The pseudogene derived from long non-coding RNA DUXAP10 promotes colorectal cancer cell growth through epigenetically silencing of p21 and PTEN

Abstract

Recently, substantial evidence has demonstrated that pseudogene derived lncRNAs are crucial regulators of cancer development and progression. DUXAP10,a pseudogene derived long non-coding RNA(lncRNA), is overexpression in colorectal cancer (CRC), but its expression pattern, biological function and underlying mechanism in CRC is still undetermined. In this study, we observed that DUXAP10 was up-regulated in CRC tissues which was positively correlated with advanced pathological stages, larger tumor sizes and lymph node metastasis. Additionally, knockdown of DUXAP10 inhibited cell proliferation, induced cell apoptosis and increase the number of G0/G1 cells significantly in the HCT116 and SW480 cell lines. Moreover, DUXAP10 silencing inhibited tumor growth in vivo. Further mechanism study showed that, by binding to histone demethylase lysine-specific demethylase 1 (LSD1), DUXAP10 promote CRC cell growth and reduced cell apoptosis through silencing the expression of p21 and phosphatase and tensin homolog (PTEN) tumor suppressor. Our findings suggested that the pseudogene-derived from lncRNA DUXAP10 promotes the biological progression of CRC and is likely to be a potential therapeutic target for CRC intervention.

Figure 1

Relative DUXAP10 expression in CRC tissues and its clinical significance. (A) The full sequence of DUXAP10 is published in the NCBI database (NR_110526.1). (B) Prediction of DUXAP10 structure based on minimum free energy (MFE) and partition function. Color scale indicates the confidence for the prediction for each base with shades of red indicating strong confidence. (http://rna.tbi.univie.ac.at/). (C) Low DUXAP10 expression levels in normal human colorectal tissues. The protein coding gene GAPDH was used as a control. (D) Relative expression of DUXAP10 in colorectal cancer tissues compared with normal tissue was analyzed by using TCGA data. (E) Relative expression of DUXAP10 in CRC tissues compared with corresponding adjacent normal tissues (n = 58), and DUXAP10 expression was classifid into two groups. (F) DUXAP10 expression was significantly higher in patients with a larger tumor size, a higher pathological stage and lymph node metastasis (shown as ΔCT). Bars: s.d., *P < 0.05, **P < 0.01.

Figure 2

Relative DUXAP10 expression levels in CRC cell lines. (A) Analysis of DUXAP10 expression levels in CRC cell lines (DLD-1, HCT116, SW480 and SW620) by qPCR. (B) qPCR analysis of DUXAP10 expression levels following treatment of HCT116 and SW480 cells with siRNAs against DUXAP10. (C) Relative DUXAP10 levels in cell cytoplasm or nucleus of HCT116 and SW480 cell lines were detected by qPCR. Bars: s.d., *P < 0.05, **P < 0.01.

Figure 3

Effects of DUXAP10 on CRC proliferation and apoptosis in vitro. (A) MTT assays were performed to determine the cell viability of HCT116 and SW480 cells after the transfection of siRNA against DUXAP10. (B) Representative results of the colony formation of HCT116 and SW480 cells transfected with the siRNA of DUXAP10. (C) Flow cytometry assays were performed to analysis the cell cycle progression when HCT116 and SW480 cells transfected with siRNA against DUXAP10. (D) Flow cytometry assays were performed to analysis the cell apoptotic in siRNA-transfected HCT116 and SW480 cells. Representative images and data based on three independent experiments. Bars: s.d, *P < 0.05, **P < 0.01.

Figure 4

Effect of DUXAP10 on CRC cell proliferation and apoptosis confirmed by Edu analysis and TUNEL assay. (A) Proliferating HCT116 and SW480 cells were labeled with Edu. The Click-it reaction revealed Edu staining (red). Cell nuclei were stained with DAPI (blue). (B) TUNEL staining assays were performed to analyze cell apoptosis after DUXAP10 knockdown. The images of TUNEL positive cells were captured by a fluorescence microscope (200×). Quantitative result of TUNEL assay was analyzed. Representative images and data based on three independent experiments. Bars: s.d, *P < 0.05, **P < 0.01.

Figure 5

DUXAP10 epigenetically silences p21 and PTEN transcription by binding with LSD1. (A) RIP assays were performed in HCT116 cells and the coprecipitated RNA was subjected to qPCR for DUXAP10. (B) Heat maps of altered genes in DUXAP10 or LSD1 knockdown HCT116 cells compared with control cells. (C) The levels of p21 and PTEN mRNA were detected by qPCR when knockdown of DUXAP10 in HCT116 cells. (D) The p21 and PTEN protein levels were determined by western blot in DUXAP10 knockdown HCT116 cells. (E,F) The expression of p21 and PTEN in HCT116 cells, after knockdown of LSD1. (G–J) ChIP-qPCR of H3K4me2 and LSD1 of the promoter region of the p21 and PTEN locus after siRNA treatment targeting si-NC or si-DUXAP10 in HCT116 cells. Representative images and data based on three independent experiments. Bars: s.d, *P < 0.05, **P < 0.01.

Figure 6

The silencing of DUXAP10 inhibited CRC growth in vivo. (A) The stable DUXAP10 knockdown HCT116 cells were used for the in vivo study. The nude mice carrying tumors from respective groups were shown. (B) Tumor volumes were calculated after injection every 3 days. (C) Tumor weights from two groups are represented. (D) qPCR was performed to detect the average expression of DUXAP10 in xenograft tumors (n = 6). (E) Images of HE staining and immunohistochemistry of the xenograft tumors. Representative Ki-67 protein levels in xenograft tumors as evaluated by IHC. (F) Summary diagram describes that DUXAP10 regulates CRC cell proliferation. Representative images and data based on three independent experiments. Bars: s.d, *P < 0.05, **P < 0.01.