Cell culture and treatment

Human renal clear cell carcinoma cell lines 786-O and Caki-1, human normal kidney proximal tubular cell line HK-2, and human monocyte cell line THP-1 were purchased from SIBS (Shanghai, China). Caki-1 cells were cultured with McCoy’s 5A medium (PM150710, Procell, China) and other three cell lines were cultured with RPMI 1640 medium (10-040-CV, CORNING, China), these medium were contained 10% fetal bovine serum (FBS, 10099-141, GIBCO, China) and 1% Penicillin–Streptomycin (E607011, Sangon, China). In addition, THP1 cells were used to induce macrophages by treating with 100 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma, USA) for 48 h. For further M2 phenotype macrophage induction, cells were treated with 20 ng/mL IL-4 for 48 h in the presence of PMA.

Exosome isolation

Exosomes extraction was completed as previous description [15]. Briefly, 12 h before harvested, culture medium 786-O and Caki-1 cells were replaced by fresh medium without FBS. After that, cell culture was collected by centrifuging at 300 g for 10 min. The supernatant was further centrifuged at 2000 g for 10 min to remove cellular and retained supernatant followed by filtering with a 0.2 mm filter and centrifuged again at 10,000 g for 1.5 h. The pellet was collected as exosomes and suspended in phosphate buffer saline (PBS) for using subsequently analysis. The concentration of exosomes was determined by the protein content using BCA protein assay.

Transmission electron microscopy (TEM)

The morphology of aliquot exosomes was observed by TEM as described previously [15]. Briefly, exosmoes were fixed with 2.5% glutaraldehyde in 0.1 mol/L PBS at 4 °C overnight followed by post-fixed in PBS 1% OsO4 for 2 h. next, exosmoes were embedded in 10% gelatin and fixed again with glutaraldehyde at 4 °C. After that, embedded exosmoes cut into blocks and stained en bloc in uranyl acetate followed by dehydrated with ethanol. Finally, sealed with epoxy resin under standard procedures. The ultra-thin sections were observed using JEM-1200EX (JEOL, Japan).

Nanoparticle tracking analysis (NTA)

The size and amount of exosomes were measured by NanoSight. In brief, exosomes were diluted in PBS buffer and resuspended again. ZetaView system was calibrated using polystyrene particles (110 nm). Then, exosomes sample were diluted onefold, tenfold, 100-fold, and 1000-fold with PBS buffer and loaded to sample chamber of ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) followed by the manufacturer's instructions. The same dilution of the same sample was repeated 3 times.

Exosomes labeling

Exosomes were labeled with PKH26 (#MINI26, Sigma) according to the manufacturer’s instructions. Briefly, exosomes from the supernatant of 1.5 × 106 cells were isolated and resuspended at 100 μL PBS followed by added 4 μL PKH26 dyes at 25 °C for staining 4 min. Using 1 mL FBS to terminate the reaction, and then added 200 μL ExoQuick-TC (SYSTEM BIOSCIENCES) on ice bath for 30 min. Finally, the tubes were centrifuged at 14,000 rpm for 3 min and removed supernatant to retain pellet exosomes. The final pellet containing exosomes was resuspended in 300 μL culture medium. PKH-26 labeled exosomes were co-culture with macrophages at 37 °C for 3 h followed by fixed with 4% paraformaldehyde at 25 °C to avoid light for 10 min. Finally, 4,6-diamidino-2-phenylindole (DAPI) solution was added and dyed for 10 min at 25 °C. The internalized exosomes were observed by fluorescence microscopy.

Western blot

Proteins concentration of cells or exosomes were measured by BCA assay after treated with RIPA buffer (Sangon Biotech) containing protease inhibitors (Roche Protease Inhibitor Cocktail). Briefly, approximately 20 μg of total protein were separated with 10% SDS-PAGE and then proteins were transferred to nitrocellulose membrane (Millipore) followed by blocked with tris buffered saline Tween-20 (TBST) containing 5% non-fat dry milk. Next, blocked membrane was incubated with primary antibodies: GAPDH (60004-1-Ig, Proteintech, 1:1000), CD63 (EXOAB-CD63A-1, SBI, 1:1000), CD9 (#13403, CST), TGF-β (sc-65378, Santa, 1:500), IL-10 (bs-0698R, Bioss, 1:1000), signal transducer and activator of transcription 3 (STAT3) (#9139, CST), p-STAT3 (#9135, CST). After washed with TBST three times, membrane was incubated with horseradish peroxidase-conjugated secondary antibody (A0261, Beyotime, 1:1000, Ab6721, Abcam, 1:5000). Bands were detected by ChemiScope (CLiNX; Shanghai), and analyzed with Image J.

