Volume 25, Issue 3, Pages 739-751 (March 2017) The Pseudogene DUXAP8 Promotes Non-small-cell Lung Cancer Cell Proliferation and Invasion by Epigenetically Silencing EGR1 and RHOB  Ming Sun, Feng-qi Nie, Chongshuang Zang, Yunfei Wang, Jiakai Hou, Chenchen Wei, Wei Li, Xiang He, Kai-hua Lu  Molecular Therapy  Volume 25, Issue 3, Pages 739-751 (March 2017) DOI: 10.1016/j.ymthe.2016.12.018 Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 1 Pseudogenes Profiling in NSCLC Tissues (A) Data mining of altered pseudogenes expression in the NSCLC microarray gene profiling results (GSE19188, GSE30219, GSE18842, and GSE31210). The results of analysis are presented as heatmaps. (B) Venn diagrams showing upregulated and downregulated pseudogenes whose dys-regulated expression pattern was shared by four microarray datasets. (C) Hierarchically clustered heatmaps of six consistently upregulated (DUXAP8, CTC-820M8.1, CDC20P1, AC006465.5, DLGAP5P1, and RP11-156J23.1) and downregulated (CSE1P1, CSE1P2, CXCR2P1, MT1JP, TNXA, and RP11-379K17.5) pseudogenes in all four NSCLC microarray genes profiling. The result is presented as a heatmap. (D) Data mining of DUXAP8, CTC-820M8.1, CDC20P1, AC006465.5, DLGAP5P1, RP11-156J23.1 expression levels in TCGA NSCLC tissues compared with non-tumor tissues. Molecular Therapy 2017 25, 739-751DOI: (10.1016/j.ymthe.2016.12.018) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 2 Pseudogene DUXAP8 Is Significantly Upregulated in NSCLC Tissues and Cell Lines (A) qRT-PCR analysis of DUXAP8 expression in 78 pairs of NSCLC tissues and corresponding non-tumor lung tissues. The DUXAP8 RNA levels were normalized to GAPDH expression. (B) Kaplan-Meier overall survival and disease-free survival analysis of the association between DUXAP8 expression level and NSCLC patient survival. (C) qRT-PCR analysis of DUXAP8 expression in HBE, 16HBE, and eight NSCLC cell lines. The DUXAP8 RNA levels were normalized to GAPDH expression. Values represent the mean ± SE from three independent experiments. (D and E) qRT-PCR analysis of DUXAP8 expression in H1299, H1975 cells transfected with DUXAP8 or NC siRNAs, and in SPCA1 and PC9 cells transfected with DUXAP8 overexpression vector. Values represent the mean ± SE from three independent experiments. *p < 0.05; **p < 0.01. Molecular Therapy 2017 25, 739-751DOI: (10.1016/j.ymthe.2016.12.018) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 3 Effects of DUXAP8 on NSCLC Cell Proliferation, Cell Cycle Progression, and Tumorigenesis (A) Growth curves of H1299 and H1975 cells after transfection with DUXAP8 siRNAs or NC were determined by MTT assays. Values represented the mean ± SE from three independent experiments. (B) Growth curves of SPCA1 and PC9 cells after transfection with DUXAP8 vector or empty vector were determined by MTT assays. (C) Cell proliferation of H1299 and H1975 cells was evaluated after transfection with DUXAP8 siRNAs or NC using EdU incorporation assays. Red represents EdU staining for proliferating cell; blue represents DAPI staining for cell nuclear. (D) The cell cycle progression of H1299 and H1975 cells was evaluated 48 hr after transfection with DUXAP8 siRNAs or NC using flow cytometry assays. The bar chart represented the percentage of cells in G0/G1, S, or G2/M phase, as indicated. (E) The cyclinD1, cyclinD3, CDK2, CDK4, and CDK6 protein levels were detected in H1299 and H1975 cells after transfection with DUXAP8 siRNAs or NC using western blot. Values represent the mean ± SE from three independent experiments. (F) The stable DUXPA8 knockdown H1299 cells were used for the in vivo study. The nude mice carrying tumors from respective groups were shown and tumor growth curves were measured after the injection of H1299 cells. Tumor volume was calculated every 3 days. Values represent the mean ± SE from three independent experiments. (G) Tumor weights are represented. Values represent the mean ± SE from three independent experiments. (H) Ki67 protein levels in tumor tissues formed from sh-DUXAP8 or empty vector-transfected H1299 cells were determined by immunohistochemistry (IHC) staining. Upper: H&E staining. Lower: immunostaining.