Human Tissue Samples and Cell Lines
All human samples were collected in compliance with the guidelines of the Ethics Committee at the Fudan University Cancer Hospital. Fresh-frozen surgical ovarian tissue samples were collected from 26 patients with ovarian cancer (aged 20-58 years) and 20 control patients with benign ovarian disease (aged 23-55 years) who were admitted to Fudan University Cancer Hospital (Shanghai, China) between July 2008 and December 2009. All cases were confirmed by pathology. The samples were defatted, immediately cut into pieces of appropriate size on ice, and stored at -80°C for later use. The ovarian carcinoma cell line, SKOV3, and the non-ovarian cancer cell line, 293T, were generous gifts from Dr. Meiqin Zhang, Laboratory of Gynecologic Oncology, Fudan University Shanghai Cancer Center, Shanghai.
Reverse-Transcription Polymerase Chain Reaction (RT-PCR) and Quantitative Real-Time PCR
Total RNA was extracted from the cells using the TRIzol reagent (Invitrogen, NY, USA) according to the manufacturer's protocol, and the RNA concentration was determined using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). RT-PCR was carried out using a SuperScript™ one-step RT-PCR kit (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized from 2 μg of total RNA at 42°C for 45 min, followed by inactivation of the reverse transcriptase at 85°C for 5 min. The cDNA was then subjected to PCR to amplify the MR-1 and GAPDH (control) genes in 50 μL reaction mixture containing the following primers: MR-1 forward 5'-CCCAGAAAGAGGG GCAAGA-3' (P1), MR-1 reverse 5'-TGAGGATGAAGAG GAGGATACCA-3' (P2), GAPDH forward 5'-GGGAGCCAAAAGGGTCATCATCTC-3' (P1) GAPDH reverse 5'-CCATGCCAGTGAGCTTC CCGTTC-3' (P2). The cycling conditions were: initial denaturation at 95°C for 3 min, followed by 36 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s. PCR products were resolved and analyzed on a 2% TAE agarose gel containing ethidium bromide. Quantitative real-time PCR for MR-1 (GAPDH as internal control) was performed in an MJ PCR system (Reno, NV, USA) using a 20 μL reaction volume containing 10 μL of SYBR® Premix Ex Taq™ reagent (Takara, Dalian, China), 0.5 pmol of each primer, 1 μg of cDNA, and the same sets of primers and cDNAs. The conditions were: an initial denaturation step at 94°C for 10 min, followed by 40 cycles of 15 s at 95°C (denaturation), 30 s at 55°C (annealing), and 30 s at 72°C (extension). Delta Ct (CtMR-1-CtGAPDH) was calculated for each set of samples and compared.
Vector Construction and Production of MR-1 Recombinant Protein
Human MR-1 cDNA was synthesized by reverse transcription from the mRNA (sequence originally published by Li et al [7]). A 429-bp fragment was amplified from the synthesized cDNA by PCR using Taq PCRx® DNA polymerase (Invitrogen) to introduce BamHI and XhoI restriction sites using the primers 5'-CGGGATCCATGGCGGCGGTGGTAGCTGC-3' (P3, BamHI site underlined) and 5'-CCGCTCGAGTCAGGTCTGCACCCCAGAC-3' (P4, XhoI site underlined). The cDNA fragment was then cloned into the PET21a (+) expression vector (Invitrogen) to yield PET21a-MR1, which was then transformed into Escherichia coli BL21 (DE3) cells for expression of the MR-1 recombinant protein [13]. The MR-1 recombinant protein contained a T7-tag at the N-terminus and 6 × His-tag at the C-terminus and was purified using Ni-resin affinity chromatography. The purified protein was confirmed by western blotting with a mouse anti-His PcAb (Invitrogen).
Antibody Production
New Zealand white rabbits were immunized four times with the MR-1 recombinant protein. Sera were harvested 3 days after the final immunization if the rabbits showed a good anti-MR-1-His titer. The anti-MR-1 antibodies R1 and R2 were then affinity-purified using a protein A column (Pierce Biotechnology Inc., Rockford, IL, USA). The antibody titers were measured using an enzyme-linked immunosorbent assay (ELISA), and immunoreactivity was confirmed by western blotting with MR-1 recombinant protein and human ovarian cancer cells (SKOV3).
Immunocytochemistry
Immunohistochemical staining was performed on 6-μm sections from formalin-fixed, paraffin-embedded human tissues. The slides were deparaffinized and rehydrated in graded ethanol solutions. Antigen retrieval was performed by heating the samples in a microwave oven for 20 min in citrate buffer (pH 6.0). The slides were then incubated with the affinity-purified MR-1 antibody (1:200) followed by incubation with a horseradish peroxidase-labeled polymer conjugated to a goat anti-rabbit immunoglobulin antibody (1:4000; R&D Systems, Minneapolis, MN, USA). Color was developed using diaminobenzidine as a chromogen and the slides were counterstained with hematoxylin. An anti-His antibody and polyclonal non-immune rabbit IgG were used as negative controls to check the specificity of the MR-1 affinity-purified antibody. Immunocytochemical staining of SKOV3 cells was performed on cover slips by fixing the cells for 20 minutes in acetone, blocking with 2% bovine serum albumin, and then incubating with affinity-purified anti-MR1 antibody followed by DAB staining and hematoxylin counterstaining. Dark brown cytoplasmic staining was defined as positive and no (or faint) staining was defined as negative. The proportion of tumor cells positive for MR-1 varied considerably: diffuse positive (> 50%), focal or heterogeneous (10%-50%), and trace (< 10%).
