Exosomal MicroRNAs As Potential Biomarkers For Cancer Cell Migration And Prognosis In Hepatocellular Carcinoma Patient-derived Cell Models

Globally,the incidence of primary liver cancer accounts for 4.7%of all malignant tumors,ranking sixth,but its mortality rate is as high as 8.2%,ranking fourth.In primary liver cancer,85-90%of the patients are hepatocellular carcinoma(HCC),and the high mortality of liver cancer is associated with the lack of anti-tumour drugs in advanced hepatocellular carcinoma that significantly improve the survival of patients.Hepatocellular carcinoma progresses to the advanced stage,mainly due to the intrahepatic and extrahepatic metastases.The exploration into high-efficiency biomarkers for predicting high-risk populations provide the feasibility of close monitoring and follow-up of high-risk groups,which might lead to early detection of metastasis,early initiation of anti-tumour therapy,and even radical treatment of adjuvant using targeted or immune-related medicine.In the field of postoperative stratification of metastatic risk in HCC patients,a large number of previous studies have focused on the biomarkers associated with postoperative metastasis and prognosis,including(1)clinical features,such as the size of maximum intrahepatic lesion,the number of tumor foci,the invasion of portal vein,etc.,(2)biochemical characteristics,such as alpha fetoprotein(AFP),carcinoembryonic antigen(CEA),etc.,(3)tumor tissue characteristics,protein expression levels,DNA variation and mRNA transcriptive levels by means of immunohistochemistry,genome sequencing,and transcriptome sequencing.Among different types of RNAs,mRNA can be translated into proteins to affect the biological characteristics of tumours,while miRNA as a class of non-translational RNA,can modulate the expression of other proteins by inhibiting the transcription of multiple genes,thus providing a wider range of regulative effect.In HCC-related studies,multiple evidences support the relationship between miRNA levels and the development of primary liver cancer.The down-regulation of several miRNAs is associated with cell proliferation,apoptosis,angiogenesis,recurrence of hepatocellular carcinoma,shorter disease-free survival and overall survival,such as miRNA miR-let-7g,miR-22,miR-26,miR-29,miR-99a,miR-122,miR-124,miR-139,miR-145 and miR-199b.In contrast,the upregulation of miR-10b,miR-17-5p,miR-21,miR-135a,miR-155,miR-182,miR-221 and miR-222 is associated with hepatocellular carcinoma metastasis,angiogenesis and poor prognosis.Although RNA is more susceptible to degradation than double-stranded DNA,its concentration is relatively stable in the in vivo circulatory system,suggesting the possibility that miRNAs serve as biomarkers for the assessment of HCC risk stratification.Tumor cells,similar to other normal cells,can secrete a small vesicle-like structure,with a diameter around 50-150 nanometers,i.e.,exosome.It can be detected in a variety of body fluids such as plasma,urine,saliva,milk and malignant effusions.The membrane of the exosomes is taken from the cell membrane,the bilayer of phospholipids.Under this coating,proteins,DNA,RNA and various other cellular components can be stably protected in the exosomes.The exosomes released by the donor cells can bind to the recipient cells and eventually fuse,thereby transferring the substances from the donor cells to the recipient cells.miRNAs are also present in exosomes and previous evidences have supported their association with tumor progression and metastasis.Thus,we propose a scientific hypothesis that miRNAs in blood exosomes of HCC patients may serve as biomarkers for assessing the invasiveness of tumours and the risk of postoperative recurrence.Patient-derived cells(PDCs)may be effective models for biomarker identification.In the present study,a wound healing assay was used to obtain 10 fast-migrated and 10 slow-migrated PDC cultures from 36 HCC samples.MicroRNA(miRNA)signatures in PDCs and PDC-derived exosomes were profiled by microRNA-sequencing.Differentially expressed miRNAs between the low-and fast-migrated groups were identified and further validated in 372 HCC profiles from The Cancer Genome Atlas(TCGA).Six exosomal miRNAs were identified to be differentially expressed between the two groups.In the fast-migrated group,five miRNAs(miR-140-3p,miR-30d-5p,miR-29b-3p,miR-130b-3p and miR-330-5p)were downregulated,and one miRNA(miR-296-3p)was upregulated compared with the slow-migrated group.Pathway analysis demonstrated that the target genes of the differentially expressed miRNAs were significantly enriched in the ’focal adhesion’ pathway,which is consistent with the roles of these miRNAs in tumor metastasis.Three miRNAs,miR-30d,miR-140 and miR-29b,were significantly associated with patient survival.These findings indicated that these exosomal miRNAs may be candidate biomarkers for predicting HCC cell migration and prognosis and may guide the treatment of advanced HCC.