The pseudogene derived long noncoding RNA DUXAP8 promotes gastric cancer cell proliferation and migration via epigenetically silencing PLEKHO1 expression

Abstract

Gastric cancer (GC) is the third leading cause of cancer death due to its poor prognosis and limited treatment options. Evidence indicates that pseudogene-derived long noncoding RNAs (lncRNAs) may be important players in human cancer progression, including GC. In this paper, we report that a newly discovered pseudogene-derived lncRNA named DUXAP8, a 2107-bp RNA, was remarkably upregulated in GC. Additionally, a higher level of DUXAP8 expression in GC was significantly associated with greater tumor size, advanced clinical stage, and lymphatic metastasis. Patients with a higher level of DUXAP8 expression had a relatively poor prognosis. Further experiments revealed that knockdown of DUXAP8 significantly inhibited cell proliferation and migration, as documented in the SGC7901 and BGC823 cell lines. Furthermore, RNA immunoprecipitation and chromatin immunoprecipitation assays demonstrated that DUXAP8 could epigenetically suppress the expression of PLEKHO1 by binding to EZH2 and SUZ12 (two key components of PRC2), thus promoting GC development. Taken together, our findings suggest that the pseudogene-derived lncRNA DUXAP8 promotes the progression of GC and is a potential therapeutic target for GC intervention.

Keywords: DUXAP8; PLEKHO1; gastric cancer; lncRNA; pseudogene.

Figure 1. Relative DUXAP8 expression in GC tissues and its clinical significance

A. Relative expression of DUXAP8 in human gastric cancerous tissues compared with noncancerous tissue via GSE58828 and GSE13861 data analysis. B. Relative expression of DUXAP8 in human GC tissues (n = 72) compared with corresponding non-tumor tissues (n= 72). DUXAP8 expression was examined by qRT-PCR and normalized to GAPDH expression (shown as Δct). C. Results are presented as the fold-change in tumor tissues relative to normal tissues, and DUXAP8 expression was classifid into two groups. D, E. Kaplan-Meier progression-free survival and overall survival curves according to DUXAP8 expression level. Error bars indicate mean ± standard errors of the mean. *P < 0.05, **P < 0.01.

Figure 2. DUXAP8 promotes GC cell proliferation in vitro

A. QRT-PCR analysis of DUXAP8 expression in the normal gastric epithelium cell line (GES1) and GC cells. B. QRT-PCR analysis of DUXAP8 expression in control (scrambled), si-DUXAP8 1#, si-DUXAP8 2# and si-DUXAP8 3# treated GC cells. C, D. MTT assays were used to determine the viability of si-DUXAP8-transfected or pcDNA-DUXAP8-transfected GC cells. Experiments were performed in triplicate. E, F. Colony formation assays were performed to determine the proliferation of sh-DUXAP8-transfected or pcDNA-DUXAP8- transfected GC cells. Colonies were counted and captured. G, H. Proliferating SGC7901 and BGC823 cells were labeled with Edu. The Click-it reaction revealed Edu staining (red). Cell nuclei were stained with DAPI (blue). Representative images and data based on three independent experiments. Error bars indicate mean ± standard errors of the mean. *P < 0.05, **P < 0.01.

Figure 3. Effect of DUXAP8 on GC cell apoptosis and migrationin vitro

A. Flow cytometry was used to detect the apoptotic rates of cells. LR, early apoptotic cells; UR, terminal apoptotic cells. B. Apoptosis in SGC7901 and BGC823 cells after DUXAP8 knockdown was detected through TUNEL staining. C. Western blot analysis of apoptosis - related proteins after scrambled siRNA, si-DUXAP8 1#, or si-DUXAP8 2# transfection in SGC7901 and BGC823 cells. β-actin protein was used as an internal control. D, E. Transwell assays were performed to investigate the changes in migratory abilities of GC cells. Error bars indicate mean ± standard errors of the mean. *P < 0.05, **P < 0.01.

