Knockdown of AGGF1 inhibits the invasion and migration of gastric cancer via epithelial–mesenchymal transition through Wnt/β-catenin pathway

Forty cases of fresh gastric cancer samples and adjacent noncancerous tissues were collected from patients that underwent curative gastric cancer resection at the Department of General Surgery in our hospital. Samples were dissected from resected specimens by a pathologist, and immediately snap-frozen in individual vials using liquid nitrogen. Frozen specimens were stored at − 70 °C in a tumor bank until further AGGF1 expression detection by western blot and qRT-PCR. Written consent was obtained from all patients, and all experiments were performed in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). The study (including the collection and use of patients’ samples) was approved by the Ethics Committee of the First Affiliated Hospital of USTC.

Four human gastric cancer cell lines (SGC-7901, MGC-803, MKN-45 and AGS) and one normal human gastric epithelium cell line (GES-1) were purchased from Oncogene Biotechnology Company (Yangzhou, P. R. China), and cultured in RPMI-1640 or DMEM medium. All cell lines were maintained at 37 °C, 5% CO₂. The characteristics and origin of GC cell lines used in the study were summarized in Additional file 1: Table S1.

The small interfering RNA (siRNA) was used for the knockdown of endogenous AGGF1 in MKN-45 and MGC-803 GC cells. The target sequence was: 5′-CGAATGAAGATCATCAAGAAT-3′, and a non-targeting sequence was used as a normal control (NC). Cells with depleted endogenous AGGF1 expression were selected by being cultured in puromycin at the final selection concentration of 2 μg/mL. To construct β-catenin overexpression in GC cells, PCR product covering β-catenin open reading frame (Gene Bank Accession No. NM001012329) was cloned into the pcDNA3.1 vector. MKN-45 GC cells after AGGF1 knockdown were transfected with the β-catenin overexpression vector by Lipofectamine 3000 (Invitrogen, USA) according to manufacturer’s protocol.

Scratch was made by a pipette tip after the formation of a monolayer of cells. Then, cells were incubated in DMEM medium containing 10% FBS at 37 °C. Gap size was measured at 0 and 24 h later. Besides, the migration distance was calculated at each time point in three independent samples.

Transwell cell migration assays were performed in 24-well plates with 8.0 µm permeable polycarbonate membrane. Matrigel at high concentration and Growth-factor lowered the Matrigel (BD Biosciences, San Diego, CA, USA) through the spread of each bottom of transwell chamber. Subsequently, cells were diluted by serum-free basic culture medium into 5 × 10⁵/mL, followed by transferring 0.5–1 mL to each transwell using pipette guns. Contrarily, we filled the lower wells by culture media with 10% FBS as a chemoattractant. Thereafter, the wells were incubated at 37 °C for 24 h in a moistened cell culture incubator. The non-invading cells on the membrane’s top side were removed by scrubbing. The invading cells on the migrated side were fixed in 10% formalin for 10 min, followed by staining with 0.1% crystal violet.

Total RNA was extracted from the cell lines using TRIzol (Invitrogen, Carlsbad, CA, USA), according to manufacturer’s instructions. The absorbance of RNA was measured at 260 nm using a NanoDrop spectrophotometer (ND-1000, Thermo Scientific, Waltham, MA, USA), in order to determine the total RNA concentration. Reverse transcription of 2 µg total RNA was conducted using the Prime Script RT reagent kit, gDNA Eraser (TaKaRa, Japan). Based on the manufacturer’s protocol, the ABI 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA, USA) together with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) were used with a first step at 95 °C for 10 min followed by 40 cycles with 95 °C for 15 s and 60 °C for 1 min, with a fluorescent reading at the end of this step to amplify the specific genes. The primers used in PCR amplification were listed in Table 1. GAPDH was used as the interval control to calculate the relative expression level of testing genes using the comparative delta Cq (2^−ΔΔCq) method. Moreover, independent determination of each sample was performed thrice, and the mean value of the expression levels was calculated.

NOD/SCID mice (6–8 weeks old) were purchased from Model Animal Research Center of Nanjing University. A lentiviral shRNA vector targeting AGGF1 was generated by inserting stranded oligonucleotides (shAGGF1, forward sequence 5′-CCGGATGGGTAGTGGAGCCTAATTTCTCGAGAAATTAGGCTCCACTACCCATTTTTTG-3′) into pLKO-puro Vector (Sigma-Aldrich). The cells were infected with AGGF1 shRNA vectors and selected with puromycin (5 μg/mL). Each mouse was injected subcutaneously into the dorsal of the mice with empty vector-transfected cells (4 × 10⁶) or with sh-AGGF1 cells (4 × 10⁶). Mice were euthanized, and the tumors were excised after 40 days. All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and the guidelines of the First Affiliated Hospital of USTC.

