Serum IncRNAs Profiles Serve As Novel Biomarkers For Early Diagnosis Of HBV-positive Hepatocarcinoma

BackgroundHepatocellular carcinoma (HCC) is the fifth most common malignant tumor aroundthe world with rising incidence and highest fatality rate in recent years. In china, HBVcontinues to be the leading contributor to high rates of chronic liver disease and cirrhosis.Chronic HBV infection generated about10-25%probability for the development of HCCaccording to the study.So there is a possibility for early diagnosis of HCC from HBVinfection to eventually HCC. Now serum AFP detection and ultrasound were used toscreened high-risk population, however, as a traditional serum marker for HCC, AFP islimited by its low sensitivity(36-64%) and specificity(79-91%), its levels often increase inthe absence of HCC (chronic hepatitis or cirrhosis) as well. Ultrasonography is operatordependent and limited in its ability to identification of less than3cm lump, which madethe early detection and treatment for HCC difficult with poor prognosis, low5-yearsurvival rate and high fatality rate. Therefore, there is an urgent need to identify anddevelop new biomarkers with high accuracy and feasibility for the early diagnosis of HCC.Highly stable cell-free circulating nucleic acids (cfCNA), both RNA and DNA species,have been discovered in the blood, plasma, and urine of humans. At present, there isevidence of a good correlation between tumor-associated changes in genomic, epigenetic,or transcriptional patterns and alterations in cfCNA levels, the ability to detect andquantitate specific DNA and RNA sequences has opened up the possibility of diagnosisand monitoring of diseases, especially in the field of cancer, such as MicroRNAsPurposeTo investigate whether the the serum tumor derived lncRNAs can be used as markersfor early diagnosis of HCC.Methods1. lncRNAs analysis of HBV-related HCC tumor tissues and paired no tumor tissuesand determination of target lncRNAs.Five HBV-related HCC tumors tissues and paired no tumor tissues were used todetected different expression lncRNAs with the12135K lncRNA Expression Microarray(p value＜0.05and fold expression change>2). Then, The68pairs of HBV-related HCCtumors tissues and corresponding no tumor liver tissues were used to validate microarrayanalysis findings by detecting5lncRNAs randomly. Among the differently expressedlncRNAs, up-regulated lncRNAs (p value＜0.05and fold expression change>5) weretested in the serum of HCC with HBV and control groups (healthy volunteers and HBVpatients).The lncRNAs existed in the serum and hold potentiality to be dignosis markerswere selected as our canditate target gens.2. The expression of target genes in retrospective training cohortThe target lncRNAs discovered were first tested with qRT-PCR in anindependent retrospective training cohort of plasma samples from379participants(N=138,HBV=104, HCC=137). A stepwise logistic regression model to estimate the risk of beingdiagnosed withHCCwas applied on the training data set (379plasma samples).3. The expression of target genes in prospective validation cohortThe parameters of the logistic model from the training phase were applied to anindependent cohort of183samples for validating the diagnostic performance of theselected lncRNA panel. In this study phase, blood samples were also obtained from threecategories of participants including healthy individuals and patients with CHB, and HCC.Results1. Gene expression profiling revealed many genes that were differentially expressedbetween the HBV-related HCC tumors tissues and paired no tumor tissues. To validatemicroarray analysis findings, we randomly selected five lncRNAs among the differentiallncRNAs and analyzed their expression, using quantitative real-time polymerase chainreaction (qRT-PCR) in68pairs of HCC and corresponding nontumor liver tissues, ourresult was similar to the microarray analysis findings. Among the up-regulated lncRNAs,we found there were23lncRNAs hold a fold expression change>5, by analyzing theirexpression, using quantitative real-time polymerase chain reaction (qRT-PCR) in379samples(N=138, HBV=104, HCC=137). We found5lncRNAs existed in serum sample.Further analysis indicated that lncRNA-uc003wbd, lncRNA-AF085935hold asignificance could be used as early diagnosis markers for HCC.2. High expression levels of lncRNA-uc003wbd，lncRNA-AF085935were observedin patients with HCC compared with those in the control group. The diagnostic accuracyof these two lncRNAs, measured by AUC, was0.8597,95%CI,0.8221~0.8974and0.8702, 95%CI,0.8309~0.9095,respectively). A stepwise logistic regression model to estimate therisk of being diagnosed withHCCwas applied on the training data set (407plasmasamples). All of the two lncRNAs turned out to be significant predictors. The predictedprobability of being diagnosed with HCC from the logit model based on the two lncRNApanel, Logit(P=HCC)=-6.859+0.719*uc003wbd+1.804*AF085935was used toconstruct theROC curve. The diagnostic performance for the established lncRNA panelwas evaluated by using ROC analysis. The AUC for the lncRNA panel was0.9432(95%CI,0.9195~0.9668; sensitivity,69.63,specificity,95.88).3. The parameters estimated from the training data set were used to predict theprobability of being diagnosed withHCCfor the independent validation data set (183plasma samples). Similarly, the predicted probability was used to construct the ROC curve.The AUC of the lncRNA panel was0.9496(95%CI,0.9201~0.9791; sensitivity,65.57,specificity,99.17).Conclusions1. Different expression of lncRNAs were detected between HBV-related HCC tumorstissues and paired no tumor tissues2. Tumor derived lncRNAs can exist in the serum.3. The dignostic panel based on the two lncRNAs can be used to discriminate the HCCpatients from the healthy people or HBV patients.Potential applications and noveltyThis study provides a new mean for early dignosi of HBV related HCC, of theregulatory actions of lncRNAs. Additional, our result indicated that lncRNAs can exist inserum.