Cell culture and transfection
Human trophoblast cell line JAR and the human cervical cancer cell line HeLa were purchased from the American Type Culture Collection (Manassas, VA, USA). HTR-8/SVneo cell was obtained from the Animal Institute of the Chinese Academy of Sciences. And cells were maintained in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (ScienCell, USA), 1% penicillin-streptomycin solution (Thermo Fisher Scientific, USA). Cultures were maintained in the cell incubator with a humidified atmosphere of 5% CO2 at 37 °C。
MiR-30a mimics and mimics negative control,miR-30a inhibitor and inhibitor negative control were purchased from Guangzhou RiboBio Co.,LTD. r. Small interfering RNA (siRNA) for STOX2 and negative control were produced by Shanghai GenePharma (Shanghai, China). The si-STOX2 sequence was 5′-AUGGGAGACAUACUGAUGGTT -3′ and the si-NC sequence was 5′-ACGUGACACGUUCGGAGAATT -3′. STOX2 DNA and corresponding negative control were composed by GeneCopoeia Inc. The cells were seeded in 6-well plates,after 70%-80% confluence, the transfection was performed by Lipofectamine® 2000 reagent according to the manufacturer's descriptions.
RNA extraction and Quantitative RT-PCR
The total RNAs were extracted from JAR and HTR-8 cells by using TRIzol reagent. cDNA is synthesized using TransScript All-in-one First-Strand Cdna Synthesis SuperMix for qPCR kit ( One-Step gDNA Removal) (TransGen, Beijing, China) according to the manufacturer's specifications. qPCR was performed by using a TransStart Top Green qPCR SuperMix (TransGen, Beijing, China) with analysis in an ABI 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR conditions were 94°C for 30 sec followed by 40 cycles of denaturation at 94°C for 5 sec and annealing/elongation at 60°C for 30 sec. U6 was used as the internal reference for miR-30a, and GAPDH was performed as the control for STOX2 expression. The PCR primer was shown as follows: STOX2 forward: 5’-AGCCTGTCCCTCCTCAAATCTCA-3’, reverse: 5’-CTCTGTGTTCTTGTTTGCCCCT-3’; GAPDH forward: 5’- GTGAAGGTCGGAGTCAACG-3’, reverse: 5’- TGAGGTCAATGAAGGGGTC -3’; the data was calculated using 2-ΔΔct method. All experiments were performed triplicate samples.
CCK-8 assay
Transfected Cells were seeded 96-well plates in 200 μl medium at the density of 5000 per well and cultured for 24, 48, 72h, cck-8 (Dojindo, Tokyo, Japan) was added into the each well according the manufacturer's specifications. After 4h in 37℃ incubation, the absorbance at 450 nm were detected using a microplate reader(BioTek, USA ).
Colony formation assay
After Transfected 48h, 3×103 Cells were seeded 6-well plates and incubated for 15 days. The colonies were fixed with methanol for 30 min and stained with 1% crystal violet for 15 min. Cells number was counted by using Image J.
EdU assay
For transfected cells, EdU reagent (Beyotime, Shanghai, China) was added into 96-well plates and cultured 2h in 37℃ incubator. Next, Cells were fixed with 4% paraformaldehyde for 30 minutes and 0.1% Triton-X 100 for 15 minutes, and incubated with Azide-488 for 30 minutes at room temperature in the dark room. Images were taken under inverted microscope (Olympus, Japan).
Transwell assay
8-μm pore size 24-well Transwell plates (Corning, USA) were used to evaluate the Migratory and invasive potential of JAR and HTR-8 cells. For migration assay, transfected cells were collected and resuspended in 1 ml serum-free RPMI-1640 medium, 5×104 cells were added to the the upper chambers, RPMI-1640 medium with 10% FBS was seeded into the lower chambers. After 24h in incubator with 5% CO2 37℃,the migratory cells were fixed with methanol for 30 min and stained with 0.2% crystal violet for 30 min. For invasion assay, the upper chambers of transwell were pre-coated with Matrigel (BD Biosciences, USA), 5s104 transfected cell were seeded into the upper chambers with serum-free RPMI-1640 medium, after incubated 30h, the cells which not invasive were wiped off with a cotton swab, and the invasive cells were fixed with methanol for 30 min and stained with 0.2% crystal violet for 30 min. All migratory and invasive chambers were counted under a light microscope (Olympus, Japan).
