The Pseudogene DUXAP8 Promotes Non-small-cell Lung ... - Cell Press

Original Article

The Pseudogene DUXAP8 Promotes Non-small-cell Lung Cancer Cell Proliferation and Invasion by Epigenetically Silencing EGR1 and RHOB Ming Sun,1,2,4 Feng-qi Nie,3,4 Chongshuang Zang,1,4 Yunfei Wang,2 Jiakai Hou,2 Chenchen Wei,3 Wei Li,1 Xiang He,1 and Kai-hua Lu1 1Department

of Oncology, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China; 2Department of Bioinformatics and Computational Biology,

UT MD Anderson Cancer Center, Houston, TX 77030, USA; 3Department of Oncology, Second Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China

Recently, the non-protein-coding functional elements in the human genome have been identified as key regulators in postgenomic biology, and a large number of pseudogenes as well as long non-coding RNAs (lncRNAs) have been found to be transcribed in multiple human cancers. However, only a small proportion of these pseudogenes has been functionally characterized. In this study, we screened for pseudogenes associated with human non-small-cell lung cancer (NSCLC) by comparative analysis of several independent datasets from the GEO. We identified a transcribed pseudogene named DUXAP8 that is upregulated in tumor tissues. Patients with higher DUXAP8 expression exhibited shorter survival, suggesting DUXAP8 as a new candidate prognostic marker for NSCLC patients. Knockdown of DUXAP8 impairs cell growth, migration, and invasion, and induces apoptosis both in vitro and in vivo. Mechanistically, DUXAP8 represses the tumor suppressors EGR1 and RHOB by recruiting histone demethylase LSD1 and histone methyltransferase EZH2, thereby promoting cell proliferation, migration, and invasion. These findings indicate that the pseudogene DUXAP8 may act as an oncogene in NSCLC by silencing EGR1 and RHOB transcription by binding with EZH2 and LSD1, which may offer a novel therapeutic target for this disease.

INTRODUCTION Lung cancer is one of the most common cancers worldwide, and non-small-cell lung cancer (NSCLC) accounts for 80% of all new lung cancer cases, including squamous cell carcinoma (SCC), adenocarcinoma, and large-cell carcinoma (LCC).1 Despite advances in chemotherapy and molecular targeting therapy, the 5-year survival rate for NSCLC patients remains less than 15%, which is largely due to most patients being at advanced stages when diagnosed and a lack of effective therapy.2–4 A lack of biomarkers for early diagnosis and prognostic markers is still one of the main challenges in NSCLC.5 Therefore, the identification of new functional genes and characterization of the molecular mechanisms behind their involvement in NSCLC are essential for the development of specific diagnostic methods and the design of more individualized and effective therapeutic strategies for NSCLC patients.

Pseudogenes are dysfunctional copies of protein-coding genes, which arise from the accumulation of natural mutations that lead to decay and degeneration of the transcript from a protein-coding standpoint.6,7 According to the latest annotation of the ENCODE project, there are 14,500 pseudogenes in the human genome, a large proportion of which is actively transcribed.8,9 Although pseudogenes were considered as non-functional genomics fossils or biologically inconsequential for a long time, recent studies have revealed that they actually play critical roles in post-transcriptional or transcriptional regulation of gene expression by functioning as endogenous competitors for microRNA (miRNA) or RNA-binding protein (RBP), and endogenous small interfering RNA (endo-siRNA or esiRNA).10–15 Interestingly, increasing evidence has indicated that some pseudogenes play critical roles in human diseases, especially cancer.16–18 For example, Poliseno et al.19,20 reported that the pseudogene PTENP1 functions in regulating PTEN by acting as a decoy for PTEN-related miRNAs such as miR-17, miR-21, miR-214, miR-19, and miR-26. Moreover, the OCT4 pseudogene OCT4-pg4 functions as a competing endogenous RNA (ceRNA) and protects the OCT4 transcript from being inhibited by miR-145, thereby promoting the growth and tumorigenicity of hepatocellular carcinoma cells.21 In addition, the rapid development of bioinformatics has led to the identification of thousands of novel transcribed pseudogenes in human cancers based on the next-generation sequencing data of cancer samples.22 For example, Han et al.23 developed a computational pipeline and detected 10,000 pseudogenes in seven cancer types using The Cancer Genome Atlas (TCGA) RNA sequencing (RNA-seq) data. Many of these pseudogenes are cancer specific, which suggests that pseudogenes may be subtype and prognostic biomarkers in cancers.23 Moreover, Welch and colleagues24 identified 440 pseudogenes that are actively transcribed in breast cancer,

Received 8 July 2016; accepted 12 December 2016; http://dx.doi.org/10.1016/j.ymthe.2016.12.018. 4

These authors contributed equally to this work.

Correspondence: Kai-hua Lu, Department of Oncology, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China. E-mail: [email protected]

Molecular Therapy Vol. 25 No 3 March 2017 ª 2016 The American Society of Gene and Cell Therapy. 739

Molecular Therapy

309 of which exhibit significant differential expression patterns among the breast cancer subtypes. However, the pseudogenes’ expression patterns and potential roles in NSCLC development and progression remain unclear. Against this background, in this study, we performed an integrative analysis of four microarray datasets from the GEO aimed at identifying novel pseudogenes for NSCLC. Then, we validated the analysis results in the TCGA datasets and focused on a pseudogene named DUXAP8, which is consistently upregulated in all datasets and associated with shorter progression-free survival (PFS). Furthermore, DUXAP8 expression was detected in a cohort of 78 paired NSCLC tissues and non-tumor tissues, and loss- and gain-of-function assays were used to investigate the biological function of DUXAP8 in NSCLC cells. Finally, a mechanistic investigation was performed to determine how DUXAP8 regulates its underlying targets.

RESULTS Comprehensive Analysis of the Pseudogene Profile in NSCLC

To characterize the profile of aberrantly expressed pseudogenes in NSCLC, we first performed comprehensive transcriptional analysis by using four independent microarray datasets (GSE19188,25 GSE30219,26 GSE18842,27 and GSE3121028) from GEO. This analysis showed that 96 pseudogenes were misregulated in GSE19188, whereas 40 were in GSE30219, 76 in GSE18842, and 54 in GSE31210 (Figure 1A; Table S2). These included six pseudogenes (CSE1P1, CSE1P2, CXCR2P1, MT1JP, TNXA, and RP11-379K17.5) that were consistently downregulated and six that were consistently upregulated (DUXAP8, CTC-820M8.1, CDC20P1, AC006465.5, DLGAP5P1, and RP11-156J23.1) in all four datasets (Figures 1B and 1C). We selected those pseudogenes that were upregulated in NSCLC tissues for further study because they may be useful for the prognosis and treatment of NSCLC. We then investigated whether these six upregulated novel candidate pseudogenes are also upregulated in TCGA lung adenocarcinoma (LUAD), squamous cell carcinoma (LUSC), and paired normal tissues using RNA sequencing data, and found that only DUXAP8 and CDC20P1 exhibited significant overexpression in LUAD and LUSC tissues (Figure 1D). Furthermore, we selected the dataset GSE30219, including a relatively wide range of clinical information, to explore the association among DUXAP8, CDC20P1, and the PFS of NSCLC patients. The results of univariate Cox proportional hazards regression analysis showed that increased DUXAP8 expression was significantly correlated with the PFS of LUAD and LUSC patients, whereas CDC20P1 was only correlated with the PFS of LUAD patients (Figures S1A and S1B). Therefore, we focused on the pseudogene DUXAP8 in this study. Because pseudogenes are highly homologous to the host gene and other pseudogenes in the same cluster, we thereby analyzed DUAP8, its host gene DUXA, and all other DUXA pseudogenes expression using TCGA sequencing data. The result showed that DUXA and all the DUXA pseudogenes are very lowly expressed except DUXAP1, DUXAP8, and DUXAP10 in lung cancer tissues, and DUXAP8 is the highest expressed DUXA pseudogene in lung cancer (Figure S1C).

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DUXAP8 Is Upregulated in NSCLC Tissues and Cells

To validate the analysis results, we determined DUXAP8 expression levels in a cohort of 78 pairs of NSCLC tissues and adjacent nontumor tissues by qPCR. The results showed that DUXAP8 expression was significantly upregulated (fold change > 1.5; p < 0.01) in 82% (64/78) of cancerous tissues compared with the level in normal tissues (Figure 2A). In addition, analysis of the association between DUXAP8 and the patients’ clinical features showed that increased DUXAP8 expression levels in NSCLC were significantly correlated with larger tumors (p = 0.041), an advanced pathological stage (p = 0.007), and lymph node metastasis (p = 0.038). However, DUXAP8 expression was not associated with other parameters such as gender (p = 0.819) and age (p = 0.651) in NSCLC (Table S3). Kaplan-Meier survival analysis was also conducted to investigate the correlation between DUXAP8 expression and the prognosis of NSCLC patients. According to the relative DUXAP8 expression in tumor tissues, the 78 NSCLC patients were classified into two groups: a high-DUXAP8 group (n = 38; fold change R 4) and a low-DUXAP8 group (n = 40; fold change % 4). The overall survival and PFS of the high-DUXAP8 group were shorter than those of the low-DUXAP8 group (Figure 2B). Moreover, univariate analysis identified four factors associated with the prognosis of the patients: tumor size, lymph node metastasis, tumor-node-metastasis (TNM) stage, and DUXAP8 expression level. Other clinicopathological features such as gender and age were not identified as statistically significant prognostic factors. Multivariate analysis of the four prognostic factors confirmed that the hazard ratio for DUXAP8 expression was 3.09 (95% confidence interval: 1.472–6.484) for progression-free survival, indicating that DUXAP8 may serve as an independent prognostic factor in NSCLC (Table S4). Next, qPCR analysis of DUXAP8 in eight NSCLC cell lines versus that in immortalized normal lung epithelial cells (HBE and 16HBE) revealed that DUXAP8 was significantly overexpressed in six NSCLC cell lines (all p < 0.01; Figure 2C). For further analysis, we knocked down DUXAP8 in H1299 and H1975 cells, which had the highest DUXAP8 expression levels, by transfection with siRNA or short hairpin RNA (shRNA) vector, and exogenously overexpressed DUXAP8 in SPCA1 and PC9 cells, which had lower DUXAP8 expression levels (Figures 2D and S2A). Knockdown of DUXAP8 Inhibits NSCLC Cell Proliferation In Vitro and Tumorigenesis In Vivo

To evaluate the potential roles of DUXAP8 in NSCLC, we performed loss- and gain-of-function experiments. The growth curves detected by MTT showed that DUXAP8 knockdown significantly impaired H1299 and H1975 cell growth (Figure 3A), whereas overexpression of DUXAP8 increased SPCA1 and PC9 cell growth (Figure 3B). Consistent with the results of MTT assays, EdU incorporation was drastically decreased following DUXAP8 downregulation and increased after DUXAP8 upregulation (Figures 3C and S2B). Moreover, colony formation assay results showed that clonogenic survival was inhibited following the downregulation of DUXAP8 in H1299

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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 RP11156J23.1) 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.

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Molecular Therapy

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) qRTPCR 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.

and H1975 cells, whereas DUXAP8 overexpression increased the colony formation ability of SPCA1 and PC9 cells (Figures S2C and S2D). To determine whether the effect of DUXAP8 on NSCLC cell growth reflected cell cycle arrest, we analyzed cell cycle progression by flow cytometry. The results showed that H1299 and H1975 cells transfected with si-DUXAP8 exhibited clear cell cycle arrest at the G1/G0 phase (Figure 3D). Moreover, the levels of some important cell cycle regulators were determined, which showed that the levels of cyclin D1, CDK2, CDK4, and CDK6 were decreased in cells with DUXAP8 knockdown (Figure 3E). To validate these results, we constructed H1299 cell lines stably expressing sh-DUXAP8 or a negative control. Then, we inoculated H1299 cells stably transfected with sh-DUXAP8 or empty vector into female nude mice. Eighteen days after the injection, the tumors formed in the sh-DUXAP8 group were substantially smaller than those in the control group (Figure 3F). Moreover, the tumor weight at the end of the experiment was markedly lower in the sh-DUXAP8 group (0.236 ± 0.055 g) than in the empty vector group (0.090 ± 0.054 g) (Figure 3G). qPCR analysis showed that the levels of DUXAP8 in tumor tissues formed from sh-DUXAP8 cells were lower than in tumors formed in the control group (Figure S2E). Tumors formed from sh-DUXAP8-transfected H1299 cells exhibited decreased positivity for Ki67 compared with those from control cells (Figure 3H). These in vivo findings complement the in vitro data and confirm that DUXAP8 is essential for regulating NSCLC cell proliferation and tumorigenesis.


