Overall, a cohort of serum samples obtained from 90 HER-2 positive patients were included from March 2012 to January 2015, to determine the expression of serum exosomal AGAP2-AS1 for trastuzumab response. Patients include 45 responding and 45 non-responding patients according to iRECIST criteria . Written-informed consent was obtained from all patients and the study protocol was approved by theResearch Scientific Ethics Committee of The First Affiliated Hospital of Zhengzhou University,The First People's Hospital of Chenzhou City and Hainan General Hospital.
Cell culture and treatment
The human HER-2 positive BC cell lines SKBR-3 and BT474 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium (Gibco, CA, USA) supplemented with 10% fetal bovine serum (Australia Origin, Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Solarbio, Beijing, China). The cells were grown in a humidified atmosphere of 5% CO2 and 95%air at 37℃. Trastuzumab (Herceptin) was purchased from Roche (Basel, Switzerland) and used by dissolving in phosphate-buffered saline (PBS). The trastuzumab-resistant cell lines, SKBR-3-TR and BT474-TR, originated from SKBR-3 and BT474 cells, respectively, were established as previously described .
RNA extraction and qRT-PCR analysis
Total RNA was isolated from the cultured cell lines or clinical samples by use of Trizolreagent (Invitrogen, Carlsbad, CA), and subjected to reverse transcription as requested(Takara, Shiga, Japan). The RNA concentration and purity were measured by Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA). The expression levels of mRNA and lncRNA were detected by the PrimeScriptTMRT reagent kit (Takara, Dalian, China). qRT-PCR was carried out using the SYBR Premix Ex TaqTMⅡ (Takara, Dalian, China) andstandardized using GAPDH as reference gene.All reactions were carried out in triplicate. The relative expression was calculated using the 2-△△ct method. PCR primers were listed in Additional file 1: Table S1.
Plasmid construction and cell transfection
For knockdown ofAGAP2-AS1 and ATG10, small interfering RNA (siRNA) was purchased from GenePharma (Shanghai, China). To construct a plasmid overexpressing AGAP2-AS1, the full-length human AGAP2-AS1 sequence was synthesized and subcloned into the pEX-3 vector (GenePharma, Shanghai, China). ThepENTER-ATG10 vector used for overexpression ofATG10 mRNA was provided by Vigene BioSciences, followed by transfection into BC cells using Lipofectamine 2000 for 48 h (Invitrogen). For stably overexpressing or silencing AGAP2-AS1, we constructed lentivirus-based vectors from Genechem (Shanghai, China). The sequences of oligonucleotides are presented in Additional file 1: Table S1.
The cells transfected accordingly were seeded in a 96-well plate, the cells were cultured for 72 h with different dose of trastuzumab. After that, 10 mg/mL of CCK8 reagent (Dojindo, Kumamoto, Japan) was supplemented for cell viability detection according to manufacturer’s protocols. The absorbance at 450 nm was measured with a microplate reader. The dose for the half survival was represented the half-maximal inhibitory concentration (IC50) of trastuzumab in SKBR-3-TR and BT474-TR cells.
Isolation and identification of exosomes
Cell supernatant was collected andsequentially centrifuged under 3000 g for 15min at 4℃ after taken out from -80℃ and filtered through a 0.45-µm pore polyvinylidene fluoride filter (Millipore, Billerica, Mass). Then, 63µl ExoQuickTM exosome precipitation solution (SBI, USA) was added into 250µl serum followed by incubation at 4℃ for 50 min. After centrifugation at 1500 g for 30 minutes, exosomes were collected. 50µl phosphate-buffered saline (PBS) was used to resuspend the exosome pellets obtained from the former step. Exosome isolated from serum of BC patients were done with similar procedures.
The sample of exosomes was identified through transmission electron microscopy, nanoparticle tracking analysis (NTA, Malvern Panalytical). Exosomes were loaded on a Formvar-carbon-coated electron microscope grid(Polysciences) for 30 min. Then the grid was washed in PBS and fixed in 2% glutaraldehyde(Sigma Aldrich) for 10 min. The grid was subsequently washed in PBS for 5 times andcounter-stained with 2% uranyl acetate (Sigma Aldrich) for 1 min. Air-dried grids wereviewed with a Hitachi transmission electron microscope.
RNA sequencing analysis
Total RNA was extracted from wild type or AGAP2-AS1-knock out SKBR-3 cells using RNeasy mini kit (Qiagen, Germany). The paired-end libraries were constructed by using TruSeq™ RNA Sample Preparation Kit (Illumina, USA) according to the manufacture’ssample preparation guide. The mRNA expression profiles of the treated cells were determined using the Illumina NovaSeq 6000 (Illumina, USA) following the manufacturer's instructions. The library construction and sequencing were performed at Shanghai Sinomics Corporation (Shanghai, China).Cuffdiff was used to evaluate differentially expressed genes. The differentially expressed genes were selected using the following filter criteria: P≤0.05 and fold change ≥2.
