Characterization of microRNA transcriptome in tumor, adjacent, and normal tissues of lung squamous cell carcinoma Jun Wang, MD, PhD, Zhi Li, MD, PhD,

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Characterization of microRNA transcriptome in tumor, adjacent, and normal tissues of lung squamous cell carcinoma Jun Wang, MD, PhD, Zhi Li, MD, PhD, Qinyu Ge, PhD, Weibing Wu, MD, Quan Zhu, MD, Jinhua Luo, MD, Liang Chen, MD The Journal of Thoracic and Cardiovascular Surgery Volume 149, Issue 5, Pages 1404-1414.e4 (May 2015) DOI: 10.1016/j.jtcvs.2015.02.012 Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 Flow chart of library preparation and data analysis of the lung cancer tissue miRNA profiling by deep sequencing. GO, Gene Ontology; miRNA, microRNA; RT & PCR, reverse transcription polymerase chain reaction; KEGG, Kyoto Encyclopedia of Genes and Genomes. The Journal of Thoracic and Cardiovascular Surgery 2015 149, 1404-1414.e4DOI: (10.1016/j.jtcvs.2015.02.012) Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 Significantly differentially expressed miRNAs in tumor, adjacent, and normal tissues. A, Upregulated and downregulated miRNAs in tumor versus normal tissues as defined by deep sequencing. B, Upregulated and downregulated miRNAs in tumor versus adjacent tissues as defined by deep sequencing. C, Upregulated and downregulated miRNAs in adjacent versus normal tissues as defined by deep sequencing. The Journal of Thoracic and Cardiovascular Surgery 2015 149, 1404-1414.e4DOI: (10.1016/j.jtcvs.2015.02.012) Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 miRNA expression level shows a gradual significant change from normal tissue to tumor tissue. A, Some of them are upregulated successively, and others are downregulated successively. B, qPCR validation of the 2 related miRNAs shows gradual changes from normal tissue to tumor tissue. The expression level of miR-218-5p shows decreasing changes, and miR-425-5p shows increasing changes. The Journal of Thoracic and Cardiovascular Surgery 2015 149, 1404-1414.e4DOI: (10.1016/j.jtcvs.2015.02.012) Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 In situ hybridization assays for miR-425-5p and miR-218-5p were performed on tumor, adjacent, and normal lung tissues from patient with SCC. Representative results are shown. Gradually upregulated miR-425-5p (A-C) and gradually downregulated miR-218 (D-F) were detected in tumor, adjacent, and normal lung tissues. The Journal of Thoracic and Cardiovascular Surgery 2015 149, 1404-1414.e4DOI: (10.1016/j.jtcvs.2015.02.012) Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 Associations between miRNA expression and different stages or tumor sizes of SCC. A, Expression levels of 4 miRNAs (miR-425-5p, 183-5p, 337-5p, and 218-5p) were significantly different and successively dysregulated from stage I to III of SCC as shown by qPCR. miR-218-5p showed downregulation, and other miRNAs showed upregulation. B, Expression levels of miR-183-5p and miR-425-5p increased with tumor size as shown by qPCR. The Journal of Thoracic and Cardiovascular Surgery 2015 149, 1404-1414.e4DOI: (10.1016/j.jtcvs.2015.02.012) Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions

Figure 6 Categories and distribution of the Gene Ontology in terms of predicted miRNA targets. A, Biological process terms are enriched in cellular, metabolic, and single-organism processes. B, Cellular component terms are enriched in cell part, organelle, and membrane. C, Molecular function terms are enriched in binding, catalytic activity, and molecular transducer activity. The Journal of Thoracic and Cardiovascular Surgery 2015 149, 1404-1414.e4DOI: (10.1016/j.jtcvs.2015.02.012) Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions

Flow chart of library preparation and data analysis of the lung cancer tissue miRNA profiling by deep sequencing. The Journal of Thoracic and Cardiovascular Surgery 2015 149, 1404-1414.e4DOI: (10.1016/j.jtcvs.2015.02.012) Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions

Figure E1 Length distribution and abundance of the small RNAs from ST, SA, and SN samples. Small RNA reads with a length of 22 nt were the most abundant, which accounted for approximately half of the total reads, followed by 21 and 21 nt reads. The Journal of Thoracic and Cardiovascular Surgery 2015 149, 1404-1414.e4DOI: (10.1016/j.jtcvs.2015.02.012) Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions

Figure E2 Hierarchical clustering analysis of miRNA expression in different tissues of lung SCC. Differential expression of miRNA in tumor and adjacent tissue compared with the normal tissue control. The data removed those low copy number miRNAs (<10). The column shows each group to the control, and the line shows the fold change of each miRNA in lung cancer tissue. The color of each pattern represents the fold change as log2, from high (red) to low (green). The Journal of Thoracic and Cardiovascular Surgery 2015 149, 1404-1414.e4DOI: (10.1016/j.jtcvs.2015.02.012) Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions