Long non-coding RNA CCAL/miR-149/FOXM1 axis promotes metastasis in gastric cancer

Abstract

Early evidence indicates that the long non-coding RNA CCAL plays a critical role in cancer progression and metastasis. However, the overall biological role and clinical significance of CCAL in gastric tumourigenesis and progression remain largely unknown. We observed that CCAL was upregulated in gastric cancer tissues and was associated with the tumour-node-metastasis stage. Functional experiments showed that CCAL promoted gastric cancer cell proliferation and metastasis in vitro and in vivo. Luciferase reporter assay indicated that CCAL directly bind to miR-149. Moreover, knockdown of CCAL significantly reduced the expression of FOXM1, a direct target of miR-149. We also showed that FOXM1 suppression by miR-149 could be partially rescued by CCAL overexpression. In addition, we identified a negative correlation between the mRNA expression of CCAL and miR-149 in gastric cancer tissues. Furthermore, we observed a negative correlation between the expression of miR-149 and FOXM1 and a positive correlation between CCAL and FOXM1 levels. These results demonstrated that the CCAL/miR-149/FOXM1 axis functions as a key regulator in gastric cancer metastasis and CCAL potentially represents a biomarker for diagnosis and potential target for therapy in the future.

Fig. 1. The expression of CCAL in gastric cancer tissues and cell lines.

a Relative CCAL expression in gastric cancer and paired adjacent normal tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR) (N = 48). b Relative CCAL expression in the gastric cancer patients for stage I + II and stage III + IV. c Relative CCAL expression in gastric cancer cell lines (BGC-823, HGC-27, MKN-28 and SGC-7901) compared to normal gastric epithelial cell line GES-1. d Relative CCAL expression level in gastric cancer cells after transfected with shRNA or sh-NC. e Relative CCAL expression level in gastric cancer cells after transfected with pcDNA-CCAL or vector. Data was presented with mean ± SD, the error bars represent the SD obtained from three independent experiments. f ROC curve for prediction of gastric cancer using CCAL expression level. The AUC was 0.6988, ROC for CEA and CA19-9 were also been showed. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2. CCAL promotes proliferation, cycle progression and inhibits apoptosis of gastric cancer cells in vitro.

a, b CCK-8 assays were used to determine the cell viability of gastric cancer cells transfected with sh-CCAL or pcDNA-CCAL with relative control. *pcDNA-CCAL; #sh-CCAL. c Colony formation assays were used to determine the cell colony formation ability of sh-CCAL or pcDNA-CCAL-transfected gastric cancer cells. The number of colonies was calculated and plotted on a histogram. d, e FACS analysis was used to determine the effect of sh-CCAL or pcDNA-CCAL on cell cycle and apoptosis. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3. CCAL promotes cell migration and invasion in gastric cancer cells.

a, b Wound-healing assay were used to investigate the horizontal migration ability with CCAL knockdown or overexpression in gastric cancer cells, and relative gap distance was calculated and plotted on a histogram. c, d Migration and invasion assays were used to investigate the vertical migration and invasion abilities with CCAL knockdown or overexpression in gastric cancer cells, and the number of cells was calculated and plotted on a histogram. e Western blot was used to analyze the expression of metastasis-related proteins with CCAL knockdown or overexpression in gastric cancer cells. Data from Western Blot assay has been represented as a quantification graph normalized to the levels of GAPDH together with the statistical tests. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4. Knockdown of CCAL inhibits gastric cancer cell tumour growth and metastasis in vivo.

a Tumour volume was measured every 5 days. b The mean weight of sh-CCAL group-derived xenograft tumours was also significantly less than sh-NC group-derived xenograft tumours. c Representative images of the xenograft tumours obtained from sh-NC or sh-CCAL group. Left, images of the xenograft tumours from the two group. Middle, images of H&E-stained tumour tissues. Right, immunohistochemical images showing the intensity of Ki-67 expression. d Representative images of the lungs obtained from sh-NC or sh-CCAL group. Left, images of the lungs from the two group. Middle, images of HE-stained lung tissues. Right, immunohistochemical images showing the intensity of vimentin expression. ***P < 0.001

Fig. 5. MiR-149 is a direct target of CCAL.

a The predicted positions of candidate miRNAs binding sites on the CCAL transcript. b QRT-PCR for miRNA levels in SGC-7901 cells after transfected with sh-CCAL. Luciferase activity in 293 T cells co-transfected with miR-149 (c) or miR-31 (d) mimics and the luciferase reporters (the mutant type CCAL) or control (the wild type CCAL). Renilla luciferase activity was measured and normalized to firefly luciferase. The experiment was repeated at least three times, and data are presented as the mean ± SD (Two-sided Student’s t-test). **P < 0.01, ***P < 0.001

Fig. 6. CCAL negatively regulates miR-149.

a Relative miR-149 expression level in gastric cancer and adjacent normal tissues was detected by qRT-PCR (N = 48). b Relative miR-149 expression in gastric cancer cell lines normalized to GES-1. c The linear correlation between the expression levels of CCAL and miR-149 in gastric cancer tissues (r = −0.2932) (P = 0.0431). Wound-healing assays were conducted in HGC-27 (d) and SGC-7901 (e) cells transfected with miR-149 mimic or co-transfected with miR-149 mimic and pcDNA-CCAL, and relative gap distance was calculated and plotted on a histogram. Migration (f)and invasion (g) assays were used to investigate the vertical migration and invasion ability in gastric cancer cells, and the number of cells was calculated and plotted on a histogram. h Western blot was used to analyze the expression of metastasis-related proteins in gastric cancer cells. *P < 0.05, **P < 0.001, ***P < 0.001

Fig. 7. The CCAL /miR-149/FOXM1 axis promotes metastasis of gastric cancer.

a MiR-149 negatively regulates FOXM1 in gastric cancer cells. 149 M, MN, IN, 149I represent miR-149 mimic, mimic control, inhibitor control, miR-149 inhibitor, relatively. b Relative FOXM1 expression in gastric cancer tissues (N = 48). c Relative FOXM1 expression in gastric cancer cell lines normalized to GES-1. d The linear correlation between the expression levels of miR-149 and FOXM1 in gastric cancer tissues (r = −0.3073) (P = 0.0336). e The linear correlation between the expression levels of CCAL and FOXM1 in gastric cancer tissues (r = 0.6652) (P < 0.001). f CCAL positively regulates FOXM1 in gastric cancer cells. g Western blot assays were performed to test FOXM1 expression after HGC-27 and SGC-7901 cells were transfected with miR-149 mimic or co-transfected with miR-149 mimic and pcDNA-CCAL. Data from Western Blot assay has been represented as a quantification graph normalized to the levels of GAPDH together with the statistical tests. *P < 0.05, **P < 0.001, ***P < 0.001

Fig. 8

Model for CCAL-regulating FOXM1 by CCAL/miR-149/FOXM1 axis