*p < 0.05; **p < 0.01. Molecular Therapy 2017 25, 739-751DOI: (10.1016/j.ymthe.2016.12.018) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 4 Knockdown of DUXAP8 Induces Cell Apoptosis and Inhibits Cell Migration and Invasion in NSCLC (A and B) The effect of DUXAP8 knockdown on H1299 and H1975 cell apoptosis was determined by measuring the percentage of Annexin V-stained cells using flow cytometry and TUNEL staining assays. Values represent the mean ± SE from three independent experiments. Values represent the mean ± SE from three independent experiments. (C and D) The effect of DUXAP8 downregulation on the migration and invasion of H1299 and H1975 cells was assessed using Transwell assays. Values represent the mean ± SE from three independent experiments. Scale bars, 100 μm. *p < 0.05; **p < 0.01. Molecular Therapy 2017 25, 739-751DOI: (10.1016/j.ymthe.2016.12.018) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 5 EGR1 and RHOB Are Downstream Targets of DUXAP8 (A) Hierarchically clustered heatmap of upregulated and downregulated mRNAs in H1299 cells after transfection with DUXAP8 or NC siRNAs. (B) GO and pathways analysis of these altered mRNAs in H1299 cells after transfection with DUXAP8 siRNA or NC. (C) qRT-PCR analysis of EGR1, RHOB, KLF2, HOXA5, ROCK2, CCND2, RARB, HMGB2, NKD2, and ADAMTS1 expression in H1299 and H1975 cells after transfection with DUXAP8 or NC siRNA. Values represent the mean ± SE from three independent experiments. (D and E) The KLF2, EGR1, and RHOB protein levels were detected in H1299 and H1975 after transfection with DUXAP8 siRNAs or NC using western blot. Values represent the mean ± SE from three independent experiments. *p < 0.05; **p < 0.01. Molecular Therapy 2017 25, 739-751DOI: (10.1016/j.ymthe.2016.12.018) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 6 DUXAP8 Represses EGR1 and RHOB Transcription via Interacting with EZH2 and LSD1 (A) qRT-PCR detection of the percentage of DUXAP8, GAPDH, and U1 in the cytoplasm and nuclear fractions of H1299 and H1975 cells. GAPDH and U1 were used as a cytoplasm and nuclear localization marker, respectively. Values represent the mean ± SE from three independent experiments. (B) DUXAP8 RNA levels in immunoprecipitates were determined by qRT-PCR. Expression levels of DUXAP8 RNA were presented as fold enrichment relative to IgG immunoprecipitates. Values represent the mean ± SE from three independent experiments. (C) The HuR, EZH2, and LSD1 protein levels in immunoprecipitates with DUXAP8 RNA were determined by western blot. AR RNA was used as positive control for HuR protein. Expression levels of HuR, EZH2, and LSD1 protein were presented. (D) qRT-PCR analysis of EGR1, RHOB, and EZH2 expression in H1299 and H1975 cells after transfection with EZH2 or NC siRNA. Values represent the mean ± SE from three independent experiments. (E) qRT-PCR analysis of EGR1, RHOB, and LSD1 expression in H1299 and H1975 cells after transfection with LSD1 or NC siRNA. Values represent the mean ± SE from three independent experiments. (F) ChIP-qPCR of EZH2 and H3K27me3 occupancy in the EGR1 promoter in H1299 cells, and IgG as a negative control. The mean values and SE were calculated from triplicates of a representative experiment. The mean values and SE were calculated from triplicates of a representative experiment. (G) ChIP-qPCR of LSD1and H3K4me2 occupancy in the RHOB promoter in H1299 and H1975 cells, and IgG as a negative control. The mean values and SE were calculated from triplicates of a representative experiment. The mean values and SE were calculated from triplicates of a representative experiment. *p < 0.05; **p < 0.01. Molecular Therapy 2017 25, 739-751DOI: (10.1016/j.ymthe.2016.12.018) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 7 DUXAP8 Exerts Oncogenic Function Partly Dependent on Silencing of EGR1 and RHOB (A) Western blot analysis of EGR1 and RHOB protein levels in H1299 and H1975 cells after transfection with EGR1 or RHOB vector. (B) Growth curves of H1299 and H1975 cells after transfection with EGR1, RHOB, or empty vector were determined by MTT assays. Values represented the mean ± SE from three independent experiments. (C) Cell proliferation of H1299 and H1975 cells was evaluated 48 hr after transfection with EGR1, RHOB, or empty vector using EdU incorporation assays. Red represents EdU staining for proliferating cell; blue represents DAPI staining for cell nuclear. Values represent the mean ± SE from three independent experiments. (D) The effect of EGR1 and RHOB upregulation on H1299 and H1975 cells’ invasive ability was assessed using Transwell assays. The mean values and SE were calculated from triplicates. (E) Growth curves of H1299 and H1975 cells after co-transfection with DUXAP8 and EGR1 or RHOB siRNAs were determined by MTT assays. Values represented the mean ± SE from three independent experiments. (F and G) The invasive ability of H1299 and H1975 cells after co-transfection with DUXAP8 and EGR1 or RHOB siRNAs was determined by transwell assays. The mean values and SE were calculated from triplicates. *p < 0.05; **p < 0.01. Molecular Therapy 2017 25, 739-751DOI: (10.1016/j.ymthe.2016.12.018) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions