| According the report in 2018,there are 18.1 million new cases and 9.6million deaths cases of cancer worldwide,1/5 of men and 1/6 of women will develop cancer,1/8 males and 1/11 females will die of cancer.Since most tumors lack of specific biomarkers and obvious clinical signs that can be used for detection,more than 60% of cancer patients are in the middle or late stages when they were diagnosed.The study showed that while the age-specific mortality rate for malignancies nationwide remained low until the age of 45,it began to rise rapidly after that age,peaking above the age of 85.With the aggravation of population aging and the severity of cancer situation in China,it is particularly important to find specific biological markers of cancer.Exosomes are lipid bilayer membranous vesicles produced by cells in a series of biological processes: endocytosis-fusion-exclusion.Because the tumor microenvironment long-term presence in a hypoxic state,the tumor cells will produce abundant signal molecules related to the pathological or physiological state of body,such as marker proteins,m RNA,mi RNA,Lnc RNA,and circular RNA.These molecules could affect the cellular physiological processes(such as tumor cells growth,migration,communication and angiogenesis,etc).Therefore,exosomal marker detection can be used for early clinical diagnosis,clinical risk or efficacy assessment,and prognosis.At present,the mainly extracting methods for exosomes were including ultracentrifugation and exosomes extraction kit.Although we could obtain higher purity exosomes by using ultracentrifugation,but the operation steps are more complicated and the price of ultracentrifuge is more expensive.Although the exosomes extraction kit is simple,the purity is not enough.If we can establish a new separation technique of exosomes,it can greatly promote the research of exosomes.With the implementation of the Human Genome Project,protein function research has become one of the core contents of the post-genome era.The mass spectrometry is one of the most powerful tools for protein identification.It has the advantages of high sensitivity,separation,and identification at the same time,which is capable to increase the reliability of the diagnosis and prognosis;currently,it is widely used in medical research.With the deepening of exosomes research,the results of exo-proteomics continue to emerge.Some studies report that it can be isolated the tumor cellular exosomes when combination of flow cytometry with tumor cells-specific membrane proteins,and according to the function of membrane proteins which related to the development of tumors to diagnose or screen tumors.However,there are few reports on the study of exosomes membrane proteins.Therefore,through the verification of exosomes and the analysis of relative quantitative proteomics results of hepatocellular carcinoma cells exosomes and hepatocyte exosomes,it is found that hepatoma cells exosomes-specific high expression membrane proteins,hoping to provide a new perspective and idea for the isolation and purification of tumor exosomes.In the first part,the cells exosomes were isolated,purified and verified,and the relative quantitative protein profiles date of the exosomes were analyzed,and the high specific membrane proteins of exosomes in hepatocellular carcinoma was selected.In the second part,the candidate membranous proteins screened in the first part of this study were verified by q RT-PCR,Western blot,immunofluorescence and bioinformatics analysis.Part Ⅰ Identification of exosomes and analysis of relative quantitative protein profilesObjective The exosomes were extracted by modified ultracentrifugation,the obtained exosomes were verified,and the relative quantitative protein profiles of the exosomes were determined.The obtained results were analyzed,and the highly expressed protein in the exosomes of hepatocellular carcinoma cells were screened out.Method 1.The starvation culture method was used to culture human normal hepatocytes cells(HL-7702),human hepatocellular carcinoma cells(Hep G2 and SMMC-7721),the cells culture supernatant was collected to isolate and purify exosomes using the modified ultracentrifugation method what studied by the research group in the previous stage.2.The morphological structure of exosomes was observed by transmission electron microscopy.Western blot was used to verify the exosomes specific proteins HSP70,CD9 and Alix.Nanosight was used to detect the particle size and concentration of exosomes.3.The exosomes protein profiles of human normal hepatocytes cells HL-7702,human hepatocellular carcinoma cells(Hep G2 and