YY1 promotes colorectal cancer proliferation through the miR-526b-3p/E2F1 axis

Abstract

We previously reported that E2F1 expression is up-regulated and positively correlated with the malignant phenotypes of colorectal cancer (CRC). However, the underlying mechanisms leading to the aberrant up-regulation of E2F1 in CRC have not been clarified. In this study, we observed that miR-526b-3p directly targets the 3'UTR of E2f1 mRNA, leading to reduced E2F1 expression. Overexpression of miR-526b-3p inhibited the proliferation of CRC cells by decreasing the level of E2F1. We also found that the Ying Yang 1 (YY1)-dependent transcriptional suppression of miR-526b-3p is responsible for the up-regulation of E2F1 in CRC, in which YY1 binds to the promoter of miR-526b gene and recruits histone deacetylase (HDAC). Knockdown of YY1 led to cell cycle arrest and diminished colony formation in CRC cells partly through relieving the miR-526b-3p suppression. Clinical analysis showed that YY1 and E2F1 were negatively correlated with miR-526b-3p in CRC tissues. Moreover, a high level of YY1 and E2F1, or a low level of miR-526b-3p, predicted poor survival of CRC patients. In conclusion, our findings highlight the dysregulation of the YY1/miR-526b-3p/E2F1 axis in CRC development, implicating a novel regulatory pathway for E2F1 as a potential therapeutic target in CRC.

Keywords: E2F1; Ying Yang 1; colorectal cancer; miR-526b-3p; proliferation.

Figure 1

The knockdown of E2F1 reduces the proliferation of CRC cells. A. The mRNA level of E2F1 in CRC and normal intestinal tissues based on TCGA database, N=327. A Mann-Whitney test was performed to compare the difference between the normal and cancer groups. B. The mRNA levels of E2F1 in 55 paired CRC and corresponding normal tissues were measured by qPCR, using β-actin as an internal control. -ΔCt (ΔCt: Ct (E2F1)-Ct (β-actin)) represented the relative expression level. A Wilcoxon matched pairs test was performed to compare the difference between the normal and cancer groups. C. Left panel: western blot analyses of E2F1 in HIEC, HCT116, SW620, and RKO cells; Right panel: the band density in the left panel was calculated by Image J software. D. Cell viability was examined in HCT116 and SW620 cells with E2F1 knockdown. *P<0.05; **P<0.01.

Figure 2

miR-526b-3p inhibits E2F1 expression by targeting the 3’UTR of E2f1 mRNA. A. The miR-526b-3p seed sequence complementary to the 3’UTR of E2f1 mRNA was predicted by TargetScan Human 7.2 and miRanda. B. The level of miR-526b-3p in HIEC, HCT116, SW620, and RKO cells was analyzed by qPCR. *P<0.05; **P<0.01. C. The mRNA level of E2F1 in HCT116 and SW620 cells transfected with scramble or miR-526b-3p mimics was analyzed by qPCR. *P<0.05. D. Western blot analyses of E2F1 in HCT116 and SW620 cells transfected with scramble or miR-526b-3p mimics. E. Left panel: the potential sequences for miR-526b-3p binding were mutated as indicated. Right panel: the function of the wild-type or mutated E2F1 3’UTR was analyzed by luciferase reporter assay in HCT116 cells transfected with scramble or miR-526b-3p mimics. **P<0.01; ***P<0.001.

Figure 3

miR-526b-3p attenuates the proliferation of CRC cells by inhibiting E2F1. A. Cell viability was measured in HCT116 and SW620 cells co-transfected with miR-526b-3p mimics or E2F1 overexpression plasmids vs scramble/EV: ***P<0.001; vs miR-526b-3p/E2F1: #P<0.05; ##P<0.01; ###P<0.001. B. DNA synthesis of HCT116 or SW620 cells was measured by EdU incorporation assay after co-transfection with miR-526b-3p mimics or E2F1 overexpression plasmids. Left panel: representative images; Right panel: percentage of cells with EdU incorporation was measured using image-J.

Figure 4

YY1 represses the transcription of the miR-526b gene. A. Transcription activity of the truncated miR-526b promoter was measured by luciferase reporter assay in HCT116 cells. **P<0.01. B. The potential sequences for YY1 binding were mutated as indicated. C. Transcription activity of the wild-type or mutated miR-526b promoter was measured by luciferase reporter assay in HCT116. **P<0.01. D. The level of miR-526b-3p in HCT116 and SW620 cells with knockdown of YY1. **P<0.01. E. Western blot analyses of E2F1 in HCT116 and SW620 cells with knockdown of YY1. F. The binding of YY1 to miR-526b promoter was examined by ChIP-PCR. Left panel: ChIP-PCR products in the input, ChIP and IgG groups were analyzed by agarose gel electrophoresis; Right panel: quantitative PCR analysis. G. The level of miR-526b-3p in HCT116 and SW620 cells transfected with YY1 overexpression plasmids or treated with 100 ng/mL TSA. **P<0.01; ***P<0.001. H. Western blot analyses of E2F1 in HCT116 and SW620 cells transfected with YY1 overexpression plasmids or treated with 100 ng/mL TSA.

Figure 5

YY1 promotes CRC cell proliferation by suppressing miR-526b-3p. A. Western blot analyses of E2F1 in HCT116 cells with stable knockdown of YY1 or miR-526b-3p. B. The level of miR-526b-3p in HCT116 cells with stable knockdown of YY1 or miR-526b-3p. **P<0.01; ***P<0.001. C. Cell viability was measured in HCT116 cells with stable knockdown of YY1 or miR-526b-3p. vs shCtrl: *P<0.05; **P<0.01; ***P<0.001. vs shYY1/shmiR-526b-3p: #P<0.05; ###P<0.001. D. DNA synthesis of HCT116 with stable knockdown of YY1 or miR-526b-3p was measured by EdU incorporation assay. Left panel: representative images; Right panel: percentage of cells with EdU incorporation was measured using image-J. E. Cell cycle profiles of HCT116 cells with stable knockdown of YY1 or miR-526b-3p were analyzed by flow cytometry. Left panel: representative images; Right panel: percentage of cells at different phases. F. Surviving colonies of HCT116 cells with stable knockdown of YY1 or miR-526b-3p. Left panel: representative images; Right panel: ratios of colony numbers relative to control.

Figure 6

Activation of YY1/miR-526b-3p/E2F1 axis is related with a poor prognosis in CRC patients. A. Representative images of YY1, E2F1, and KI67 staining in CRC and adjacent normal tissues. B. The correlation between the immunostaining scores of YY1 and E2F1 in CRC tissues. Nonparametric spearman test was applied in analyzing the correlation. C. The miR-526b-3p levels in 55 paired CRC and corresponding normal tissues were measured by qPCR, using U6 as an internal control. The expression value (ΔCt (N)-ΔCt (T)) represented the difference in the miR-526b-3p level between normal tissue and tumor. An expression value >0 indicated that miR-526b-3p level was increased in tumors. An expression value <0 indicated that miR-526b-3p level was decreased in tumors. Wilcoxon matched pairs test was applied to compare the difference between the normal and cancer groups. D. The correlation among the levels of Yy1 mRNA, E2f1 mRNA, and miR-526b-3p in 55 CRC tissues. A Pearson test was applied in the correlation analysis. E. The overall survival of CRC patients with low and high expression of Yy1 protein, E2f1 protein, or miR-526b-3p was illustrated.

Figure 7

Schema indicating the promotive effect of YY1/miR-526b-3p/E2F1 axis on CRC proliferation. In CRC cells, highly expressed YY1 recruits HDAC to silence the transcription of the miR-526b gene, leading to the downregulation of miR-526b-3p, which targets the 3’UTR of E2f1 mRNA. The resulting increased E2F1 level promotes CRC proliferation.