Reagents
DMEM medium, foetal bovine serum (FBS), 0.02% EDTA and 0.25% trypsin were purchased from Gibco (St. Louis, MO, USA). Quantitative polymerase chain reaction (qPCR) master mix was purchased from Promega (Madison, WI, USA) and a Hairpin–it Real-Time PCR Kit was purchased from GenePharma (Shanghai, China). The Epithelial-Mesenchymal Transition Antibody Sampler Kit was purchased from Abcam (Cambridge, MA, UK). The anti-TGFβR2 rabbit monoclonal antibody and anti-β-actin mouse monoclonal antibody were purchased from Abcam (Cambridge, MA, UK). Peroxidase-conjugated AffiniPure goat anti-mouse IgG and peroxidase-conjugated AffiniPure goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Dual-Luciferase® Reporter Assay System was purchased from Promega (Madison, WI, USA).
Cell culture
Human placental choriocarcinoma cell lines (JAR and JEG-3) were obtained from the State Key Laboratory of Reproductive Biology (SKLRB), Institute of Zoology (IOZ), Chinese Academy of Sciences (CAS). HTR8-SVneo was purchased from Jennio Biotech Co., Ltd (Guang Zhou, China). Cells were cultured in DMEM supplemented with 10% FBS in an incubator with an atmosphere of 5% CO2 at 37˚C. The cells were then subcultured using 0.25% trypsin and 0.02% EDTA when the cells were approximately 80% confluent. Experiments were performed using cells at a logarithmic growth phase.
Paraffin-embedded human choriocarcinoma tissue
We included ten formalin-fixed paraffin-embedded (FFPE) choriocarcinoma samples from the pathological archives of the Affiliated Hospital of Chengde Medical College, Chengde Third Hospital and the Second Affiliated Hospital of Hebei Medical University. Ten women with normal early pregnancy and a single gestational sac were randomly selected, and the gestational villus were obtained at Chengde Women's and Children's Hospital.
miRNA microarray analysis
Total RNA in formalin-fixed paraffin-embedded (FFPE) sections was extracted using a Recover All™ Total Nucleic Acid Isolation Kit (Ambion-1975). MiRNA expression analysis was conducted on total RNA samples containing small RNAs using the human miRNA microarray, Release19.0, 8x60K (Agilent Technologies). For control and test RNAs, the synthesis of target miRNA probes and hybridization was performed using a miRNA Labeling Reagent and Hybridization Kit (Agilent Technology) according to the manufacturer’s instructions. The hybridized microarrays were washed according to the manufacturer’s washing protocol (Agilent Technology). Images of hybridized arrays were obtained using a DNA microarray scanner (Agilent Technology) and were quantified with Feature Extraction Software (Agilent Technology). Data normalization and the selection of genes with a fold change (FC) > 2 were performed using GeneSpring GX software (Agilent Technology).
Quantitative real-time PCR analysis (qRT-PCR)
Total RNA was isolated using the RNAgents® Total RNA Isolation System (Promega) with DNase I (Invitrogen) treatment. Then, 2 µg RNA, oligo (dt) 20 primers and the M‑MLV First‑Strand Synthesis System kit was used to synthesize cDNA. Real-time PCR was performed using the SYBR® Green I on GoTaq® qPCR Detection System (Promega) according to the manufacturer’s instructions. The results were analysed using the comparative threshold cycle method with GAPDH as an internal control. The results were normalized to GAPDH levels using the formula ΔCt (Cycle threshold) = Ct of target gene – Ct of GAPDH. The mRNA level of the control group was used as the baseline; therefore, ΔΔCt was calculated using the formula ΔΔCt = ΔCt of target gene – ΔCt of the baseline. The fold change in the mRNA level was calculated as fold = 2-ΔΔCt. Primers used in this study were synthesized by Sangon Biotech Technology (Shanghai, China) (see Table 1).
