Small protein DDX11-AS1-ORF encoded by lncRNA DDX11-AS1 promotes colorectal cancer progression through VEGFA-activated p38-MAPK pathway

Abstract

This study aims to investigate the expression, function, and mechanism of action of the small protein DDX11 antisense RNA 1 - open reading frame (DDX11-AS1-ORF), encoded by the long non-coding RNA (lncRNA) DDX11 antisense RNA 1 (DDX11-AS1), in the progression of colorectal cancer (CRC). The expression levels of DDX11-AS1 were assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis in 10 pairs of colorectal cancer tissues and corresponding non-tumor tissues. Functional evaluations of DDX11-AS1 and DDX11-AS1-ORF were conducted using cell counting kit-8 (CCK8) assays, colony formation assays, Transwell migration assays, and in vitro tube formation assays. The coding potential of DDX11-AS1 was validated by western blot and immunofluorescence. The activation of the p38 mitogen-activated protein kinase (p38-MAPK) pathway by DDX11-AS1-ORF through VEGFA was analyzed using western blot. The results showed that DDX11-AS1 was significantly upregulated in colorectal cancer tissues and cells, promoting cancer cell proliferation, migration, and angiogenesis. DDX11-AS1 translated into a functional small protein, DDX11-AS1-ORF, which independently enhanced the malignant behaviors of tumor cells. DDX11-AS1-ORF promoted colorectal cancer progression by activating the p38-MAPK signaling pathway through Vascular Endothelial Growth Factor A (VEGFA). The critical role of the p38-MAPK pathway in DDX11-AS1-ORF mediated tumor promotion was confirmed using the p38-MAPK pathway inhibitor SB203580. In conclusion, the small protein DDX11-AS1-ORF, encoded by DDX11-AS1, plays a crucial role in the development of colorectal cancer by promoting tumor proliferation, migration, and angiogenesis through the activation of VEGFA and the p38-MAPK signaling pathway. These findings provide a novel potential target for molecular targeted therapy in colorectal cancer.

Keywords: Colorectal cancer; VEGFA; lncRNA; p38-MAPK pathway; small protein.

Figure 1

Expression of DDX11-AS1 in colorectal cancer tissues and its impact on cell proliferation and migration. A. qRT-PCR analysis of DDX11-AS1 mRNA expression in 10 pairs of colorectal cancer tissues. Results show significant upregulation of DDX11-AS1 in cancerous tissues. Statistical analysis was performed using independent samples t-test. B. Transfection of interference fragments of DDX11-AS1 in colorectal cancer cell lines SW480 and HCT116, with proliferation assessed by CCK8 assay. Statistical analysis was performed using repeated measures ANOVA. C. Colony formation assay results indicating a significant decrease in the proliferation of SW480 and HCT116 cells following DDX11-AS1 knockdown. One-way ANOVA was used for statistical analysis. D. Transwell migration assay demonstrating a significant reduction in the migratory ability of colorectal cancer cells post-DDX11-AS1 interference. One-way ANOVA was used for statistical analysis. *P<0.05, ***P<0.001, ****P<0.0001.

Figure 2

Effects of DDX11-AS1 on migration and angiogenesis in colorectal cancer cells. A. Wound healing assay results showing a significant reduction in the migratory ability of colorectal cancer cells following DDX11-AS1 interference. One-way ANOVA was used for statistical analysis. B. In vitro tube formation assay indicating that DDX11-AS1 interference inhibited capillary tube formation in human umbilical vein endothelial cells (HUVECs). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. **P<0.01, ***P<0.001, ****P<0.0001.

Figure 3

Verification of peptide DDX11-AS1-ORF translated from DDX11-AS1. A. Western blot analysis to detect whether DDX11-AS1-ORF can be translated into a peptide. Results confirm the translation of an 89 amino acid peptide, DDX11-AS1-ORF. B. Immunofluorescence experiments further verifying the expression of DDX11-AS1-ORF in HCT116 and SW480 cells. Results demonstrate the translation of peptide DDX11-AS1-ORF.

Figure 4

Functional validation of peptide DDX11-AS1-ORF. A. Overexpression of DDX11-AS1-ORF following interference of DDX11-AS1. Western blot analysis performed to verify protein expression of DDX11-AS1-ORF. B. CCK8 assay to assess the proliferative capability of DDX11-AS1-ORF in colorectal cancer cells HCT116 and SW480. C. Colony formation assay confirming the impact of DDX11-AS1-ORF on the proliferation of colorectal cancer cells. D. Transwell migration assay evaluating the influence of DDX11-AS1-ORF on the migratory ability of colorectal cancer cells. For repeated data measured at multiple time points from the same group of subjects, use Repeated Measures ANOVA for analysis, Statistical analysis for other comparisons between groups was performed using one-way ANOVA followed by Dunnett’s test. **P<0.01, ***P<0.001, ****P<0.0001.

Figure 5

Impact of DDX11-AS1-ORF on migration and angiogenesis in colorectal cancer cells. A. Wound healing assay verifying the effect of DDX11-AS1-ORF on the migratory ability of colorectal cancer cells HCT116 and SW480. Statistical analysis was performed using repeated measures ANOVA. B. In vitro tube formation assay assessing whether DDX11-AS1-ORF can independently promote capillary tube formation in human umbilical vein endothelial cells (HUVECs). One-way ANOVA followed by Dunnett’s test was used for statistical analysis. ***P<0.001, ****P<0.0001.

Figure 6

Oncogenic activation of the p38-MAPK pathway by peptide DDX11-AS1-ORF via VEGFA. A. Western blot analysis of VEGFA protein expression following overexpression of DDX11-AS1-ORF. Statistical analysis was performed using independent samples t-test. B. Western blot analysis evaluating the activation of p38-MAPK and PI3K pathways by DDX11-AS1-ORF. Independent samples t-test was used for statistical analysis. C. Post-transfection of DDX11-AS1-ORF plasmid and interference with VEGFA expression, Western blot analysis assessing the impact on p38-MAPK pathway activation. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. D. CCK8 assay evaluating the proliferation of colorectal cancer cells following inhibition of the p38-MAPK pathway using the inhibitor SB203580. Statistical analysis was performed using repeated measures ANOVA. E. Colony formation assay showing that inhibition of the p38-MAPK pathway reduces the proliferative effect of DDX11-AS1-ORF on colorectal cancer cells. One-way ANOVA was used for statistical analysis. F. Transwell migration assay examining the effect of DDX11-AS1-ORF on colorectal cancer cell migration following p38-MAPK pathway inhibition. One-way ANOVA was used for statistical analysis. G. Wound healing assay further confirming the impact of DDX11-AS1-ORF on the migratory ability of colorectal cancer cells post-p38-MAPK pathway inhibition. Statistical analysis was performed using repeated measures ANOVA. H. Tube formation assay indicating that inhibition of the p38-MAPK pathway reduces the angiogenic capabilities of DDX11-AS1-ORF in human umbilical vein endothelial cells (HUVECs). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. **P<0.01, ***P<0.001, ****P<0.0001.