Western blot assay
After the cells were transfected for 48h, total protein was cleaved using ProteinExt® Mammalian Total Protein Extraction Kit (TransGen, Beijing, China ), and the protein was determined by the BCA method (TransGen, Beijing, China ). 30µg or 60µg protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10%) and transferred onto a nitrocellulose membrane (Millipore, Bedford, MA, USA) using cold transfer buffer. The membrane was stained with Ponceau S (Beyotime, Shanghai, China) and washed with TBST. 5% non-fat milk was used to block the membrane for 2h at room temperature. The membrane was incubated with the primary antibody overnight 4℃. The list of primary antibodies is as follows: STOX2 (1:1000, Abcam), ERK (1:1000, Beyotime), p-ERK (1:500, Beyotime), AKT (1:1000, Beyotime), p-AKT (1:500, abcam), P38 (1:1000, Elabscience, wuhan, China), p-P38 (1:500, Elabscience, wuhan, China), GAPDH (1:4000, proteintech). Next day, the membrane was washed and the secondary antibody was incubated at room temperature for 1 h. Blots were then developed by chemiluminescence with Pierce ECL kits (Thermo scientific, USA). The data was used to analysis by Image J software.
Luciferase activity assay
The miRNA prediction website (miRBase, TargetScan, PicTar) was assessed the binding site of miR-30a and STOX2 3’UTR. The 3’-UTR of STOX2 were synthesised by PCR and cloned into the Xhol site downstream of Renilla luciferase genes in the PmiR report vector (Promega, USA), the 3’UTR of STOX2 primer forward: 5’-CCGCTCGAGCGGCAGATCTTCTGTCTCATTCGATACA-3’
Reverse: 5’-CCGCTCGAGCGGCAGATCTTCTGTCTCATTCGATACA-3’. WT or Mut seed sequences of 3’UTR of STOX2 were constructed onto the PmiR report vector. HeLa cells were seeded into 12-well plates and cotransfected with miR-30a mimics or negative control and WT or Mut vector, after incubation of 24h, the Dual-Luciferase® Reporter Assay was implemented with the manufacturer's manual (Promega, USA). The luciferase activities were measured by a Fluorescence/Multi-Detection Microplate Reader (BioTek, USA)
Cell cycle analysis
48h after transient transfection, the cells was collected and washed with PBS two times, then fixed with cool 70% ethanol overnight at 4℃. The fixed cells were stained with PI/RNase solution (Sungene, Tianjin, China) for 30 min at room temperature and analyzed using a FACS Calibur (BD Biosciences, USA). The percentage of cells in each period was analysed by ModFit software.
Immunohistochemistry (IHC)
Tissue sections were dewaxed in xylene and dehydrated gradient ethanol, the activity of endogenous peroxidase was activated and blocked with 3% H2O2 for 20 min in the dark room. Goat serum was added on the tissues by using a dropwise way for 20 min at room temperature. Then, the sections were incubated with the following primary antibodies overnight at 4℃:STOX2 (1:100, Abcam), AKT (1:70, Beyotime), ERK (1:100, Beyotime), p-ERK (1:50, Beyotime), p-AKT (1:70, abcam), P38 (1:100, Elabscience, wuhan,China), p-P38 (1:50, Elabscience, wuhan,China). Next, the second antibody and horseradish peroxidase streptavidin were maintained for 30 min at 37℃, respectively. The sections were stained by DAB coloration (OriGene Technologies, Beijing, China) and hematoxylin stain (KeyGEN BioTECH, Jiangsu, China). Tissues were imaged with a light microscope.
Hematoxylin and eosin (HE) staining
The Hydatidiform mole and normal placenta tissues were fixed in 4% formaldehyde and embedded in paraffin for Hematoxylin and eosin (HE) staining. The slices were stained with hematoxylin for 20 min and eosin for 30 s−1 min. Finally, the slices were analyzed under a light microscope.
Statistical Analysis
All data were presented as mean ± SD and analysised by using GraphPad Prism 6.0 software. All experiment was repeated three times independently. Significance of differences between the two groups was assessed via One-way ANOVA and P value <0.05 was considered statistically significant.