FFPE samples were cut into 1-3 mm cores. Total RNA was isolated using the Qiagen RNeasy FFPE protocol. For analysis of miRNA expression, miRNA-specific reverse transcription was performed with a GenePharma MicroRNA Reverse Transcription Kit according to the manufacturer’s instructions. Quantitative RT-PCR was performed using GenePharma PCR Master Mix; U6 small nuclear RNA was used as an internal control. All reactions were performed in triplicate.
Western blot analysis
Total proteins were extracted from each group using RIPA buffer (Thermo Scientific, Rockford, IL, USA) and were quantified using a BCA kit (Thermo Scientific). The proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidenedifluoride (PVDF) membranes. After blocking with 5% bovine serum albumin, the blots were probed with the appropriate primary antibodies overnight at 4°C. The antibodies used included antibodies against E-cadherin (1:1000 dilution), N-cadherin (1:1000 dilution), snail (1:1000 dilution), slug (1:1000 dilution), Vimentin (1:1000 dilution), α smooth muscle actin (SMA) (1:1000 dilution), TGFβR2 (1:1000 dilution) and β-actin (1:500 dilution). The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse or anti-rabbit; 1:1000 dilution). Immunoreactive bands were detected using enhanced chemiluminescence (ECL) substrate (Pierce, Rockford, IL, USA) and imaged using the Image Quant LAS4000 system (GE Company, Pittsburgh, PA, USA).
In vitro migration, invasion, and wound scratch assays
The cells were collected and resuspended in serum-free DMEM at a concentration of 4×105 cells/ml, as determined by cell counts. Then, the cell suspension solution was seeded into the upper chambers (200μl/well), and the bottom chambers were filled with DMEM containing 10% FBS (1ml/well). We used CORNING (USA) Transwell chambers (Lot 3422, 6.5mm Diameter Inserts; 8-μm pore size; polycarbonate membrane) to quantify cell migration. For the Transwell invasion assay, CORNING (USA) Transwell chambers (Lot 354480, polycarbonate membrane, Matrigel-coated) were used. After they were cultured at 37°C for 24h, the cells that had not penetrated the polycarbonate membrane were wiped off with a cotton swab. The membrane was removed, fixed in 4% paraformaldehyde and stained with crystal violet solution. Cell migration and invasion were determined by counting five random high-power fields (Olympus Corp, Tokyo, Japan).
For the wound scratch assay, cells were plated and grown until confluence after which the cells were scratched using sterile tips. Cellular migration (toward the scratched area) was assessed after 24 h.
Transient transfection
Hsa-miR-373-3p inhibitor (miR-373i), hsa-miR-373 mimics (miR-373) and a negative control (NC) were purchased from GenePharma (Shanghai, China). The miRNA sequences are shown in Table 1. Cells were transiently transfected using jetPRIME® in vitro DNA & siRNA transfection reagent (Poly plus) according to the manufacturer’s instructions.
Luciferase assay
In the luciferase reporter vector, the wild-type or mutant 3'-UTR of human TGFβR2 was cloned into the downstream of the renilla luciferase gene in pGL3 vectors (GenePharma, Shanghai, China). Cells were seeded onto 24-well plates (1x105 cells per well) the day before transfection. Cells were then transfected with the firefly luciferase reporter plasmid including either the wild-type or mutant 3'-UTR of TGFβR2 (50 ng per well) and the pRL-TK Renilla luciferase reporter (10 ng per well). The cells were then transfected with miR-373-3p mimics or negative control (20 µM) using jetPRIME® in vitro DNA & siRNA transfection reagent (Poly plus).After 24 h of transfection, cells were lysed with 1× reporter lysis buffer, and firefly and renilla luciferase activities were measured using the Dual-Luciferase® Reporter Kit (Promega) according to the manufacturer’s instructions. Firefly luciferase activity was standardized to renilla activity as a control.
Statistical analysis
All experiments were repeated at least three times. Data are presented as the mean± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) and Student's t test. A P-value <0.05 was considered statistically significant and is indicated by asterisks in the figures.
