Long noncoding RNA MAPKAPK5-AS1 promotes colorectal cancer proliferation by partly silencing p21 expression

Abstract

Colorectal cancer (CRC) is the third most common malignancy in the world, and long noncoding RNA (lncRNA) plays a critical role in carcinogenesis. Here, we report a novel lncRNA, MAPKAPK5-AS1, that acts as a critical oncogene in CRC. In addition, we attempted to explore the functions of MAPKAPK5-AS1 on tumor progression in vitro and in vivo. Quantitative RT-PCR was used to examine the expression of MAPKAPK5-AS1 in CRC tissues and cells. Expression of MAPKAPK5-AS1 was significantly upregulated in 50 CRC tissues, and increased expression of MAPKAPK5-AS1 was found to be associated with greater tumor size and advanced pathological stage in CRC patients. Knockdown of MAPKAPK5-AS1 significantly inhibited proliferation and caused apoptosis in CRC cells. We also found that p21 is a target of MAPKAPK5-AS1. In addition, we are the first to report that MAPKAPK5-AS1 plays a carcinogenic role in CRC. MAPKAPK5-AS1 is a novel prognostic biomarker and a potential therapeutic candidate for CRC cancer.

Keywords: MAPKAPK5-AS1; apoptosis; colorectal cancer; lncRNA; proliferation.

Figure 1

MAPKAPK5‐AS1 is upregulated in human colorectal cancer (CRC) tissues and is significantly correlated with larger tumor size, advanced TNM stage, lymph node metastasis, and poor prognosis. A, Relative expression of MAPKAPK5‐AS1 in CRC tissues (n = 50) compared with the corresponding non‐tumor tissues (n = 50); the patients were classified into two groups according to the median level of MAPKAPK5‐AS1. MAPKAPK5‐AS1 expression was examined by quantitative RT‐PCR (qRT‐PCR) and normalized to GAPDH expression. B‐D, MAPKAPK5‐AS1 expression was significantly higher in patients with larger tumor size, higher pathological stage, and lymph node metastasis. E, Kaplan‐Meier overall survival curves according to MAPKAPK5‐AS1 expression level. Cum., cumulative. F, MAPKAPK5‐AS1 expression was assessed by qRT‐PCR analysis in CRC cells (DLD‐1, HCT116, HT29, and SW480) and the normal human colonic epithelial cell line (HCoEpiC). G, Quantitative PCR analysis of MAPKAPK5‐AS1 expression levels following transfection of DLD‐1 and SW480 cells with siRNAs against MAPKAPK5‐AS1. Representative images and data based on 3 independent experiments. Bars: ± SD. **P < .01. siNC, negative control

Figure 2

MAPKAPK5‐AS1 promotes colorectal cancer cell proliferation in vitro. A, CCK‐8 assays were used to determine the proliferation of DLD‐1 and SW480 cells following treatment with si‐MAPKAPK5‐AS1 2#, 3#, and the negative control (si‐NC). B, Results of the colony formation of DLD‐1 and SW480 cells transfected with MAPKAPK5‐AS1 siRNA. C, Flow cytometry assays were used to analyze cell apoptosis in siRNA‐transfected DLD‐1 and SW480 cells. Representative images and data based on 3 independent experiments. Bars: ± SD. *P < .05, **P < .01

Figure 3

MAPKAPK5‐AS1 downregulation promotes cell cycle arrest and apoptosis in colorectal cancer cells in vitro. A, Bar chart represents the percentage of cells in G₀/G₁, S, or G₂/M phase, as indicated. B, Proliferating DLD‐1 and SW480 cells were labelled with 5‐ethynyl‐2′‐deoxyuridine (EdU). The Clickit reaction revealed EdU staining (red). Cell nuclei were stained with DAPI (blue). C, Apoptosis assay verifies apoptosis. Representative images and data based on 3 independent experiments. Bars: ± SD. **P < .01. siNC, negative control

Figure 4

Silencing of MAPKAPK5‐AS1 inhibits colorectal cancer growth in vivo. A, Stable MAPKAPK5‐AS1 knockdown DLD‐1 cells were used for the in vivo study. Nude mice carrying tumors from respective groups are shown. B, Tumor volumes were calculated after injection every 2 days. C, qRT‐PCR experiments were performed to detect the average expression of MAPKAPK5‐AS1 in xenograft tumors. D, Tumor weights from 2 treatment groups are represented. E, Images of H&E staining and immunohistochemistry of the xenograft tumors. Representative Ki‐67 protein levels in xenograft tumors as evaluated by immunohistochemistry. Representative images and data based on 3 independent experiments. Bars: ± SD, *P < .05, **P < .01

Figure 5

MAPKAPK5‐AS1 overexpression induces promotion of colorectal cancer cell proliferation. A, Level of MAPKAPK5‐AS1 in HCT116 cells transfected with pcDNA‐MAPKAPK5‐AS1 detected by quantitative PCR analysis. B‐D, MTT, 5‐ethynyl‐2′‐deoxyuridine (EdU) staining, and colon formation assays were used to determine cell viability. Representative images and data based on 3 independent experiments. Bars: ± SD, *P < .05, **P < .01

Figure 6

MAPKAPK5‐AS1 partly silences p21 transcription by binding to enhancer of zeste homolog 2 (EZH2). A, Expression levels of MAPKAPK5‐AS1 in cell nucleus and cytoplasm in DLD‐1 and SW480 cells investigated by quantitative RT‐PCR (qRT‐PCR). U6 was used as a nuclear marker, and GAPDH was used as a cytosol marker. B, FISH analysis of the location of MAPKAPK5‐AS1 (red) in the cytoplasm and nuclear fractions (blue) of SW480 cells. C, Eight classical mRNA expression levels D, Expression of p21 was determined after knockdown of MAPKAPK5‐AS1 by qRT‐PCR and western blot assays after transfection. E, RNA immunoprecipitation assays were carried out in DLD‐1 and SW480 cells, and the coprecipitated RNA was subjected to qRT‐PCR for MAPKAPK5‐AS1. F, DUXAP10 was used as negative control. G, ChIP‐qRT‐PCR of EZH2 occupancy and H3K27me3 binding in the p21 promoter in DLD‐1 and SW480 cells treated with si‐MAPKAPK5‐AS1 (48 hours) or the negative control (si‐NC); IgG as negative control. H, ChIP‐qRT‐PCR of EZH2 occupancy and H3K27me3 binding in the p21 promoter in HCT116 cells treated with pcDNA‐MAPKAPK5‐AS1; IgG as negative control. I, qRT‐PCR assays were used to determine the expression of EZH2 in DLD‐1 cells after EZH2 knockdown. p21 expression was investigated in DLD‐1 cells after knockdown of EZH2 through qRT‐PCR and western blot assays. Representative images and data based on 3 independent experiments. Bars: ± SD, *P < .05, **P < .01

Figure 7

Knockdown of p21 promotes colorectal cancer cell proliferation. A,B, DLD‐1 cells were transfected with si‐p21, and p21 expression was analyzed by western blotting. C,D, CCK‐8 and 5‐ethynyl‐2′‐deoxyuridine (EdU) assays were used to determine the viability of si‐p21‐transfected DLD‐1 cells. E, Colony formation assays were used to determine the cell viability of sh‐MAPKAPK5‐AS1 and sh‐p21 cotransfected DLD‐1 cells. Bars: ± SD, *P < .05, **P < .01