Identification Of CircRNA Expression Profile In PBMcs Of Hepatocellular Carcinoma Patients And Prediction Of Diagnostic Value

ObjectiveA growing number studies have revealed that circular RNAs（circRNAs）are significantly related to the occurrence and development of cancer,participating in the proliferation,invasion,apoptosis,invasion and metastasis of cancer cells.In addition,the stress resistance,stability and disease specificity of circRNAs endow they distinct advantages as a biological label for many diseases,especially in cancer.The morbidity and mortality of hepatocellular carcinoma（HCC）are in the forefront of all kinds of malignant tumors in the world,especially in China.The major reason may be that the early diagnosis rate of HCC is low and there is no specific therapeutic target for HCC for a long time,so patients lack the best time for early treatment.Therefore,it is urgent to develop a non-invasive,sensitive and specific biomarker for HCC diagnosis.In this study,high throughput sequencing（RNA-seq）was used to analyze the expression profiles of circRNAs in PBMCs between HCC patients and normal controls,and to discover the significantly different circRNAs.Quantitative Reverse Transcription Polymerase Chain Reaction（qRT-PCR）was used to validate the significantly different circRNAs and bioinformatics predicted its potential functional mechanism.Finally,the clinical potential diagnostic value of circ₀000798 as a biomarker of HCC was further explored,paving the way for exploring circRNA as a potential target for HCC diagnosis and treatment.MethodsPBMCs were isolated from fresh peripheral blood between 4 primary HCC patients and 3 normal controls.Total RNA was extracted and the expression profile of circRNA in PBMCs was analyzed by RNA-seq.Significant difference expression（fold change,FC）≥ 2 or ≤ 0.05,P < 0.05)circRNA were detected and further analyzed the potential function by GO and KEGG.qRT-PCR was followly used to validate the six significant difference circRNAs in PBMCs from 72 HCC patients and 30 healthy controls.Analyzing the correlation between circ₀000798 and clinicopathological parameters of HCC patients,and fuether evaluating the potential of circ₀000798 as a biomarker of early screening for HCC patients by Receiver Operating Characteristic（ROC）and Area Under Curve（AUC）.Kaplan_Meier was ultimately used to analyze the correlation between circ₀000798 and prognosis of HCC patients.ResultsA total of 5609 circRNAs differentially expressed in PBMCs between HCC patients and normal controls were identified by RNA-seq analysis.Among them,58（21 up-regulated and 37 down-regulated）were significantly different in PBMCs of HCC patients.GO and KEGG analysis showed that these circRNAs were mostly involved in immune and immune-related signaling pathways.Endogenous competitive RNA（ceRNA）network analy indicates that a circRNA can bind to multiple miRNAs,and a single miRNA can bind to multiple circRNAs and to regulate immune and immune-related molecules and signaling pathways.3 up-regulated circRNAs（hsa_circ₀005505,hsa_circ₀001394 and hsa_circRNA₀000798）and 3 down-regulated circRNAs（hsa_circ₀004771,hsa_circ₀001074 and hsa_circRNA₀067735）were further verified by qRT-PCR.The results showed that the expression of these 6 circRNAs in HCC group was consistent with the sequencing results.The expression level of circ₀000798 was significantly correlated with the major clinicopathological parameters of HCC patients.Area Under Curve（AUC）of circ₀000798 in PBMCs between HCC patients and normal controls was 0.703(（95% confidence interval（CI）: 0.604-0.803;P = 0.001）,while the AUC of circ₀000798 in PBMCs of only HCC patients was 0.909（（95% confidence interval（CI）: 0.840-0.978）;P < 0.001）.ConclusionThe study is the first time to systematically identify the circRNA expression profiles in PBMCs between HCC patients and normal controls.Further clinical analysis and bioinformatics prediction reveal that circ₀000798 can be used as a non-invasive biomarker for HCC diagnosis,providing a new idea for early diagnosis and treatment of HCC.