Reagents
DMEM medium, foetal bovine serum (FBS), 0.02% EDTA and 0.25% trypsin were purchased from Gibco (St. Louis, MO, USA). Quantitative polymerase chain reaction (qPCR) master mix was purchased from Promega (Madison, WI, USA) and a Hairpin–it Real-Time PCR Kit was purchased from GenePharma (Shanghai, China). The Epithelial-Mesenchymal Transition Antibody Sampler Kit was purchased from Abcam (Cambridge, MA, UK). The anti-TGFβR2 rabbit monoclonal antibody and anti-β-actin mouse monoclonal antibody were purchased from Abcam (Cambridge, MA, UK). Peroxidase-conjugated AffiniPure goat anti-mouse IgG and peroxidase-conjugated AffiniPure goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Dual-Luciferase® Reporter Assay System was purchased from Promega (Madison, WI, USA).
Cell culture
Human placental choriocarcinoma cell lines (JAR and JEG-3) were obtained from the State Key Laboratory of Reproductive Biology (SKLRB), Institute of Zoology (IOZ), Chinese Academy of Sciences (CAS). HTR8-SVneo was purchased from Jennio Biotech Co., Ltd (Guang Zhou, China). Cells were cultured in DMEM supplemented with 10% FBS in an incubator with an atmosphere of 5% CO2 at 37˚C. The cells were then subcultured using 0.25% trypsin and 0.02% EDTA when the cells were approximately 80% confluent. Experiments were performed using cells at a logarithmic growth phase.
Paraffin-embedded human choriocarcinoma tissue
We included ten formalin-fixed paraffin-embedded (FFPE) choriocarcinoma samples from the pathological archives of the Affiliated Hospital of Chengde Medical College, Chengde Third Hospital and the Second Affiliated Hospital of Hebei Medical University. Ten women with normal early pregnancy and a single gestational sac were randomly selected, and the gestational villus were obtained at Chengde Women's and Children's Hospital.
miRNA microarray analysis
Total RNA in formalin-fixed paraffin-embedded (FFPE) sections was extracted using a Recover All™ Total Nucleic Acid Isolation Kit (Ambion-1975). MiRNA expression analysis was conducted on total RNA samples containing small RNAs using the human miRNA microarray, Release19.0, 8x60K (Agilent Technologies). For control and test RNAs, the synthesis of target miRNA probes and hybridization was performed using a miRNA Labeling Reagent and Hybridization Kit (Agilent Technology) according to the manufacturer’s instructions. The hybridized microarrays were washed according to the manufacturer’s washing protocol (Agilent Technology). Images of hybridized arrays were obtained using a DNA microarray scanner (Agilent Technology) and were quantified with Feature Extraction Software (Agilent Technology). Data normalization and the selection of genes with a fold change (FC) > 2 were performed using GeneSpring GX software (Agilent Technology).
Quantitative real-time PCR analysis (qRT-PCR)
Total RNA was isolated using the RNAgents® Total RNA Isolation System (Promega) with DNase I (Invitrogen) treatment. Then, 2 µg RNA, oligo (dt) 20 primers and the M‑MLV First‑Strand Synthesis System kit was used to synthesize cDNA. Real-time PCR was performed using the SYBR® Green I on GoTaq® qPCR Detection System (Promega) according to the manufacturer’s instructions. The results were analysed using the comparative threshold cycle method with GAPDH as an internal control. The results were normalized to GAPDH levels using the formula ΔCt (Cycle threshold) = Ct of target gene – Ct of GAPDH. The mRNA level of the control group was used as the baseline; therefore, ΔΔCt was calculated using the formula ΔΔCt = ΔCt of target gene – ΔCt of the baseline. The fold change in the mRNA level was calculated as fold = 2-ΔΔCt. Primers used in this study were synthesized by Sangon Biotech Technology (Shanghai, China) (see Table 1).
