Clinical samples
Histologically confirmed RCC tissues and paired adjacent normal tissue were collected from 80 patients who underwent radical nephrectomy at Peking University First Hospital between 2012 and 2013; after collection, the tissues were snap-frozen in liquid nitrogen. The Ethics Committee of Peking University First Hospital approved this study. Written informed consent was obtained from all the patients.
Cell lines and reagents
Human RCC cell lines 769P and 786O were purchased from ATCC (MA, USA). The human acute monocytic leukemia cell line THP-1 and human umbilical vein endothelial cells (HUVECs) were obtained from the China Center for Type Culture Collection (Wuhan, China). Pri-let-7d lentivirus stably transfected 786O and 769P cell lines overexpressing let-7d, as well as the corresponding vehicle-transfected control cells, were established as previously described [17]. The transformed human embryonic kidney 293FT cells were purchased from Invitrogen. The 786O and 769P cells were maintained in RPMI 1640 medium. The HUVECs were maintained in DMEM/F12 (50:50, v/v) medium. The 293FT cells were routinely cultured in DMEM. All the culture media were supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin sodium and 100 μg/ml streptomycin sulfate, and the cells were incubated at 37 °C in a standard humidified incubator containing 5% CO2.
Phorbol-12-myristate-13-acetate (PMA) was purchased from Sigma-Aldrich. THP-1 cells were seeded at 2 × 105 cells/cm2 and treated with PMA (200 nM) for 48 h to differentiate the cells into resting macrophages (M0 cells) as previously described [18].
In vivo assay
Approval of the animal studies was obtained from the Ethics Committee of Tsinghua Changgung Hospital. Specific pathogen-free (SPF) 5-week-old female BALB/C-nu/nu nude mice (Vital River Laboratory Animals, Beijing, China) were used in this study and maintained in accordance with institutional guidelines for the use of laboratory animals. A total of 3 × 106 786O cells stably transfected with lentivirus and 3 × 106 THP-1 cells treated with PMA (M0) were subcutaneously injected into the right flank of each mouse in a 1:1 mixture of Matrigel (BD Biosciences, San Jose, CA). Five mice were included in each group. After 7 weeks, the mice were euthanized, and the tumors were dissected, weighed and prepared for RNA isolation.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. To analyze the mature miRNA, 1μg total RNA was polyadenylated to add a polyA tail with polyA polymerase (NEB, Beverly, MA, USA) and then reverse-transcribed with an oligo-dT adapter primer into cDNA for qRT-PCR quantification [17]. To detect the protein-coding mRNA, cDNAs were synthesized from 2 µg of total RNA using Moloney murine leukemia virus reverse transcriptase and oligo(dT)15 primers (Invitrogen). Quantitative RT-PCR was performed using the SYBR Select Master Mix (Life) in a final volume of 10 μL on an ABI7500 Fast PCR machine (Applied Biosystems, Foster City, CA, USA). U6 and GAPDH were selected as the endogenous references for miRNA and mRNA, respectively. Relative quantification (RQ) was calculated based on the 2−ΔCt method, where ΔCt=Ct (target)-Ct (reference). The fold change was calculated by the 2−ΔΔct method. For a list of all the primers, see Supporting Information, Table S1.
Lentiviral vector construction, virus packaging and cell infection
All the constructs were generated by standard DNA recombination techniques as previously described [17]. Briefly, the open reading frames of the IL-10 and IL-13 cDNAs were obtained by RT–PCR and cloned into the vector pcDNA3.1. All of the constructs were verified by sequencing. The sequences of the primers are listed in Table S1 (see Supporting Information). Lentiviruses were produced using the ViraPowerTM Packaging Mix (Invitrogen) in 293FT packaging cells. The virus-infected cells were selected with 5 μg/ml blasticidin + 500 μg/ml G418 (Invitrogen). The antibiotic-resistant clones were pooled and used for the subsequent assays.
In vitro cell coculture and conditioned medium preparation
RCC cells were seeded in 6-well plates at a density of 1 × 105 cells/well in serum-containing medium. Twenty-four hours later, the medium was replaced with serum-free RPMI 1640 medium, and the cells were incubated for an additional 48 h. The conditioned medium was collected and centrifuged at 3000×g for 5 min to remove the cells or cell debris.
Transwell cellculture inserts (Corning Incorporated, Corning, NY) with 12-mm polyester (PET) membranes (pore size: 0.4 μM, density: 1.6 × 106 pores/cm2) were used for the noncontact coculture of RCC cells and THP-1 cells in 6-well plates, as previously described [19]. Briefly, 2 × 105 PMA-pretreated TPH-1 cells were cocultured with RCC cells in serum-free RPMI 1640 medium at 37°C in an atmosphere containing 5% CO2. Twenty-four hours later, the culture medium from the lower compartments was collected, centrifuged and used as conditioned medium. In the in vitro rescue experiment, human recombinant IL-10 and IL-13 (R&D Systems, Minneapolis, MN) were added to the lower chamber of the coculture system at a concentration of 100 ng/mL and incubated for 24 h before collecting the conditioned medium.
