Study On Screening And Validation Of Differentially Expressed Proteins In Exosomes Of Liver Cancer Cells

Tumor is one of the diseases that threaten human health and survival the most.The incidence and mortality of malignant tumors are increasing day by day,which brings heavy burden of economic and disease to families and society.Liver cancer(HCC)is one of the most common malignancies in the world.The global cancer statistics report(2015)shows that about 600,000 people are diagnosed with liver cancer each year.The number of new cases and deaths in China are accounted for approximately 50% of the total cases in Europe and North America.The mortality of liver cancer currently ranks as the second in all types of cancers.Primary liver cancer generally does not have any clinical symptoms in the early stage,and it is frequently found and diagnosed in the middle and late stages.Therefore,the prognosis is extremely poor.Early detection is one of the best ways to improve the five-year survival rate of liver cancer.At present,the diagnostic methods of liver cancer include serum tumor markers(especially the level of AFP expression),medical imaging(including Bultrasound,CT,etc.)and pathological biopsy.As the evaluation standard of the health effect of population,biomarker detection is an ideal method for the diagnosis of liver cancer because of its minimal invasion,simplicity,economy,and ease of operation.At present,the most widely used serum marker of liver cancer is AFP,but as the number of AFP-negative patients increased,the number of patients with liver cancer are missed detection increased as well,which directly affects the early diagnosis and prognosis evaluation.Therefore,discovering and evaluating serum markers of liver cancer is an urgent task to improve the diagnosis and prognosis of liver cancer.Proteins are directly responsible for life functions,and serum protein marker is the most common type of marker.Serum components are complex and susceptible to other substanceswithin human body's.Improper preservation and repeated freezing and thawing of serum after collection will lead to the degradation of the protein in the serum,making it extremely difficult to detect protein markers.In 2007,Valadi H found that the cells can exchange genetic material between each other throughout RNAs in exosomes and participate in cell-to-cell communication.The exosomes have attracted researchers' attention since then.Exosomes are vesicles with bilayer lipid membrane that are secreted by cells and distributed in peripheral blood,urine,saliva,ascites and other body fluids.The pathological or physiological markers of the source cells carry signal molecules such as m RNA,mi RNAs,etc.These specific small molecules may be involved in the specific organ diseases.The presence of bilayer lipid membrane reduces the interference to exosomes carrying substances from the complex components within the serum,and it is a well-studied marker material.Subsequently,In 2014,Melo SA found that exosomes derived from cells and serum patients with breast cancer could induce non-neoplastic epithelial neoplasms in a Dicer-independent manner.In 2015,Melo SA discovered that glypican-1 protein(GPC1)screened from tumor exosomes may be used as a potential non-invasive diagnostic tool and screening tool for early pancreatic cancer with diagnostic sensitivity and specificity closed to 100%.In 2018,researchers Fuhrmann G used exosomes as drug carriers for enzyme prodrug therapy.Studies have shown that exosomes have outstanding prospect as drugtargeted carriers.The discovery and confirmation of exosomes have profoundly affected people's understanding of the physiology and pathological processes of organism.Almost all of the biomedical biochemistry and molecular biological tests will be opened a whole new dimension in exosome analysis.In this study,the exosomes of hepatocellular carcinoma(HCC)cells were extracted and subjected to mass spectrometry detection and highthroughput sequencing.The differential proteins of HCC cells were discovered,screened,and validated in order to obtain the target protein.Simultaneously,the level of target protein in the serum of healthy controls and patients with HCC was detected.The study of exosomes that are produced by HCC may provide new perspectives and ideas for the molecular mechanism of hepatocarcinogenesis.Part I Screen of Differentially Expressed Proteins in Hepatocellular Carcinoma Exosomes Using Mass Spectrometry and SequencingObjective:The extraction method for exosomes of HCC was established,and the extracted precipitates were verified.The differentially expressed proteins of HCC exosomes were screened by mass spectrometry and high-throughput sequencing techniques.Methods:1.HL-7702,SMMC-7721,Hep G2 cells were cultured until 80% of the bottom of the petri dish were covered by cells.They were rinsed three times with PBS and replaced with serum-depleted medium.The culture supernatant was collected after 48 hours of hunger culture.Modified ultracentrifugation was used for extraction and purification of the exosomes.2.The morphology was observed by transmission electron microscopy.Nanosight was used for particle size analysis.Western blot was used to detect the exosome markers CD9 and Hsp70 for multiplex verification.3.LC-MS/MS mass spectrometry was used to detect and analyze HL-7702,SMMC-7721,and Hep G2 cell exosome proteins.The proteins that specifically expressed in HCC cells were selected.4.Serum exosomes were extracted from 6 patients with HCC and 4 healthy controls.The library was constructed and high-throughput sequencing technology was conducted to analyze exosomal differential m RNA in serum in healthy controls and HCC patients.5.The overlapping of differentially expressed protein that were selected by proteomics technique and differentially expressed m RNA that were selected by high-throughput sequencing were marked as the differentially expressed proteins in exosomes in HCC.Results:1.The method of starvation culture and modified ultracentrifugation could effectively extract exosomes.The exosomes extracted by ultracentrifugation had a cleaner background,and its lipid membrane structure was clear.2.The vesicles with double lipid membrane structures between 50-90 nm in size could be observed by transmission electron microscopy;Nanosight particle size analysis showed a peak particle size at 58.6 nm,the average was 96.0 nm.Particle scatter plots and distribution plots showed that the diameters of particle were concentrated,and most of them were in the size range between 30-120 nm,which was consistent with the characteristics of the expected exosomes.HL-7702,SMMC-7721,and Hep G2 cell pellets were detected by Western blot,and the exosomal specific proteins Hsp70 and CD9 were detected.3.The result of mass spectrometry showed that Hep G2 exosome expressed a total of 2875 proteins,SMMC-7721 exosome expressed a total of 2042 proteins,and 845 proteins were co-expressedin both Hep G2 and SMMC-7721 hepatoma cell exosome.4.Sequencing the serum exosomes of 6 healthy controls and 4 HCC patients,and 34,791 transcripts were obtained.Further screening was performed under the conditions of |log2FC |> 1 and q-value < 0.05,which resulted in 9440 differential transcripts.8,963 differential m RNAs were up-regulated,and 477 differential m RNAs were down-regulated.5.182 proteins were screened by proteomics and high-throughput sequencing.Screening was performed under the condition of log2 FC of m RNA in high-throughput sequencing greater than 2,and 30 up-regulated proteins were therefore selected.Eight of the secreted proteins were selected as the candidate proteins for further validation analysis.Conclusion:1.This study has established a method for effectively extracting exosomes by using starvation culture combined with modified ultracentrifugation.2.The results from transmission electron microscopy,Nanosight particle size analysis,and Western blot detection demonstrated that the extracted exosomes satisfied the requirements.3.Proteomics and transcriptome sequencing were used to screen the differentially expressed proteins in liver cancer exosomes.Combined with bioinformatics analysis,8 secreted proteins(APOA4,ECM1,CDCP1,PXDN,AHSG,S100A13,COL7A1,FBLN1)were screened as the candidate markers for liver cancer for and the further analysis.Part 2 Analysis of differentially expressed proteins in exosomes of hepatocellular carcinomaObjective:To verify the expression level of candidate proteins,screen the target proteins,and detect their expression level within serum samples in order to find out the potential biomarker for HCC.Methods:1.HL-7702,SMMC-7721,and Hep G2 cells were cultured,total RNA was extracted from the cells,and reverse transcription was carried out.RT-PCR was used to detect the expression level of the m RNA in candidate protein of HCC by using c DNA as a template.2.The HL-7702,SMMC-7721,and Hep G2 cells were cultured,and the culture supernatant was collected after 48 hours of starvation culture.The exosomes were isolated and purified by ultracentrifugation.Exosomal total RNA was extracted and then reversely transcribed.The expression level of candidate protein m RNA was detected by RT-PCR and adopted c DNA as the template,and the target protein was screened.3.HL-7702,SMMC-7721,Hep G2,Huh-7 cells were cultured,counted and seeded on the slides.After 24 hours of incubation in the cell culture incubator,cells were removed for immunohistochemistry experiments to detect the level of expression of target protein COL7A1 in normal liver cells and HCC cells.4.Serum gradient ELISA was performed to detect one sample from HC and one sample from HCC;The sample from a HC and a HCC patient were tested in a 2-fold dilution of ELISA in order to detect protein COL7A1.30 healthy controls and 30 patients with HCC with gender and age matched were selected.RT-PCR screening was used to detect the m RNA level of COL7A1 from the mixed serum.The serum from 35 healthy control and 47 patients with HCC with gender and age matched were obtained.RT-PCR screening was used to detect the serum m RNA level of COL7A1.Results:1.Compared to normal hepatocyte HL-7702,all of the m RNAs of candidate proteins were up-regulated in hepatoma cells(P < 0.05).APOA4,PXDN,S100A13,COL7A1,and FBLN1 were up-regulated in both hepatoma cells SMMC-7721 and Hep G2(P < 0.05).2.Compared with the exosomal mRNA expression level of HL-7702,the m RNAs of four candidate proteins PXDN,S100A13,COL7A1,and FBLN1 were all up-regulated in exosomes of hepatoma cells(P < 0.05).Combined with the current studies and bioinformatics analysis,COL7A1 was selected as the target protein for further analysis and validation.3.The results of immunohistochemistry showed that the expression of COL7A1 in hepatoma cells SMMC-7721,Hep G2,and Huh-7 was up-regulated compared to normal liver cell HL-7702(P < 0.01),which was consistent with the expression levels within hepatoma cells and the exosomes of hepatoma cell.4.After the initial screening,the mixed serum of 30 healthy controls and 30 patients with HCC indicated that the m RNA expression level of COL7A1 in AFP-negative patients with HCC was found to be lower than healthy controls;47 HCC patients with AFP < 20 ?g/L and 35 healthy controls were selected and tested,and the results showed that the m RNA expression level of COL7A1 in serum was significantly down-regulated in AFP-negative patients(P < 0.001).Conclusion:1.COL7A1 was up-regulated in HCC cells and its exosomes.COL7A1 may be closely related to carcinogenesis of HCC,and its mechanism needs further study.2.The m RNA expression level of COL7A1 in the serum of HCC with AFPnegative was significantly down-regulated.Whether the m RNA of COL7A1 can be a marker of AFP-negative HCC,the confirmation by multi-center verification is needed.