Cancer-Associated Fibroblasts Promoting "Stemness" Of Hepatocellular Carcinoma Cells Via Exosomes

Purpose:Hepatocellular carcinoma(HCC)is the sixth most common malignancy in the world,and patients with hepatocellular carcinoma often have a poor prognosis due to its pro-angiogenic ability and high rate of recurrence and metastasis.The efficacy of current comprehensive therapies such as interventional,immunological and molecularly targeted drugs for metastatic or recurrent hepatocellular carcinoma is not satisfactory.Therefore,it is crucial to investigate the mechanism of liver cancer invasion and metastasis,and to find new ways and targets to stop the metastasis and recurrence of liver cancer to improve the survival rate of liver cancer patients.In recent years,the involvement of tumor microenvironment in tumor development has become a frontier of tumor research.Cancer-associated fibroblasts(CAFs),a major component of hepatocellular carcinoma microenvironment,play an important role in promoting hepatocellular carcinoma invasion and metastasis.The aim of this study is to explore the role of CAFs in promoting the invasion and migration of hepatocellular carcinoma cells,to further explore and elucidate the molecular mechanism of CAFs in promoting the biological behavior of hepatocellular carcinoma cells,and to explore the new mechanism of tumor microenvironment to deter the recurrence and metastasis of hepatocellular carcinoma.Methods:(1)CAFs and normal fibroblasts(NFs)were obtained from surgical specimens of patients with hepatocellular carcinoma or hepatic hemangioma,respectively.CAFs and NFs were isolated,purified and identified;(2)Exosomes were extracted from conditioned medium(CM)of CAFs and NFs,and the quality of the extracted exosomes was verified by Western blot,NTA particle size analysis and electron microscopy;(3)Cell co-culture,conditioned medium(3)To observe the promotion effect of CAFs on the "stemness" of hepatocellular carcinoma cells through exosomes by using cell co-culture,conditioned medium treatment and exosome treatment.(4)We used si RNA to interfere with the expression of lnc RNA NEAT1 in CAFs,and then used cell co-culture,conditioned medium treatment and exosome treatment to observe the effect of lnc RNA NEAT1 in CAFs-derived exosomes on the "stemness" of hepatocellular carcinoma cells.(5)Finally,immunofluorescence assay and RIP assay were used to demonstrate that lnc RNA NEAT1 can bind to YAP protein and may promote the "stemness" of hepatocellular carcinoma cells by driving the liquid-liquid phase separation of YAP protein.Results:(1)Transwell invasion and migration assay,scratch assay and floating sphere formation assay were applied at three levels of cell co-culture,conditioned medium treatment and exosome treatment,and the ability of CAFs to promote the "stemness" of hepatocellular carcinoma cells via exosomes was found to be stronger than that of NFs;(2)In a mouse tumor metastasis model,it was found that CAFs-derived exosomes promoted the invasion ability of hepatocellular carcinoma cells and had significant metastatic foci in the lung.(2)in a mouse tumor metastasis model,CAFs-derived exosomes were found to promote the invasive ability of hepatocellular carcinoma cells more strongly,and had obvious metastatic foci in the lung;(3)after interfering with the expression of lnc RNA NEAT1 in CAFs by si RNA and extracting si NEAT1-CAFs-derived exosomes to treat hepatocellular carcinoma cells,the invasive migration ability of hepatocellular carcinoma cells was reduced;(4)using RIP experiments,it was found that lnc RNA NEAT1 was associated with NFs.(4)using RIP assay,we found that lnc RNA NEAT1 combined with YAP protein may trigger the liquid-liquid phase separation of YAP protein in cells,thus promoting the "stemness" ability of hepatocellular carcinoma cells.Conclusion: This study shows that tumor-associated fibroblasts(CAFs)in the tumor microenvironment can promote the "stemness" of hepatocellular carcinoma cells through lnc RNA NEAT1 in exosomes.Preliminary mechanistic studies suggest that this effect may be accomplished through the ability of lnc RNA NEAT1 to bind to YAP proteins,triggering liquid-liquid phase separation of YAP proteins in the cells.It is expected that the role and mechanism of tumor microenvironment in promoting the "stemness" of hepatocellular carcinoma can be elucidated from the perspective of exosomal regulation of transcription factor traits in cells,which may provide new targets for blocking the invasion and metastasis of hepatocellular carcinoma by interfering with lnc RNA-regulated transcription factor traits in CAFs-derived exosomes.