Dusp6 inhibits epithelial-mesenchymal transition in endometrial adenocarcinoma via ERK signaling pathway

Tumor and the corresponding adjacent tumor tissues (more than 5 cm from the tumor site without cancer cell infiltrations) were obtained from 35 endometrial adenocarcinoma patients from 2016 to 2018 in the Department of Obstetrics and Gynecology, Shandong Provincial Hospital. The study was authorized by the Ethics Committee of Shandong University, China. Written consents were obtained from all patients. Additionally, Ishikawa 3H12 (Purchased from ATCC, USA) high-differentiated endometrial adenocarcinoma cell line was used in in vitro study. The cells were cultured in DMEM supplemented with 5% charcoal-stripped fetal bovine serum (FBS), 100μ/ml of penicillin and 100 μ/ml of streptomycin at 37°C in a humidified environment with 5% CO² in air.

Short hairpin RNA (shRNA) plasmids which targets the coding region of DUSP6; or by transfecting cells with non-target shRNA control vector were constructed by GenePharma, Shanghai and cloned into pGPU/GFP/Neo. The constructs were transfected into Ishikawa cells by Lipofectamine 3000 according to the manufacturer’s protocol. cDNA of DUSP6 was obtained and synthetized by GenePharma, Shanghai and cloned into pcDNA3.1 plasmid. Ishikawa 3H12 cells were transiently transfected with either pcDNA3.1-Dusp6 plasmid or empty vector (EV) using Lipofectamine 3000 according to the manufacturer’s protocol. Whole cell lysates were collected at the indicated time points after transfection to verify the expression level of DUSP6.

Cell viability was evaluated with MTT (Qiagen, German) assays according to the manufacturer’s instructions. Transfected Ishikawa 3H12 cells were seeded at a density of 6.000 cells per well in 96-well plates in RPMI 1640 containing 10% FBS overnight and then maintained in 0.5% FBS media for 96h before testing. The absorbance was measured at a wavelength of 490 nm on a Synergy Multi-Mode Microplate Reader (Biotek, USA). Each assay was performed on five replicate wells.

Fresh endometrial adenocarcinoma tumor tissue samples were washed using phosphate buffered saline (PBS) and fixed using formalin. After dehydration and paraffin-embedding, samples were cut and mounted onto glass slides. Formalin-fixed sections were deparaffinized and rehydrated and incubated with 0.6% hydrogen peroxide in methanol. Then antigen retrieval was performed and the sections were blocked and incubated overnight with rabbit anti-human DUSP6/E-cad/N-cad primary antibody (1:100, Abcam, USA) at 4°C. HRP-conjugated goat anti-rabbit IgG secondary antibody was deployed afterwards. DAB was used as chromogenic agent. Same sections incubated with non-immune serum instead of primary antibody were used as a negative control. The experiments were performed in duplicate. The intensity of the staining of DUSP6/E-cad/N-cad was scored by a clinical pathologist who was blinded with any knowledge of the pathological data.

To evaluate the migration of cells, Ishikawa 3H12 cells overexpressed or knockdown of DUSP6 were cultured until confluent beforehand. Then the cell monolayer was scratched with a pipette tip (Scar bar: 1mm) and incubated for 24 h, the relative wound areas were calculated through: migrated distance of DUSP6 expression or knockdown clones/ migrated distance of vector-alone stable clones x 100.

A micropore chamber assay was used to assess the invasion ability of Ishikawa 3H12 cells that with the overexpression/knockdown of DUSP6. Matrigel was diluted by pre-cold serum free DMEM medium at 1: 5 before use. A total number of 3x104 cells were seeded into the top chamber of a 24-well Matrigel-coated micropore polycarbonate membrane filter with 8μm pores (Becton Dickinson Labware, Franklin Lakes, NJ) for invasion assays. The DMEM containing 10% FBS was added to the bottom chamber as a chemoattractant. The cells were then incubated at 37°C for 22 h. The members were washed and fixed followed by stained by crystal violet. The cells migrated through the membranes were counted under the inverted light microscope at 10x magnification. All tests were performed in triplicate.

To verify the expression of DUSP6 in transfected cell, total RNA was isolated with TRIzol reagent (Life Technologies, USA) and converted to first-strand cDNA using M-MLV Reverse Transcriptase (USB, Cleveland, OH). The mRNA level was analyzed with quantitative real-time PCR assay. Specific primers were synthesized as the following sequences show: DUSP (forward) 5’-GAACTGTGGTGTCTTGGTACATT-3′ and (reverse) 5′-GTTCATCGACAGATTGAGCTTCT-3’.22 The GAPDH gene was used as an internal control and was amplified from the same cDNA samples for 25 cycles. The cDNA of a normal endometrial cell line was used as a positive control.

