Data collection and bioinformatics analysis

Based on our previous sequencing results [17], differentially expressed lncRNAs in exosomes from breast cancer cells and normal breast epithelial cells were obtained using the threshold value (log2 (fold change, FC) > 1.25 for upregulated lncRNAs and P < 0.05). mRNAs with differential expression in breast cancer and normal tissues were identified using |log2 (FC)| > 1.25 and P < 0.05 in the TCGA database (https://cancergenome.nih.gov). DAVID was utilized to perform gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Cytoscape version 3.6.1 was used to establish a network.

Cell culture

The breast cancer cell line MDA-MB-468 was purchased from the Chinese Academy of Sciences (Shanghai, China). The breast cancer cell line MDA-MB-231, MCF-7 and normal breast epithelial cell MCF-10A were purchased from Procell (Wuhan, China). The human umbilical vein endothelial cells (HUVECs) were purchased from iCellbioscience (Shanghai, China). MDA-MB-231 cells, and MCF-7 cells were cultured in the Dulbecco’s Modified Eagle’s Medium (Corning, #10-013-CVR). MDA-MB-468 cells were cultured in the L-15 Medium (Solarbio, # LA9510). MCF-10A cells were cultured in the MCF-10A cell specific medium (Procell, #CM-0525). HUVECs were cultured in the ScienCell 1001 medium. All medium was added with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Sangon). All cells were incubated at 37 °C with 5% CO2.

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from cells and exosomes using TRIzol reagent (Invitrogen, USA). cDNA was prepared through the reverse transcription reaction with the cDNA Synthesis Kit (Thermo). SYBR Green PCR Master Mix (Roche) was applied for qRT-PCR analysis. The lncRNA and mRNA expression were normalized to GAPDH. The primer sequences are listed in Table S1.

Isolation and characterization of exosomes

MDA-MB-231 cells and MCF10A cells were cultured in medium without FBS. The culture supernatants were centrifuged at 300×g for 10 min, followed by centrifugation at 2000×g for 20 min to remove the cells. Cell debris was removed by centrifuging the supernatant at 10,000×g for 30 min. The exosomes were extracted by ultracentrifugation at 100,000×g for 1.5 h, and the precipitates were collected. Transmission electron microscopy (TEM) was used to observe exosome morphology and particle size. In brief, exosome was resuspended in 4% paraformaldehyde, and images were obtained using the JEOL 100 CX electron microscope (Tokyo, Japan). The protein concentration of exosomes was determined using a Modified BCA Protein Assay Kit (C503051, Sangon Biotech).

Western blot

RIPA lysis buffer (Thermo Fisher) was used to extract proteins from MDA-MB-231 cells, MCF-10A cells, corresponding exosomes, and HUVECs. The samples were washed with 1× PBS and resuspended in SDS lysis buffer, and then centrifuged at 16,000×g for 30 min and the supernatant was collected. The remaining steps were carried out as reported previously [18]. The following antibodies were used: GAPDH (Proteintech, #60004-1-Lg, 1:2000); CD63, (Abcam, #ab216130, 1:1000); PBRM1 (Proteintech, #12563-1-AP, 1:1000); goat anti-rabbit IgG H&L(HRP) (Beyotime, #A0208, 1:1000); and goat anti-mouse IgG H&L(HRP) (Beyotime, #A0216, 1:1000).

CCK-8 assay

A CCK-8 assay was utilized to evaluate the proliferation ability of HUVECs. Exosome-treated HUVECs were digested into a single-cell suspension (1 × 104/mL). The cells were seeded into 6-well plates at a density of 103 cells/well and cultured overnight at 37 °C. The cells were then added with 10 μL CCK-8 solution. After 2 h, the absorbance value was measured at 450 nm.

Wound healing assay

A wound healing assay was utilized to evaluate the migration ability of HUVECs. HUVECs were seeded into 24-well tissue culture plates at a density of 8 × 104 cells/well and cultured overnight. After the cells were scratched, they were washed with PBS to remove the detached cells. Subsequently, serum-free medium was added, followed by exosomes derived from MDA-MB-231 and MCF-10A for 24 h of incubation. At 0 and 24 h, the monolayer cells were photographed using a Nikon microscope.

Tube formation assay

A tube formation assay was utilized to evaluate the tube formation ability of HUVECs. The cooled 24-well plates were filled with 200 μL of cooled Matrigel (10 mg/mL) and placed in a 37 °C incubator for 1 h. HUVECs were digested into cell suspensions, and 8 × 104 cells were seeded on coagulated Matrigel gel, followed by the addition of exosomes for 24 h of incubation (37 °C, 5% CO2). The 24-well plate was taken out and photographed under a microscope.

Cell transfection

The pcDNA3.1-CMV-SNHG12, pcDNA3.1-CMV-PBRM1 and pcDNA3.1-CMV-MMP10 plasmids were purchased from Hanbio Biotechnology. SiRNA-SNHG12 and siRNA-MMP10 were designed and synthesized by RiboBio (Guangzhou, China). Lipofectamine 2000 (Invitrogen) was used to transfect the plasmids into MDA-MB-231 cells or HUVECs, and DharmaFECT4 (Dharmacon, USA) was used to transfect the siRNAs.

