Circulating LncRNAs Serve As Novel Potential Biomarkers For The Diagnosis And Prognosis Of Esophageal Squamous Cell Carcinoma

BackgroudEsophageal squamous cell carcinoma(ESCC)is one of the most common malignancies worldwide,with about 477,900 new diagnosed cases and 375,000 deaths occurred in China.Early detection and timely intervention of ESCC could reduce the incidence and improve the patient outcome.In clinical practice,barium esophagography is the most widely used tool in the early detection of ESCC.However,this test is not prone to detect minimal lesion due to low sensitivity.Endoscopic or surgical biopsy is the gold-standard technique for diagnosis of ESCC,but they are invasive and uncomfortable for patients,which rendered them impractical for mass cancer screening.Currently,squamous cell carcinoma antigen(SCCA)and CYFRA 21-1 are the classic tumor markers commonly used in the management of ESCC.Owing to low diagnostic sensitivity and specificity,they have limited utility for the early detection of ESCC.Thus,novel and reliable molecular biomarkers to complement and improve on current ESCC screening strategies are urgently needed.Long noncoding RNA(lncRNA)are also closely associated with tumor initiation progression invasion and immigration.Meanwhile,lncRNAs may enter body circulating system in the form of microvesicle or exosome,or in combination with RNA binding protein.Interestingly,the circulating IncRNAs are widely existed in serum and other body fluids.Therefore,this provides a new idea for searching circulating serum or exosomal IncRNA as noninvasive diagnosis and predicting prognosis of tumor.In this study,lncRNAs which were previously reported to be dysregulated in ESCC were selected as candidate IncRNAs.The expression level of candidate lncRNAs were evaluated in ESCC and matched adjacent normal tissues by quantitative real-time PCR(qRT-PCR).After data analysis,differentially expressed lncRNAs were selected and further measured in circulating exosomes or serum.A diagnostic model for ESCC using logistic regression analysis was constructed and evaluated.At the same time,the analysis of over survival was estimated by Kaplan-Meier method and comparison was made using the long-rank test.The Cox proportional hazards regression model was used to identify the independent prognostic factor.Finally,the most valuable lncRNA UCA1 was selected in serum to detect its expression and evaluate its diagnostic efficacy in ESCC.This discovery of the novel IncRNA biomarkers in circulating could open up new avenues for investigating the ESCC diagnosis and prognosis.Part One.Evaluation of serum exosomal lncRNAs as potential diagnostic and prognostic biomarkers for esophageal squamous cell carcinomaPurposesEsophageal squamous cell carcinoma is one of the most aggressive malignancies worldwide and effective diagnosis are urgently needed.Accumulating evidence indicates that long non-coding RNAs(lncRNAs)in exosomes have been recognised as promising stable biomarkers in cancers.The aim of this study was to identify serum-derived exosomes lncRNA signatures for diagnosis and prognosis of esophageal squamous cell carcinoma(ESCC).Methods1.Exosomes from serum of ESCC and healthy subjects were isolated and identified.2.Twenty-four ESCC-related lncRNAs were chosen and detected in thirty-four ESCC vs.adjacent tissues using quantitative real-time PCR(qRT-PCR).3.Exosomal lncRNA profiles from serum of 202 ESCC and 202 controls was performed and established new models for ESCC detection in training set.4.The diagnostic accuracy of candidate lncRNAs was further validated with an additional 222 individuals using receiver operating characteristic curve.The Cox proportional hazards regression model was used to identify the independent prognostic factor.Results1.There were twenty differentially expressed lncRNAs in ESCC compared to healthy volunteers.2.In the training set,four lncRNAs(UCA1,POU3F3,ESCCAL-1 and PEG10)were dramaticly upregulated in serum Exosomes of ESCC patients.The AUCs for UCA1,POU3F3,ESCCAL-1 and PEG10 were 0.733(95%confidence interval[CI]=0.687-0.776),0.717(95%CI=0.670-0.760),0.676(95%CI=0.628-0.720),0.648(95%CI=0.599-0.694),respectively.A four-lncRNA signature(UCA1,POU3F3,ESCCAL-1 and PEG10)for diagnosis of ESCC was finally developed by logistic regression model.The diagnostic accuracy of four-lncRNA panel was evaluated with AUC value of 0.844(95%CI=0.805-0.878)for training stage.3.In the validation phase,the diagnostic accuracy of four-lncRNA panel was 0.853(95%CI=0.799-0.897)with the sensitivity of 80.20%and specificity of 80.20%.The corresponding AUCs for patients with TNM stage Ⅰ-Ⅱ and Ⅲ were 0.820 and 0.935,significantly higher than squamous cell carcinoma antigen(SCCA)(P<0.001),which were 0.652 and 0.642,respectively.4.In addition,Kaplan-Meier analysis indicated that patients with high levels of UCA1 and POU3F3 had significantly lower survival rate(P<0.001).In Cox regression analysis,IncRNA POU3F3 was independently associated with ESCC prognosis(P=0.004).Conclusions1.A four-lncRNA panel(UCA1,POU3F3,ESCCAL-1 and PEG10)in serum exosome was identified as a novel and reliable biomarker for ESCC diagnosis.2.Exosomal lncRNA POU3F3 may serve as a independent prognostic factor of ESCC patients.Part two.Expression and clinical significance of serum lncRNA UCAl in esophageal squamous cell carcinomaObjectiveEsophageal squamous cell carcinoma(ESCC)is one of the most aggressive malignancies worldwide.Early diagnosis and effective treatment are urgently needed.Accumulating evidence indicates that circulating long noncoding RNAs(lncRNAs)have been recognised as promising stable biomarkers in cancers.The aim of this study was to explore the expression level of lncRNA UCAl in serum of ESCC investigate the potiential clinical significance.MethodsA total of 96 ESCC cases and another 96 age and gender matched healthy controls were involved.The serum samples were collectedto determine the expression of UCAl with qRT-PCR.The UCAl expression of 12 patients with ESCC before and after operation was compared.The correlation between UCAl expression and clinical pathological parameters of ESCC were analyzed,and the diagnostic value of serum lncRNA UCA1 was evaluated with the receiver operating characteristic curve(ROC).ResultsCompared with controls,UCAI expression levels were significantly upregulated in ESCC serum and decreased significantly after operation(P<0.001).In addition,the area under the curve(AUC)was 0.816,providing a sensitivity of 77.1%and a specificity of 76.0%.Moreover,lncRNA UCA1 was statistically superior to squamous cell carcinoma antigen(AUC=0.679)in discriminating ESCC patients from healthy controls(P<0.001).ConclusionSerum lncRNA UCA1 may serve as a potential tumor biomarker for clinical diagnosis and postoperative monitoring of ESCC patients.