Study Approval
Approval for this research was obtained from the Ethics Committee of Xi’an Jiaotong University (Xi’an, China). All parents of the participants signed written informed consent. The experiments were carried out in accordance with the ethical guidelines of the Declaration of Helsinki.
Subjects
The study enrolled 100 children with ASD (between 2.5 and 6 years of age; 90 males) and 60 age-matched TD children (54 males) as control. The ASD children were recruited from Xi’an Children’s Hospital, Xi’an, China. The healthy children were recruited from the same region to minimize the influence of different environments. The ASD children were examined by a developmental behavioral pediatrician and a pediatric neurologist or psychiatrist. All the consultants agreed on the diagnosis of ASD according to DSM-V criteria. Children with tuberous sclerosis complex, Rett syndrome, Prader Willi syndrome, Angelman syndrome, or Fragile X syndrome were excluded. All the participants were screened via a parental interview for current and past physical illnesses. Those who had any type of infection or disease within two weeks before the time of examination were excluded. ASD was evaluated with the autism diagnostic observation schedule (Table 1).
Collection and preparation of serum samples
Venous blood samples were collected by a pediatric nurse. The blood was allowed to clot at room temperature for 30 min, and the clot was then removed by centrifuging at 1,500×g for 10 minutes. The resulting supernatant is immediately transferred to a clean polypropylene tube, and EDTA-free inhibitor cocktail (Halt protease inhibitor; Thermo Scientific Pierce Protein Research Products, Rockford, IL, USA) was added at a concentration of 10 μ L/mL serum. The obtained serum was aliquoted into small portions and was immediately frozen on dry ice and stored at −80 °C. To tolerate individual variation, 25 μL of each serum sample was collected and every 20 samples were pooled into one subgroup. Altogether, we got 5 ASD subgroups and 3 TD subgroups (n=20). To avoid bias caused by gender difference, proportion of males in each subgroup was the same (90%). The remaining serum in each sample was maintained for further individual validation.
Isolation of serum neuron-derived exosomes (NDEs)
NDEs in the serum samples were isolated as described previously [20,21] with minor modifications. Briefly, 0.5 ml of serum was incubated with 0.15 ml of thromboplastin-D (Fisher Scientific, Inc., Hanover Park, IL) at room temperature for 60 min. Then 0.5 ml of calcium- and magnesium-free Dulbecco’s balanced salt solution (DBS−2) with protease inhibitor cocktail (Roche Applied Sciences, Inc., Indianapolis, IN) and phosphatase inhibitor cocktail (Pierce Halt, Thermo Scientific, Inc., Rockford, IL) was added and the mixture was centrifuged at 1,500×g for 20 min. The supernatant was mixed with 252 μl of ExoQuick exosome precipitation solution (EXOQ; System Biosciences, Inc., Mountainview, CA), and incubated for 1 hr at 4°C. The resultant exosome suspension was centrifuged at 1,500×g for 30 min at 4°C and the pellet was re-suspended in 150 μl of DBS−2 with the inhibitor cocktails before immunochemical enrichment of NDEs. Each sample received 100 μL of 3% BSA (1:3.33 dilution of Blocker BSA 10% solution in DBS−2 [Thermo Scientific, Inc.]) and was incubated with 1 μg of mouse anti-human CD171 (L1CAM neural adhesion protein) biotinylated antibody (clone 5G3, eBioscience, San Diego, CA) for 1 hr at 4°C. Following that, 25 μl of streptavidin-agarose resin (Thermo Scientific, Inc.) plus 50 μL of 3% BSA was added and the sample was incubated at 4°C for 30 min. After centrifugation at 200×g for 10 min at 4°C and removal of the supernate, 3% BSA was added again, and centrifugation and supernatant removal were repeated. Each pellet was suspended in 50 μl of 0.05 M glycine-HCl (pH 3.0), incubated at 4°C for 10 min, and re-centrifuged at 4,000×g for 10 min at 4°C. The obtained supernatant was transferred to a new Eppendorf tube containing 5 μL of 1 M Tris-HCl (pH 8.0) and stored at −80°C.
Nanoparticle tracking analysis (NTA)
NDEs suspension at a concentration between 1 x 107/ml and 1 x 109/ml was examined using a Nanosight NS300 (NanoSight Ltd., Amesbury, UK), equipped with a 405 nm laser to determine the size and quantity of particles isolated.
Transmission electron microscopy (TEM)
NDEs solution (20-40 μl) was placed on a copper mesh, post-negatively stained with 2% phosphotungstic acid for 10 min, and then dried for 2 min under incandescent light. The copper mesh was observed and photographed under a transmission electron microscope (H-7650 Hitachi microscope; Hitachi, Tokyo, Japan).
Western blot analysis
The NDEs suspension was denatured in 5X sodium dodecyl sulfonate (SDS) buffer and subjected to western blot analysis (10% SDS-polyacrylamide gel electrophoresis; 50 μg protein/lane) using mouse monoclonal antibody CD63 (ab59479), mouse monoclonal antibody CD81 (ab79559) and rabbit polyclonal antibody L1CAM (ab232894; all from Abcam, Shanghai, China). The proteins were visualized on the chemiluminescence detection Syngene GBox (Syngene Europe).