Knockdown of DUXAP8 Induces Cell Apoptosis and Inhibits Cell Migration and Invasion

To determine whether NSCLC cell proliferation was influenced by apoptosis, we performed TUNEL staining and flow cytometry apoptosis analysis. The results showed that NSCLC cells transfected with DUXAP8 siRNA exhibited a higher rate of apoptosis than control cells (Figures 4A and 4B). Cancer cell migration and invasion are a significant part of cancer progression, so we investigated the effect of DUXAP8 on these features in NSCLC by performing Transwell assays. The results showed that the knockdown of DUXAP8 expression impeded H1299 and H1975 cell migration and invasion compared with those of controls, whereas the upregulation of DUXAP8 promoted the migration and invasion of SPCA1 and PC9 cells (Figures 4C, 4D, and S2F). Taken together, these findings indicate that DUXAP8 has important roles in NSCLC progression. EGR1 and RHOB Are Key Downstream Targets of DUXAP8 in NSCLC Cells

To explore the entire repertoire of DUXAP8-affected genes in NSCLC, we further performed an RNA sequencing analysis after silencing DUXAP8 in H1299 cells. The results showed that the expression of 65 genes was upregulated (log2 fold change > 2) and that of 99 genes was downregulated (Figure 5A; Table S5) in H1299 cells with DUXAP8 knockdown compared with that in control cells. Gene Ontology (GO) and pathway enrichment analysis showed that these genes are involved in the biological processes of cell growth, cell proliferation, and cell death, among others (Figure 5B). Among these


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.

genes with altered expression, we chose 10 cancer-associated genes involved in cancer cell proliferation, apoptosis, migration, and invasion for further study. This further study using qPCR showed that the alterations in the expression of seven genes were consistent with the sequencing data in H1299 and H1975 cells. Among them, the knockdown of DUXAP8 significantly increased the expression of RHOB in both H1299 and H1975 cells, while it increased the expression of EGR1 in H1299 cells (Figure 5C). EGR1 and RHOB are newly identified tumor suppressors, and their loss is frequently observed during lung cancer development and progression.29,30 Therefore, we assumed that DUXAP8 contributes to NSCLC by repressing EGR1 and RHOB expression. Accordingly, we used western blot analysis to examine their protein levels in H1299 and H1975 cells transfected with DUXAP8 or control siRNA. Consistent with the qPCR results, knockdown of DUXAP8 also significantly increased

EGR1 and RHOB protein levels in NSCLC cells (Figures 5D and 5E). These findings suggest that EGR1 and RHOB are important downstream targets of DUXAP8 that are relevant to DUXAP8-mediated promotion of NSCLC cells proliferation and invasion. DUXAP8 Epigenetically Represses EGR1 and RHOB Transcription by Binding EZH2 and LSD1

Mechanistically, most of the previously characterized pseudogenes regulate the expression of their parental genes by functioning as ceRNAs, such as PTENP1 and BRAFP1. However, whether pseudogenes can regulate their targets through other mechanisms is unclear. To resolve this issue, here we first analyzed the distribution of the DUXAP8 transcript in NSCLC cells and found that it mostly localized in the nucleus, which suggested that it may regulate targets at the transcriptional level (Figure 6A). Recently, a few pseudogenes


Molecular Therapy

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 mm. *p < 0.05; **p < 0.01.

expressing long non-coding RNAs (lncRNAs) have been reported to regulate gene expression by interacting with specific RNA binding proteins. To determine whether DUXAP8 regulates the potential targets EGR1 and RHOB through binding with RBPs, we performed RIP assays for some well-known RBPs including PRC2 and LSD1 in NSCLC cells. The results showed that DUXAP8 RNA could bind with EZH2, LSD1, SUZ12, TDP43, and HuR, but its interaction with LSD1 and EZH2 was stronger, whereas AGAP2-AS1, which could also simultaneously bind with EZH2 and LSD, was used as a control (Figures 6B, S3A, and S3B). RNA pull-down analysis also showed that DUXAP8 RNA could directly bind with EZH2 and LSD1 in H1299 cells (Figure 6C). According to the RNA immunoprecipitation (RIP) and RNA pulldown results, we investigated whether EZH2 or LSD1 is involved in the silencing of EGR1 and RHOB in NSCLC cells. The results


of qPCR showed that EGR1 expression was increased in H1299 cells transfected with si-EZH2 (Figure 6D), whereas RHOB expression was increased in H1299 and H1975 cells with LSD1 knockdown (Figure 6E). To investigate further whether DUXAP8 repressed EGR1 and RHOB expression by interacting with EZH2 or LSD1, we performed chromatin immunoprecipitation (ChIP) analysis. The results showed that EZH2 could directly bind to EGR1 promoter regions and mediate H3K27me3 modification, whereas LSD1 could bind to the RHOB promoter regions and mediate H3K4me2 demethylation. However, knockdown of DUXAP8 reduced their binding ability and induced modification of H3K27me3 methylation and H3K4me2 demethylation (Figures 6F and 6G). These findings indicate that DUXAP8 contributes to NSCLC cell proliferation, migration, and invasion, partly by recruiting EZH2 and LSD1 to repress EGR1 or RHOB transcription.


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.

Silencing of EGR1 and RHOB Is Partly Involved in the Oncogenic Function of DUXAP8

To investigate whether EGR1 and RHOB are involved in the DUXAP8induced promotion of NSCLC cell proliferation and invasion, we first analyzed their expression levels in NSCLC and paired normal tissues

using datasets from Oncomine. The analysis results showed that both EGR1 and RHOB are significantly downregulated in NSCLC tissues (Figure S4). Next, we performed gain-of-function assays by transfecting EGR1 and RHOB overexpression vectors into H1299 cells and RHOB vectors into H1975 cells. The results of western blot showed


Molecular Therapy

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.

that EGR1 and RHOB expression were significantly upregulated in H1299 or H1975 cells transfected with EGR1 or RHOB vector compared with that in control cells (Figure 7A). MTT and EdU assays revealed that EGR1 and RHOB overexpression inhibited H1299 cell proliferation, and RHOB overexpression also inhibited H1975 cell proliferation (Figures 7B and 7C). Moreover, Transwell assays showed that increased levels of EGR1 and RHOB significantly inhibited the invasiveness of H1299 and H1975 cells (Figure 7D). Furthermore, to determine whether DUXAP8 regulates NSCLC cell proliferation and invasion in a manner dependent on repressing EGR1 and RHOB expression, we performed rescue experiments. Accordingly, H1299 and H1975 cells were co-transfected with si-DUXAP8, si-EGR1, or si-RHOB (Figure S5). The results of


MTT assays indicated that co-transfection could partially rescue DUXAP8-promoted growth or si-DUXAP8-impaired proliferation in H1299 and H1975 cells (Figure 7E), whereas Transwell analysis showed that the knockdown of EGR1 or RHOB could partially reverse si-DUXAP8-impaired invasion (Figures 7F and 7G). Finally, correlation analysis revealed that DUXAP8 expression was inversely correlated with EGR1 and RHOB in 20 paired NSCLC tissues (Figure S6). These findings indicate that DUXAP8 regulates NSCLC cell proliferation and invasion in a manner partly dependent on the silencing of EGR1 and RHOB expression.

DISCUSSION The function and mechanisms of action of the vast majority of pseudogenes in human cancers, and whether they are actively transcribed


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.

and exert any activity, remain largely unknown. In the current study, we thus attempted to identify and characterize novel candidate pseudogenes that contribute to NSCLC development and progression by using public microarray profiling data from GEO. The obtained results revealed a new oncogenic pseudogene, DUXAP8, which is significantly upregulated in NSCLC tissues, and showed that higher DUXAP8 is associated with shorter PFS. In agreement with these results, qRT-PCR showed that DUXAP8 levels were significantly higher in NSCLC tissues than in non-tumor lung tissues. Similarly, compared with the normal HBE cells, DUXAP8 expression was strik-

ingly higher in six different NSCLC cell lines, but not the SPCA1 and PC9 cell lines. Moreover, loss- and gain-of-function experiments demonstrated that the pseudogene DUXAP8 plays a key role in cell proliferation and invasion. Knockdown of DUXAP8 significantly decreased cell proliferation and invasion, and induced cell G0/G1 arrest and apoptosis, whereas forced DUXPA8 expression promoted cell growth and invasion. Taken together, these findings suggest that the pseudogene DUXAP8 functions as an oncogene, and its overexpression could contribute to NSCLC tumorigenesis and progression.


Molecular Therapy

The major type of regulation between a pseudogene and its cognate gene is the ceRNA mechanism, and a number of pseudogenes have been found to act as sponges for specific miRNAs.31–33 Researchers have ascribed this pattern to their high homology to the parental genes. However, other potential mechanisms by which pseudogenes regulate their underlying targets remain unclear. Here, we provide evidence that pseudogenes can also regulate the expression of other genes, not their cognate genes, by interacting with RNA binding proteins. We found that DUXAP8 RNA accumulates in the nucleus, and RIP assays revealed that it could directly bind with histone methyltransferase EZH2 and histone demethylase LSD1, suggesting that DUXAP8 regulates gene transcription through epigenetic modification. Moreover, RNA sequencing in cells treated with DUXAP8 siRNA and control siRNA showed that the expression of two newly identified tumor suppressors, EGR1 and RHOB, is significantly upregulated after the knockdown of DUXAP8. Interestingly, ChIP assays revealed that DUXAP8 could recruit EZH2 to the EGR1 promoter region and repress its transcription via the induction of H3K27 trimethylation, whereas LSD1 could be recruited to the RHOB promoter by DUXAP8 RNA, resulting in the silencing of RHOB expression through H3K42 demethylation. Our findings indicated that pseudogene RNAs could also regulate gene expression through other mechanisms, such as interacting with some well-known RBPs, as well as the regulation of targets by lncRNAs. EGR1 is a zinc finger transcription factor that can bind to a GC-rich consensus sequence to regulate multiple cellular responses. Several previous studies showed that EGR1 exhibits prominent tumor suppressor function in lung cancer,34 colon cancer,35 and glioma.36 Ferraro et al.37 reported that EGR1 is a significant and strong predictor of PTEN, and reduced EGR1 levels are associated with poor overall survival (OS) and DFS in NSCLC. In addition, loss of EGR1 leads to increased tumor transformation and subsequent patient morbidity and mortality.38 In this study, we also found that EGR1 expression is downregulated in NSCLC tissues, and that EGR1 functions as a tumor suppressor by inhibiting NSCLC cell proliferation and invasion. Interestingly, EZH2 recruited by the pseudogene DUXAP8 could repress EGR1 transcription in NSCLC cells, which may partly explain its loss in NSCLC. As another target of DUXAP8, RHOB is a member of the small GTPase Rho family, which has been widely implicated in cell transformation, invasion, and metastasis.39 In contrast with its close relatives RhoA and RhoC, loss of RhoB expression has been reported in several cancers, suggesting that it may be an important tumor suppressor.40 Mazieres et al.41 found that the level of the RhoB protein dramatically decreased over the course of lung cancer progression and was lost in 96% of invasive tumors, whereas the ectopic expression of RHOB in the lung cancer cell line A549 suppressed cell proliferation both in vitro and in vivo. Moreover, RhoB loss could induce Rac1-dependent mesenchymal cell invasion in lung cells through the control of PP2A activity,29 and promote the invasion of human bronchial cells via the activation of AKT1.42 Furthermore, Mazieres et al.43 also reported that the loss of RhoB expression was induced via epigenetic regulation by histone deacetylation in lung cancer, whereas we found that LSD1 recruited by DUXAP8-mediated


histone demethylation also contributed to RHOB loss of expression in NSCLC cells. Interestingly, rescue experiments demonstrated that the pseudogene DUXAP8 exerts oncogenic function in a manner partly dependent on the repression of RHOB and EGR1 expression in NSCLC cells. In summary, we identified a novel pseudogene, DUXAP8, which is significantly upregulated in NSCLC tissues and cells. Increased DUXAP8 expression is associated with poor prognosis and shorter OS and PFS survival in NSCLC patients. Its effects of promoting cell proliferation, migration, and invasion suggest that it exhibits oncogenicity in NSCLC tumorigenesis and progression. This oncogenicity occurs partly through the epigenetic silencing of EGR1 and RHOB expression by interacting with EZH2 and LSD1. Our findings extend our understanding of the roles of pseudogenes in the pathogenesis of NSCLC. However, other possible targets and mechanisms that underlie the regulatory behaviors of DUXAP8 were not investigated in this study, and these remain incompletely understood and warrant further investigation.