Figure 4. DUXAP8 knockdown increases the expression of genes involved in cell proliferation and migration

A. Mean-centered, hierarchical clustering of 448 transcripts altered in scrambled siRNA-treated cells and si-DUXAP8-treated cells, with three repeats. B. Gene Ontology analysis for all genes with altered expressions between the scrambled siRNA-treated and si-DUXAP8-treated cells in vitro. Cell apoptosis and migration were both among the significant biological processes for genes whose transcripts level were changed in the DUXAP8-depleted GC cells. C, D. QRT-PCR analysis in si-DUXAP8- treated or pcDNA-DUXAP8-treated GC cells reveal altered mRNA level of genes involved in cell proliferation and migration upon DUXAP8 depletion. Error bars indicate mean ± standard errors of the mean. *P < 0.05, **P < 0.01.

Figure 5. DUXAP8 epigenetically silences PLEKHO1 transcription by binding with PRC2

A. DUXAP8 expression levels in cell nucleus or cytoplasm of SGC7901 and BGC823 cells were detected by qRT-PCR. U6 was used as a nucleus marker and GAPDH was used as a cytosol marker. B. RIP experiments were performed in SGC7901, BGC823 cells and the coprecipitated RNA was subjected to qRT-PCR for DUXAP8. The fold enrichment of DUXAP8 in EZH2/SUZ12/LSD1 RIP is relative to its matching IgG control. C. QRT-PCR analysis of PLEKHO1, DPM3, HABP4, RBMS3, RARRES1 and HNRNPM expression levels in control, si-EZH2 and si-SUZ12 treated GC cells. D. ChIp-qRT-PCR of EZH2 occupancy, SUZ12 occupancy and H3K27me3 binding in the PLEKHO1 promoters in SGC7901, BGC823 cells treated with control or si-DUXAP8 2# (48h); IgG as a negative control. Error bars indicate mean ± standard errors of the mean. *P < 0.05, **P < 0.01.

Figure 6. Down-regulation of PLEKHO1 promotes GC cell proliferation and is involved in the oncogene function of DUXAP8

A. QRT-PCR analysis of PLEKHO1 expression in 72 paired human GC tissues and adjacent noncancerous tissuesin. B. QRT-PCR analysis of PLEKHO1 expression in the normal gastric epithelium cell line (GES1) and GC cells. C. MTT assays were used to determine the cell viability for pcDNA-PLEKHO1-transfected GC cells. Experiments were performed in triplicate. D. Colony-formation assays were used to determine the cell proliferation for pcDNA-PLEKHO1-transfected GC cells. Experiments were performed in triplicate. E. Transwell assays were performed to investigate the changes in migratory abilities of GC cells. F. MTT assays were used to determine the cell viability for pcDNA-DUXAP8 and pcDNA-PLEKHO1 co-transfected GC cells. Experiments were performed in triplicate. G. Colony-formation assays were used to determine the cell viability for pcDNA-DUXAP8 and pcDNA-PLEKHO1 co-transfected GC cells. Experiments were performed in triplicate. Error bars indicate mean ± standard errors of the mean. *P < 0.05, **P < 0.01.

Figure 7. DUXAP8 promotes tumorigenesis of GC cells in vivo

A. Empty vector or sh-DUXAP8 were transfected into SGC7901 cells, which were injected in the nude mice (n = 6), respectively. Tumors formed in sh-DUXAP8 group were dramatically smaller than the control group. B. Tumor volumes were calculated after injection every three days. Points, mean (n = 6); bars indicate SD. C. Tumor weights were represented as means of tumor weights ± SD. D. QRT-PCR was performed to detect the average expression of DUXAP8 in xenograft tumors (n = 6). E. The tumor sections were under H&E staining and IHC staining using antibodies against ki-67. Error bars indicate mean ± standard errors of the mean. *P < 0.05, **P < 0.01.