The Role Of CD147~+ Small Extracellular Vesicles In The Diagnosis And Angiogenesis Of Hepatocellular Carcinoma

Objective:Hepatocellular carcinoma(HCC)is a common malignant tumor of the digestive system,accounting for 70%to 90%of all liver cancer types and ranking as the third leading cause of cancer-related death.Early screening for HCC is mainly done by abdominal ultrasound and serum alpha-fetoprotein(AFP).However,many patients with HCC have unclear imaging and negative AFP results,which will make the diagnosis more difficult and even delay early treatment.Small extracellular vesicles(s EVs)are nanoscale bilayer membrane structured vesicles secreted by cells,which can deliver bioactive molecules to recipient cells,affect their biological properties,alter the tumor microenvironment and produce long-distance effects,and are widely distributed in various body fluids in the human body.Tumor-derived s EVs are considered to be potential therapeutic agents and disease biomarkers that have been shown to play a role in cancer progression.CD147 is a transmembrane glycoprotein that is overexpressed in a variety of human malignancies and correlates with tumor size,cancer stage,progression and prognosis.However,the function and molecular mechanisms of CD147-positive(CD147~+)s EVs of hepatocellular carcinoma origin in the progression of hepatocellular carcinoma are unknown.This study investigated the feasibility of CD147 in plasma-derived small extracellular vesicles from HCC patients as a clinical diagnostic marker and examined the mechanism of CD147~+small extracellular vesicles on angiogenesis in the progression of hepatocellular carcinoma.Methods:1.Small extracellular vesicles were isolated and obtained by ultracentrifugation.Isolated s EVs were identified by Nano-flow cytometry(Nano FCM),transmission electron microscopy and Western Blot.2.Plasma CD147~+s EVs expression levels in HCC patients,liver cirrhosis(LC)patients and healthy donors(HD)were analyzed by Nano-flow cytometry.3.The diagnostic efficacy of CD147 as a diagnostic marker for HCC was evaluated by receiver operating characteristic(ROC)curve.4.The expression levels of CD147~+s EVs of HCC cell line Hep G2 cells and normal hepatocyte line LO2 cells origin were analyzed by Nano FCM.5.Hep G2-derived s EVs were co-incubated with human umbilical vein endothelial cells(HUVEC),and the uptake of s EVs by HUVEC cells was observed by laser confocal microscopy.6.Construction of stable Hep G2 cell line with knockdown of CD147 by synthesizing a lentivirus targeting CD147 interference.7.HUVEC cells were treated with Hep G2 cells and CD147 knockdown Hep G2 cell-derived s EVs.The proliferation,migration and invasion ability of HUVEC cells were detected by CCK-8,wound healing,transwell assays and angiogenesis assay.Results:1.Particle sizes of s EVs were detected by Nano FCM,ranging from 30 to 180 nm;s EVs were vesicular with intact membrane under transmission electron microscopy;three positive marker proteins CD9,CD63 and TSG101 were detected in s EVs by Western Blot,while the negative marker protein GM130 was negative.2.Compared with healthy donors,plasma CD147~+s EVs expression was significantly higher in HCC patients(p<0.001)rather than plasma CD147~+s EVs expression in LC patients(p=0.862).3.The combined CD147~+s EVs and AFP assay had good diagnostic efficacy for HCC,with the sensitivity of 88.39%,the specificity of 100%,and area under the curve(AUC)up to 0.959.4.Compared with LO2-derived s EVs,CD147~+s EVs in Hep G2-derived s EVs were significantly increased(p<0.01).5.CCK-8,wound healing,Transwell assay and angiogenesis assay showed that Hep G2-derived s EVs could significantly promote the proliferation,migration,invasion and angiogenesis of HUVEC cells(p<0.05);however,stable CD147 knockdown Hep G2-derived s EVs treated with HUVEC cells reduced proliferation,migration,invasion and angiogenesis capacity(p<0.05).Conclusion:1.Morphologically intact s EVs from peripheral blood can be extracted by ultracentrifugation successfully.2.Plasma CD147~+s EVs levels were significantly higher in HCC patients than in the healthy population and increased significantly with progression of HCC staging.3.High expression of CD147~+s EVs in plasma is a potential diagnostic biomarker for HCC.4.HCC-derived s EVs may have a pro-angiogenic function by promoting the proliferation,migration,invasive and angiogenesis ability of HUVEC cells via CD147.