MicroRNA‑223‑induced inhibition of the FBXW7 gene affects the proliferation and apoptosis of colorectal cancer cells via the Notch and Akt/mTOR pathways

Abstract

The tumour suppressor gene F‑box and WD repeat domain‑containing 7 (FBXW7) plays an important role in human cancer by regulating cell division, proliferation and differentiation. However, the exact regulatory mechanisms of microRNA (miR)‑223 in colorectal cancer (CRC) cells are still unknown. The present study aimed to investigate the effect and mechanism of miR‑223 inhibiting FBXW7 on the proliferation and apoptosis of CRC cells. HCT116 cells were transfected with miR‑223 mimics or small interfering RNA (siRNA) targeting FBXW7 (siFBXW7), and the effects of these treatments on cell proliferation and apoptosis were examined. The downstream Notch and Akt/mTOR pathways were also assessed. Following miR‑223 overexpression, the mRNA and protein expression levels of FBXW7 were downregulated. Transfection with miR‑223 mimics or siFBXW7 promoted the proliferation of HCT116 cells and inhibited apoptosis by promoting the Notch and Akt/mTOR signalling pathways. Conversely, miR‑223 mimics transfection with FBXW7 overexpression inhibited cell viability and restored apoptosis. Thus, the present study demonstrated that miR‑223 could bind to the FBXW7 gene and inhibit its expression, ultimately increasing the proliferation and preventing the apoptosis of CRC cells through the Notch and Akt/mTOR signalling pathways.

Keywords: colorectal cancer; F‑box and WD repeat domain containing 7; microRNA‑223; Notch pathway; Akt/mTOR pathway; proliferation; apoptosis.

Figure 1.

FBXW7 mRNA and protein expression levels in CRC tissues and adjacent normal tissues. (A) Differences in FBXW7 expression in 27 types of tumour tissue and normal tissue samples in The Cancer Genome Atlas and the Genotype Tissue Expression database. The red arrow marks the expression in CRC tissue, compared with healthy tissue. (B) FBXW7 mRNA expression in CRC tissues and normal adjacent tissue. n=20 in each group. (C) FBXW7 protein expression in CRC tissue and normal adjacent tissue. (D) Western blotting results normalized to those of GAPDH levels. (E) Immunohistochemical staining of FBXW7 expression in CRC and normal adjacent tissues. Magnification, ×200. Data are presented as the mean ± SD of three replicates. **P<0.01, ***P<0.001, vs. adjacent (paired Student's t-test). CRC, colorectal cancer; N, normal; T, tumour; FBXW7, F-box and WD repeat domain containing 7; log₂(TPM+1), normalization of gene expression; TPM, transcripts per million.

Figure 2.

miR analysis and detection in CRC tissues and HCT116 cells. (A) miR molecules targeting the FBXW7 gene were predicted using TargetScan and miRanda. miR sequences are marked in blue, and the FBXW7 3′UTR sequence was under them. The putative binding site is shown in red. (B) RT-qPCR detection of miR-223 levels in CRC tissues and adjacent normal tissues. n=20. ***P<0.001 (paired two-tailed Student's t-test). miR, microRNA; CRC, colorectal cancer; FBXW7, F-box and WD repeat domain containing 7; RT-qPCR, reverse transcription-quantitative PCR.

Figure 3.

miR-223 inhibits the expression of FBXW7 in HCT116 cells. (A) miR-223 binding to FBXW7 was detected using a luciferase reporter assay. Comparisons were made using Tukey's test. (B) miR-223 expression in HCT116 cells transfected with miR-223 mimics or inhibitor. (C) FBXW7 protein expression levels in HCT116 cells following transfection with miR-223 mimics or inhibitor. (D) FBXW7 mRNA expression levels in HCT116 cells transfected with miR-223 or inhibitor. Data are presented as the mean ± SD of three replicates. **P<0.01, ***P<0.001 (one-way ANOVA). miR, microRNA; FBXW7, F-box and WD repeat domain containing 7. miCtr, mimics control; Ctr, inhibitor control; NC negative control; n.s., not significant.

Figure 4.

Effect of miR-223 on cell viability and apoptosis of HCT116 cells. (A) Effect of miR-223 mimics transfection on the activity of HCT116 cells. Cells were detected using the Cell Counting Kit-8 reagent after transfection with miR-223 mimics or inhibitor from 0 to 72 h. *P<0.05 vs. miCtr (one-way ANOVA and Tukey's post hoc test). (B) Effect of miR-223 mimics and inhibitor transfection on the early apoptosis of HCT116 cells. (C) Apoptosis rates were recorded following Annexin V-FITC/PI double staining. Data are presented as the mean ± SD of three replicates. **P<0.01 (one-way ANOVA and Tukey's post hoc test). miR, microRNA; FBXW7, F-box and WD repeat domain containing 7; miCtr, mimics control; Ctr, inhibitor control; FITC, fluorescein isothiocyanate; PI, propidium iodide; UL, upper left; UR, upper right; LL, lower left; LR, lower right.

Figure 5.

Effect of FBXW7 knockdown on the viability and apoptosis of HCT116 cells. (A) FBXW7 mRNA expression levels in HCT116 cells transfected with siRNA targeting FBXW7 or si-NC. (B) FBXW7 protein expression levels were detected by western blotting. (C) HCT116 cell viability following transfection with siFBXW7-1 and −2, or si-NC. (D) Cell viability following transfection with miR-223 mimics, miCtr or co-transfection with miR-223 mimics and FBXW7 overexpression plasmid. (E) HCT116 cell apoptosis following transfection with siFBXW7. Apoptotic rates are calculated as the frequency of Annexin V⁺PI⁺ cells relative to total cells. (F) FBXW7 protein expression levels in HCT116 cells transfected with miR-223 mimics and FBXW7 overexpression plasmid. The upper panel shows overexpression of FBXW7 induced by pTriEx-FBXW7 alone, the lower panel shows expression of FBXW7 in cells transfected with pTriEx-FBXW7 and miR-223 mimics. (G) Apoptosis of HCT116 cells following transfection with miR-223 mimics, miCtr or co-transfection with miR-223 mimics and FBXW7 overexpression plasmid. Data are presented as the mean ± SD of three replicates. *P<0.05, **P<0.01 (one-way ANOVA and Tukey's post hoc test). miR, microRNA; FBXW7, F-box and WD repeat domain containing 7; miCtr, mimics control; si, small interfering RNA; NC, negative control; FITC, fluorescein isothiocyanate; PI, propidium iodide; UL, upper left; UR, upper right; LL, lower left; LR, lower right.

Figure 6.

FBXW7 regulates the progression of CRC through the Notch and AKT/mTOR signalling pathways. Relative mRNA expression levels of Notch and Hes-1 in HCT116 cells following (A) miR-223 mimics or (B) siFBXW7−2 transfection. miCtr + si-NC co-transfection was used as a control. (C) Protein expression levels of NICD and Hes-1 in HCT116 cells following miR-223 mimic or siFBXW7−2 transfection. (D) Protein expression levels of total Akt, p-Akt, mTOR and p-mTOR in HCT116 cells following miR-223 mimic or siFBXW7−2 transfection. (E) Protein expression levels of Bax and Bcl-2 after transfection with miR-223 mimics or siFBXW7−2 transfection. (F) Ratios of p-Akt/Akt, p-mTOR/mTOR and Bax/Bcl-2 calculated using the densitometric value of the protein bands, after normalization to GAPDH. Data are presented as the mean ± SD of three replicates. *P<0.05, **P<0.01, ***P<0.001, vs. control (one-way ANOVA and Tukey's post hoc test). miR, microRNA; FBXW7, F-box and WD repeat domain containing 7; si, small interfering RNA; Hes-1; hes family bHLH transcription factor 1; p, phosphorylated; NICD, Notch intracellular receptor domain.