MiR-200c/FUT4 axis prevents the proliferation of colon cancer cells by downregulating the Wnt/β-catenin pathway

Abstract

Background: MicroRNA (miR)-200c has been widely reported to be involved in colon cancer progress. However, the mechanisms of miR-200c in regulating tumor metastasis and growth remain to be fully elucidated. This study aimed to investigate the mechanism of miR-200c targets fucosyltransferase 4 (FUT4) on the proliferation of colon cancer.

Methods: The miR-200c and FUT4 mRNA levels in LoVo and SW480 cells were measured by real-time quantitative polymerase chain reaction. Further, miR-200c mimic, FUT4 siRNA and FUT4 mimic were transfected into cells, separately. Cell counting kit-8, plate colony formation and transwell assays were used to analyse the cells biological behaviour.. Immunofluorescence was used to analyse the Ki-67 expression Moreover, the Wnt/β-catenin pathway-related proteins were detected by western blots. A double luciferase experiment was performed to confirm the relationship between miR-200c and FUT4. In vivo, tumour growth and Wnt/β-catenin pathway-related proteins were also analysed.

Results: In vitro, the expression of miR-200c and FUT4 were negatively correlated in LoVo and SW480 cells (correlation coefficients were - 0.9046 and - 0.9236, respectively). MiR-200c overexpression inhibited the proliferation, migration and invasion of LoVo and SW480 cells by downregulating FUT4. The Ki67-positive cells and Wnt/β-catenin signalling pathway-related proteins were reduced in the miR-200c overexpression and FUT4 silencing groups. A dual luciferase reporting system identified FUT4 as the target of miR-200c. The results in vivo were further confirmed the foundation of cells study.

Conclusions: In summary, miR-200c overexpression inhibits proliferation of colon cancer targeting FUT4 to downregulate the Wnt/β-catenin pathway, which promises molecular targets to inhibit metastasis for colon cancer therapy.

Keywords: Colon cancer; FUT4; Wnt/β-catenin pathway; miR-200c.

Fig. 1

The mRNA expression of miR-200c and FUT4 in colon cancer cells. a The mRNA expression of miR-200c and FUT4 in LoVo cells was analysed by RT-qPCR; b The mRNA expression of miR-200c and FUT4 in SW480 cells was analysed by RT-qPCR; c The correlation between miR-200c and FUT4 in LoVo and SW480 cells was determined using Pearson correlation analysis. Data are expressed as the mean ± SD. The t-test was used for data analysis between two groups. In this experiment, the analyses were repeated 3 times. The resolution of figure is 300 pixels

Fig. 2

FUT4 is a downstream target gene of miR-200c in colon cancer. a Sequence alignment between miR-200c and FUT4. The WT binding sites of miR-200c on FUT4 and its mutated binding sequences are shown. b Luciferase activity in cells following cotransfection with miR-200c mimic and luciferase reporters containing WT- or mut-FUT4 transcript. In this experiment, the analyses were repeated 3 times. Data are expressed as the mean ± SD. ^*p < 0.05. The resolution of figure is 300 pixels

Fig. 3

MiR-200c overexpression inhibits the proliferation of colon cancer cells by downregulating FUT4. a The transfection efficiency of miR-200c and FUT4 in LoVo cells was measured by RT-qPCR; b The transfection efficiency of miR-200c and FUT4 in SW480 cells was measured by RT-qPCR; c The cell viability was measured by CCK-8 assays; d The proliferation of cells was analysed using plate colony formation assays. ^*p < 0.05 vs. the BC group; ^#p < 0.05 vs. miR-200c; ^&p < 0.05 vs. miR-200c + the NC1 group. Data are expressed as the mean ± SD. ANOVA with Turkey’s t test was used for data analysis among groups. Each group was analysed in triplicate. The resolution of figure is 300 pixels. BC: blank control group; miR-200c: miR-200c overexpression group; si-FUT4: FUT4 silencing group; miR-200c + NC1: miR-200c overexpression and FUT4 negative control group; miR-200c + FUT4: miR-200c overexpression and FUT4 overexpression group

Fig. 4

MiR-200c overexpression inhibits the migration and invasion of colon cancer cells by downregulating FUT4. Transwell assays were used to analyse the effect of miR-200c overexpression on the migration and invasion of LoVo cells (a) and SW480 cells (b). Magnification: × 400. scale = 50 μm ^*p < 0.05 vs. the BC group; ^#p < 0.05 vs. miR-200c; ^&p < 0.05 vs. the miR-200c + NC1 group. Data are expressed as the mean ± SD. ANOVA with Turkey’s t test was used for data analysis among groups. Each group was analysed in triplicate. The resolution of figure is 300 pixels. BC: blank control group; miR-200c: miR-200c overexpression group; si-FUT4: FUT4 silencing group; miR-200c + NC1: miR-200c overexpression and FUT4 negative control group; miR-200c + FUT4: miR-200c overexpression and FUT4 overexpression group

Fig. 5

Immunofluorescence was used to analyse the effect of miR-200c overexpression on the expression of Ki67 in colon cancer cells (magnification, × 400, scale = 50 μm). Ki-67 was labelled using green fluorescence (FITC), nuclei were labelled using blue fluorescence (DAPI), and merge indicates the two fluorescence superpositions. ^*p < 0.05 vs. the BC group; ^#p < 0.05 vs. miR-200c; ^&p < 0.05 vs. the miR-200c + NC1 group. Data are expressed as the mean ± SD. ANOVA with Turkey’s t test was used for data analysis among groups. Each group was analysed in triplicate. The resolution of figure is 300 pixels. BC: blank control group; miR-200c: miR-200c overexpression group; si-FUT4: FUT4 silencing group; miR-200c + NC1: miR-200c overexpression and FUT4 negative control group; miR-200c + FUT4: miR-200c overexpression and FUT4 overexpression group

Fig. 6

Western blot analysis was used to detect the effect of miR-200c overexpression on the expression of Wnt/β-catenin-related proteins in LoVo cells (a) and SW480 cells (b). ^*p < 0.05 vs. the BC group; ^#p < 0.05 vs. miR-200c; ^&p < 0.05 vs. the miR-200c + NC1 group. p-GSK-3β/GSK-3β: the ratio of phosphorylated GSK-3β (p-GSK-3β) and total GSK-3β. Data are expressed as the mean ± SD. ANOVA with Turkey’s t test was used for data analysis among groups. Each group was analysed in triplicate. The resolution of figure is 300 pixels. BC: blank control group; miR-200c: miR-200c overexpression group; si-FUT4: FUT4 silencing group; miR-200c + NC1: miR-200c overexpression and FUT4 negative control group; miR-200c + FUT4: miR-200c overexpression and FUT4 overexpression group. The full length blots were presented in Supplementary Figure 1

Fig. 7

MiR-200c overexpression inhibits tumour growth by downregulating FUT4. a Tumour volume; b tumour weight; c tumour image; d the expression of Ki-67 was analysed by immunohistochemistry (magnification,× 400, scale = 50 μm). The resolution of figure is 300 pixels. ^*p < 0.05 vs. the model group; ^#p < 0.05 vs. the miR-200c group; ^&p < 0.05 vs. the miR-200c + NC1 group. Model: blank control group; miR-200c: miR-200c overexpression group; si-FUT4: FUT4 silencing group; miR-200c + NC1: miR-200c overexpression and FUT4 negative control group; miR-200c + FUT4: miR-200c overexpression and FUT4 overexpression group

Fig. 8

MiR-200C overexpression inhibits the Wnt/β-catenin pathway by downregulating FUT4 in tumour tissues. a Wnt/β-catenin pathway-related proteins in LoVo tumour tissues of mice; b Wnt/β-catenin pathway-related proteins in SW80 tumour tissues of mice. The resolution of figure is 300 pixels. ^*p < 0.05 vs. the model group; ^#p < 0.05 vs. the miR-200c group; ^&p < 0.05 vs. the miR-200c + NC1 group. Model: blank control group; miR-200c: miR-200c overexpression group; si-FUT4: FUT4 silencing group; miR-200c + NC1: miR-200c overexpression and FUT4 negative control group; miR-200c + FUT4: miR-200c overexpression and FUT4 overexpression group. The full length blots were presented in Supplementary Figure 2