Screening And Identification Of Differential Phospholipids In Urinary Exosomes Of Patients With Hepatocellular Carcinoma

Background:Hepatocellular carcinoma(HCC)is the most common primary liver cancer,accounting for more than 90%of the pathological types of primary liver cancer;it is also the most common clinical type of liver cancer.The clinical manifestations of patients with HCC are diverse and complex,with large individual differences,and early diagnosis is difficult.The commonly used technical methods for clinical diagnosis of liver cancer are divided into laboratory-related index detection and imaging examination,and the cytological type of confirmed liver cancer depends on pathological means.With the development of science and the in-depth study of tumors such as HCC,potential small molecular markers have gradually entered the perspective of researchers,such as exosomes.Exosome is a membranous vesicle containing a large number of biologically active molecules,which has certain guiding significance for the diagnosis,staging,treatment and prognosis of diseases.Urine is one of the few clinical specimens that can be used to isolate exosomes by non-invasive methods,but the large amount of TH protein is a major difficulty in interfering with the separation and extraction of exosomes.Therefore,it is urgent to optimize the separation and extraction method to obtain both concentration and purity.Qualified urine exosomes.Metabolomics technology can be used to analyze the dynamic changes of small molecule metabolites,especially lipid composition,which can intuitively reflect the overall metabolic change trajectory of the body’s physiology and pathological processes.It has important implications for the body’s health,disease progression,diagnosis and treatment Significant.Objective:The purpose of this study is to provide a simple and specific method for obtaining exosomes in urine,and to analyze HCC and liver by a new type of mass spectrometry-extractive electrospray ionization mass spectrometry(EESI-MS).The phospholipid composition of exosomes in the urine of 4 groups including sclerosis,chronic hepatitis B,and healthy controls was analyzed for changes in specific components.Provide a new idea for the early non-invasive diagnosis of HCC.Methods:Urinary exosomes were extracted using ultra-high speed centrifugation(200,000 g),laboratory self-research methods,and exosome extraction kits.Western Blot was used to detect exosomal surface markers and compare the concentration and heterogeneous protein distribution of the three methods.Situation;after selecting the optimal method to extract exosomes,use transmission electron microscope(TEM),tunable resistive pulse sensing(TRPS),and Western Blot to observe its morphology,particle size and protein markers concentration.The modified Folch method was used to extract lipid components from exosomes.Test different voltages and extractants on EESI-MS,optimize parameters such as signal peak intensity,signal value breadth,and signal stability,and select the most suitable voltage and extractant.Using EESI-MS for 4 groups of specimens including HCC,cirrhosis,chronic hepatitis B,and healthy controls(experimental cohort,10 cases in each group,40 cases in total,each sample was divided into 3 samples for a total of120 samples;validation cohort 20 cases in each group,a total of 80 cases,each sample is divided into 3 samples,a total of 240 samples)to detect,compare the fingerprint spectrum differences;use MATLAB R2015a software for principal component analysis(PCA)and screening If there is a difference in phospholipids,the collision-induced dissociation(CID)test is used to identify and confirm the target ions.The heat map was used to analyze the intragroup correlation of the four groups of phospholipids,and the area under curve(AUC)and the Youden index were used to determine the diagnostic efficacy of different phospholipid components and combinations for HCC.At the same time,Metaboanalyst online software was used to enrich the metabolic pathways of differential phospholipids.Results:(1)Western Blot showed that ultra-high-speed centrifugation and laboratory self-research methods were qualified for removal of TH protein,and TH-proteins remained in the kit method,and the exosomes concentration of the laboratory self-research method was higher.Existence of exosomes isolated by laboratory self-research methods.(2)EESI-MS detects the exosome phospholipids in urine at a voltage of+3kV,the extractant is methanol/distilled water/glacial acetic acid(1/1/0.02),the signal peak is the strongest,and the signal value is captured in the range of m/z50~1000 Widest and most stable signal.(3)The first-order fingerprints showed that the m/z of the four groups were significantly different,showing the specificity of their respective disease types.(4)The PCA results showed that the 4 groups(HCC group,cirrhosis group,chronic hepatitis B group,and control group)could be distinguished independently.Among them,HCC patients could be clearly distinguished from healthy controls,and the most significant difference in the mass spectrum peaks in the experimental cohort It is m/z754,m/z787,m/z832,and m/z877.The most obvious peaks in the verification cohort are m/z726,m/z787,m/z832,and m/z877.(5)The relative abundance of target ions in each difference was significantly different from the other groups in the HCC group(P<0.05).The intergroup correlation analysis showed that the HCC group showed a negative correlation with the other 3 groups.(6)The seven target phospholipids with significant differences identified by the CID test are[SM(d18:1/16:0)+Na]~+(m/z726),[SM(d18:0/18:1)+Na]~+(m/z754),[PC(16:0/16:0)+Na]~+(m/z757),[PC(18:1/18:1)+H]~+(m/z787),[PG(18:0/20:3)+H]~+(m/z803),[PC(18:0/20:4)+Na]~+(m/z832),[PC(20:4/20:0)+K]~+(m/z877),2 of which are sphingomyelin,4 are phosphatidylcholines and 1 is phosphatidylglycerol.(7)The area under the curve(AUC)of m/z726,m/z754,m/z757,m/z787,m/z803,m/z832,and m/z877 in the experimental cohort were 0.852,0.873,0.778,0.884,0.708,0.817,0.751,and the area under the curve(AUC)of m/z726,m/z754,m/z757,m/z787,m/z803,m/z832,and m/z877 in the verification queue were 0.818,0.741,0.780,0.890,0.715,0.821,0.820.The AUCs of the seven phospholipids combined diagnosis are 0.989(the experimental cohort)and 0.985(the verification cohort).(8)Different phospholipids participate in 5 metabolic pathways,including sphingomyelin metabolism,glycerophospholipid metabolism,arachidonic acid metabolism,linoleic acid metabolism,andα-linolenic acid metabolism.Conclusion:EESI-MS detection of urinary exosomes phospholipids in patients with hepatocellular carcinoma is reproducible,rapid and efficient;it may provide a new idea for non-invasive and non-radiative detection of clinical diseases.