SMMC-7721)were analyzed by LC-MS/MS relative quantitative proteomics detection.4.The co-expressed proteins of two hepatocellular carcinoma cells(SMMC-7721 and Hep G2)were screened.Uniprot standardization protein name,DAVID gene functional annotation and enrichment of KEGG pathway analysis the co-expressed proteins.The co-expressing proteins were compared with normal hepatocytes cells HL-7702 exosomes proteins and candidate protein with high specific expression of hepatocellular carcinoma cells exosomes was obtained.5.GO classification analysis was conducted on the screened candidate proteins to understand the subcellular location and distribution of each candidate protein.Result1.Transmission electron microscopy showed that the exosomes of hepatocellular carcinoma cells Hep G2 were observed to be elliptic in shape,with typical lipid bilayer membrane structure.The results of western blot showed that the expression of exosomes specific proteins CD9,Alix and HSP70 could be detected in the supernatant particle sediments extracted from HL-7702 and Hep G2 cells culture by modified ultracentrifugation.The particle concentration detected by Nanosight was 1.12×109 / m L,with the peak value of58.2 nm,and the median value of 74.8 nm.All the above three experiments show that the modified ultracentrifugation method can effectively isolated high-purity exosomes.2.According to the Uniprot database,there were 2879 proteins of Hep G2 cells exosomes,2426 proteins of SMMC-7721 cells exosomes detected by the relative quantitative proteomics.Among them,845 co-expressed proteins were both in Hep G2 and 7721 cells exosomes,removal of 9 proteins with the same name,co-expressed proteins left in 836.3.The 836 proteins were analyzed by KEGG pathway,and 34 pathways related to hepatocellular carcinoma were enriched,75 proteins involved in these34 pathways but not expressed in HL-7702 normal hepatocytes cells exosomes.Included 8 proteins with PSMs greater than 10,which were KPNB1,NRAS,CD81,AP2M1,ITGA2,IGF2 R,FN1,and ACTC1.4.GO classification results showed that 8 candidate proteins KPNB1,NRAS,CD81,AP2M1,ITGA2,IGF2 R,FN1,ACTC1 were mainly distributed in the organelle membrane or cells membrane,and they were involved in important physiological and biochemical functions.Conclusion1.Transmission electron microscopy,western blot,and Nanosight results showed that high-purity exosomes were obtained by modified ultracentrifugation.2.The protein expression profile of hepatocellular carcinoma cells exosomes was different from that of normal hepatocyte exosomes.The 8proteins that were highly expressed in hepatoma cells exosomes but not expressed by normal hepatocyte exosomes with KPNB1,NRAS,CD81,AP2M1,ITGA2,IGF2 R,FN1,and ACTC1,all of them are organelle membranes or cells membrane proteins,with are candidate proteins for further study.Part Ⅱ Verification of exosome-specific expression membranous protein in hepatocellular carcinomaObjective The 8 candidate membranous proteins screened by the relative quantitative protein expression profiling analysis were verified to analyze their m RNA and protein expression levels in hepatocellular carcinoma tissues,hepatocellular carcinoma cells and hepatocellular carcinoma cells exosomes,and their relationship with prognosis,providing new perspectives and ideas for the study of exosomes membrane proteins.Method1.HL-7702,Hep G2,Huh7 and Hep3 B cells were cultured.After total RNA was extracted,the m RNA expression levels of 8 candidate membranous proteins KPNB1,NRAS,CD81,AP2M1,ITGA2,IGF2 R,FN1 and ACTC1 were verified by q RT-PCR.2.HL-7702,Hep G2,Huh7,Hep3 B cells were cultured,the total cells proteins were extracted to verify the protein expression levels of candidate membranous proteins ITGA2 and IGF2 R in hepatocellular carcinoma cells.3.The HL-7702,Hep G2,Huh7 and Hep3 B cells were counted on seed plates.After cultured for 12 h,the distribution and expression of candidate membranous proteins ITGA2 and IGF2 R in HL-7702,Hep G2,Huh7 and Hep3 B cells were verified by immunofluorescence.4.HL-7702,Hep G2,Huh7,Hep3 B cells were cultured,and the cells exosomes were extracted and purified by modified ultracentrifugation,and the total protein of the cells exosomes was extracted.The protein expression levels of candidate membranous proteins ITGA2 and IGF2 R in hepatocellular carcinoma cells exosomes were verified,and the target membranous protein IGF2 R of this study was screened.5.The Gene Expression Profiling Interactive Analysis(GEPIA),Onco Lnc website and the cancer genome atlas(TCGA)database were used to analyze the expression level of target membranous protein