With in-depth research on sEVs, the compositions and functions of sEVs have gradually received attention in tumor biology and developmental biology. Among the numerous sEV omics studies, most of them have focused on the contents of miRNAs, lncRNAs, and circRNAs, but few have focused on proteins. Proteomics research on sEVs falls far behind transcriptomics research. Therefore, applying proteomics analysis, we aimed to comprehensively detect the differentially accumulated proteins in the sEVs to discover potential biomarkers for HCC evaluation. In our study, we launched a systematic approach for biomarkers in plasma sEVs of HCC, and the following components were included: (1) isolation and identification of sEVs, (2) determination of the profiles of differentially accumulated proteins with label-free quantification, (3) performance of domain annotations, enrichment analysis, and network analysis of the differentially accumulated proteins, and (4) validation of the significantly accumulated proteins in the sEVs of HCC patients.
Our results described the protein signature of plasma sEVs of HCC, the upregulated pathway of complement cascades (C1QB, C1QC, C4BPA, and C4BPB) and the coagulation cascade (F13B, FGA, FGB, and FGG). The coagulation factor XIII A chain is the last enzyme in the coagulation cascade, while the coagulation factor XIII B chain protects F13A from removal. It is clear that FGG fibrinogen and F13A are highly expressed in the plasma of HCC patients, but the relationship between F13B and HCC is unknown. The mRNA level of FGG and the elevated level of plasma fibrinogen are related to clinical stage, tumor thrombosis, and prognosis of HCC(14, 15). Ander Arbelaiz(16) found that compared to the primary sclerosing cholangitis (PSC) patients, fibrinogen gamma chain (FGG), alpha-1-acid glycoprotein 1 (A1AG1), and S100A8 proteins showed the best differential diagnostic capacity in the sEVs of Cholangiocarcinoma(CCA) patients. Most notably, ficolin-2(FCN2), inter-alpha-trypsin inhibitor heavy chain H4, and FGG showed higher diagnostic value than CA19-9 in CCA I-II versus PSC patients. In addition, Asad Uzzaman et al. found similar results that FGA, FGB, and FGG were dysregulated in liver cancer sEVs and verified that the expression of fibrinogen, fibulin-1(FCN1), and thrombospondin-1 could differentiate controls from patients with liver cancer or cirrhosis (17). However, they focused on the fibrinogen and didn’t verify the fibrinogen alpha, beta, and gamma chains in more detail. Besides, Sen Wang analyzed the sEV proteins between the MHCC97-H and MHCC97-L cell lines to find that the filamin A, talin 1 and fibulin 1 in the sEVs were strongly related to HCC metastasis(18). Their results were in strong agreement with our results, which supported the above perspective of fibrinogen in HCC
As the fundamental defense system, the complement cascade is central to immunological networks which tightly regulate humoral and cellular responses to noxious stimuli. Complement components have been linked with several cancers, which indicates that complement regulation might be a factor for oncogenesis(19). Importantly, the liver is the major source for the biosynthesis of complement components and expresses a variety of complement receptors. (20). Therefore, the progression of HCC might be largely influenced by the complement system. As previous studies have shown, upregulation of C1QB or C1QC is related to prostate cancer(21), melanoma(22), renal cell carcinomas(23), and HCC(24, 25). The upregulated C1qTNF6 activated the Akt pathway to promote HCC angiogenesis(26). Upregulation of C4BP is related to colorectal cancer(27, 28), epithelial ovarian cancer(29), non-small cell lung cancer(30), and HCC(31, 32). Interestingly, HCC cells can be protected from complement attack by upregulating C4b-binding protein alpha through binding to the transcription factor Sp1(31). Ardakani et al. found that C4b-binding protein was significantly related to HCC and cirrhosis. (32). Thus, the ability of C4BP to regulate tumorigenesis in multiple organs and the liver as a primary source strongly suggests a role for C4BP in HCC. However, the relationship between sEVs and the complement system in HCC has yet to be clarified. Our results showed that the components of the complement system (C1QB, C1QC, C4BPA, and C4BPB) were more highly expressed in the HCC group sEVs than in the control group sEVs, which might be significant for the evaluation of HCC.
In summary, our results highlighted the protein signatures of plasma sEVs from HCC patients, the upregulated pathway of complement cascades and the coagulation cascade. These factors might be used for noninvasive diagnosis. This hypothesis has been confirmed by previous studies and our experiments. Our data provide useful insight into the cargos from plasma sEVs, which might be candidates for evaluating and diagnosing liver cancer.