| Objective: Patients with advanced hepatocellular carcinoma(HCC)often face the high postoperative recurrence and poor prognosis.Especially the advanced patients,it is easy to drug resistance.In addition,HCC is highly related to HBV(Hepatitis B Virus)infection where has a high morbidity in China.Therefore,a better understanding of the underlying mechanisms in the development of HCC could help us identify novel targets for the treatment of HCC.Exosomes are small vesicles secreted by cells.These vesicles are 40-100 nm in diameter and contain a variety of biomolecules,such as lipids,proteins,and nucleic acids.Exosomes are nanoscale vesicles secreted by cells through exocytosis,which can be absorbed by target cells and transmit biological signals between local cells and distant cells.Exosomes are widely involved in and regulate the basic physiological processes between different cells,such as neuronal communication,antigen presentation,immune response,organ development and reproductive performance.They are also involved in the development of pathologic diseases,including cancer progression,cardiovascular disease,inflammation,and neurodegenerative disease.In recent years,it has been found that secretion of exosomes promoted the malignant biological process of tumor.At the same time,many studies have been focused on exosomes as biomarkers,vaccine and drug carriers or to interfere with exosome secretion,and finally to carry out therapeutic intervention and targeted regulation of diseases.Rab protein is a functional member of the evolutionally conserved Ras superfamily with guanosine triphosphoatase(GTPase),which regulates intracellular transport in all eukaryotic cells.Studies have shown that Rab35 is an important molecular in the endocytosis process,regulating a variety of intracellular processes,including cell division,cell migration and neuronal axon growth.There is a growing evidence that Rab-associated proteins are involved in the development of cancers,but the exact mechanism is unclear.It has been found that it can regulate the proliferation,migration,signal transduction and secretion of exosomes in tumor cells.As a member of the Rab GTPase family,Rab35 is widely involved in intracellular vesicle transport and material transport.Rab35 is abnormally highly expressed in tumor tissues of breast,prostate and HCC,and promotes invasion and metastasis of tumor cells through a variety of cell survival signals.Therefore,the identification and differentiation of exosomes from different cell types is crucial to understanding their signaling properties.The purpose of this experiment was to investigate whether Rab35 promoted the malignant capacity of HCC and whether exosomes were involved in the progression of HCC.Methods: In our study,the mRNA expression of Rab35 between the adjacent normal and tumor tissues was compared by the Cancer Genome Atlas(TCGA);We compared the effect of Rab35 on the survival and prognosis of patients by Kaplan Meier survival analysis;We identified the possible function of Rab35 involved in the regulation of HCC by pathway enrichment function analysis.The pathway enrichment function analysis of differentially expressed genes showed that the high expression of Rab35 was related to cell proliferation,matrix remodeling and membrane transport.Huh7 and Hep G2 cells were transfected with pcDNA3.1-Rab35 to overexpress Rab35.The proliferation of cells was evaluated by CCK8 assay;the ability of migration and invasion was detected by Transwell assay.In order to determine the effect of Rab35 on the release of exosomes,after overexpression of Rab35,the exosomes were isolated by ultracentrifugation and identified by electron microscopy(EM)and nanoparticle tracking analysis(NTA).Western blot was used to detect the expression of CD63 and TSG101,and the exosomes secreted by Rab35 were co cultured with the receptor cells.CCK8 and Transwell were used to detect the proliferation,migration and invasion of the receptor hepatoma cells.In order to determine how Rab35 is involved in the regulation of exocrine secretion,the intracellular distribution and localization of CD63 and Rab35 were observed by fluorescence confocal assay after over expression of Rab35.Results: We identified that Rab35 mRNA expression was up-regulated in tumor tissues compared with the normal tissues and associated with poor prognosis(P<0.05).In vitro assays,overexpression of Rab35 could promote cell proliferation,migration and invasion in HCC cells(P<0.05).In addition,we found that Rab35 could promote exosome secretion of HCC cells and the secreted exosomes contribute to the proliferation,migration and invasion of receptor HCC cells.(P<0.05).In further study,we demonstrated that Rab35 induced colocalization with CD63 on MVB membrane(P<0.05).Conclusion: Rab35 mRNA expression is overexpressed in HCC,and may promote the proliferation,invasion and migration of HCC cells by promoting the secretion of exosomes induced by MVB.Our findings are expected to provide a new strategy for targeted treatment of HCC. |
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