target protein,glial cell-derived neurotrophic factor family receptor α(GDNF family receptor alpha like,GFRAL),which could interact with GDF15.The regulation of activated CAF on autophagy and proliferation of hepatoma cells mediated by GDF15/GFRAL signal axis was studied by subcutaneous tumor bearing model in nude mice,immunofluorescence,immunohistochemistry,Western blot and flow cytometry experiment.Result: 1.Activated CAF promoted the proliferation and viability of HCC cells compared with PAF,LX-2 and HFL as shown by plate clone formation,CCK8 cell viability and nude mouse tumorigenesis experiment.2.The results of Western blotting,real time PCR,immunofluorescence and transmission electron microscopy showed that CAF induced autophagy in hepatoma cells.3.Once autophagy was inhibited by autophagy blocker,the proliferation and survival ability of CAF CM on hepatoma cells decreased.4.The results of Western blotting and real time PCR showed that activated CAF induced autophagy of hepatoma cells by paracrine GDF15.5.Knockdown of CAF GDF15 expression attenuated the proliferation promoting ability of CAF and survival ability of hepatoma cells was shown by plate cloning experiment,CCK8 experiment and tumorigenesis experiments in nude mice.6.The results of Western blotting and flow cytometry showed that GFRAL mediated the regulation of autophagy in hepatoma cells.7.CCK8 experiment showed that knockdown of GFRAL expression in hepatoma cells inhibited the protective effect of GDF15 on hepatoma cells.Conclusion: 1.Activated CAF can promote the proliferation of hepatoma cells by inducing autophagy.2.GDF15 participate in the regulation of activated CAF on autophagy of hepatoma cells by acting on hepatoma cell receptor GFRAL.