Study On The Function And Mechanism Of Exosomes In The Sorafenib Resistance And Metastasis Of HCC

Hepatocellular carcinoma(HCC)is one of the most prevalent tumors and it is the second most frequent cause of cancer deaths worldwide.Many patients are not eligible surgical resection due to the advanced stage at diagnosis.Combined with the high invasion and metastasis degree of HCC,the prognosis of HCC patients is extremely poor.Currently,the 5-year survival rate of patients with HCC is less than 20%.Previous studies suggested that exosomes as a carrier of bioactive molecules in tumor microenvironment play an important role in the progression,metastasis and drugs resistance of HCC.Moreover,different cellular exosomes contain a different set of molecules(proteins,lipids,mRNAs and miRNAs)which may reveal genetic information of their parent cells.Therefore,increasing observations suggest that exosomes are potential new molecular biomarkers for diagnosis and prognosis of HCC.However,the role and mechanism of different sources of exosomes in HCC cells sorafenib resistance and EMT are not very clear.In addition,there are few studies about exosomal miRNA in HCC diagnosis,prognosis and tumor development.Our research focus on HCC cells derived exosomes,and investigate the roles and molecular mechanism of different HCC exosomes in cells sorafenib resistance,invasion,metastasis and clinical diagnosis of HCC.Firstly,we extracted the HCC cells with different invasive characteristics exosomes by ultracentrifugation method,and identified exosomes with TEM and western blot methods.Then,we explored their roles in regulating sorafenib resistance in HCC cells as well as the underlying potential molecular mechanisms.We found that the exosomes were of the expected size and expressed the exosomal markers CD9 and CD63.In addition,they were able to induce sorafenib resistance in vitro by activating the HGF/c-Met/Akt signaling pathway and inhibiting sorafenib induced apoptosis both in vitro and vivo.Moreover,exosomes derived from tumor cells with higher invasiveness had greater efficacy.These results may provide a new direction for improving the effectiveness of sorafenib in treating HCC.Then we investigated the role and mechanism of HCC cells derived exosomes in tumor invasion and metastasis.Through cells scratch,Transwell and other experiment methods,we examined the changes in receptor cells migration and phenotype protein expression.We found that HCC cells derived exosomes could transfer TGF-β to receptor cells and regulate tumor microenvironment,further effect HCC cells phenotype and tumor progression,metastasis.Last,we sought to explore the feasibility of using serum exosomal miRNAs as novel serological biomarkers for HCC patients diagnosis and prognosis..In the current study,miRNA microarray was employed to screen out miR-665,which demonstrated different expression levels among exosomes derived from different invasive hepatoma cells.We found that exosomal miR-665 levels in HCC patients were significantly higher than that in healthy subjects.Furthermore,the exosomal miR-665 levels of HCC patients in larger tumor size,local invasion of tumor and advanced clinical stage of HCC group were significantly higher than that of control groups.In addition,the survival time of exosomal miR-665 high expression group was significantly shorter than that of low expression group.In vitro and vivo studies we found that HCC cells derived exosomes can promote the proliferation of hepatoma cells,and up-regulating the protein expression level of MAPK/ERK pathway.After application of anti-miR-665,the cell proliferation promoting effects of exosomes were weakened.Our study suggested that exosomal miR-665 may be an effective biomarkers in HCC diagnosis and prognosis.Furthermore,we detected the protein in HCC cells derived exosomes,we found VEGFR contained in exosomes which is closely related to malignant proliferation and angiogenesis of HCC cells.In summary,we systematic studied the HCC cells derived exosome’ roles and mechanisms in HCC sorafenib resistance,metastasis,clinical diagnosis and prognosis.We found that exosomes play an important roles in HCC cells proliferation,metastasis and drug resistance through delivering bioactive molecules to the target cells.HCC cells with different invasive characteristics derived exosomes contained different expression of biological molecules.Exosomes and exosomal content may be novel diagnostic and therapeutic approaches of HCC.