Establishment of Stable Cell Line Overexpressing MR-1
The full-length coding sequence for the MR-1 cDNA was subcloned into the pMX-puro(+) vector (Invitrogen) to yield pMX-MR-1, which was then transfected into 293T cells using Fusion 6® (Roche, Mannheim, Germany) according to the manufacturer's instructions. A pMX-mock vector (pMX-puro(+) containing a random non-MR-1 DNA sequence) was used as the control. Puromycin (0.5 μg/ml; Invitrogen) was added to the cells 48 h after transfection to select stably transfected clones. Single clones were picked from both pMX-MR-1 and pMX-mock transfected samples, and RNA and protein were extracted to check the expression levels of MR-1 by real-time PCR and western blotting. Positive clones were designated pMX-MR-1-293T, and control clones as pMX-mock-293T. 293T cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with high glucose, 10% fetal bovine serum (FBS, Gibco BRL, Grand Island, NY, USA), and 1% penicillin/streptomycin at 37°C in 5% CO2.
Cell Proliferation and Invasion Assays
For the cell proliferation assay, pMX-MR-1-293T and pMX-mock-293T cells were grown at a density of 1000 cells/ml in 24-well plates with regular medium changes. The cell number was counted every 24 hours for 6 days as follows: cells were detached by brief exposure to 0.025% trypsin containing 2 mM EDTA in PBS, washed in culture medium without FBS, and then resuspended in the same medium for manual cell counting. For the invasion assays, Matrigel was thawed at 4°C overnight, diluted in cold serum-free culture medium, plated onto 24-well plates preloaded with Transwell™ culture inserts (12 mm diameter, 8 μm pore size; Costar, Cambridge, MA, USA) and incubated for 5 hours at 37°C for gelling. The cells were then plated onto the Transwell™ inserts (50,000 cells/well) and cultured at 37°C in 5% CO2. After 16 hours, the cells on the upper side of the well were removed and fixed prior to staining with hematoxylin and eosin. Cells migrating to the underside were counted under a microscope. All experiments were repeated at least three times.
MR-1 Knockdown Using RNAi
SKOV3 cells were cultured in Gibco® RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO2. When the cells reached 70-80% confluence, they were transfected with pGPU6-MR-1 short hairpin DNA (shDNA) targeting MR-1. The sequences of the shDNAs were as follows: shDNA 232: 5'-CACCGCTAACAAGGCTTCTCATAACTTCAAGAGAGT TATGAGAAGCCTTGTTAGCTTTTTG-3'; shDNA 442: 5'-CACCGACCGTGTGA AGCAGATGAAGTTCAAGAGACTTCATCTGCTTCACACGGTCTTTTTTG-3'; shDNA 584: 5'-CACCGACCCTCCTAGGCTATTGACTTTCAAGAGAAGTCAATA GCCTAGGAGGGTCTTTTTTG-3'; mock sequence: 5'-CACCGTTCTCCGAACGT GTCACGTCAAGAGATTACGTGAGACGTTCGGAGAATTTTTTG-3'. The pGPU6-shDNA plasmids were constructed by cloning the respective shDNAs into the pGPU6/GFP/Neo vector (Invitrogen). The cells were exposed to 1500 μg/ml G418 (Invitrogen) for 2 days after transfection to select for stably transfected clones. Cells were then plated at a lower density in 96-well plates containing RPMI 1640 and G418 (750 μg/ml) until single colonies were formed. Colonies from the SKOV3-MR-1-shDNA and SKOV3-mock-shDNA-transfecetd cells were then picked and expanded. Real-time PCR and western blotting were used to test the expression levels of MR-1. GAPDH was used as the internal control.
Effects of Anti-Tumor Drugs on MR-1 Expression
SKOV3 cells (50,000 cells/well) were cultured in six-well plates in RPMI 1640 under the conditions described above. After 24 hours, 10 μL of serially diluted paclitaxel (equivalent to 0, 12.5, 25, 50, 100 nmol/L; Bristol-Myers Squibb Inc., USA) or carboplatin (equivalent to 0, 40, 80, 160, 320 mg/L) (Qilu Pharmacy Inc., China) were added to the medium and the cells were cultured for an additional 48 hours. One set of cells was treated with TUNEL reagent (Roche) and counterstained with DAPI, and the number of apoptotic cells (stained by Annexin V-FITC/PI) was determined by flow cytometry (Becton Dickinson, USA). The other set of cells was harvested for RNA and protein extraction, and the expression levels of MR-1 were determined by real-time RT-PCR and western blotting.
Statistical analysis
Statistical analysis was performed using Analysis of Variance (ANOVA) and the post-hoc Student-Newman-Keuls test, with SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). The results were expressed as the mean ± SD. A P value < 0.05 was considered significant.
