Identification Of Long Noncoding RNAs As Potential Novel Diagnosis And Prognosis Biomarkers In Colorectal Cancer

Objective:Colorectal cancer（CRC）is prevalent worldwide and improvements in timely and effective diagnosis are imperatively needed.Recent evidences have revealed the new role of lncRNAs in oncogenesis and indicated the possibility of using them for the diagnosis of diverse cancers,including CRC.This study aimed to identify potential long noncoding RNA（lncRNA）biomarkers for CRC diagnosis along with prognosis prediction.Methods:1.Primary CRC tissues along with corresponding adjacent non-tumor（NM）tissues from 6 different CRC patients were collected and then subjected to microarray analysis for an initial screen of lncRNAs biomarkers.Candidate lncRNAs were then selected according to the result of the analysis and previous studies.2.In the training phase,lncRNA candidates were firstly verified by RT-qPCR in 80 pairs of tissue specimens,involving primary CRC tissues and matched NM tissues.Subsequently,the verified lncRNA were further examined in an independent cohort of serum samples obtained from 120 CRC patients and 120 controls.The data from this was used to construct the diagnostic panel,based on the logistic regression model for differential diagnosis between the CRC and the control group.3.In the validation phase,the diagnostic lncRNA panel constructed in the training phase was applied to another independent cohort of serum samples from 240 patients（120 CRC patients and 120 controls）to validate its diagnostic performance,while serum samples from the same cohort were obtained for CEA test.4.In the validation phase,the correlation between lncRNAs and disease-specific survival rate of CRC patients was assessed.Survival curve was estimated with the Kaplan-Meier method and comparison was made using the log-rank test.The Cox proportional hazards regression model was used to identify the independent prognostic factors.5.The correlation of lncRNAs expression between tissue and serum sample was analyzed.The expression stability of the IncRNAs was also assessed after exposing to harsh condition.Results:1.The microarray analysis with 6 pairs of primary colorectal and adjacent normal tissues was conducted with a microarray targeting 33000 IncRNAs.5873 lncRNAs were identified with significant differential expression（fold change≥2.0 and p<0.05）.18 candidate lncRNAs were selected in total including 10 lncRNAs based on the result of the microarray analysis and 8 lncRNAs from previous published articles.2.In the training phase,four IncRNAs（BANCR,NR₀26817,NR-029373,and NR₀34119）were identified to be significantly dysregulated in both tissues and serum samples with consistent pattern（all p<0.01）.BANCR was upregulated in CRC.NR₀26817,NR₀29373,and NR₀34119 were downregulated in CRC.The corresponding AUCs of the 4 lncRNAs（BANCR,NR₀26817,NR₀29373 and NR₀34119）were 0.638,0.708,0.812 and 0.724,respectively.A diagnostic IncRNA panel based on the four lncRNAs for CRC was finally developed by logistic regression model with an area under the receiver operating characteristic curve（AUC）of 0.891（95%CI,0.844-0.927,sensitivity = 81.67%,specif-icity =80.00%）in the training phase.3.In the validation phase,the AUC of the diagnostic lncRNA panel was 0.881（95%CI,0.833 to 0.919,sensitivity = 89.17%,specificity = 75.83%）.Moreover,the AUC of this panel for patients with TNM stage Ⅰ,Ⅱ and Ⅲ were 0.774,0.844 and 0.949,respectively,significantly higher than those of CEA,which were 0.588,0695 and 0.861,respectively（all at p<0.001）.4.Kaplan-Meier analysis showed that patients with low levels of NR₀29373 and NR₀34119 had significantly lower disease-specific survival rate（p=0.013 and 0.044,respectively）.Multivariate Cox analysis demonstrated that NR₀29373 and NR₀34119 were both independently associated with disease-specific survival rate（p=0.013 and 0.038,respectively）.5.A significant correlation was observed for all 4 lncRNAs（BANCR,NR₀26817,NR₀29373 and NR₀34119）between their expression levels in tissue and serum.No obvious alterations in the expression level of these 4 IncRNAs were observed after exposing to harsh conditions.Conclusion:1.Our study established a distinctive 4-lncRNA panel（BANCR,NR₀26817,NR₀29373,NR₀34119）with considerable value in diagnosing CRC.2.Expression level of NR₀29373 and NR₀34119 may function as potential biomarkers for CRC prognosis prediction.