Transwell assay

A transwell assay was utilized to evaluate the migration ability of HUVECs. After removing the medium, cells were washed with an appropriate amount of PBS, and added 0.25% Trypsin–EDTA for digestion 3 min. After the cells shrank and became round, the digestion was terminated by adding culture medium. Thereafter, cells were centrifuged at 200×g for 3 min, and the supernatant was removed. Cells were resuspended with serum-free medium. Next, 700 μL of medium containing 10% FBS was added to the lower chamber, and 500 μL of cell suspension and 20 μg/mL exosomes were added to the upper chamber. The culture was continued in the CO2 incubator for 24 h. Subsequently, the upper chamber was added with 4% polyformaldehyde and fixed at room temperature for 30 min, followed by crystal violet staining. Finally, the cells were counted under a microscope (OLYMPUS, CKX53).

Tumor formation in nude mice

All animal experiments were approved by the Animal Care Committee of Ningbo University. Female athymic BALB/c nude mice (5 weeks, 20 ± 2 g) were obtained from the Chinese Academy of Science. MDA-MB-231 cells were seeded subcutaneously on each flank of the nude mice. A tumor with a size of 100 mm3 was formed subcutaneously, and the collected exosomes of knockdown SNHG12 and the corresponding empty vector were injected into the tumor site once a week (n = 5). After 26 d, the mice were sacrificed, the tumor volume and weight were measured, and samples were collected to detect SNHG12 and microvessel density (MVD).

Immunofluorescence analysis

Immunofluorescence analysis was performed as previously described [19]. Briefly, the tumor tissue was fixed, followed by dehydration, embedding, sectioning (4 μm), dehydration, antigen retrieval, and blocking. Immunofluorescence analysis was performed using anti-CD31 (Abcam, #ab222783), anti-CD34 (Abcam, #ab81289), and Cy3 conjugated Donkey anti-rabbit IgG (H + L) (Servicebio, #GB21403) antibodies. After DAPI staining, the images were observed under a fluorescence microscope (Nikon).

RNA sequencing

The RNA of HUVECs transfected with the SNHG12-overexpressing vector or control vector was extracted for library construction using KAPA Stranded RNA-seq library Prep Kit (Illumina, USA). The Illumina NovaSeq 6000 Sequencing system (Illumina) was applied to pair-end sequence the library. The transcriptome sequencing experiment was completed by Mingma Technologies Co., Ltd. (Shanghai, China). Adapter sequences were filtered to obtain clean reads. The sequencing results were then aligned to a reference genome GRCh38, and non-coding transcripts longer than 200 nt were selected as lncRNAs. The differentially expressed lncRNAs were screened according to the screening criteria (log2 FC > 0.585 or < −0.585, p < 0.05), and analyzed through heat map.

RNA-pull down

The Magnetic RNA–protein Pull-down Kit (Thermo) was used for lncRNA pull-down, according to the manufacturer’s instruction. In brief, SNHG12 or antisense RNA was labeled with biotin, streptavidin magnetic beads were added and incubated for 30 min at 24 °C with stirring. Subsequently, we washed unbound RNA with Tris and added RNA–protein binding buffer containing total protein to tubes containing streptavidin magnetic beads. After the magnetic beads were incubated with rotation at 4 °C for 90 min, they were washed with washing buffer and then incubated with elution buffer for 15 min at 37 °C with agitation. The supernatant was collected for silver staining.

RNA immunoprecipitation (RIP)

RIP lysis buffer was used to lyse the cells. After cell lysis, 50 μL magnetic beads and 0.5 mL RIP wash buffer were added, vortexed briefly and mixed, and the supernatant was aspirated. Subsequently, 100 μL of RIP wash buffer was added to resuspend the magnetic beads and 5 μg of PBRM1/1 μg IgG antibody, and the beads were incubated at room temperature for 30 min. After brief centrifugation, the supernatant was removed and mixed with 0.5 mL RIP wash buffer. Then, 900 μL of RIP immunoprecipitation buffer was added to the magnetic beads-antibody mixture, and centrifuged at 4 °C at 14,000 rpm for 10 min. The supernatant was sucked into the magnetic bead antibody tube, incubated at 4 °C overnight for co-immunoprecipitation reaction, and 10 μL of the supernatant was sucked as “input.” The remaining portion of the magnetic beads was washed with RIP wash buffer, proteinase K buffer was added and incubated at 55 °C for 30 min before RNA extraction. qRT-PCR was performed for the detection of lncRNA.

Chromatin immunoprecipitation (ChIP)

The ChIP assay kit (Millipore, New Bedford, MA) was used according to the manufacturer’s instructions. Proteins were cross-linked to DNA by fixing in formaldehyde. Anti-PBRM1 antibody was used to extract chromatin from the cells, and the chromatin-crosslinked DNA was sheared into 250–500 bp fragments. Putative PBRM1-binding fragments were detected using qRT-PCR.

Statistical analysis

Statistical data are presented as the mean value ± standard deviation (SD). All data analyses in this study were performed using the GraphPad Prism 8.0 software. Student’s t test and analysis of variance were applied for comparison of differences between two groups or more than two groups, respectively. P value <0.05 was considered significant.