Long Non-Coding RNA LOXL1-AS1 Enhances Colorectal Cancer Proliferation, Migration and Invasion Through miR-708-5p/CD44-EGFR Axis

Abstract

Introduction: Colorectal cancer (CRC), the third most common cancer worldwide, involves a physiological and pathological long non-coding RNA (lncRNA) paradigm shift. It has been reported that the lncRNA LOXL1-AS1 affects tumor development for many kinds of cancers, but its functions and mechanisms in CRC remain unknown.

Methods: Expression levels of LOXL1-AS1 and miR-708-5p within CRC tissues and cell lines were measured using qRT-PCR. The performance of gain-of-function and loss-of-function assays was aimed at examining the effects of LOXL1-AS1 and miR-708-5p; colony formation and cell viability assays were carried out to measure cell multiplication; and Transwell migration and wound-healing assays were carried out for the measurement of cell migration and invasion. Luciferase reporter assay was used to verify the interactions between LOXL1-AS1 and miR-708-5p and between miR-708-5p and the CD44-EGFR signaling pathway. Finally, expression of CD44 and EGFR proteins was measured by Western blot and immunofluorescence assays.

Results: In this study, we reveal that the regulation of lncRNA LOXL1-AS1 occurs within CRC based on the correlation with poor clinical outcomes. LOXL1-AS1 knockdown along with miR-708-5p overpresentation in CRC cell lines inhibited cell multiplication, migration, and invasion. The inhibiting effect of LOXL1-AS1 knockdown on CRC was reversed by upregulating the CD44-EGFR signal pathway. From the perspective of mechanism, LOXL1-AS1 imposes sponging upon miR-708-5p and thereby promotes the CD44-EGFR signal pathway in CRC cells.

Discussion: This study demonstrated that lncRNA LOXL1-AS1 enhances multiplication, migration, invasion, and progression of CRC by sponging miR-708-5p to regulate the CD44-EGFR signal pathway.

Keywords: CD44-EGFR signal pathway; LOXL1-AS1; colorectal cancer; miR-708-5p; migration.

Figure 1

LOXL1-AS1 was downregulated in CRC and acted as an oncogene. (A) Expression of LOXL1-AS1 in CRC tissue (n = 40) as determined with qRT-PCR. (B and C) Expression of LOXL1-AS1 in cultured CRC cells as determined with qRT-PCR. (D and E) Cell viability as evaluated with CCK-8 assay. (F and G) Assessment of clonogenic capacity evaluated with the cell colony formation assay. (H and I) Invasion capability evaluated by Transwell invasion assays. (J and K) Migratory capability evaluated by wound-healing assays. Photomicrographs show typical images of Lovo and SW480 cells (400×). Data are presented as mean ± SD of 3 independent experiments. *P < 0.01.

Figure 2

MiR-708-5p binds directly to LOXL1-AS1 and expression of miR-708-5p was downregulated in CRC. (A) Expression of miR-708-5p in CRC tissues (n = 40) as determined with qRT-PCR. (B) Presentation of miR-708-5p in CRC cells as determined with qRT-PCR. (C) Prediction of binding site of LOXL1-AS1 within the 3ʹUTR of miR-708-5p according to TargetScan. (D and E) Luciferase activity of LOXL1-AS1 as detected with the dual-luciferase reporter assay. (F) Expression of miR-708-5p in CRC cells as detected by qRT-PCR. Data are presented as mean ± SD of 3 independent experiments. *P < 0.01.

Figure 3

miR-708-5p inhibition completely blocks the effect of LOXL1-AS1 downregulation in Lovo and SW480. (A) Expression of LOXL1-AS1 in CRC tissues as determined by qRT-PCR. (B and C) Evaluation of cell viability by CCK-8 assays. (D and E) Clonogenic capacity assessed by cell colony formation assays. (F and G) Invasion capability evaluated by Transwell invasion assays. (H and I) Migratory capability evaluated by wound-healing assays. Photomicrographs show typical appearance of the Lovo and SW480 cells (400×). Data are presented as mean ± SD of 3 independent experiments. *p < 0.01 versus the si-NC group. ^#p < 0.01 versus the si-LOXL1-AS1 group.

Figure 4

CD44-EGFR was the target pathway of miR-708-5p. (A) The predication of binding site featured by miR-708-5p within the 3ʹUTR of CD44 was made using TargetScan. (B and C) Luciferase activity of the CD44 as detected by dual-luciferase reporter assay. (D–F) Expression of CD44 and EGFR in Lovo and SW480 cells as demonstrated with Western blot assays. (G–J) Luciferase activity of CD44 and EGFR in Lovo and SW480 cells examined by immunofluorescence assay. Representative images of the Lovo and SW480 cells (400×). Data are presented as mean ± SD of 3 independent experiments. *p < 0.01 versus the si-NC group. ^#p < 0.01 versus the si-LOXL1-AS1 group.

Figure 5

CD44 overexpression completely blocks the effect of LOXL1-AS1 downregulation in Lovo and SW480 cells. (A and B) Expression of CD44 in Lovo and SW480 cells by Western blot assays. (A and C) EGFR expression in Lovo and SW480 cells by Western blot assays. (D and E) Evaluation of cell viability of Lovo and SW480 cells by CCK-8 assays. (F and G) Clonogenic capacity of Lovo and SW480 cells by cell colony formation assays. Representative images of the Lovo and SW480 cells are (400×). Data are presented as mean ± SD of 3 independent experiments. *p < 0.01 versus the si-NC group. ^#p < 0.01 versus the si-LOXL1-AS1 group.

Figure 6

CD44 overexpression completely blocks the effect of LOXL1-AS1 downregulation in Lovo and SW480 cells. (A and B) Invasion capability of Lovo and SW480 cells in Transwell invasion assays. (C and D) Migratory capability of Lovo and SW480 cells by wound-healing assays. Representative images of the Lovo and SW480 cells are (400×). Data are presented as mean ± SD of 3 independent experiments. *p < 0.01 versus the si-NC group. ^#p < 0.01 versus the si-LOXL1-AS1 group.