In vitro cell proliferation, migration, and tube formation assay
HUVECs were seeded in 96-well plates (5 × 103 cells/well) and cultured in conditioned medium from RCC cells alone or THP-1 cells cocultured with RCC cells. Cell proliferation was evaluated by a Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) at various time points for up to four days according to the manufacturer’s instructions. The cell migration assays were performed using a Boyden chamber as previously described [17]. Briefly, 2× 105 HUVECs in 100 μl were added to the upper chamber of the 24-transwell apparatus, and 800 μl conditioned medium was added to the lower chamber. After 24 h of incubation, the cells that migrated though the membrane were fixed with 100% methanol, stained with 0.1% crystal violet stain for 5 min, and counted under a microscope. Five high-power fields (magnification, 200×) per well were randomly selected and manually counted. The Fibrin In Vitro Angiogenesis Assay (Millipore) was used to analyze new blood vessel formation according to the manufacturer's instructions. Briefly, 96-well plates were coated with cold liquid ECMatrix (50 µL/well) and incubated at 37 °C for 1 h. Then, HUVECs were seeded at a density of 5×103 cells/well on the polymerized ECMatrix and incubated with conditioned media at 37 °C for 6 to 8 h. Branch number was observed under a phase-contrast microscope. The enclosed networks in 5 random fields per well were measured by ImageJ software, and the average value was calculated. The experiment was repeated in triplicate.
Immunohistochemistry (IHC)
Paraffin-embedded tissues were analyzed using immunohistochemical staining with an anti-CD31 antibody (DAKO, Carpinteria, CA) and the microvessel density (MVD) was evaluated as previously described [20]. CD31 was used as a vascular endothelial cell marker. Any morphologically identifiable vessels with a lumen and CD31-positive endothelial cell clusters that were clearly separate from the adjacent microvessels in the selected field were all counted as individual vessels. Five independent areas with the highest numbers of discrete microvessels were selected, digitally photographed, and manually counted under a microscope using a 20× objective lens (Olympus IX71, Olympus Optical Co. Ltd, Tokyo, Japan). The average vessel counts for each tumor tissue were used for the statistical analysis. Two pathologists who were blinded to the patients' clinical data independently examined all the slides.
ELISA
The conditioned medium from the cocultured M0 cells was used to detect PIGF by a ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. The optical density (OD) was measured at 450 nm with a Multiskan microplate spectrophotometer (ThermoLab Systems, Milford, MA).
Western blot analysis
Total protein was extracted from the tumor tissues and cultured cells using a radioimmunoprecipitation assay (RIPA) kit (ShineGene Molecular Biotech, Inc., Shanghai, China), as instructed by the manufacturer. After determining the protein concentration using a bicinchoninic acid (BCA) kit (Pierce, USA), equal amounts of the total protein were subjected to 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE). Thereafter, the proteins were blotted onto polyvinylidene difluoride (PVDF) membranes (Pall, Pensacola, FL). After the nonspecific binding sites were blocked, the blots were incubated with antibodies against IL-10 or IL-13 (Abcam, Cambridge, UK) for 1 h at 37 °C. After three washes with TBST and incubation for 2 h with the appropriate HRP-conjugated secondary antibodies (Applygen, China), the ImmobilonTM Western Chemiluminescent HRP substrate (Millipore) was used to detect the proteins of interest on the blots. The blotted membranes were then scanned using GeneSnap (Syngene, Cambridge, UK) acquisition software.
Flow cytometry
Cells were centrifuged at 1500 rpm for 5 min, washed with PBS and filtered through a 100-μm mesh for flow cytometry. The cells were subsequently stained with an antibody against an M2 macrophage surface markers (PE-labeled anti-human CD204 (R&D Systems, Minneapolis, MN)) for 30 min at 4 °C. Isotype control antibodies (Biolegend) were used as negative controls. The cells were analyzed using a flow cytometer (FAC-SCalibur, BD Biosciences). The collected data were processed by the CellQuest program (BD Biosciences). The experiment was repeated in triplicate.
Dual luciferase activity assay
The 3’-untranslated regions (UTRs) of human IL-10 and IL-13 containing putative binding sites of let-7d and the corresponding mutant binding sites were chemically synthesized and inserted immediately downstream of the firefly luciferase cDNA in the pGL3-control vector (Promega, Madison, WI) by GenePharma (Shanghai, China) to generate the pGL3–IL-10, pGL3–IL-13, pGL3–IL-10–Mut, and pGL3–IL-13–Mut constructs. 293FT cells were plated at 1.5 × 105 cells/well in 24-well plates 24 h before the transfection.ThepGL3 constructs (0.3 μg) plus the pRL-TK plasmid that expressed Renilla luciferase (26 ng; Promega) were transfected in combination with 60 pmol of either a stability-enhanced non targeting oligonucleotide control or let-7d mimics (GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega) on the Varioskan™ Flash Multimode Reader (Thermo Fisher Scientific, Inc., Waltham, MA) according to the manufacturer's instructions. For each transfected well, the firefly luciferase activity was normalized to the Renilla luciferase activity. The results were obtained from three independent experiments, and each experiment was performed in quadruplicate.
Statistical analysis
All the data are presented as the mean ± SD of at least three independent experiments and were analyzed using SPSS 20.0 statistical software (IBM, Chicago, IL, USA). The significance of the difference between two groups was determined using double-sided Student's t-test. In the case of multiple tests, one-way ANOVA followed by the Bonferroni–Holm procedure was used unless otherwise specified. Correlations were evaluated using two-tailed Spearman’s test. p<0.05 was considered statistically significant.