Identification Of Serum Exosomal MiRNA That Predicts Hepatocellular Carcinoma Recurrence After Liver Transplantation

Background Liver cancer is one of the most common malignancies with high morbidity and mortality in the world.Hepatocellular carcinoma(HCC)is the main type of liver cancer.Due to lack of specific symptom at the early stage,about 80% of HCC patients are diagnosed at advanced stages.When diagnosed,they have lost the optimal opportunity for resection,so the prognosis is extremely dismal.For the patients with early HCC,liver transplantation(LT)is the most effective treatment,but HCC recurrence after transplantation is still a severe problem.Even after strict selection of recipients,5-30% of recipients will occur HCC recurrence.The recurrence rate will increase significantly as the recipient's standard expands,affecting the prognosis of patients.Thus,it is necessary to provide early warning and monitoring of HCC recurrence after liver transplantation.Exosomes are small extracellular vesicles derived from endosomes,which are widely distributed in body fluids.The contents in exosomes can reflect the source cells and disease states,thus,they can be used as biomarkers for diagnosis and prognosis.At present,exosomal micro RNAs(miRNAs)have been reported to participate in HCC development.However,no research has been conducted to investigate the value of exosomal miRNAs in predicting HBV-related HCC recurrence after liver transplantation.Method The clinical information,follow-up information and preoperative serum were collected from the HCC patients received LT at the first affiliated hospital of Zhejiang University,School of medicine,from 2015 Janaury to 2016 December.According to whether the recipients occurred recurrence with 3 years,the recipients were divided into recurrence group and non-recurrence group.The comparision between the two groups was conducted.Serum exosomes were separated by precipitation method,and the exosomes were characterized by transmission electron microscopy(TEM),cryo-electron microscopy,Western blotting(WB)and nanoparticle trace analysis(NTA).HCC-related miRNAs were identified as the miRNAs dysregulated in both HCC tissues and circulating exosomes of HCC patients,as well as HCC cell-derived exosomes,by using bioformatic tools,inculding TCGA database,and GEO datasets and pubmed.Real-time quantitative PCR(RT-PCR)was used to detect the expression level of HCC-related miRNAs in circulating exosomes from pre-transplant serum.The differences of exosomal miRNAs in recurrent and non-recurrent groups were compared(Mann-Whitney test or t test).ROC curves were used to evaluate the predictive ability of exosomal miRNAs for recurrence.GO analysis and KEGG analysis were used to evaluate the potential function of exosome miRNAs.The miRNA-overexpressed HCC cell line was constructed.CCK8 and transwell were used to evaluate the proliferation and migration of HCC cells.Western blotting was used to detect the changes of epithelial-mesenchymal transition(EMT)related proteins to observe the effect of miRNA on the biological behavior of hepatoma cells.Results 1.The product isolated by precipitation has a typical cup-shaped morphology,and expresses exosome-associated markers such as CD9,CD63 and Tsg101,with a diameter of 50-200 nm,consistent with previous reports.2.A total of 9 miRNAs were dysregulated in both HCC tissues and circulating exosomes from HCC patients,as wells as HCC cell-derived exosomes.5 of them were members of miR-17-92 cluster and miR-106b-25 cluster(miR-19a?miR-19b?miR-92a?miR-93?miR-25),which were considered as HCC-related miRNAs and entered the next step of analysis.3.The levels of exosomal miR-19a?miR-19b?miR-92a?miR-93?miR-25 was significantly elevated in the recurrent group,and the predictive ability of exosomal miR-25 for recurrence was best,and the area under ROC curve(AUC)was 0.825.4.The functions of target genes of miR-25 was closely related to cell proliferation,cell cycle,cell death and immune regulation,and may be involved in the regulation of oncogenic pathways such as MAPK,Hippo-YAP and TGF-?.Overexpression of miR-25 enhanced the proliferation and migration of hepatoma cells,activating epithelial-mesenchymal transition,which promote HCC recurrence.Conclusion 1.The exosomes were successfully isolated by precipitation from pre-operative serum.2.The members of miR-17-92 cluster and miR-106b-25 cluster(miR-19a?miR-19b?miR-92a?miR-93?miR-25)were HCC-related miRNAs.3.HCC-related miRNAs were upregulated in circulating exosomes from recurrence group.The predictive ability of miR-25 in circulating exosomes was the best.4.Exosomal miR-25 might promote cell proliferation and migration by autocrine or paracrine way to result in HCC recurrence.