LncRNA DICER1-AS1 promotes colorectal cancer progression by activating the MAPK/ERK signaling pathway through sponging miR-650

Abstract

Background: Colorectal cancer (CRC) is a disease with high morbidity and mortality rates globally. Long noncoding RNAs (lncRNAs) play a fundamental role in tumor progression, and increasing attention has been paid to their role in CRC. This study aimed to determine the function of lncRNA DICER1 antisense RNA 1 (DICER1-AS1) in CRC and confirm its potential regulatory mechanisms in CRC.

Methods: The publicly available dataset was used to assess DICER1-AS1 function and expression in CRC. RT-qPCR or western blot assays were performed to verify DICER1-AS1, miR-650, and mitogen-activated protein kinase 1 (MAPK1) expression in CRC cells or tissues. To determine the function of DICER1-AS1, we performed CCK-8, colony formation, transwell, cell cycle, and in vivo animal assays. Using RNA sequence analysis, luciferase reporter assays, and bioinformatics analysis, the connection between DICER1-AS1, MAPK1, and miR-650 was investigated.

Results: DICER1-AS1 was significantly upregulated in CRC tissue compared to normal colon tissue. High DICER1-AS1 expression suggested a poor prognosis in CRC patients. Functionally, upregulation of DICER1-AS1 effectively promoted CRC proliferation, migration, and invasion ex vivo and tumor progression in vivo. Mechanistically, DICER1-AS1 functions as a competitive endogenous RNA (ceRNA) that sponges miR-650 to upregulate MAPK1, promotes ERK1/2 phosphorylation, and sequentially activates the MAPK/ERK signaling pathway.

Conclusion: Our investigations found that upregulation of DICER1-AS1 activates the MAPK/ERK signaling pathway by sponging miR-650 to promote CRC progression, revealing a possible clinically significant biomarker and therapeutic target.

Keywords: DICER1-AS1; MAPK/ERK pathway; MAPK1; colorectal cancer; malignant proliferation; miR-650.

FIGURE 1

DICER1‐AS1 is upregulated in CRC and correlates with poor patient prognosis. (A) NCBI results of downregulated DICER1‐AS1 in normal colon tissues. (B) DICER1‐AS1 expression in human CRC and normal tissues was analyzed from TCGA database. (C) DICER1‐AS1 expression in all stages of CRC (p < 0.05). (D, E) Kaplan–Meier analysis of overall survival (D) and disease‐free survival (E) in two groups stratified by low and high expression of DICER1‐AS1 in CRC patients based on clinical data derived from the GEPIA database. (F‐G) DICER1‐AS1 overexpression is found in circulating cancer cells (CTCs) from CRC. (H) DICER1‐AS1 expression in CRC cell lines (SW480, RKO, LOVO, and HCT116) compared with the normal colorectal epithelial cell line NCM460 measured by RT–qPCR. (I) RT–qPCR analysis of DICER1‐AS1 expression in eight pairs of CRC and corresponding normal tissues. The data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

DICER1‐AS1 promotes CRC cell progression in vitro. (A) The relative DICER1‐AS1 expression level in SW480 and LOVO cells transfected with DICER1‐AS1‐OE or the negative control (NC). (B) CCK‐8 assay of SW480 and LOVO cells transfected with DICER1‐AS1‐OE or the negative control (NC). (C‐D) Representative images (C) and quantification (D) of the colony formation assay in SW480 and LOVO cells transfected with DICER1‐AS1‐OE or the negative control (NC). (E‐F) Representative images (left) and quantification (right) of transwell migration and invasion assays in SW480 and LOVO cells transfected with DICER1‐AS1‐OE or the negative control (NC) (magnification, ×100, scale bar, 500 μm). (G‐H) Representative images (G) and quantification (H) of flow cytometric analysis of the cell cycle in SW480 and LOVO cells transfected with DICER1‐AS1‐OE or the negative control (NC).The data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

