Study participants and blood sample collection
Blood samples were collected from 5 PD patients (age: 66.20 ± 4.02; female: 2) and 4 healthy controls (age: 65.25 ± 3.77; female: 2) for microarray analysis and from 50 PD patients (age: 64.68 ± 9.82; female: 23) and 40 healthy controls (age: 64.83 ± 9.41; female: 22) for real-time quantitative PCR (RT-qPCR). There were no significant differences in age or gender between PD patients and healthy controls. All subjects were enrolled from the Movement Disorder Clinic at the Department of Neurology, Ruijin Hospital affiliated with Shanghai Jiao Tong University School of Medicine. Patients were assessed for eligibility when they were diagnosed with idiopathic Parkinson’s disease according to Parkinson’s disease Queen Square Brain Bank criteria [23]. The control group was recruited from healthy subjects exhibiting no disease symptoms who came for a routine physical examination. Peripheral venous blood (6 ml) was collected in heparin anticoagulant tubes, and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll Plus (Solarbio, Beijing, China) density gradient centrifugation. For this, blood was diluted 1:1 with phosphate-buffered saline (PBS, Gibco, USA) before layering onto Ficoll Plus. After centrifugation for 20 min at 1200 g, PBMCs were collected from the plasma-Ficoll interphase. Cells were washed twice with PBS and counted before analysis. This study was approved by the ethics committee of Ruijin Hospital. All participants signed the informed consent.
Microarray analysis
We performed enrichment of lncRNAs from PBMCs using the miRNeasy Micro Kit and the RNeasy Mini Kit (QIAGEN, CA, USA) according to the manufacturer’s protocols. Microarray hybridization was performed based on the manufacturer’s standard protocols. Microarray slides were sequenced on an Agilent Microarray Scanner, and the raw data were normalized by the quantile algorithm GeneSpring Software 12.6.1 (Agilent Technologies). To identify differentially expressed lncRNAs between PD patients and healthy controls, filtering criteria were applied (≥ 2-fold change, P < 0.05). Differentially expressed lncRNAs with statistical significance between the two groups were identified through volcano plot filtering. The microarray work was performed by Kang Cheng Biotech, Shanghai, China.
Cell culture, stimulation, and transfection
Human monocytic THP-1 cells and mouse microglial BV2 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco, USA) at 37 °C and 5% CO2. To induce the polarization of macrophages, cells were treated with 100 ng/ml lipopolysaccharide (LPS) for 12 h and 20 ng/ml interleukin-4 (IL-4) for 6 h. Cells were plated 24 h prior to transfection at a confluency of 70 to 80%. The plasmid vectors were transfected using Lipofectamine™ 3000 (Invitrogen, USA) reagent according to the manufacturer’s protocol. Small RNA interference (siRNA) transfection was performed using Lipofectamine RNAiMAX (Invitrogen, USA) according to the manufacturer’s instructions. Full-length complementary cDNAs of HOXA-AS2 and PGC-1α were synthesized and cloned into the expression vector pcDNA3.1 (Invitrogen, China). HOXA-AS2 siRNA (HOXA-AS2i), PGC-1α siRNA (PGC-1αi), and scrambled negative control siRNA (Scr) were synthesized by GenePharma Co. (Shanghai, China). The siRNA sequences are listed in Supplementary Table S1.
Real-time quantitative PCR
Total RNA was isolated from tissues or cell cultures using TRIzol reagent (Invitrogen, USA). Complementary DNA (cDNA) was synthesized using PrimeScript RT reagent (Takara, Dalian, China). RT-qPCR was performed on an Applied Biosystem 7500 Real-time PCR system (Applied Biosystems, CA, USA) using SYBR-Green Master Mix (Takara, Tokyo, Japan). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control, and the RT-qPCR result was calculated by the 2-ΔΔCT method. The primer sequences for all genes are provided in Supplementary Table S2.
Western blot
The cells were homogenized in RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors to extract proteins. The protein samples were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. Then, the membranes were incubated with the primary antibodies. Anti-PGC-1α (sc13067, Santa Cruz, 1:1000), anti-EZH2 (ab3748, Abcam, 1:1000), and anti-β-actin (A5441, Sigma-Aldrich, 1:2500) antibodies. The protein bands were analyzed using Image Lab software (Bio-Rad), and the gray values of experimental proteins were detected using ImageJ software.