The whole cell lysates were extracted using RIPA buffer with protein inhibitors cocktail (Complete Mini, Roche Diagnostics, Switzerland). The concentration of total cellular protein was measured using BCA assay kit (Pierce, IL, USA) according to manufacturer’s instructions. 30 μg of proteins from each sample were electrophoresed to separate on 10% sodium dodecysulfate-polyacryl-amide gels (SDS-PAGE) and then transferred to polyvinylidene difluoride membrane (PVDF, CA, USA). The PVDF membranes were blocked with 5% non-fat dry milk for 1 h at room temperature and incubated at 4°C overnight with the primary antibodies against: DUSP6, E-cadherin, N-cadherin, Vimentin, ERK, p-ERK, GAPDH (1:1000, Cell Signaling, USA). The membranes were then washed and incubated with the secondary antibody against rabbit at 37°C for 1 h and protein bands were detected by the chemiluminescence method.

Data are presented as means ±SD from three independent experiments. All statistical analyses were performed using SPSS 13.0 (SPSS Inc, USA). For comparisons between groups, one-way analysis of variance was performed. Western blot analysis was quantified by Image J. P values <0.05 were considered as statistically significant.

By determining the expressions of DUSP6, E-cadherin and N-cadherin in both endometrial adenocarcinoma cancerous and the corresponding adjacent non-tumor tissues using western blot and immunohistochemistry analysis, down-regulation of DUSP6 (Figure 1A) and E-cadherin (Figure 1B) were obeserved in cancerous specimens compared to corresponding normal counterparts. The results of immunohistochemical staining were in line with the results of western-blot in terms of the expressions of DUSP6 and E-cadherin. Contrarily, significant up-regulation of N-cadherin was observed in cancerous tissues than that in their non-cancerous counterparts (Figure 1C)

Figure 1

To investigate the role of DUSP6 in the progress of endometrial adenocarcinoma, proliferation, invasion and migration of Ishikawa 3H12 cell line was assessed after overexpressing DUSP6 by transfecting pcDNA3.1-Dusp6 vector or empty vector control. The efficiency of transfection was validated by western-blot (Figure 2A) The MTT assay demonstrated that the cell viability was significantly decreased in DUSP6 overexpressed Ishikawa 3H12 cells than that of vector (Figure 2B) Further in vitro Transwell Matrigel assay (Figure 2C) and wound healing assay (Figure 2D) showed the attenuated invasion and migration ability in DUSP6 overexpressed tumor cell lines. DUSP6 overexpressed clones in Matrigel culture exhibited a non-cohesive and disorganized morphology compared to polarized and well-organized spheroids shown in vector-alone clones and controls. The width of wound formed in cultures of cells overexpressing DUSP6 significantly superior to that in cultures of vector-alone clones and controls together referred to differed EMT-associated properties.23 Increased protein level of E-cadherin and decreased Vimentin were observed in DUSP6 overexpressed cells (Figure 2E)

Figure 2

Cell growth, migration and invasion were evaluated after silencing DUSP6 expression by shRNA in Ishikawa cell line. Cells were transfected with shRNA targeting DUSP6 or NC. The efficiency of knockdown was assessed by western blot (Figure 3A) Cell viability of DUSP6 knock down cell was increased significantly compared to that of NC (Figure 3B) The enhanced cell invasion and migration ability were also observed in tumor cells with the absence of DUSP6 expression based on transwell matrigel (Figure 3C) and wound healing assay (Figure 3D) respectively. EMT process was promoted in the DUSP6 knock down tumor cells indicated by decreased protein level of E-cadherin and increased level of Vimentin (Figure 3E)

Figure 3

The activation of ERK pathway has been validated in tumorigenesis in varied human carcinoma and played an important role in endometrial adenocarcinoma. The present study confirmed that the dysregulation of DUSP6 induced aberrant activation of ERK signaling in Ishikawa 3H12 cell lines (Figure 4, Figure 5). According to western-blot results, the level of p-ERK/ERK was significantly down-regulated in overexpressed DUSP6 cells, indicating activation of ERK signaling was inhibited by DUSP6 (Figure 4). In contrary, the level of p-ERK/ERK was significantly up-regulated in DUSP6 knockdown cells compared to that in sh-NC (Figure 5).

Figure 4

Figure 5