EGR1 mediates miR-203a suppress the hepatocellular carcinoma cells progression by targeting HOXD3 through EGFR signaling pathway

Abstract

EGR1 plays a critical role in cancer progression. However, its precise role in hepatocellular carcinoma has not been elucidated. In this study, we found that the overexpression of EGR1 suppresses hepatocellular carcinoma cell proliferation and increases cell apoptosis by binding to the miR-203a promoter sequence. In addition, we investigated the function of miR-203a on progression of HCC cells. We verified that the effect of overexpression of miR-203a is consistent with that of EGR1 in regulation of cell progression. Through bioinformatic analysis and luciferase assays, we confirmed that miR-203a targets HOXD3. Silencing HOXD3 could block transition of the G2/M phase, increase cell apoptosis, decrease the expression of cell cycle and apoptosis-related proteins, EGFR, p-AKT, p-ERK, CCNB1, CDK1 and Bcl2 by targeting EGFR through EGFR/AKT and ERK cell signaling pathways. Likewise, restoration of HOXD3 counteracted the effects of miR-203a expression.In conclusion, our findings are the first to demonstrate that EGR1 is a key player in the transcriptional control of miR-203a, and that miR-203a acts as an anti-oncogene to suppress HCC tumorigenesis by targeting HOXD3 through EGFR-related cell signaling pathways.

Keywords: EGR1; HOXD3; cell progression; hepatocellular carcinoma; miR-203a.

Figure 1. EGR1 induces miR-203a promoter activity in HCCs

A. The expression levels of EGR1 mRNA in HCC and healthy tissues were analyzed by qRT-PCR. B. The relationship between EGR1 and miR-203a were assayed by Pearson's r. C. Schematic diagram of the putative miR-203a promoter with one potential EGR1 response element. D. Luciferase activity of reporter constructs spanning the putative EGR1 binding site or a negative control sequence. E. The interaction of EGR1 with miR-203a was shown using ChIP assays with control (rat IgG) or anti-EGR1 antibody. F. qRT-PCR analysis was performed with primers spanning predicted EGR1 of miR-203a. G. The expression of miR-203a was analyzed by qRT-PCR after transfection with EGR1 vector or empty vector in SMMC-7721 cells. H-J. MTT assay, cell cycle and cell apoptosis were performed to determine the impact of HCCs treated with EGR1 expression vector. K. Proposed model for HOXD3-mediated effects of miR-203a on the EGFR-AKT/MAPK pathways. The expression of EGFR, AKT, p-AKT, ERK, p-ERK, CCNB1, CDK1, p-P27, Bcl-2 and Bax was detected by western blot (*: p < 0.05, **: p < 0.01.).

Figure 2. MiR-203a inhibits HCC cell proliferation and promotes apoptosis in vitro

A. SMMC-7721/Hep3B cells were transfected with miR-203a vector or control vector and the effects were determined by MTT assay after 24, 48, and 72 h. B. Representative results of a colony formation assay for SMMC-7721/Hep3B cells after miR-203a overexpression. C. SMMC-7721/Hep3B cells were transfected with miR-203a vector and control vector. After 24 h, cell cycle distribution was analyzed by flow cytometry. A histogram indicates the percentage of cells in G0/G1, S, and G2/M cell cycle phases. D. The expression of EGFR, AKT, p-AKT, ERK, p-ERK, p-P27, CCNB1 and CDK1 was analyzed by western blot. E. Apoptosis was detected by annexin-V/propidium iodide combined labeling flow cytometry in SMMC-7721/Hep3B cells 24 h after transfection with miR-203a or control vector. Apoptotic evaluation was carried out by calculating the percentage of apoptotic cells. F. Analysis of the expression of apoptosis-related proteins in SMMC-7721/Hep3B cells after transfection with miR-ctrl or a miR-203a overexpression construct. G. MTT assay of SMMC- 7721/Hep3B cells after miR-203a overexpression. H. The growth of SMMC-7721/Hep3B cells was detected by colony formation. I. Cell cycle was determined in SMMC-7721/Hep3B cells transfected with ASO-miR-203a or a negative control. J. The expression of EGFR, AKT, p-AKT, ERK, p-ERK, CCNB1 and CDK1 was analyzed by western blot. K. Apoptosis was determined in SMMC-7721/Hep3B cells transfected with ASO-miR-203a or a negative control. L. the expression of Bcl-2 and Bax were detected by western blot (*: P < 0.05, **: P < 0.01).

Figure 3. MiR-203a inhibits tumor proliferation by directly targeting HOXD3 in hepatocellular carcinoma

A. miR-203a is highly conserved across species and it has binding sites within the 3′-UTR of human HOXD3. A luciferase assay was performed in SMMC-7721 cells in which miR-203a was co-transfected with pGLO-HOXD3 or pGLO-HOXD3 mutant vector. B. mRNA expression levels of HOXD3 were measured by RT-PCR in tumor tissues and normal tissues. C. There is an inverse correlation between miR-203a and HOXD3 expression in HCC tissues (r = −0.38, *: P < 0.05). D. mRNA and protein expression levels of HOXD3 were measured by RT-PCR and western blot in SMMC-7721/Hep3B cells transfected with miR-203a, ASO-miR-203a, or a negative control. E. HOXD3 protein expression levels were measured by IHC and ICC in HCC tissues and SMMC-7721 transfected with miR-203a. F. HOXD3 protein levels were measured by western blotting for 4 paired HCC and healthy tissue samples (*: P < 0.05, **: P < 0.01).

Figure 4. Expression levels of HOXD3 affect HCC progression

A. The expression levels of HOXD3 were measured by qRT–PCR (upper panel) and western blot (lower panel) in SMMC-7721/Hep3B cells transfected with siHOXD3. B. An MTT assay was performed to determine the growth of HCCs treated with siHOXD3 or a negative control (si-ctrl). Data are reported as mean ± s.d. for 3 independent experiments. C. The colony formation assay was performed 7 days after transfection of HCCs with siHOXD3 or a negative control (si-ctrl). D. Cell cycle progression was determined in HCCs 48 h after transfection with siHOXD3 by propidium-iodide staining and flow cytometry. The histogram indicates the percentage of cells in G0/G1, S, and G2/M cell-cycle phases. E. Apoptosis was determined in HCCs 48 h after transfection with siHOXD3. F. Protein expression of HOXD3-dependent cell cycle or apoptosis-related proteins in HCCs transfected with siHOXD3 or si-ctrl-HCCs was analyzed by western blot. G-K. qRT-PCR, MTT assay, cell cycle, cell apoptosis and western blot were performed to determine the impact of HCCs treated with HOXD3 expression vector (*: P < 0.05, **: P < 0.01).

Figure 5. MiR-203a inhibits hepatocellular carcinoma progression in vivo

A. Tumor growth was analyzed through imaging of small animals at day 25. B. Tumor growth curves. C. Tumor weight. D. The expression levels of miR-203a were analyzed by qRT-PCR analysis in the tumor tissues from the animal. E. The expression levels of HOXD3, EGFR, p-AKT, p-ERK, CCNB1, and Bcl2 were analyzed by western blot in tissues from the animal (*: P < 0.05, **: P < 0.01, Student's t-test).

Figure 6. Proposed model for MiR-203a was directly activated by the EGR1 transcription factor

Expression of miR-203a downregulated the target genes of HOXD3. HOXD3 is essential for the activation of direct EGFR and indirect (p-ERK1/2 and p-AKT through PI3K or MAPK) signaling pathways in HCC cells.