ART1 knockdown decreases the IL-6-induced proliferation of colorectal cancer cells

Abstract

Colorectal cancer (CRC) is a worldwide health concern. Chronic inflammation is a risk factor for CRC, and interleukin-6 (IL-6) plays a pivotal role in this process. Arginine-specific mono-ADP-ribosyltransferase-1 (ART1) positively regulates inflammatory cytokines. ART1 knockdown reduces the level of glycoprotein 130 (gp130), a key transducer in the IL-6 signalling pathway. However, the relationship between ART1 and IL-6 and the resulting effects on IL-6-induced proliferation in CRC cells remain unclear. The aims of this study were to investigate the effects of ART1 knockdown on IL-6-induced cell proliferation in vitro and use an in vivo murine model to observe the growth of transplanted tumours. The results showed that compared with the control, ART1-sh cancer cells induced by IL-6 exhibited reduced viability, a lower rate of colony formation, less DNA synthesis, decreased protein levels of gp130, c-Myc, cyclin D1, Bcl-xL, and a reduced p-STAT3/STAT3 ratio (P < 0.05). Moreover, mice transplanted with ART1-sh CT26 cells that had high levels of IL-6 displayed tumours with smaller volumes (P < 0.05). ART1 and gp130 were colocalized in CT26, LoVo and HCT116 cells, and their expression was positively correlated in human CRC tissues. Overall, ART1 may serve as a promising regulatory factor for IL-6 signalling and a potential therapeutic target for human CRC.

Keywords: ART1; Cell proliferation; Colorectal cancer; IL-6; gp130.

Fig. 1

IL-6 induced the proliferation of CT26 cells. (A) Proliferation rates of CT26 WT cells after incubation with different concentrations of IL-6 (0, 5, 10, 20, 50 and 100 ng/ml) for different durations (24, 48 and 72 h). (B-D) Expression levels of gp130, c-Myc, cyclin D1, and Bcl-xL and the p-STAT3/STAT3 ratio in CT26 WT cells after induction with IL-6 (25 ng/ml) for different durations. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. WT, wild-type; p-, phosphorylated; gp130, glycoprotein 130

Fig. 2

ART1 knockdown decreased IL-6-induced cell proliferation. (A) Proliferation rates of WT, NC and ART1-sh CT26 cells after incubation with different concentrations of IL-6 (0, 5, 10, 20, 50 and 100 ng/ml) for 48 h. (B and C) Protein expression levels of ART1 in CT26 and LoVo cells transfected with ART1-sh. (D and E) Protein expression levels of c-Myc, cyclin D1 and Bcl-xL in WT, NC and ART1-sh CT26 cells after induction with IL-6 (25 ng/ml) for 24 h. (F and H) DNA synthesis in WT, NC and ART1-sh CT26 cells after incubation with IL-6 (25 ng/ml) for 24 h. Red fluorescence represents DNA synthesis, and blue fluorescence represents the nuclei. The scale bar is 50 μm. (G and I) Colony formation rate of WT, NC, and ART1-sh CT26 cells after incubation with IL-6 (25 ng/ml) for 2 weeks. ^*P < 0.05, ^**P < 0.01. WT, wild-type; sh, short hairpin RNA; NC, negative control; ART1, mono-ADP-ribosyltransferase-1

Fig. 3

ART1 knockdown inhibited IL-6-induced gp130 and p-STAT3 expression. (A and C) Protein expression level of gp130 and ratio of p-STAT3/STAT3 in NC and ART1-sh CT26 cells after induction with IL-6 (25 ng/ml) for 0.5 or 24 h. (B and D) Protein expression level of gp130 and the p-STAT3/STAT3 ratio in NC and ART1-sh LoVo cells after induction with IL-6 (20 ng/ml) for 0.5 or 24 h. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. WT, wild-type; p-, phosphorylated; gp130, glycoprotein 130; sh, short hairpin RNA; NC, negative control; ART1, mono-ADP-ribosyltransferase-1.

Fig. 4

Knocking down ART1 influenced the IL-6 signalling pathway at the molecular level. (A-D) Protein expression level of gp130 and ratio of p-STAT3/STAT3 in WT, NC and ART1-sh CT26 and LoVo cells. (E and F) Immunofluorescence staining of p-STAT3 in WT, NC and ART1-sh CT26 cells. Red fluorescence represents p-STAT3 in both the nucleus and cytoplasm, and blue fluorescence represents the nuclei. The scale bar is 100 μm. (G and H) mRNA expression level of gp130 in WT, NC and ART1-sh CT26 and LoVo cells. (I) mRNA expression level of IL-6 in WT, NC and ART1-sh CT26 cells. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. WT, wild-type; p-, phosphorylated; gp130, glycoprotein 130; sh, short hairpin RNA; NC, negative control; ART1, mono-ADP-ribosyltransferase-1.

Fig. 5

Double immunofluorescence staining of ART1 and gp130. (A and B) Double immunofluorescence staining of ART1 and gp130 in WT CT26, LoVo and HCT116 cells. Green fluorescence represents ART1, red fluorescence represents gp130, and blue fluorescence represents the nuclei. The scale bar is 25 μm. The Pearson correlation coefficients were 0.588, 0.632 and 0.782, respectively. WT, wild-type; gp130, glycoprotein 130; ART1, mono-ADP-ribosyltransferase-1.

Fig. 6

Expression levels of ART1 and gp130 in colorectal cancer tumour and control tissues. The scale bars are 50 and 100 μm. gp130, glycoprotein 130; ART1, mono-ADP-ribosyltransferase-1.

Fig. 7

ART1 knockdown inhibits tumour growth in a colitis mouse model. (A) H&E staining of normal and colitis mouse colon tissues. The scale bar represents 50 μm. (B and C) Volume of transplanted tumours formed in colitis model mice by NC and ART1-sh CT26 cells. (D and E) Protein expression levels of gp130, c-Myc, cyclin D1, and Bcl-xL and the ratio of p-STAT3/STAT3 in the transplanted tumours formed by NC and ART1-sh CT26 cells in colitis model mice. ^**P < 0.01, ^***P < 0.001. p-, phosphorylated; gp130, glycoprotein 130; sh, short hairpin RNA; NC, negative control; ART1, mono-ADP-ribosyltransferase-1.