MATERIALS AND METHODS Microarray and TCGA Dataset Processing and Pseudogene Profile Mining

All of the microarray raw data (.CEL files) for four NSCLC cohorts were obtained from the GEO database. The four datasets are based on the HG-U133 Plus 2 microarray platform. All probes were reannotated according to the GENCODE V19 pseudogene annotation file, and a total of 47,362 probes were mapped to 5,052 pseudogenes. The GSE30219 dataset was used for survival analysis. The samples were evenly split into two groups, a high-risk group and a low-risk group, according to the expression levels of the gene of interest. Disease-free survival time (in months) was compared between these two groups. For TCGA pseudogene analysis, pseudogene expression levels were calculated from aligned BAM files using the RNASeqQC software. Expression levels were compared between primary solid tumor (TP) and solid tissue normal (NT) samples using the Mann-Whitney U test. NSCLC Sample Collection

A total of 78 paired NSCLC and adjacent non-tumor tissues were collected from patients who underwent surgery at Jiangsu Province Hospital between 2010 and 2011, and were diagnosed with NSCLC based on histopathological evaluation. Clinicopathological characteristics including TNM staging were recorded. These patients had not undergone local or systemic treatment before surgery. All collected tissue samples were immediately snap-frozen in liquid nitrogen and stored at 80 C until required. Our study was approved by the Research Ethics Committee of Nanjing Medical University, China. Written informed consent was obtained from all patients. Cell Culture and Transfection

Five NSCLC adenocarcinoma cell lines (PC9, SPC-A1, NCI-H1975, H1299, and A549) and three NSCLC squamous carcinomas cell lines (H520, H1703, and SK-MES-1) were purchased from the Chinese


Academy of Sciences Cell Bank. All cell lines were authenticated by short tandem repeat DNA profiling. A549, H1975, H1299, H1703, and H520 cells were cultured in RPMI 1640; 16HBE, SK-MES-1, PC9, and SPC-A1 cells were cultured in DMEM (Invitrogen) medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen) at 37 C/5% CO2. The transient transfection of small interfering RNAs was performed using RNAiMAX (Invitrogen), following the manufacturer’s instructions. The different siRNAs are listed in Table S1. Human DUXAP8 cDNA and short hairpin RNA directed against DUXAP8 were ligated into the pCDNA3.1 and BLOCK-iT U6 shRNA vector. Plasmid vectors for transfection were prepared using DNA Midiprep or Midiprep kits (QIAGEN), transfected using X-tremeGENE HP (Roche Applied Science), and selected using G418. RNA Extraction and qPCR Assays

Total RNA was isolated with TRIzol reagent (Invitrogen), in accordance with the manufacturer’s instructions. One microgram of total RNA was reverse-transcribed in a volume of 20 mL under standard conditions, in accordance with the instructions of the PrimeScript RT Reagent Kit (TaKaRa). SYBR Premix Ex Taq (TaKaRa) was used to determine the expression levels of DUXAP8 and its targets, following the manufacturer’s instructions. Results were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The specific primers are shown in Table S1. Cell Proliferation Assay

Cell proliferation was monitored using a Cell Proliferation Reagent Kit I (MTT) (Roche Applied Science) and EdU assay kit (Life Technologies). For MTT assays, cells were seeded into a 96-well plate. The cells in each well were supplemented with 20 mL MTT solution. Plates were incubated for 6 hr; then the absorbance at 490 nm was measured. For the EdU incorporation assay, cells were cultured in 24-well plates. Then, 10 mM EdU was added to each well and the cells were cultured for an additional 2 hr. Then, the cells were fixed with 4% formaldehyde for 30 min. After washing, EdU can be detected with a Click-iTR EdU Kit for 30 min, and the cells were stained with DAPI for 10 min and visualized using a fluorescent microscope (Olympus). The EdU incorporation rate was expressed as the ratio of EdU-positive cells to total DAPI-positive cells (blue cells), which were counted using Image-Pro Plus (IPP) 6.0 software (Media Cybernetics). Flow Cytometry

Cells were harvested 48 hr after transfection by trypsinization, and double-stained with FITC-Annexin V and propidium iodide (PI) using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences). Then, the cells were analyzed with a flow cytometer (FACScan; BD Biosciences) equipped with CellQuest software (BD Biosciences). Cells for cell cycle analysis were stained with PI using the CycleTEST PLUS DNA Reagent Kit (BD Biosciences), following the manufacturer’s protocol, and analyzed by FACScan. The proportions of cells in the G0/G1, S, and G2/M phases were determined and compared.

