NFE2L3 Inhibition Induces Cell Cycle Arrest at the G0/G1 Phase in Colorectal Cancer Cells through Downregulating CCND1 and pRb1-ser807/811

Abstract

The molecular mechanism for colorectal cancer to develop remains unelucidated. To find biomarkers related to colorectal cancer development, we analyzed the gene expression profile of 380 colorectal cancer patients and 51 healthy controls by R software. Finally, 1579 upregulated differential expression genes (DEGs) and 3218 downregulated DEGs were identified. Then, the top 20 upregulated DEGs were compared with 181 upregulated DEGs that we reported previously, and 11 overlapped DEGs were found. NFE2L3 (nuclear factor, erythroid 2-like 3) was among those overlapped DEGs and was rarely reported in colorectal cancer. Real-time polymerase chain reaction (PCR) results showed that higher NFE2L3 expression levels were identified in paired tumor samples than in paratumor samples (48 paired samples). Flow cytometry analysis revealed that the cell cycle was arrested at the G0/G1 phase after inhibition of NFE2L3 in both HCT116 and SW480 cell lines. Western blot detection showed that CCND1 and phosphorylated Rb transcriptional corepressor 1 at ser-807/811 (pRb1-ser807/811) expression levels were downregulated when NFE2L3 was inhibited in those two cell lines. A significant positive correlation was observed between NFE2L3 and CCND1 expression levels in colorectal tissue samples. These evidences indicate that downregulation of NFE2L3 induces cell cycle arrest at the G0/G1 phase through downregulation of CCND1 and pRb1-ser807/811.

Figure 1

Screening and validation of differential expression genes. (a) Volcano plot of differential expression genes in TCGA COADREAD gene expression dataset. x-axis: log2 fold change; y-axis: -log10 (FDR p value) for each probe; vertical dotted lines: fold change ≥ 2 or ≤2. It showed that 1579 significantly upregulated genes (red color) and 3220 significantly downregulated genes (green color) were selected in this dataset. (b) 11 merged genes were found with the intersection of the top 20 upregulated genes from the COADREAD dataset and 181 upregulated genes from our previous research. (c) NFE2L3 expression levels in 51 normal and 370 tumor samples. NFE2L3 showed significantly higher expression levels in tumor samples than in normal samples (p < 0.0001). (d) Validation of NFE2L3 expression levels with 48 paired tumor and paratumor samples. The NFE2L3 mRNA expression level was significantly upregulated in tumor samples (p < 0.0001).

Figure 2

Changes in cell cycle distribution in HCT116 and SW480 cells after transfection with siRNA for 48 hours. (a–d) Cell cycle distribution in the HCT116 cell line. In HCT116 cells, the cell percentage in NC, si-1, and si-2 groups was 41.07 ± 5.18, 79.14 ± 5.75, and 59.53 ± 4.59, respectively, at the G0/G1 phase; 31.68 ± 2.98, 11.59 ± 4.77, and 21.08 ± 5.37, respectively, at the S phase; and 27.25 ± 2.79, 9.27 ± 1.52, and 19.4 ± 2.06 at the G2 phase. The cell percentage of the si-1 and si-2 groups at the G0/G1 phase was reduced compared to that of the NC group (p = 0.0003, p = 0.0216). The cell percentage at the S phase was decreased in the si-1 and si-2 groups compared to the NC group (p = 0.0044, p = 0.0180). The cell percentage at the G2/M phase was reduced in the si-1 group compared to the NC group (p = 0.0026). (e–h) Cell cycle distribution in the SW480 cell line. In SW480 cells, the cell percentage in NC, si-1, and si-2 groups was 47.18 ± 4.56, 74.77 ± 3.31, and 50.39 ± 3.65, respectively, at the G0/G1 phase; 27.70 ± 5.65, 12.80 ± 2.64, and 26.56 ± 3.98, respectively, at the S phase; and 25.12 ± 1.09, 12.43 ± 0.79, and 23.05 ± 1.38, respectively, at the G2 phase. The cell percentage at the G0/G1 phase was reduced in the si-1 group compared to the NC group (p = 0.0012). The cell percentage at the S phase was decreased in the si-1 group compared to the NC group (p = 0.0136). The cell percentage at the G2/M phase was reduced in the si-1 group compared to the NC group (p = 0.0064).

Figure 3

CCND1 mRNA and protein expression levels in colorectal cancer cell lines after NFE2L3 knockdown. (a) The mRNA expression levels of NFE2L3 in HCT116 cells after NFE2L3 knockdown. Inhibition of the NFE2L3 mRNA level was displayed in the si-1 and si-2 groups (p = 0.0225, p = 0.0184). (b) CCND1 mRNA expression levels in HCT116 cells after NFE2L3 knockdown. Inhibition of CCND1 mRNA expression was detected in the si-1 and si-2 groups (p = 0.0189, p = 0.0291). (c) Protein expression changes of CCND1 in HCT116 cells when inhibited by NFE2L3 siRNA. Downregulation of CCND1 and pRb1-ser807/811 proteins was observed in the si-1 and si-2 groups. (d) The mRNA expression levels of NFE2L3 in SW480 cells after NFE2L3 knockdown. The NFE2L3 mRNA expression level was significantly inhibited by si-1 and si-2 (p = 0.0258, p = 0.0232). (e) CCND1 mRNA expression levels in SW480 cells after NFE2L3 knockdown. The CCND1 mRNA level was significantly downregulated in the si-1 group (p = 0.0374). (f) Protein expression changes of CCND1 in SW480 cells after transfection with NFE2L3 siRNA. CCND1 and pRb1-ser807/811 protein expression levels were downregulated in the si-1 and si-2 groups.

Figure 4

The expression correlation between CCND1 and NFE2L3 in colorectal tissue samples. (a) CCND1 was upregulated in 380 tumor samples compared to 51 normal samples (p < 0.0001). (b) Expression level correlation analysis between NFE2L3 and CCND1 in 431 samples (normal and primary tumor samples) of TCGA COADREAD gene expression dataset. A significant positive correlation between NFE2L3 and CCND1 expression was found (r = 0.5698, p < 0.0001).