Identification and Validation of Long Noncoding RNA Biomarkers in Human Non–Small- Cell Lung Carcinomas Hui Yu, MD, Qinghua Xu, PhD, Fang Liu, PhD, Xun.

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Identification and Validation of Long Noncoding RNA Biomarkers in Human Non–Small- Cell Lung Carcinomas Hui Yu, MD, Qinghua Xu, PhD, Fang Liu, PhD, Xun Ye, PhD, Jialei Wang, MD, Xia Meng, PhD Journal of Thoracic Oncology Volume 10, Issue 4, Pages 645-654 (April 2015) DOI: 10.1097/JTO.0000000000000470 Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 Schematic overview of the work flow. lncRNA expression profiles were retrieved from Affymetrix HG-U133Plus2 microarray through a bioinformatics approach. lncRNA expression analyses were performed in the training set (GSE19188) first and then validated in the test set (GSE18842). lncRNAs, long noncoding RNAs. Journal of Thoracic Oncology 2015 10, 645-654DOI: (10.1097/JTO.0000000000000470) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 PAM analysis identified lncRNA signatures to distinguish normal lung tissues from NSCLC tumors. Upper graph The minimum overall misclassification error was observed at a threshold value of 4.31 with 64 lncRNAs. Lower graph The profile for misclassification error variation for normal lung tissues (indicated as “0”) and NSCLC tumors (indicated as “1”) as a function of threshold value and the number of genes involved. PAM, prediction analysis of microarrays; lncRNAs, long noncoding RNAs; NSCLC, non–small-cell lung cancer. Journal of Thoracic Oncology 2015 10, 645-654DOI: (10.1097/JTO.0000000000000470) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 Venn diagram of differentially expressed lncRNAs between subtypes of NSCLC. NSCLC, non–small-cell lung cancer; SCLC, small-cell lung cancer; SCC, squamous cell carcinoma; ADC, adenocarcinoma; lncRNAs, long noncoding RNAs. Journal of Thoracic Oncology 2015 10, 645-654DOI: (10.1097/JTO.0000000000000470) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 4 Clinical validation of six candidate lncRNAs by qRT-PCR analysis. The expression profiles of six candidate lncRNAs were further evaluated by qRT-PCR in an independent sample of 57 NSCLC tumors and 16 normal lung tissues. Points Fold changes of target lncRNA's expression intensity in tumors compared with normal tissue. Horizontal bars The median value of fold changes in each sample set. The qRT-PCR analyses confirmed that the expression level of ADAMTS9-AS2, C1401f132, and LINC00312 in NSCLC tumors were indeed significantly down-regulated when compared with those in normal lung tissues (p = 0.004, p = 0.003, and p = 0.05, respectively), whereas LINC00673 was significantly up-regulated in NSCLC tumors compared with normal lung tissues (p = 0.05). Furthermore, the levels of LINC00261 and TP73-AS1 were significantly differently expressed in subgroups of NSCLC samples (p = 0.004 and p = 0.03, respectively). lncRNAs, long noncoding RNAs; NSCLC, non–small-cell lung cancer; qRT-PCR, quantitative real-time polymerase chain reaction. Journal of Thoracic Oncology 2015 10, 645-654DOI: (10.1097/JTO.0000000000000470) Copyright © 2015 International Association for the Study of Lung Cancer Terms and Conditions