Long non-coding RNA ABHD11-AS1 promotes colorectal cancer development through regulation of miR-133a/SOX4 axis

Abstract

Recently, lncRNA has been verified to regulate the development and progression of tumor. LncRNA ABHD11-AS1 has been proven to serve as an oncogene in several cancers. However, the role of ABHD11-AS1 in colorectal cancer remains totally unknown. In the present study, qRT-PCR assay revealed that ABHD11-AS1 expression was markedly higher in colorectal cancer tissues and cell lines. In addition, patients who displayed overexpression of ABHD11-AS1 showed a significantly poorer progression free survival (PFS) and overall survival (OS) by Kaplan-Meier analysis. Loss-of-function experiments suggested that silencing of ABHD11-AS1 expression could significantly reduce the proliferation, colony formation, migration and invasion of colorectal cancer cells, and increase cell apoptosis. Moreover, bioinformatics analysis, biotin pull-down assay, luciferase reporter assay, and RIP assay disclosed that ABHD11-AS1 straightly interacted with miR-133a. We also found that SOX4 was a downstream target of miR-133a and ABHD11-AS1 subsequently exerted its biological effects via modulating the expression of SOX4 in colorectal cancer cells. Collectively, these findings manifested that the ABHD11-AS1/miR-133a/SOX4 axis may be a cogitable and promising therapeutic target for colorectal cancer.

Keywords: colorectal cancer; large intervening non-coding RNA; microRNA; tumorigenesis.

Figure 1. Screening out ABHD11-AS1 (WBSCR26) from the Cancer Genome Atlas (TCGA) in cancers by an interactive web server ‘GEPIA’

RNA-Seq data from TCGA of lncRNA ABHD11-AS1 in different cancers were analyzed, involved in colon cancer tissues (n = 275), paired normal colon tissue (n = 349) and rectal cancer tissues (n = 92), and paired normal rectal tissue (n = 316).

Figure 2. LncRNA ABHD11-AS1 expression was up-regulated in CRC and associated with poor prognosis of CRC patients

(A) The expression of ABHD11-AS1 in CRC tissues (Tumor = 132) was significantly higher than the pair-matched normal adjacent colorectal tissues (Normal = 132) (**P < 0.01). (B) The expression of ABHD11-AS1 was down-regulated in a series of CRC cell lines compared with human colonic epithelial cell (HcoEpiC), **P < 0.01 compared with HcoEpiC. (C) The expression of ABHD11-AS1 in tumor tissues with high clinical stage (III–IV) was significantly higher than that of low clinical stage (I–II) (**P < 0.01). (D) The expression of ABHD11-AS1 in tumor tissues with lymph node metastasis was significantly higher than that without lymph node metastasis (**P < 0.01). (E) and (F) Higher expression of ABHD11-AS1 was associated with shorter the PFS or OS by Kaplan–Meier analysis and log rank test. *P < 0.05, **P < 0.01.

Figure 3. Knockdown of ABHD11-AS1 abrogated cell proliferation, colony formation, migration, and invasion, promoted cell apoptosis in CRC cells

(A) ABHD11-AS1 expression levels were examined by qRT-PCR in siRNA transfected CRC cells. **P < 0.01 compared with Si-NC. (B) and (C) Knockdown of ABHD11-AS1 significantly inhibited cell proliferation of CRC cells by performing CellTiter-Glo assay. (D) Colony formation assay showed that knockdown of ABHD11-AS1 significantly reduced the colony-forming ability of CRC cells. (E) Knockdown of ABHD11-AS1 significantly promoted cell apoptosis by caspase-3 ELISA assay kit. (F) Knockdown of ABHD11-AS1 significantly inhibited migration and invasion of CRC cells by using transwell assay. Data were expressed as mean ± SD from three independent assay. * P <0.05, **P < 0.01.

Figure 4. ABHD11-AS1 directly interacts with miR-133a

(A) Expression of miRNAs was determined by using qRT-PCR in the sample pulled down by biotinylated ABHD11-AS1 probe. (B) Predict binding sites between ABHD11-AS1 and miR-133a/b. (C) The relative expression of miR-133a in tumor tissues was significantly down-regulated compared with that in adjacent normal tissues. (D) Pearson correlation analysis demonstrated that there was a remarkable negative correlation between miR-133a and ABHD11-AS1 expression in CRC tissues. (E) The relative luciferase activities were inhibited in the HEK-293 cells transfected with the reporter vector WT-ABHD11-AS1, not Mut-ABHD11-AS1. (F) and (G) The RNA immunoprecipitation assay was used to confirm that ABHD11-AS1 and miR-133a could directly bind to AGO2 in CRC cells. (H) Relative expression of miR-133a was determined by qRT-PCR in CRC cells transfected with si-ABHD11-AS1. (I) Relative expression of ABHD11-AS1 in CRC cells transfected with miR-133a mimics was examined by qRT-PCR. Data were expressed as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01.

Figure 5. SOX4 was a novel target of miR-133a and positively regulated by ABHD11-AS1

(A) The 3′-UTR of SOX4 harbored miR-133a cognate site. (B) HEK-293 cells were co-transfected with miR-133a mimic and wild-type (WT) or mutant SOX4 3′-UTR for 48 h, the luciferase activity was measured. (C) and (D) SW480 and HCT-116 cells were transfected with miR-133a mimic for 48 h, the mRNA and protein level of SOX4 was measured. (E) HEK-293 cells were co-transfected with pcDNA-ABHD11-AS1 (ABHD11-AS1), miR-133a mimic and SOX4-5 3′-UTR for 48 h, the luciferase activity was measured. **P < 0.01, compared with NC-mimic. ##P < 0.01, compared with miR-133a mimic. (F) and (G) ABHD11-AS1 positively regulated the level of SOX4 mRNA and protein in SW480 and HCT-116 cells. **P < 0.01.

Figure 6. miR-133a mediated the tumorigenic effects of ABHD11-AS1 in CRC cells

(A) and (B) miR-133a knockdown increased the mRNA and protein expression of SOX4 in ABHD11-AS1 knockdown. (C) Quantify the signal intensity of Western blots. (D) and (E) CellTiter-Glo assays showed that miR-133a could largely reverse the suppressive effects of ABHD11-AS1 knockdown on proliferation. (F) miR-133a silencing partly rescued the colony formation of CRC cells after ABHD11-AS1 knockdown. (G) Down-regulation of miR-133a largely decreased the promotion of apoptosis in CRC cells transfected with si-ABHD11-AS1. (H) and (I) miR-133a silencing partly reversed the suppressive effects of ABHD11-AS1 on migration and invasion. *P < 0.05, **P < 0.01.