DICER1‐AS1 activates the MAPK/ERK signaling pathway. (A) Gene expression profiles of SW480 cells transfected with DICER1‐AS1‐OE or the negative control (NC). (B) GO functional annotation clustering of genes regulated by DICER1‐AS1 in SW480 cells is shown. The ten most enriched groups according to GO analysis are ranked based on p values. (C) Enriched KEGG pathway analysis showing the most enriched pathways. (D, E) Western blot analysis of p‐ERK1/2 expression in SW480 (D) and LOVO (E) cells transfected with DICER1‐AS1‐OE or the negative control (NC). (F, G) Western blot analysis of p‐ERK1/2 expression in SW480 (F) and LOVO (G) cells cotransfected with DICER1‐AS1‐OE or the negative control (NC) together with ERK inhibitor (80 nM). The data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

DICER1‐AS1 is predominantly distributed in the cytoplasm and competitively binds miR‐650. (A) DICER1‐AS1 was predicted to be located mainly in the cytoplasm using the bioinformatics tools in lncLocator. (B) A nuclear‐cytoplasmic fractionation assay indicated that DICER1‐AS1 was mainly localized in the cytoplasm of LOVO cells. (C) The localization of DICER1‐AS1 was observed in SW480 and LOVO cells by FISH. The nuclei were stained with DAPI (magnification, ×400, scale bar, 20 μm). (D) Two miRNAs were predicted to harbor complementary sequences to DICER1‐AS1 according to the lncbase, starBase and miRDB databases. (E) Relative levels of miR‐3612 and miR‐650 in SW480 and LOVO cells transfected with DICER1‐AS1‐OE or the negative control (NC). (F) Schematic diagram showing the sequence of miR‐650 and the 3′‐UTR of DICER1‐AS1, containing the wild type and mutant types. (G) Luciferase reporter assay indicated that DICER1‐AS1 WT activity was inhibited following transfection of SW480 and LOVO cells with miR 650 mimics. The data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

MiR‐650 inhibits CRC cell proliferation. (A) Expression level of miR‐650 in SW480 and LOVO cells transfected with miR‐650 mimics or miR‐Ctrl. (BC) CCK‐8 assay of SW480 and LOVO cells transfected with miR‐650 mimics or miR‐Ctrl. (D, E) Representative images (left) and quantification (right) of the colony formation assay in SW480 and LOVO cells with miR‐650 mimics or miR‐Ctrl. (F, G) CCK‐8 assays of SW480 (F) and LOVO (G) cells cotransfected with DICER1‐AS1‐OE or negative control (NC) together with miR‐650 mimics or miR‐Ctrl. (H, I) Representative images (H) and quantification (I) of the colony formation assay in SW480 and LOVO cells cotransfected with DICER1‐AS1‐OE or negative control (NC) together with miR‐650 mimics or miR‐Ctrl. The data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

DICER1‐AS1 sponges miR‐650 to upregulate MAPK1 expression, promoting ERK1/2 phosphorylation and activating the MAPK/ERK pathway. (A) Venn diagram exhibiting the overlapping target genes of miR‐650 predicted by starBase, miRDB and PicTar and the uptrend of mRNA in RNA sequencing of SW480 cells transfected with DICER1‐AS1‐OE compared with the negative control (NC). (B) The GEPIA database showed an upregulated expression level of MAPK1 in CRC. (C) Schematic diagram showing the sequence of miR‐650 and the 3′‐UTR of MAPK1, containing the wild type and mutant types. (D) Luciferase reporter assay indicated that MAPK1 WT activity was inhibited following transfection of the cells with miR 650 mimics in LOVO cells. (E) Relative levels of MAPK1 in SW480 and LOVO cells transfected with DICER1‐AS1‐OE or the negative control (NC). (G) Relative levels of MAPK1 in SW480 and LOVO cells transfected with miR‐650 mimics or miR‐Ctrl. (H, I) Western blot analysis of p‐ERK1/2 expression in SW480 cells cotransfected with DICER1‐AS1‐OE or the negative control (NC) together with miR‐650 mimics or miR‐Ctrl. The data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

DICER1‐AS1 facilitates CRC cell tumorigenesis in vivo. (A) Imaging of mice (left) and xenograft tumors (right) showed that overexpression of DICER1‐AS1 led to larger tumors. (B, C) The nude mouse xenograft model showed that overexpression of DICER1‐AS1 promoted tumor growth (B) and tumor weights (C) compared with those of the negative control (NC) cells. (D) Relative expression levels of p‐ERK1/2 were observed in subcutaneous tumor tissues by western blot assays. The data are presented as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.