A Proteomic Investigation Of Plasma-derived Exosomes In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the most common type of primary liver cancer,accounting for >75% of the total cases.The incidence rate of HCC ranks sixth among all malignant tumors around the world,and the mortality rate of HCC ranks fourth.In2018,the estimated global incidence rate of HCC was 9.3/100,000,while the mortality rate was 8.5/100,000.In China,the incidence and mortality account for about 50% of all the cases around the world.The main risk factors of HCC include chronic hepatitis virus infection,alcohol abuse,and non-alcoholic fatty liver disease(NAFLD).More than80% of HCC patients are diagnosed at advanced stage.As the therapies for advanced HCC are lacking,the five-year survival rate of advanced HCC is less than 15%.Thus,HCC patients may benefit from early diagnosis,which could improve the treatments for HCC.Currently,the methods for HCC screening and early diagnosis include imaging examination and tumor marker detection.For tumor marker detection,serum alpha-fetoprotein(AFP)level detection is widely used in HCC in clinical.Nevertheless,the sensitivities and specificities of the AFP detection were reported as 58%～68% and80%～94%,respectively.In addition,the effect of AFP detection on small HCC is poor,which further limits the application of AFP detection.Therefore,the development of novel biomarker for the early diagnosis of HCC is urgently needed.Exosome is one kind of extracellular vesicles,ranging from 40 to 150 nanometer(nm)in diameter.Exosomes are widely distributed in body fluids,including blood,saliva and urine.Exosomes contain constituents from cells(such as protein,DNA,RNA,etc.),which could be captured by other cells through endocytosis.Evidence indicates that exosomes could play important roles in the development of common diseases,including cancers.Thus,exosomes are considered to be promising biomarkers.The plasma exosomes from cancer patients harbor biomolecules from cancer cells,such as proteins,mi RNAs and circle RNAs.These biomolecules may be used in the screening and early diagnosis of cancer.However,few studies have focused on exosomes in HCC,plasma exosomal biomarkers for HCC are needed.To identify candidate biomarkers for HCC early diagnosis,and to reveal a proteomic profile of plasma exosomes of HCC,we collected plasma samples from 7HCC patients,7 cancer-free individuals with liver cirrhosis and 7 healthy individuals.We extracted and purified plasma exosomes,using Izon q EV Kits.We further performed quality controls using transmission electron microscopy analysis,nanoparticle tracking analysis and western blot analysis.In addition,we quantified the exosomes proteins using high performance Liquid chromatography-mass spectrometry(LC-MS).We revealed the proteomics profile of plasma exosomes in HCC,and identified differentially expressed proteins between HCC group and liver cirrhosis or healthy individuals.Transmission electron microscopy analysis showed that our extracts are round morphology.Nanoparticle tracking analysis showed that the sizes of our extracts are30～150 nm.Western blotting analysis showed that the exosome surface specific markers(TSG101,CD63 and CD9)are detected in our extracts.These results indicated that most of the extracts were exosomes.Furthermore,we extracted proteins from exosomes,and perform proteome analysis by high performance LC-MS.A total of 267 proteins were detected.We then inquired the public exosome databases: Vesiclepedia and Exo Carta.Among the 267 detected proteins,226 and 203 ones were verified in the Vesiclepedia and Exo Carta databases,respectively.The Gene Ontology(GO)annotations suggested that the 267 detected proteins were mainly enriched in biological processes including cytoskeleton,extracellular matrix and lysosome,and are mainly enriched in cellular components including extracellular areas and vesicles.Taken together,these results indicated that most of the detected proteins were exosomal proteins.In order to identify serum biomarkers of HCC,we performed differential analysis among the 267 detected proteins.We defined differentially expressed proteins as those with P < 0.05 and fold change > 1.2.We found 8 differentially expressed proteins between HCC group and healthy individuals group,while we found 4differentially expressed proteins between HCC group and liver cirrhosis group.We found that SIGIR protein showed significantly lower expressions in HCC than that in liver cirrhosis group and healthy individuals group.The specificity and sensitivity of SIGIR protein are high.These results suggested that SIGIR might be the candidate biomarkers for HCC.In summary,this study presented a landscape of the HCC plasma exosomal proteins.Differential expression analyses identified that SIGIR was a candidate biomarker of HCC.The results of this study need further verified in large-scale clinical cohorts.