Extraction of total RNA in NDEs
Total RNA in NDEs was isolated using the Exosomal RNA isolation kit (Norgen Biotek, 58000) according to the manufacturer’s instructions. Briefly, 200 μl of the transferred supernatant containing purified NDEs was incubated with 300 uL Lysis Buffer A and 37.5 uL Lysis Additive B at room temperature for 10 min, following which 500 uL of 96-100% Ethanol was added to the mixture and mixed well via 10-second vortexing. Then, 500 uL of the mixture was transferred into a Mini Spin column and centrifuged at 3,000×g for 1 min, and the remaining mixutre was transfered and centrifuged by repeating the steps. After that, 600 uL Wash Solution A was applied and the column was centrifuged at 3,300×g for 30 seconds twice.The spin column was then moved to a fresh 1.7 mL Elution tube, and 50 uL Elution Solution A was added. Finally, centrifugation was performed at 400×g for 1 min and 5,800×g for 2 min to obtain total RNA.
Human lncRNA microarray and data analysis
Total RNA was purified using a RNeasy Mini Kit (Qiagen, Germany) and was checked for a RIN number to inspect RNA integration with an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US). LC Biotech Human lncRNA Microarray 4×180 K (Agilent Technologies; Santa Clara, CA) was utilized to detect the expression of mRNAs and lncRNAs in NDEs. The microarray slide contains 26,083 mRNA probes and 1,05,135 lncRNA probes, and lncRNA sequencing data are available from Gencode, UCSC, Ensembl, Refseq, LNCIpedia, NONCODE, LNcRNA Disease, Ernas, NRED and other databases. Amplification of cRNA, fluorescent labeling and hybridization of the microarray were performed by following the protocol of Agilent Technologies. Briefly, equal amount of RNA from each subgroup was reversely transcribed into cDNA, which was then labeled with Cy3 (GE Healthcare; Biosciences, Piscataway, NJ, USA) and hybridized with the microarray slide. After that, the slide was scanned on the Agilent Microarray Scanner G5761A (Agilent Technologies). Data were extracted with Feature Extraction software 12.0.3.1 (Agilent Technologies), and raw data were normalized by Quantile algorithm. Genes with p value<0.05 and a fold change of at least 2 were selected for further analysis. GO/KEGG pathway enrichment analyses of the target genes were performed using Fisher's exact test. The function of lncRNAs was predicted by analyzing the functional annotations of mRNAs that were highly co-expressed with lncRNAs.
Small RNA library construction, sequencing and data processing
Approximately 1 ug total RNA was used to prepare small RNA library according to the protocol of TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, USA). Single-end sequencing (36 bp) was performed with an Illumina Hiseq2500 at LC-BIO (Hangzhou, China). Briefly, the raw reads were subjected to the Illumina pipeline filter (Solexa 0.3), and the dataset was further processed with an in-house program, ACGT101-miR (LC Sciences, Houston, Texas, USA), to remove adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, snoRNA) and repeats. Subsequently, unique sequences with a length of 18~26 nucleotides were mapped to Homo species precursors in miRBase 20.0 by BLAST search to identify known miRNAs and novel 3p- and 5p- derived miRNAs. The hairpin RNA structures containing the sequences were predicated from the flank 80 snt sequences using RNAfold software (http://rna.tbi.univie.ac. at/cgi-bin/RNAfold.cgi). miRNA differential expression based on normalized deep-sequencing counts was analyzed using the Fisher exact test and Student t test, and the significance threshold was set to be 0.01 or 0.05. To predict the genes targeted by most abundant miRNAs, two computational target prediction algorithms (TargetScan 50 and miRanda 3.3a ) were used to identify miRNA binding sites. Finally, the data predicted by both algorithms were combined and the overlaps were calculated. The GO terms and KEGG pathways of these most abundant miRNAs, miRNA targets were also annotated.
Quantitative real-time PCR
All primers were designed and synthesized by Takara (TakaraBiotechnology, Dalian, China). To avoid false-positive amplification of contaminating genomic DNA in the mRNA samples, all the primers spanning different exons were designed (Table 2). For mRNA, cDNA was synthesized using a PrimeScript RT reagent kit (Takara Biotechnology Co, Ltd, Dalian, China). Quantitative real-time PCR (qRT-PCR) was performed using the IQ5 real-time PCR detection system, and GADPH was taken as a control. Relative quantification of mRNA expression levels was performed using SYBR Premix Ex Taq II on an FTC-3000TM System (Funglyn Biotech Inc., Toronto, Canada). For miRNA,cDNA was synthesized by servicebio RT First strand cDNA Synthesis Kit (Servicebio, Wuhan, China), qRT-PCR was carried out using the SYBR Premix Ex TaqTM II (TaKaRa),and U6 was taken as a control. PCR conditions consisted of a 5 min preincubation at 95◦C, followed by 40 repeats of 95°C for 10 s and 60°C for 20 s. All samples were run in triplicate and the average values were calculated. The relative levels of mRNAs EDNRA, SLC17A6, HTR3A, OSTC and TMEM165, as well as miRNAs PC-5p-139289_26 and hsa-miR-193a-5p were calculated using the 2−ΔΔCt method.
Statistics
All statistical analyses were performed using the software SPSS (version 17) and group statistics are presented as mean ± SD. The t-test for independent variables was used to examine the inter-group differences, and a significance level of 0.05 was adopted.