Long non-coding RNA FOXD2-AS1 contributes to colorectal cancer proliferation through its interaction with microRNA-185-5p

Abstract

Emerging evidence has indicated that long non-coding RNA plays an important role in carcinogenesis at the transcriptional and post-translational levels. The regulation of carcinogenesis-related effectors is potent in the determination of tumor initiation and progression. In the current study, FOXD2-AS1 was found to interact with microRNA (miR)-185-5p to modulate proliferation, migration, and invasion of colorectal cancer (CRC) cells. Interestingly, expression of cell division control 42 was significantly influenced by FOXD2-AS1 and miR-185-5p. In CRC patients, the expression level of FOXD2-AS1 in CRC tissue was closely associated with miR-185-5p and cell division control 42, and implicated in the overall survival rate. Therefore, our study suggests that long non-coding RNA FOXD2-AS1 plays a positive role in CRC and could be developed and used as a potential biomarker for the diagnosis and therapy of CRC. This will greatly improve the prevention and treatment of the third most common cancer worldwide.

Keywords: FOXD2-AS1; biomarker; colorectal cancer; long non-coding RNA; microRNA-185-5p.

Figure 1

Increased mRNA expression levels of FOXD2‐AS1 in colorectal cancer (CRC). The mRNA level was determined by quantitative RT‐PCR in human CRC tissue (A) and in four CRC cell lines (B) compared with non‐tumor tissue and normal CRC cells, respectively. **P < .0002, unpaired t‐test

Figure 2

FOXD2‐AS1 promotes cell proliferation, migration, and invasion. Overexpression of FOXD2‐AS1 in HCT116 and SW620 colorectal cancer cells induced accelerated cell growth, evaluated by MTT assay (A) and crystal violet staining (B). Cell migration and invasion were determined by wound healing assay (C) and Transwell assay (D), respectively. Conversely, reduced expression of FOXD2‐AS1 in HT‐29 and LOVO cells by siRNA knockdown suppressed cell growth (A,B), migration (C) and invasion (D). *P < .05, **P < .01. two‐way ANOVA test. siNC, scrambled control siRNA

Figure 3

FOXD2‐AS1 interacts with microRNA (miR)‐185‐5p in colorectal cancer cells. A, Predicted interaction between human miR‐185‐5p and FOXD2‐AS1 in Starbase. B, Synthetic biotin‐labeled microRNAs were transfected into HCT116 and SW620 cells and mRNA levels of FOXD2‐AS1 were tested by quantitative RT‐PCR in precipitates pulled down by streptavidin (B). **P < .01, two‐way ANOVA test

Figure 4

FOXD2‐AS1 negatively regulates microRNA (miR)‐185‐5p and positively regulates cell division control protein 42 (CDC42) expression. A,B, miR‐185‐5p levels were measured by quantitative RT‐PCR in HCT116 and SW620 cells overexpressing FOXD2‐AS1 (A) and in HT‐29 and LOVO cells with knockdown of FOXD2‐AS1 expression (B). C‐E, CDC42 mRNA levels (C,D) and protein levels (E) were evaluated in these cells. F,G, Luciferase assay was carried out to investigate the regulatory effects of FOXD2‐AS1 on CDC42 regulatory 3′‐UTR region in these cells. **P < .01, two‐way ANOVA test

Figure 5

Antagonistic effects of FOXD2‐AS1 and microRNA (miR)‐185‐5p in colorectal cancer. SW620 cells were transfected with control (NC) miRNA mimics, pCDNA3‐FOXD2‐AS1 + NC mimics, or pCDNA3‐FOXD2‐AS1 + miR‐158‐5p mimics. A‐D, Cell proliferation (A,B), migration (C), and invasion (D) were investigated. E,F, Cell division control protein 42 (CDC42) level was induced by FOXD2‐AS1 overexpression at protein (E) and mRNA (F) levels. *P < .05, two‐way ANOVA test; **P < .01, one‐way ANOVA test

Figure 6

Correlation of FOXD2‐AS1 with cell division control protein 42 (CDC42), microRNA (miR)‐185‐5p, and colorectal cancer patient survival. Linear regression analysis was used to illustrate the correlation between FOXD2‐AS1 and CDC42 (A) and miR‐185‐5p (B). C, FOXD2‐AS1 expression is positively correlated with 6‐y survival rate in colorectal cancer patients, P = .4629