Cell Migration and Invasion Assays

For cell migration and invasion assays, 24-well Transwell chambers with an 8-mm pore size polycarbonate membrane were used (Corning Incorporated). Cells were seeded on the top of the membrane precoated with Matrigel (BD) (without Matrigel for the cell migration assay). After incubation for 24 hr, cells inside the upper chamber were removed with cotton swabs, whereas cells on the lower membrane surface were fixed and then stained with 0.5% Crystal violet solution. Five randomly selected fields were counted in each well. In Vivo Tumor Formation Assay

Four-week-old female athymic BALB/c nude mice were maintained under pathogen-free conditions and manipulated according to protocols approved by the Shanghai Medical Experimental Animal Care Commission. H1299 cells were stably transfected with sh-DUXAP8 or empty vector and harvested, washed with PBS, and resuspended at a concentration of 1 108 cells/mL. A total of 100 mL of suspended cells was subcutaneously injected into each mouse. Tumor growth was examined every 3 days, and tumor volumes were calculated using the following equation: volume = length width2/2. At 18 days postinjection, the mice were euthanized and the subcutaneous growth of each tumor was examined. The protocol was approved by the Committee on the Ethics of Animal Experiments of Nanjing Medical University. RNA Immunoprecipitation

For the immunoprecipitation (IP) of endogenous EZH2 and LSD1 complexes from whole-cell extracts, the cells were lysed. The supernatants were incubated with protein A/G Sepharose beads coated with antibodies that recognized EZH2, SUZ12, LSD1, and SNRNP70, or with control IgG (Millipore) for 6 hr at 4 C. After the beads had been washed with washing buffer, the complexes were incubated with 0.1% sodium dodecyl sulfate (SDS)/0.5 mg/mL Proteinase K (30 min at 55 C) to remove proteins. The RNA isolated from the IP materials was further assessed by qRT-PCR analysis. RNA Pull-Down Assays

DUXAP8 RNAs were in vitro transcribed using T7 RNA polymerase (Ambio Life), followed by purification using the RNeasy Plus Mini Kit (QIAGEN) and treatment with RNase-free DNase I (QIAGEN). Transcribed RNAs were biotin-labeled with the Biotin RNA Labeling Mix (Ambio Life). Positive, negative, and biotinylated RNAs were mixed and incubated with H1299 cell lysates. Magnetic beads were added to each binding reaction, followed by incubation at room temperature. Then, the beads were washed with washing buffer. Finally, the eluted proteins were detected by western blot analysis. Chromatin Immunoprecipitation

H1299 and H1975 cells were treated with formaldehyde and incubated for 10 min to generate DNA-protein crosslinks. Cell lysates were then sonicated to generate chromatin fragments of 200–300 bp and immunoprecipitated with LSD1-, H3K4me2-, EZH2-, and H3K27me3-specific antibody (Millipore) or IgG as a control. Precipitated chromatin DNA was recovered and analyzed by qPCR.


Molecular Therapy

Western Blot and Antibodies

The proteins from cell lysates were separated by 10% SDS-PAGE, transferred to 0.22-mm negative control (NC) membranes (Sigma), and incubated with specific antibodies. ECL chromogenic substrate was used for quantification by densitometry (Quantity One software; Bio-Rad). GAPDH antibody was used as a control. Anti-GAPDH, -P21, -CDK2, -CDK4, -CDK6, -cyclin D1, and -cyclin D3 antibodies (1:1,000) were purchased from Cell Signaling Technology. Anti-EGR1 and -RHOB antibodies were purchased from ProTech. Statistical Analysis

All statistical analyses were performed using SPSS 17.0 software (IBM). The significance of differences between groups was estimated by Student’s t test, Wilcoxon’s test, or the c2 test. DFS and OS rates were calculated by the Kaplan-Meier method with the log rank test applied for comparison. Pearson’s correlation analyses were used to investigate the correlation among DUXAP8, ERG1, and RHOB expression. Two-sided p values were calculated, and a probability level of 0.05 was selected to represent statistical significance.

SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and five tables and can be found with this article online at http://dx.doi.org/10.1016/j.ymthe. 2016.12.018.

AUTHOR CONTRIBUTIONS M.S. and K.L. performed acquisition of data, drafting of the manuscript, and critical revision of the manuscript for important intellectual content. Y.W. and J.H. provided analysis and interpretation of data and statistical analysis. C.Z., F.N., W.L., and X.H. provided technical or material support. K.L. approved the final version of the manuscript.

CONFLICTS OF INTEREST No potential conflicts of interest were disclosed.

ACKNOWLEDGMENTS This work was supported by National Natural Scientific Foundation of China grants 81372397, 81672949(to K.L.), and 81602013 (to F.N.) and Priority Academic Program Development of Jiangsu Higher Education Institutions grant JX10231801 (to K.L.). M.S. was supported by the Odyssey Program.

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