Subcutaneous tumorigenicity assay
4-week-old female BALB/c nude mice were purchased from the Beijing Vital River Laboratory Animal Technology and housed in specific pathogen-free barrier facilities. Fifteen mice were randomly assigned to two exosome-treated groups and one control group (5 mice/group). Mice were subcutaneous injected SKBR-3-TR cells (1×106 cells in 0.1ml physiological saline) in the flank. Each group received PBS, SKBR-3-TR-EXOVector, or SKBR-3-TR-EXOAGAP2-AS1treatmentsevery 3 days for 5 weeks. Meanwhile, each group received intraperitoneal treatment of trastuzumab once every two days for 4 weeks. After 4 weeks post-injection, mice were imaged using a luminescence imaging system. At the end of imaging, all mice were euthanized by carbon dioxide inhalation, followed by cervical dislocation to ensure death. Then, tumors were surgically dissected and weighted. Tumor volume (mm3) = 0.5 × width2 × length.
To test the therapeutic role of AGAP2-AS1, ASO was used for treatment of xenograft tumors through tail vein injection. Briefly, 5×105 SKBR-3-TR cells in 100μL of sterile PBS were injected directly into the mammary fat pads of mice to establish in situ xenograft. Then, mice were divided into 3 groups (n =5 in each group) after the xenograft was established. ASO was used at the concentration of 10 nmol, twice a week.
All experimental procedures were conducted in conformity with the ethical standards of national and international guidelines and policies. This study was approved by the Institute Animal Care and Use Committee of The First Affiliated Hospital of Zhengzhou University (Zhengzhou, China).
AGAP2-AS1 was transcribed using T7 RNA polymerase in vitro (Ambio Life) followed by the purification with RNeasy Plus Mini Kit (Qiagen) treatment with DNase I (Qiagen). The cell lysates were freshly prepared using Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, Cat# 20164). Cell protein extracts were collected and mixed with the biotinylated
RNA probes for AGAP2-AS1 in magnetic beads. Resultswere analyzed by sliver staining and mass spectrometry (ekspertTMnanoLC; AB Sciex TripleTOF 5600-plus; SCIEX). Data were analyzed using Proteinpilot software (https://omictools.com/proteinpilot-tool). The retrieved protein ELAVL1 was further validated by standard western blot.
RNA immunoprecipitation (RIP) and Chromatin immunoprecipitation (ChIP)
Using EZ-Magna RIP RNA Binding Protein Immunoprecipitation Kit,RIP assay was implemented as instructed by supplier (Millipore, Billerica,MA). Cell lysates were reaped for incubation with the ELAVL1 (cat. no. ab200342, Abcam, Cambridge, MA) antibody in magnetic beads, using IgG antibody as negativecontrol. All precipitates wereexamined by qRT-PCR.
For ChIP assay, the Millipore EZ ChIP Kit (Millipre) was used according to the manufacture’s guideline. After fixation with 4%formaldehyde, cells were lysed by using RIPA buffer (Beyotime, Shanghai, China). Then, DNA fragments (length of 200-1000 bp) were obtained by treatment with ultrasonic followed byimmunoprecipitation usinganti-H3K27ac antibody (Abcam, cat. no. ab4729), H3K4me3 antibody (Abcam, cat. no. ab8580) and the negative control IgG antibody (EMD Millipore, cat. no. 12-371) overnight at 4°C. The cross-linked chromatin was then acquired and detected via qRT-PCR after route elution. Experiments were performed in triplicate.
RNA fluorescent in situ hybridization (RNA-FISH)
The colocalization of AGAP2-AS1 and ELAVL1 were confirmed by fluorescence staining. Briefly, SKBR-3 cells were seeded on a glass-bottomed confocal plate and cultured overnight. After fixation with 4% PFA and permeabilization with 0.5% Triton, hybridization was carried out overnight with the AGAP2-AS1 probes conjugated with Alexa Fluor 555 (Invitrogen, CA,USA) at 37°C in 2×SSC, 10% formamide and 10% dextran. Subsequently, Anti-ELAVL1 was incubated in the dark overnight followed by incubation with secondary antibody for 1h. Finally, the nuclei were stained by DAPI and the images were captured under a Nikon A1Si Laser Scanning Confocal Microscope (Nikon Instruments Inc, Japan).
Immunohistochemistry (IHC) analysis
For IHC, the formalin-fixed, paraffin-embedded sections were dewaxed and rehydrated aspreviously described , and then treated with 3% hydrogen peroxide followed by EDTA buffer for antigenretrieval. The sections were then blocked in goat serum for 30 min, incubated with rabbit anti ATG10 antibody (Abcam, cat. no. ab229728) at 4°C overnight and subsequent with horseradish peroxidase-conjugatedsecondary antibodies for 30 min at room temperature. Finally, the sections were stained withthe DAB substrate and hematoxylin. Images were recorded by Nikon Eclipse Ti microscope.
Western Blot analysis
Protein extraction were performed using RIPA lysis buffer (Pierce, IL, USA) containingprotease inhibitor (Roche, CA, USA). Protein extracts were subjected to 10%SDS-polyacrylamide gel electrophoresis followed by electro-transfer to polyvinylidenedifluoride membrane. After 1h of pre-membrane blocking with 5% BSA, the proteins wereincubated with respective primary antibodies at 4°C overnight followed by secondaryantibodies incubation at room temperature for 1 h. The detection of proteins was carried outusing ECL reagent.
Each experiment contained at least 3 individual bio-replications. Datawere all given as the mean ± standard deviation (SD) and processed withPrism Version 5.0 (GraphPad Software, La Jolla, CA). Statistical analysisin form of Student’s t-test or one‐way analysis of variance (ANOVA),was considered to be significant when p-values below 0.05.