Study On The Diagnostic Value Of Plasma Exsomal Circular RNA 0006037 In Hepatocellular Carcinoma

ObjectiveHepatocellular carcinoma(HCC)is the most common primary liver tumor,accounting for75～85% of all primary liver tumors.Since most patients with HCC are diagnosed in the middle or late stages and lack of effective treatment methods,the prognosis of them is poor.Therefore,early diagnosis of HCC is very important.Circular RNA(circ RNA)is a group of RNAs featured by the presence of a covalent bond linking 3’and 5’ ends produced by back-splicing,which has clear advantages as a biomarker.Exosomes are extracellular vesicles which participate in tumor angiogenesis and tumor metastasis in varying degrees.Besides,the multiple roles of exosomes mainly depend on their donor cells and their components,including various RNAs(including circ RNAs)and proteins.This study takes the advantage of the characteristics of circ RNA to explore its diagnostic value in HCC,hoping to provide new biomarker for early diagnosis of HCC.Methods1.The high throughput sequencing technology was used to screen out differential circ RNAs in HCC plasma exosome.We selected circular RNA 0006037(hsa＿circ＿0006037)in this study.2.The 100 paired cancerous tissues and paracancerous tissues of HCC and the tissue of 37 patients with chronic hepatitis(CH)were collected.The plasma of 58 patients with HCC and 58 patients with CH were collected.3.RNA extraction,reverse transcription and qRT-PCR were used to determine the expression level of hsa＿circ＿0006037 in HCC tissues、 adjacent tissues and CH tissues.The correlation between hsa＿circ＿0006037 and the clinicopathological factors of HCC was analyzed.4.The exosomes in plasma were extracted by ultrahigh speed centrifugation techniques.The morphology and particle size were identified by transmission electron microscope(TEM)and nanoparticle tracking analysis(NTA).We extracted of total RNA from plasma exosomes,determined hsa＿circ＿0006037 expression level,analyzed correlation between hsa＿circ＿0006037 expression level and clinicopathological factors,and constructed receiver operating characteristic curve(ROC)to analysis the diagnostic value.Results1.In this study,exosomes were successfully isolated by ultrahigh speed centrifugation techniques.By TEM and NTA identification,we found that the exosomes were cup-shaped and vesicle-like structures with a particle size of about 115 nm.2.Hsa＿circ＿0006037 was down-regulated in HCC tissues compared with adjacent tissues(P<0.001),and its expression level was related to alkaline phosphatase(P= 0.033).3.Hsa＿circ＿0006037 was up-regulated in plasma exosomes(P<0.001),and its expression level was related to the international normalized ratio(P=0.038)、 gamma-glutamyl transferase(P=0.004)、 ascites(P=0.007)、 Barcelona Clinic Liver Cancer stages(P=0.011)and tumor node metastasis stages(P=0.003).Further analysis of ROC curve revealed that hsa＿circ＿0006037 had an AUC of 0.711,a sensitivity of 79.3%,and a specificity of 56.9%when differentiating HCC from CH.It was verified that hsa＿circ＿0006037 can be used as a molecular marker for HCC.ConclusionsOur study found that hsa＿circ＿0006037 expression was reduced in tissues,while exosomederived hsa＿circ＿0006037 was significantly increased in the plasma of HCC patients,suggesting that it can be used as a new biomarker for the diagnosis of HCC.