FFPE samples were cut into 1-3 mm cores. Total RNA was isolated using the Qiagen RNeasy FFPE protocol. For analysis of miRNA expression, miRNA-specific reverse transcription was performed with a GenePharma MicroRNA Reverse Transcription Kit according to the manufacturer’s instructions. Quantitative RT-PCR was performed using GenePharma PCR Master Mix; U6 small nuclear RNA was used as an internal control. All reactions were performed in triplicate.
Western blot analysis
Total proteins were extracted from each group using RIPA buffer (Thermo Scientific, Rockford, IL, USA) and were quantified using a BCA kit (Thermo Scientific). The proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidenedifluoride (PVDF) membranes. After blocking with 5% bovine serum albumin, the blots were probed with the appropriate primary antibodies overnight at 4°C. The antibodies used included antibodies against E-cadherin (1:1000 dilution), N-cadherin (1:1000 dilution), snail (1:1000 dilution), slug (1:1000 dilution), Vimentin (1:1000 dilution), α smooth muscle actin (SMA) (1:1000 dilution), TGFβR2 (1:1000 dilution) and β-actin (1:500 dilution). The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse or anti-rabbit; 1:1000 dilution). Immunoreactive bands were detected using enhanced chemiluminescence (ECL) substrate (Pierce, Rockford, IL, USA) and imaged using the Image Quant LAS4000 system (GE Company, Pittsburgh, PA, USA).
In vitro migration, invasion, and wound scratch assays
The cells were collected and resuspended in serum-free DMEM at a concentration of 4×105 cells/ml, as determined by cell counts. Then, the cell suspension solution was seeded into the upper chambers (200μl/well), and the bottom chambers were filled with DMEM containing 10% FBS (1ml/well). We used CORNING (USA) Transwell chambers (Lot 3422, 6.5mm Diameter Inserts; 8-μm pore size; polycarbonate membrane) to quantify cell migration. For the Transwell invasion assay, CORNING (USA) Transwell chambers (Lot 354480, polycarbonate membrane, Matrigel-coated) were used. After they were cultured at 37°C for 24h, the cells that had not penetrated the polycarbonate membrane were wiped off with a cotton swab. The membrane was removed, fixed in 4% paraformaldehyde and stained with crystal violet solution. Cell migration and invasion were determined by counting five random high-power fields (Olympus Corp, Tokyo, Japan).
For the wound scratch assay, cells were plated and grown until confluence after which the cells were scratched using sterile tips. Cellular migration (toward the scratched area) was assessed after 24 h.
Transient transfection
Hsa-miR-373-3p inhibitor (miR-373i), hsa-miR-373 mimics (miR-373) and a negative control (NC) were purchased from GenePharma (Shanghai, China). The miRNA sequences are shown in Table 1. Cells were transiently transfected using jetPRIME® in vitro DNA & siRNA transfection reagent (Poly plus) according to the manufacturer’s instructions.
Luciferase assay
In the luciferase reporter vector, the wild-type or mutant 3'-UTR of human TGFβR2 was cloned into the downstream of the renilla luciferase gene in pGL3 vectors (GenePharma, Shanghai, China). Cells were seeded onto 24-well plates (1x105 cells per well) the day before transfection. Cells were then transfected with the firefly luciferase reporter plasmid including either the wild-type or mutant 3'-UTR of TGFβR2 (50 ng per well) and the pRL-TK Renilla luciferase reporter (10 ng per well). The cells were then transfected with miR-373-3p mimics or negative control (20 µM) using jetPRIME® in vitro DNA & siRNA transfection reagent (Poly plus).After 24 h of transfection, cells were lysed with 1× reporter lysis buffer, and firefly and renilla luciferase activities were measured using the Dual-Luciferase® Reporter Kit (Promega) according to the manufacturer’s instructions. Firefly luciferase activity was standardized to renilla activity as a control.
Statistical analysis
All experiments were repeated at least three times. Data are presented as the mean± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) and Student's t test. A P-value <0.05 was considered statistically significant and is indicated by asterisks in the figures.