IGF2 R in hepatocellular carcinoma tissues and its influence on the survival rate of hepatocellular carcinoma patients.6.The expression of the target membranous protein IGF2 R in other tumor cells were analyzed: the total RNA of prostate cancer cells PC-3 and neuroblastoma cells SH5 Y were extracted,and the expression level of target membranous protein IGF2 R m RNA was analyzed by q RT-PCR;the total proteins of PC-3 and SH5 Y cells were extracted,and the protein expression of IGF2 R in the cells was analyzed by western blot.Result1.The results of q RT-PCR showed that compared with normal hepatocyte HL-7702,the m RNA expression levels of 5 proteins KPNB1,NRAS,CD81,ITGA2 and IGF2 R were highly expressed in Hep G2,Huh7 and Hep3 B cells(P<0.05).and the candidate membranous proteins ITGA2 and IGF2 R were particularly significant in Hep3 B hepatocellular carcinoma cells,IGF2R (HL-7702 vs Hep3 B,1.000 ± 0.000 vs 7.875 ± 1.009,P = 0.000),ITGA2(HL-7702 vs Hep3 B,1.016 ± 0.024 vs 5.842 ± 1.122,P = 0.002).2.Western blot results showed that compared with HL-7702 normal hepatocyte cells,the proteins expression levels of candidate membranous protein IGF2 R in Huh7 and Hep3 B hepatocellular carcinoma cells were 0.936 ± 0.310(P = 0.029),1.577 ± 0.551(P = 0.012),respectively,and IGF2 R expression was increased in hepatocellular carcinoma cells.The protein expression levels of candidate membranous proteins ITGA2 in Huh7 and Hep3 B hepatocellular carcinoma cells were 1.591 ± 0.372(P = 0.028)and 1.690 ± 0.542(P = 0.021),respectively,and ITGA2 expression was increased in hepatocellular carcinoma cells.3.The results of cells immunofluorescence showed that compared with HL-7702 normal hepatocyte cells,candidate membranous proteins ITGA2 and IGF2 R were expressed in Hep G2,Huh7 and Hep3 B hepatocellular carcinoma cells,and the expression of IGF2 R was higher in hepatocellular carcinoma cells,especially in Hep3 B hepatocellular carcinoma cells.4.Western blot results showed that compared with the expression of candidate membrane protein IGF2 R in the exosomes of HL-7702 normal hepatocyte exosomes(0.179 ± 0.066),the expression of IGF2 R in Huh7 and Hep3 B hepatocellular carcinoma cells exosomes were increased by 1.044 ±0.153(P = 0.009),1.174 ± 0.088(P = 0.001),respectively.Compared with the expression of candidate membrane protein ITGA2 in the exosomes of HL-7702 normal hepatocyte exosomes(0.888 ± 0.106),the expression of ITGA2 in the exosomes of Huh7 and Hep3 B hepatocellular carcinoma cells was increased by1.502 ± 0.234(P = 0.014),1.921 ± 0.234(P = 0.000),respectively.5.Combined with the above experimental results,the IGF2 R was used as the target membranous protein.After search keyword IGF2 R,GEPIA website data showed that IGF2 R higher expression in hepatocellular carcinoma tissues compared with adjacent tissues.The Kaplan-Meier survival curve in the Onco Lnc website showed that the survival rate in the high-expression IGF2 R hepatocellular carcinoma group was significantly lower than that in the low-expression group(P = 0.022).6.Compared with normal hepatocytes cells and hepatocellular carcinoma cells,target membranous proteins IGF2 R was expressed in PC-3 and SH5 Y cells,and its m RNA expression was lower than that of normal hepatocytes(HL-7702 vs SH5Y: 1.000 ± 0.008 vs 0.000 ± 0.000,P = 0.000;HL-7702 vs PC-3: 1.000 ±0.008 vs 0.473 ± 0.270,P = 0.000)and Hep G2 cells(Hep G2 vs SH5Y: 1.3546 ±0.197 vs 0.000 ± 0.000,P = 0.000;Hep G2 vs PC-3: 1.3546 ± 0.197 vs 0.473 ±0.270,P = 0.020);Huh7(Huh7 vs SH5 Y,3.069 ± 0.250 vs 0.000 ± 0.000,P =0.000;Huh7 vs PC-3,3.069 ± 0.250 vs 0.473 ± 0.270,P = 0.000);Hep3B(Hep3B vs SH5 Y,7.542 ± 1.218 vs 0.000 ± 0.000,P = 0.000;Hep3B vs PC-3,7.542 ± 1.218 vs 0.473 ± 0.270,P = 0.001).Compared with normal hepatocytes cells or hepatocellular carcinoma cells,target membrane protein IGF2 R was expressed in PC-3 and SH5 Y cancer cells,but its protein expression was lower than Hep3 B cells(Hep3B vs PC-3,1.372 ± 0.128 vs 0.263 ± 0.007,P = 0.000;Hep3B vs SH5 Y,1.372 ± 0.128 vs 0.502 ± 0.044,P = 0.000).Conclusion1.The m RNA and protein expression level of IGF2 R in hepatocellular carcinoma were up-regulated;2.The protein expression level of IGF2 R in exosomes of hepatocellular carcinoma cells was up-regulated;3.High expression of IGF2 R predicts lower survival rate in patients with hepatocellular carcinoma;4.IGF2 R was expressed to some extent in most tumors,but it was significantly increased in hepatocellular carcinoma cells Hep3 B,which can be used as an important tool protein to isolate its exosomes. |
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