Cell lines and culture conditions

JEG-3 choriocarcinoma cells (HBT-36) and NK-92MI cells (CRL-2408) were obtained from American Type Culture Collection (Manassas, VA). The JEG-3 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, BRL, Gaithersburg, MD), containing 10% fetal bovine serum (FBS). The NK-92MI cells were maintained in alpha minimum essential medium (α-MEM, Gibco) supplemented with 2 mM L-glutamine, 0.2 mM i-inositol, 0.02 mM folic acid, 0.1 mM 2-mercaptoethanol, 12.5% FBS, and 12.5% horse serum. All cells were cultured at 37 ^o C in a humidified atmosphere of 5% CO ₂ and 95% air.

Transient overexpression of hsa-miR-152

The miR-152 precursor (pre-miR-152) and Cy3 dye labeled pre-miR negative control #1 (pre-miR-control) were purchased from Ambion (Austin, TX). The JEG-3 cells were transfected with pre-miR-152 using siPORT NeoFx reagent (Ambion) according to the manufacturer’s protocol.

To confirm the efficiency of transfection, the Cy3-labeled control #1 was also transfected. The medium was replaced with fresh growth medium after 12 hours. Forty-eight to 72 hours after transfection, cells were used for reverse-transcription polymerase chain reaction (RT-PCR) analysis, Western blot analysis, transwell invasion assay, and NK cell cytotoxicity assay.

RT-PCR analysis

Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA). RNA integrity was confirmed by electrophoresis in a 1.5% agarose denaturing gel. For the detection of HLA-G gene, 1 μg of the total RNA was subsequently reverse transcribed into complementary deoxyribonucleic acid (cDNA) primed by oligo (dT) with the use of the RevertAid first-strand cDNA synthesis kit (MBI; Fermentas, Vilnius, Lithuania). β-Actin was used as an endogenous control.

For the detection of mature miRNA, 4 μg of the total RNA was reverse transcribed to cDNA with gene-specific RT primer, which could fold to a stem-loop structure. The highly conserved and universally expressed small nuclear RNA U6 was used as an endogenous control. All primer sequences and the reaction conditions are shown in the Table . A 25 μL PCR master mix was prepared as follows: 1 μL RT products, 200 μmol/L deoxyribonucleoside triphosphates, 2 mmol/L MgCl ₂ , 1 IU Taq polymerase, and 10 pmol of each primer.

The amplification was ensured within the exponential phase of PCR by preliminary experiments. PCR products were subjected to electrophoresis on agarose gels. The relative densities of target genes normalized with control genes were analyzed with the Image-Pro Plus (software version 6.0; Media Cybernetics, Silver Spring, MD).

Western blot analysis

Infected cells were washed 3 times with ice-cold phosphate-buffered saline (PBS) and then lysed with 1× lysis buffer (Cell Signaling Technology, Beverly, MA). The supernatant was collected after centrifugation.

Protein estimation was carried out by the Bradford method. Thirty micrograms of protein per lane was separated by 12% sodium dodecyl sulfate–polyacry1-amide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA) by semidry electroblotting. The membrane was blocked in 5% nonfat dry milk in PBST for 1 hour at room temperature and then incubated with monoclonal antibody MEM-G/1 (Serotec, Oxford, UK) in the PBS at 4°C overnight.

On the next day, the membrane was washed with PBST and incubated with horseradish peroxidase–conjugated goat antimouse immunoglobulin G (Sigma, St Louis, MO) for 1 hour at room temperature. Signals were detected with SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). After stripping, the same membrane was reprobed with β-actin antibody (NeoMarkers, Fremont, CA) as internal loading control. The relative intensity of HLA-G was normalized to β-actin, which was determined by densitometric analysis.

Target in vitro assay

For the luciferase reporter experiment, the 3′-UTR segment of HLA-G (accession number NM_002127 ) was amplified by PCR. The cDNA transcribed from total RNA of JEG-3 cells was used as template. Primer sequences designed to carry the Spe I and Hind III sites are shown in the Table .

The PCR product was inserted into the Spe I and Hind III cloning sites of the pMIR-PEPORT luciferase expression reporter vector (Ambion) to generate pMIR-UTR. Transfection was performed with siPORT XP-1 reagent (Ambion). For the test group, transfections of JEG-3 cells were performed with pMIR-UTR and pre-miR-152.

For the blank control group, transfections were performed with pMIR-UTR but no pre-miR-152. For the negative control group, transfections were performed with pMIR-UTR and pre-miR-control. All cells were also transfected with pMIR-PEPORT β-galactosidase (pMIR-β-gal) for normalizing variability because of transfection differences. After 48 hours, the cells were lysed and measured for luciferase activity and β-gal activity with the use of the luciferase assay system and β-galactosidase enzyme assay system (Promega, Madison, WI) according to the manufacturer’s instructions.

Transwell invasion assay

In vitro cellular invasion was assayed by determining the ability of cells to invade a synthetic basement membrane. Briefly, infected cells were plated at 4 × 10 ⁴ cells in transwell inserts (8-μm pore size; Costar, Cambridge, MA) precoated with type I collagen (Col I; 80 μg/mL; cell matrix type I A; Institute of Biochemistry, Osaka, Japan) and incubated with DMEM containing 0.2% FBS. Lower chambers were loaded with DMEM medium containing 10% FBS.

The cells were incubated at 37°C and allowed to invade through the collagen barrier for 24 hours. Following incubation, the invading cells were fixed with 4% paraformaldehyde and stained with hematoxylin, and noninvading cells were removed with a cotton swab. Cell invasion index was determined by counting the number of stained cells in 10 randomly selected nonoverlapping fields of the membranes with a light microscope. Images were captured with a Nikon microscope (TE2000S; Nikon, Tokyo, Japan; original magnification, ×200).

NK cell cytotoxicity assay

Cytotoxicity analysis was performed with CytoTox96 nonradioactive cytotoxicity assay (Promega) as the protocol instructed. Briefly, effector/target ratio (E/T) was optimized. The NK-92 cells (effector cells) were mixed with 1 × 10 ⁵ JEG-3 cells (target cells) at different E/T ratios (ranging from 2.5:1 to 20:1). Target cell spontaneous release and maximal release of lactic dehydrogenase (LDH) and the effector cell spontaneous LDH release were determined by incubating these cells in medium alone. After incubation for 4 hours, the LDH measurement was performed by a standard 96-well plate reader. The following formula was used to compute percent cytotoxicity: percent cytotoxicity = (experimental – effector spontaneous – target spontaneous)/(target maximum – target spontaneous) × 100.

Statistical analysis

All values are presented as the mean ± SD of 3 individual experiments done in triplicate. Comparison of the values between groups was performed with 1-way analysis of variance by SPSS 11.0 software (SPSS Inc, Chicago, IL), and P < .05 was taken to be statistically significant.