qRT-PCR

Total RNA and exosome RNA were extracted with TRIzol reagent (Invitrogen, USA), and cDNA was synthesized by PrimeScript RT reagent Kit (Takara). Quantitative PCR was carried out on an ABI Q6 (Applied Biosystems Inc., USA) with the primers, as shown in Additional file 1: Table S1. The PCR protocol was: 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s, 60 °C for 60 s. Actin was used as an endogenous normalizer, and data were quantified by the 2−ΔΔCt method.

Cell migration assay

The migration ability of tumor cells was assessed by Transwell. The 0.8 μm pore size 24-well plate membranes were used in this assay (Corning, USA). In the upper chamber, 60,000 cells were seeded in culture medium (Gibco, USA). The lower chamber was filled with culture medium containing 20% FBS. After the incubated with 24 h, the filters were stained with Crystal Violet (Sangon Biotech) at 25 °C for 30 min. After sealed with neutral gum, the migration cell was counted of each filter under six different randomly fields.

Cell transfection

To overexpression of lncRNA AP000439.2, we constructed full length of AP000439.2 into pcDNA3.1 vector (oe-AP000439.2), and the empty vector was set as a negative control (oe-NC). To interfere with lncRNA AP000439.2 expression, we designed three siRNA targeting lncRNA AP000439.2 and a control siRNA (siNC). To interfere with STAT3 expression, we also designed three siRNA targeting STAT3 and a control siRNA. The siRNAs against AP000439.2 or STAT3 were purchased from Genepharma (Suzhou, China). The sequences of siRNAs were shown in Additional file 1: Table S1. For overexpression and interference, these vector and siRNA were transfected into cells by using Lipofectamine™ 2000 (Sigma-Aldrich) according to the kit instructions. In brief, 2 μg vector or siRNA diluted to 50 μL OPTI-MEM reagent, and 5 μL Lipofectamine™ 2000 diluted to 45 μL OPTI-MEM reagent. Next, the 50 μL sample mixture was mixed with the 50 μL transfection reagent and incubated at 25 °C for 20 min. After incubation, the mixture was added to the cell samples and incubated for 6 h until subsequent experiments.

Nuclear-cytoplasmic separation

Tumor cells were harvested and washed with ice cold PBS and then resuspended in buffer (consisted of 10 mM Tris–HCl pH 7.4, 5 mM MgCl2, 10 mM NaCl, 1 mM DTT, proteinase inhibitors, RNase inhibitor), and incubated on ice and mixed every 5 min for 20 min. Then, a final concentration of 0.5% NP-40 was added into the tube and mixed on ice for incubation another 1 min. Next, cells were lysed by frequent vortexing for 10 min followed by centrifuged at 3000 rpm at 4 °C for 5 min. Finally, the supernatant was collected as the cytoplasm fraction while the sediment was considered as nuclear fraction. The total RNA was extracted from these two fractions and detected by qRT-PCR.

RNA pull-down assay

The AP000439.2 and antisense were transcription by in vitro transcription system of T7 RNA polymerase (Ambion Life, USA) to obtain AP000439.2 RNA and antisense RNA, after purified by RNeasy Plus Mini Kit (QIAGEN, USA), then, it was labeled with biotin. Labeled RNA was captured by pre-clear streptavidin magnetic beads (Life Technologies). Next, this complex was incubated with cell lysate to capture target protein at 4 °C for 50 min. The RNA–protein precipitation was washed with wash buffer and eluted. The captured protein was detected by silver staining, mass spectrometry and western blot. To determine which specific region of AP000439.2 interacts with STAT3, we truncated AP000439.2 into two parts: △1 represents the region between 1 and 320 nt, △2 represents the region between 321 and 726 nt. We constructed two recombinant plasmid of pcDNA3.1 (+)-AP000439.2 1-320 nt and pcDNA3.1 (+)-AP000439.2 321-726 nt. To determine which specific region of STAT3 interacts with AP000439.2, we truncated STAT3 into three parts: △1 represents the region between 1 and 319 amino acid (aa), △2 represents the region between 320 and 583 aa, △3 represents the region between 584 and 770 aa. And then the recombinant plasmids were processed RNA pull-down assay.

