WT1 Promotes Invasion of NSCLC via Suppression of CDH1

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WT1 Promotes Invasion of NSCLC via Suppression of CDH1 Chen Wu, PhD, Weiyou Zhu, MD, PhD, Jing Qian, MS, Shaohua He, MS, Changping Wu, MD, PhD, Yijiang Chen, MD, PhD, Yongqian Shu, MD, PhD Journal of Thoracic Oncology Volume 8, Issue 9, Pages 1163-1169 (September 2013) DOI: 10.1097/JTO.0b013e31829f6a5f Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 1 Expression of WT1 and CDH1 transcripts and correlation between expressions of these two genes in non–small-cell lung cancer patients. A, Significantly higher RNA expression of WT1 in tumor specimens than in adjacent tissue samples (p < 0.0001). B, Significantly decreased RNA expression of CDH1 in tumor specimens in comparison with adjacent tumor-free tissues (p < 0.0001). C, A negative correlation was found between RNA expression of WT1 and CDH1 in tumor samples (R = −0.697; p < 0.0001). Journal of Thoracic Oncology 2013 8, 1163-1169DOI: (10.1097/JTO.0b013e31829f6a5f) Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 2 WT1 promotes migration and invasion of NSCLC cells. The result of Transwell assay showed that the invasive ability of H1568 was significantly enhanced after WT1 overexpression (B versus A) and suppressed after WT1 silencing (C versus A) at a 48-hour time point, which was confirmed by integrated optical density (IOD) value evaluation (D). Similar effect of WT1 was observed in H1650 NSCLC cells (E–H). The result of wound-healing assay showed that migration ability of H1568 (I) and H1650 (J) cells were promoted by WT1 overexpression and restrained by WT1 silencing at 48-hour time point. The cell-proliferation assay test showed no distinct differences on proliferation after manipulation of WT1 in both H1568 (K) or H1650 cells (L) at 12-hour, 24-hour, and 48-hour time points. *p < 0.05. NSCLC, non–small-cell lung cancer; WT1, Wilms’ tumor gene. Journal of Thoracic Oncology 2013 8, 1163-1169DOI: (10.1097/JTO.0b013e31829f6a5f) Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

FIGURE 3 CDH1 was a direct target of WT1. Via PCR array, nine metastasis-related genes varied larger than twofolds (including Cdh1) in H1568 cells after WT1 overexpression and silencing (A); Western blot assay validated the result of PCR array of negative trend of WT1 and E-cadherin in both H1568 and H1650 non–small-cell lung cancer cells (B); A schematic diagram showed the CDH1 promoter containing WT1 binding motif and its relationship with transcriptional start site (C); Luciferase reporter assay demonstrated that at 0-hour, 6-hour, 12-hour, and 24-hour time points, pcDNA3.1-WT1 down-regulated (D) and WT1-siRNA enhanced (E) Cdh1 transcription (versus empty plasmid) both in a time-dependent manner. In control group (wild-type CDH1 promoter), pcDNA-3.1-WT1 could inhibit and WT1-siRNA could enhance CDH1 transcription, but no regulatory function of WT1 in mutant group (mutant CDH1 promoter) were detected in H1568 (F). *p < 0.05. PCR, polymerase chain reaction; WT1, Wilms’ tumor gene. Journal of Thoracic Oncology 2013 8, 1163-1169DOI: (10.1097/JTO.0b013e31829f6a5f) Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions