E2A predicts prognosis of colorectal cancer patients and regulates cancer cell growth by targeting miR-320a

Abstract

Background and objective: Transcriptional factor E2A is crucial for the normal development and differentiation of B and T lymphocytes. Dysregulation of E2A leads to leukemia and tumorigenesis of some solid tumors. The expression and clinical significance of E2A as well as its role in colorectal cancer (CRC) are still unknown. This study aims to assess E2A expression in CRC tissues, evaluate its prognosis value, and investigate its role in colon cancer cell growth.

Methods: E2A expression in CRC tissues and normal mucosa was detected by immunohistochemical staining; Kaplan-Meier survival curve and Cox regression model were used to evaluate the prognostic value of E2A. Lentivirus was used to construct E2A stably knocked-down cells. MTT assay was employed to detect cell proliferation change; cell cycle was analyzed by flow cytometry; and chromatin immunoprecipitation (ChIP) assay was used to validate the predicted binding target of E2A.

Results: Expression of E2A was lower in CRC tissues than normal mucosa; low E2A expression correlated with advanced TNM stage and larger tumor size, and predicted poor prognosis of CRC patients. E2A knockdown resulted in increased cell proliferation rate and cell cycle acceleration. ChIP assay showed miR-320a was a direct target of E2A and upregulation of miR-320a in E2A downregulated cells could reverse cell proliferation and cell cycle changes caused by E2A deficiency.

Conclusions: E2A is an independent prognostic factor for CRC patients and targets miR-320a to regulate cell proliferation of colon cancer cells.

Figure 1. IHC staining of E2A in CRC tissues and normal mucosa.

Representative images of IHC are shown as: (A) HE staining of normal mucosa; (B) HE staining of CRC tissue; (C) high E2A expression (brown color) presented as nuclear and cytoplasmic staining in normal mucosa; (D) low E2A expression in normal mucosa; (E–H) stage I (E) to stage II (F), III (G), and IV (H) CRC tissues with different E2A staining intensities; E2A appeared predominantly in nucleus. Images were taken under 200× magnification.

Figure 2. Kaplan-Meier survival curves for OS and DFS stratified by E2A expression.

(A) OS for high and low E2A expression patients; (B) DFS for high and low E2A expression patients. Patients with high E2A expression have more favorable OS and DFS (P = 0.000, for OS and DFS both).

Figure 3. Knockdown of E2A promotes colon cancer cell growth.

(A) Expression of E2A in normal human colon mucosal epithelium cell NCM460 and CRC cells lines was shown by western blot. GAPDH was used as loading control; (B) and (D) Cells with downregulated E2A expression (SW480/shE2A, Caco-2/shE2A) showed higher cell proliferation rate than that of the negative control (SW480/shNC, Caco-2/shNC) and wild type (SW480/WT, Caco-2/WT) cells; (C) and (E) Parental cells (SW480/shE2A, Caco-2/shE2A) were co-transfected with E12 or E47 plasmid (SW480/shE2A_E12 and SW480/shE2A_E47; Caco-2/shE2A_E12 and Caco-2/shE2A_E47) to restore E2A expression. Co-transfected cells showed decreased cell proliferation rate than that of the parental or vector control (SW480/shE2A_VC, Caco-2/shE2A_VC) transfected cells. All data represented as mean value ± SD from 3 independent experiments. (*, P<0.05).

Figure 4. Effect of E2A on cell cycle progression.

(A) Downregulation of E2A in SW480 cells led to an increase of cells at S phase and concomitantly a decrease of cells at G1/G0 phase; (B) Ectopic expression of E12 or E47 increased cells at G1/G0 phase of SW480/shE2A cells and reduced S phase cells. Data represents means ± SD from 3 independent experiments. (*, P<0.05).

Figure 5. miR-320a is a direct target of E2A to regulate cell cycle and proliferation.

(A) miR-320a expression was decreased in SW480/shE2A cells as shown by qRT-PCR; (B) Co-transfection of E12 or E47 into SW480/shE2A cells could normalize the expression of miR-320a; (C) Upper panel: sequence of the miR-320a gene showing the E-box, the predicted binding site of E2A; lower panel: ChIP assay showed E2A bound to miR-320a at its E-box, GCAGGTG; Co-transfection of miR-320a mimics reversed the cell cycle changes (D) and cell growth (E) of SW480/shE2A cells; (F) Co-transfection of miR-320a mimics decreased cell proliferation rate of Caco-2/shE2A cells. Data is expressed with the means ± SD from 3 independent experiments. (*, P<0.05).