All cells were lysed completely in lysis buffer at 4 °C. Then, total protein concentration was calculated using the BCA protein assay kit (Beyotime, Jiangsu, China). To break the structure of protein, the protein was heated at 100 °C for 10 min. Following that, equivalent amount of total protein was added into each well of 10% polyacrylamide gels. Subsequent to electrophoresis, the protein was transferred to nitrocellulose membranes, followed by soaking the same in 5% bovine serum albumin (BSA) or 5% non-fat milk diluted in Tris Buffered Saline Tween (TBST) for 2 h. Thereafter, the blocked membranes were incubated in primary antibodies AGGF1 (1:1000; Proteintech, IL, USA), E-cadherin (1:1500; Cell Signaling Technology Danvers, MA, USA), vimentin (1:1000; Cell Signaling Technology Danvers, MA, USA), snail (1:1000; Cell Signaling Technology Danvers, MA, USA), β-catenin (1:1000; Cell Signaling Technology Danvers, MA, USA), GSK-3β (1:1000; Cell Signaling Technology Danvers, MA, USA), Phosphorylated GSK-3β (1:1000; Cell Signaling Technology Danvers, MA, USA), and GAPDH (1:1000; Abmart, Shanghai, China) at 4 °C overnight. Subsequent to washing the membranes with TBST, the membranes were incubated with a secondary antibody (dilution, 1: 5000; Cat. No. KC-RB-035; KangCheng Bio-tech, Shanghai, China) for a period of approximately 60 min at room temperature. After washing the membranes with TBST, the membranes were ameliorated through the ECL kit (Thermo Scientific), followed by capturing the emitted signals using KODAK X-OMAT BT Film (Kodak, Rochester, NY). The gray value of the protein was measured by performing the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

All data were analyzed using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Two-tailed Student’s t-test was used to determine the differences between groups. P < 0.05 was considered statistically significant.

Initially, the protein and mRNA levels of AGGF1 in 40 pairs of frozen GC and corresponding normal tissues were detected by western blot and qRT-PCR, respectively. As shown in Fig. 1a, AGGF1 protein levels were upregulated in GC tissues compared with the matched adjacent normal tissues (P < 0.001). Besides, AGGF1 mRNA levels were increased in GC tissues compared with the matched adjacent normal tissues (P < 0.01, Fig. 1b). Then, AGGF1 expression levels in 4 GC cell lines (SGC-7901, MGC-803, MKN-45 and AGS) and GES-1 (normal control) were examined. The results showed that both mRNA and protein levels of AGGF1 were upregulated in GC cell lines compared with GES-1 (all P < 0.05, Fig. 1c, d).

To further explore the mechanistic role of AGGF1 in GC, knockdown of AGGF1 gene expression by small RNA interfering method was performed in the MKN-45 and MGC-803 cell lines (Additional file 2: Figure S1). The results showed that MKN-45 and MGC-803 cells with AGGF1 knockdown had significantly slower closure of the wound area compared to their controls by wound-healing assay (both P < 0.01, Fig. 2). Besides, effects of ectopic AGGF1 expression on cell invasion and migration potential of MKN-45 and MGC-803 cells were also analyzed. In the transwell invasion and migration assay, cells with AGGF1 knockdown exhibited decreased invasion and migration abilities in MKN-45 (both P < 0.01, Fig. 3a) and MGC-803 cells (both P < 0.01, Fig. 3b) compared with those in the control groups.

To evaluate the influence of AGGF1 knockdown on tumor growth, an in vivo nude mice xenograft model was established by using MKN-45 and MGC-803 cells. As shown in Fig. 4, both subcutaneous xenograft groups using transfected cells with sh-AGGF1 had dramatically decreased abilities to form tumors compared to those in control groups, as indicated by the final xenograft tumor size.

To explore whether AGGF1 is associated with EMT, we examined the expression levels of epithelial and mesenchymal markers (E-cadherin, Vimentin, Snail) in the MKN-45 and MGC-803 cells with or without AGGF1 inhibition. The results showed that epithelial markers, E-cadherin, were increased compared with the controls, whereas mesenchymal marker Vimentin and related transcription factors Snail were significantly decreased in MKN-45 and MGC-803 cells with AGGF1 knockdown by qRT-PCR (Fig. 5a, b) and western blot (Fig. 5c, d) analyses. Consistently, gene silencing of AGGF1 retarded the development of EMT in MKN-45 and MGC-803 GC cells.

To evaluate the relationship between AGGF1 and Wnt/β-catenin signaling in GC, the activation status of GSK3β and β-catenin was firstly investigated. As shown in Fig. 6, the amount of phosphorylated GSK3β was significantly reduced by suppression of AGGF1 as well as the total β-catenin expression levels compared with control (all P < 0.01). To further demonstrate that Wnt/β-catenin acts as a critical pathway linking AGGF1 and EMT, we conducted the overexpression of β-catenin in the MKN-45 cells transfected with si-AGGF1. As shown in Fig. 7, E-cadherin level was increased together with decrease of snail, vimentin and β-catenin after AGGF1 knockdown in MKN-45 (all P < 0.05). Contrarily, while transfected with β-catenin plasmid in MKN-45 cell line with si-AGGF1, the above EMT markers showed a remarkable reversal (all P < 0.05).