RNA immunoprecipitation (RIP) assay

The EZMagna RNA immunoprecipitation Kit (17-701; Millipore) was used to investigate whether AP000439.2 could interact potential binding proteins STAT3. 786-O cells were lysed using RIP lysis buffer to harvest cell lysates and 10% lysates were used as input. Then, left cell lysates individually incubated with magnetic beads conjugated with anti-STAT3 (SAB1406670; Millipore) antibody and control IgG (Millipore) at 4 °C for 12 h. After that, the beads were washed three times with RIP buffer. Next, added Proteinase K into tubes and incubated at 55 °C for 30 min to remove the proteins. Finally, the expression of AP000439.2 in purified RNA was detected by RT-qPCR, and U6 was served as control.

RNA-Seq

Two groups of 786-O cell-derived exosmes containing oe-AP000439.2 or oe-NC were subjected to incubate with macrophages, and then these pretreated macrophages were performed transcriptome sequencing with triple biological repetitions. The extraction of total RNA was prepared with NEBNext Poly(A) mRNA kits (E7490, NEB) followed by treated with NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module kit (E6111, NEB) to synthesis the second strand of cDNA. Next, the cDNA libraries were constructed using Vazyme kit (ND604, Vazyme). Libraries were sequenced by Illumina Hiseq X Ten platform. Raw data were quality controlled using Fast-QC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Clean data were mapped to the human genome (hg38) by TopHat (version 2.1.1, http://ccb.jhu.edu/software/tophat/index.shtml). The gene expression level was calculated by Cufflinks, and when the gene expression satisfied the criteria for |log2 fold change|> 1 and false discovery rates < 0.05, it was considered a differentially expressed gene (DEG). The pathway analysis of DEG was performed by DAVID (https://david.ncifcrf.gov/).

Tumor-bearing mouse model

5-week-old female athymic BALB/c nude mice were divided into two groups (5 mice per group): siNC-exo group and siAP000439.2-exo group. All mice with established subcutaneous ccRCC xenograft tumor model through injected 5 × 106 Caki-1 cells at the flank of mice. In parallel, a dosage of 5 mg exosomes were injected into the tail vein of mice, injected every 3 days for 2 weeks. For siAP000439.2-exo group, mice were injected by AP000439.2-deleted exosome; for siNC-exo group, mice were injected by NC exosome. The growth rate of a tumor was monitored by measuring the tumor size using a digital calliper. After injected Caki-1 cells, the tumor size was continue measured every 3 days for 4 weeks. Tumor volume was calculated using the following formula: Tumor volume = length × width × width/2. The mice experiments were approved by the Animal Ethics Committee of the Jinling Hospital, Nanjing University Medical School.

Flow cytometry assay

Tumor tissues were triturated and digested with trypsin–EDTA (11668-500, Invitrogen) to obtain single-cell suspension. Cells were cultured in ultralow attachment six-well plates (CLS3471-24EA, Corning) overnight. Appropriately 1 × 106 cells were taken used for flow cytometry and then incubated with 50 μL primary antibodies (anti-F4/80, 86007S, CST; anti-NOS2, 12-59520-82, Thermo Fisher; anti-CD163, 11-1631-82, Thermo Fisher) in an ice bath for 30 min in the dark. After incubation, cells were concentrated at 200 g for 5 min and removed supernatant. Finally, the precipitation was resuspended in PBS solution after washing and subjected to flow cytometry analysis within 1 h by FACSVerse™ (BD).

Statistical analysis

The comparisons of means among groups were analyzed by one-way ANOVA followed by Tukey’s test. The comparisons of means between two groups were analyzed by Two-tailed Student’s t-test. A value of p < 0.05 was considered statistically significant. Statistical analyses were performed with the SPSS package.