ELF3 promotes epithelial-mesenchymal transition by protecting ZEB1 from miR-141-3p-mediated silencing in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is one of the most common malignant cancers and currently the third leading cause of cancer-related deaths, worldwide. Epithelial-mesenchymal transition (EMT) plays a major role in HCC progression. In this study, we first found that the expression of E74-like ETS transcription factor 3 (ELF3), a member of the E-twenty-six family of transcription factors, was increased in HCC tissues, and that ELF3 overexpression was associated with poor prognoses for HCC patients. Gain-of-function and loss-of-function studies revealed that increased ELF3 expression promoted HCC cell proliferation, migration, and invasion, while these processes were inhibited when ELF3 was silenced. Additionally, ELF3 was found to promote EMT, which we demonstrated through decreased E-cadherin expression and increased N-cadherin and fibronectin expression. ELF3 knockdown reversed EMT via repressing ZEB1 expression through miR-141-3p upregulation. Chromatin immunoprecipitation assays revealed that ELF3 bound to the miR-141-3p promoter, suppressing miR-141-3p expression. Taken together, our data show that ELF3 repressed E-cadherin and promoted EMT in HCC cells by suppressing miR-141-3p, thereby activating ZEB1. Thus, ELF3 may be a potential prognostic biomarker and/or therapeutic target for HCC.

Fig. 1. Expression of analyses of ELF3 mRNA and protein in HCC cell lines and tissues.

a ELF3 expression in four HCC cell lines analyzed by qPCR and western blot. b ELF3 expression is upregulated in HCC tissues analyzed by qPCR and western blot. c Upregulation of ELF3 expression in HCC tissues at the mRNA level compared with normal liver tissues revealed using the two Roessler liver datasets from the Oncomine dataset. d Representative IHC image of ELF3 expression in HCC and ANT (magnification ×100, scale bar:200μm). The region in the black square was further enlarged (magnificantion ×400, scale bar:50μm). OS and DFS of HCC patients with low and high ELF3 expression in all HCC patients (e, f) and HCC patients with early-stage tumor (g, h). Survival curve was calculated with the log-rank test. Each error bar represents the mean ± SD of three replicate samples. p < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01 based on the Student's t test

Fig. 2. ELF3 overexpression promoted the proliferation, migration, and invasion of HCC cell in vitro.

a Western blot and qPCR evaluated the protein expression and mRNA of ELF3 in Huh7-ELF3 cells and Huh7-control cells. b The colony formation and c CCK-8 assay revealed that ELF3 overexpression promoted cell growth in vitro. d Transwell migration and invasion assays indicated that ELF3 ectopic expression promoted cell migration and invasion (magnification ×100, scale bar: 200 μm). Each error bar represents the mean ± SD of three replicate samples. p < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, and ***p < 0.001 based on the Student's t test

Fig. 3. Down-regulation of ELF3 inhibited the proliferation, migration, and invasion of HCC cell in vitro.

a Western blot and qPCR evaluated the protein expression and mRNA of ELF3 in MHCC-LM3-siELF3-1, MHCC-LM3-siELF3-2, MHCC-LM3-siELF3-3 cells and MHCC-LM3-sicontrol cells. b The colony formation and c CCK-8 assay revealed that down-regulation of ELF3 inhibited MHCC-LM3-shELF3 cells' growth in vitro. d Transwell migration and invasion assays indicated that ELF3 knockdown inhibited cell migration and invasion (magnification ×100, scale bar: 200 μm). Each error bar represents the mean ± SD of three replicate samples. p < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, and ***p < 0.001 based on the Student's t test

Fig. 4. ELF3 promotes EMT by regulating ZEB1 activation in HCC cells.

a mRNA and protein expression of ELF3, E-cadherin, ZEB1, fibronectin, N-cadherin in MHCC-LM3-shELF3 cells and in Huh7-ELF3 cells (b) and their control cells were analyzed by qPCR and western blot. c Protein expression of E-cadherin and ZEB1 in MHCC-LM3 and Huh7 cells with ZEB1 knockdown were analyzed by western blot. d Representative IHC images of ELF3 and E-cadherin expression in HCC tissues (magnification ×400, scale bar: 50 μm). e The correlation analysis between ELF3 and E-cadherin in HCC samples. Each error bar represents the mean ± SD of three replicate samples. p < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, and ***p < 0.001 based on the Student's t test

Fig. 5. Knockdown of ELF3 decreased ZEB1 by upregulating miR-141-3p.

a miRNAs of expression in MHCC-LM3-shELF3 cells and MHCC-LM3-shcontrol cells were analyzed by sqRT-PCR. b Protein expression of ZEB1 and E-cadherin in MHCC-LM3-shELF3 cells and MHCC-LM3-shcontrol cells with miR-141-3p inhibitor or control were analyzed by western blot. c Migration and invasion assay indicated that 141-3p inhibitor increased MHCC-LM3-shELF3 cells' migration and invasion (magnification ×100, scale bar: 200 μm). d Predicted ELF3 binding site in miR-141-3p promoter from JASPER. e CHIP assay showed that ELF3 could bind to the promoter of miR-141-3p. f promoter reporter assay and mutation rescue assay suggested that ELF3 could inhibit miR-141 promoter activity by specifically interacting with the predicted genomic region. Each error bar represents the mean ± SD of three replicate samples. p < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, and ***p < 0.001 based on the Student's t test

Fig. 6. Knockdown of ELF3 inhibited MHCC-LM3 cell metastasis in vivo.

a IVIS images of BALB/c nu/nu mice injected with MHCC-LM3-shELF3 cells and MHCC-LM3-shcontrol cells. b The total photon flux were measured and analyzed using the Living Image software, and the results showed less total metastatic foci in the MHCC-LM3-shELF3 group. c Representative images of hematoxylin and eosin staining of xenograft tumors (magnification ×100, scale bar:200μm). The region in the black square was further enlarged (magnificantion ×400, scale bar:50μm). d Representative IHC images of ELF3 and E-cadherin in xenograft tumors (magnification ×100, scale bar:200μm). The region in the black square was further enlarged (magnification ×400, scale bar:50μm). p < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, and ***p < 0.001 based on the Student t test