Experimental animals and protocols
Male C57BL/6 mice (20–25 g) were purchased from Shanghai Model Organisms Center, Inc. (Shanghai, China). The mice were kept under environmentally controlled conditions (ambient temperature: 20 ± 2 °C; humidity: 50–65%) on a 12 h light/dark cycle and provided ad libitum access to food and water. The mice were substantially anesthetized and placed under a stereotaxic apparatus (David Kopf Instrument, CA, USA) for intracerebroventricular (ICV) injection into the left lateral ventricle of mice (bregma: − 2 mm, lateral: 2 mm, dorsoventral: 3 mm) using a Hamilton microsyringe. HOXA-AS2 siRNA and control siRNA were carefully diluted with equal volumes of transfection reagent siPORT NeoFX (Invitrogen, CA, USA), and the mixtures were then incubated at 25 °C for 15 min according to the guidelines supplied by the manufacturer for five consecutive days. The mice were injected intraperitoneally with LPS (1 mg/kg) after the last ICV injection. Open-field tests were carried out 4 h after LPS challenge to evaluate behavioral activity, as previously described [24].
Fluorescence in situ hybridization and immunocytofluorescence staining
Fluorescence in situ hybridization (FISH) assays were performed using a Fluorescent In Situ Hybridization Kit (RiboBio, Guangzhou, China) according to the protocol. Probe sequences targeting HOXA-AS2 were designed and generated by RiboBio, and experiments were performed according to the manufacturer’s instructions. Immunocytofluorescence staining (ICF) was performed according to the previous report. Briefly, tissue sections were fixed, permeabilized, and incubated with primary rabbit anti-Iba-1 antibody (ab5076, Abcam, 1:800) overnight at 4 °C, followed by 2 h of incubation with FITC-conjugated secondary antibody (Jackson ImmunoResearch). Fluorescence images were captured using an Olympus laser scanning microscope FV3000 (Olympus Corporation, Japan) and analyzed using Image-Pro Plus (Media Cybernetics).
Cytoplasmic and nuclear RNA isolation
Cytoplasmic and nuclear RNA was extracted using nuclear/cytoplasmic isolation reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cytoplasmic and nuclear fractions were split for RNA extraction. The expression levels of HOXA-AS2, NEAT1, and GAPDH in the nucleus or cytoplasm were detected by RT-qPCR.
RNA immunoprecipitation and RNA pull-down assay
RNA immunoprecipitation (RIP) was performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer’s protocols. Cells were collected and lysed in RIP lysis buffer and then incubated with magnetic beds and anti-EZH2 (ab3748, Abcam), anti-SUZ12 (ab236322, Abcam), and anti-RbAp48 (ab1765, Abcam) antibodies overnight at 4 °C. After the precipitate was washed with RIP wash buffer six times, the coprecipitated RNA was detected by qPCR analysis. Immunoglobulin G RIP of cells served as the negative control. RNA pull-down assays were performed using the Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific). The retrieved proteins were detected by Western blotting with an anti-EZH2 antibody.
Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assays were performed using the ChIP Assay Kit (Beyotime, China) following the manufacturer’s protocol. Briefly, cells were cross-linked with 1% formaldehyde solution for 10 min at 37 °C and quenched with 125 mM glycine. DNA fragments ranging from 200 to 500 bp were yielded via sonication. Then, the lysates were immunoprecipitated with anti-EZH2 and anti-H3K27me3 antibodies (Millipore, USA). IgG was used as negative control. The precipitate DNA was extracted and analyzed by RT-qPCR. ChIP primers are listed in Supplementary Table S2.
Statistical analysis
Statistical analyses were performed using SPSS software v.13.0 (Chicago, IL, USA) and GraphPad Prism software (La Jolla, CA, USA). The differences between two or multiple groups were analyzed by t test or one-way analysis of variance (ANOVA) if the data were normally distributed. Nonparametric Mann-Whitney U or Kruskal-Wallis tests were used for continuous variables if the data were not normally distributed. Spearman’s correlation coefficient was used for correlation analysis. Receiver operating characteristic (ROC) curves were constructed to detect biomarkers for differentiating PD from healthy controls; the predictive accuracies of the parameters for detecting PD were quantified by using the area under the curve (AUC). Data are shown as the mean ± SD. Statistical significance was defined as P < 0.05.