Long non-coding RNA LINC01503 promotes colorectal cancer cell proliferation and invasion by regulating miR-4492/FOXK1 signaling

Abstract

Increasing evidence indicates that long non-coding RNAs (lncRNAs) are closely associated with the progression of human cancer, including colorectal cancer (CRC). A previous study suggested that lncRNA LINC01503 promotes squamous cell carcinoma progression. However, the function of LINC01503 in CRC has remained elusive. The present study indicated that LINC01503 was significantly upregulated in CRC tissues compared with that in adjacent normal tissues as detected by reverse transcription-quantitative polymerase chain reaction. It was demonstrated that knockdown of long intergenic non-protein coding RNA (LINC)01503 markedly inhibited the proliferation and invasion of CRC cells, whereas overexpression of LINC01503 had the opposite effects, as indicated by Cell Counting kit-8 and Transwell assays. Mechanistically, it was revealed that LINC01503 serves as a sponge for microRNA (miR)-4492, which targets forkhead box K1 (FOXK1) in CRC cells. In addition, luciferase reporter assays demonstrated the direct binding of miR-4492 mimics to LINC01503 and to a sequence in the 3'-untranslated region of FOXK1. Furthermore, it was demonstrated that overexpression of LINC01503 reduced the availability of miR-4492 in CRC cells. Furthermore, miR-4492 mimics inhibited FOXK1 expression, while simultaneous overexpression of LINC01503 abolished this effect. Finally, it was demonstrated that restoration of FOXK1 abolished the inhibitory effect of LINC01503 knockdown on CRC cell proliferation and invasion. Taken together, the present results suggested that LINC01503 promotes CRC progression via acting as a competing endogenous RNA for miR-4492/FOXK1.

Keywords: LINC01503; colorectal cancer; forkhead box K1; invasion; microRNA-4492; proliferation.

Figure 1.

LINC01503 is highly expressed in CRC tissues. (A) Relative expression levels of LINC01503 in CRC tissues (n=47) and adjacent normal tissues (n=28) were determined by RT-qPCR. (B) Relative expression levels of LINC01503 in 28 pairs of CRC tissues and adjacent normal tissues were analyzed by RT-qPCR. (C) The expression levels of LINC01503 in CRC cell lines and the non-cancerous cell line FHC were examined by RT-qPCR. Values are expressed as the mean ± standard deviation. *P<0.05. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CRC, colorectal cancer.

Figure 2.

Silencing of LINC01503 inhibits CRC cell proliferation and invasion. (A) Relative expression levels of LINC01503 in HCT8 and SW620 cells transfected with siLINC01503 or siNC were measured by reverse transcription-quantitative polymerase chain reaction. (B) A Cell Counting kit-8 assay was used to determine CRC cell proliferation. (C) A Transwell assay was utilized to measure CRC cell invasion (magnification, ×100). Values are expressed as the mean ± standard deviation. *P<0.05. CRC, colorectal cancer; siLINC01503/siLINC, small interfering RNA selective for LINC01503; siNC, control siRNA; OD, optical density.

Figure 3.

LINC01503 overexpression promotes colorectal cancer cell proliferation and invasion. (A) Relative expression levels of LINC01503 in HCT8 and SW620 cells transfected with pCDNA3-LINC01503 or empty vector were analyzed by reverse transcription-quantitative polymerase chain reaction. (B) A Cell Counting kit-8 assay was used to detect the proliferation in HCT8 and SW620 cells transfected with pCDNA3-LINC01503 or empty vector. (C) A Transwell assay was used to assess the invasion of HCT8 and SW620 cells transfected with pCDNA3-LINC01503 or empty vector (magnification, ×100). Values are expressed as the mean ± standard deviation. *P<0.05. OD, optical density; oeLINC, LINC01503 overexpression vector.

Figure 4.

LINC01503 facilitates FOXK1 expression via sponging miR-4492. (A) Predicted binding sites of miR-4492 in LINC01503 and the 3′-UTR of FOXK1. (B) Relative expression of miR-4492 in HCT8 and SW620 cells transfected with miR-4492 mimics or controls. (C and D) Luciferase reporter assays were used to verify the interaction between miR-4492 with LINC01503 or the 3′-UTR of FOXK1 in HCT8 and SW620 cells. (E) Overexpression of LINC01503 inhibited the expression of miR-4492 available in HCT8 and SW620 cells. (F) miR-4492 mimics inhibited the mRNA expression of FOXK1 in HCT8 and SW620 cells, while ectopic expression LINC01503 abrogated this effect. (G) Relative expression of miR-4492 in CRC tissues and adjacent normal tissues. (H) Relative expression of FOXK1 in CRC tissues and adjacent normal tissues. (I) Assessment of the correlation between miR-4492 and FOXK1 in CRC tissues by reverse transcription-quantitative polymerase chain reaction. Values are expressed as the mean ± standard deviation. *P<0.05. CRC, colorectal cancer; UTR, untranslated region; miR, microRNA; FOXK1, forkhead box K1; oeLINC, LINC01503 overexpression vector; NC, negative control; mut, mutated; WT, wild-type.

Figure 5.

Restoration of FOXK1 abrogates the effects of LINC01503 knockdown. HCT8 and SW620 cells subjected to LINC01503 knockdown and optional transfection with FOXK1 overexpression vector. (A) The relative expression levels of FOXK1 were measured by reverse transcription-quantitative polymerase chain reaction. (B) A Cell Counting kit-8 assay was used to determine the proliferation of the cells. (C) Transwell assays were utilized to analyze the invasion of the cells (magnification, ×100). Values are expressed as the mean ± standard deviation. *P<0.05. FOXK1, forkhead box K1; siLINC01503/siLINC, small interfering RNA selective for LINC01503; siNC, control siRNA; oeLINC, LINC01503 overexpression vector; OD, optical density.