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The NONRATT023402.2/rno-miR-3065-5p/NGFR axis affects levodopa-induced dyskinesia in a rat model of Parkinson’s disease – Cell Death Discovery


Animals

Male Sprague-Dawley rats weighing 200–250 g were obtained from Vital River Experimental Animal Technology (Beijing, China) and were housed in a temperature-controlled room (23 °C) on a 12-h light/dark cycle with food and water available ad libitum. All animal experiments were conducted according to the Chinese Animal Welfare Act, Beijing Guidelines for the Care and Use of Laboratory Animals and Guidance for Animal Experimentation of Capital Medical University. The study protocol was approved by the Ethics Committee of Beijing Neurosurgical Institute, Capital Medical University (protocol no. AEEI-2018-200).

Adeno-associated virus (AAV vector)

An adeno-associated virus (AAV) vector was used to genetically overexpress NONRATT023402.2 (AAV2/9-NONRATT023402.2-mCherry, 1.5 × 1012 vg/mL), inhibitor rno-miR-3065-5p (AAV2-rno-miR-3065-5p-EGFP, 1.6 × 1012 vg/mL), or silence NGFR (AAV2/9-NGFR shRNA- EGFP, 6.3 × 1012 vg/mL). The empty AAV vector coding mCherry was used as the control. The shRNA sequence targeting NGFR was 5′-GCAGATGTGCCTATGGCTACT-3′ and the scrambled sequence was 5′-GAAGCAACTCGTCTGGACAGT-3′, which were based on a previous study [28]. The AAV vector was constructed by HanBio Biotechnology (Shanghai, China). The inhibitor sequence of rno-miR-3065-5p was 5′-AGCATCAGTGATTTTGTTGA-3′ and the scrambled sequence was 5′-TTCTCCGAACGTGTCACGT-3′. The AAV vector was constructed by Genepharma (Shanghai, China).

Surgical procedures and pharmacology

The intracerebral microinjections were performed via stereotaxic injections, as previously described [9]. Briefly, rats were anesthetized with 2–3% isoflurane using an animal anesthesia ventilator system (RWD Life Science, Shenzhen, China) before stereotactic surgery. A total of 4 μL AAV was infused into the right striatum through a microsyringe (Hamilton, Reno, NV, USA) at a speed of 0.2 μL/min. The needle remained in place for an additional 5 min before it was slowly retracted. The coordinates for the two sites of striatum were anteroposterior (AP) = 0.5 mm, mediolateral (ML) = −3.0 mm relative to bregma, and dorsoventral (DV) = −4.5/−6.0 mm from the skull surface. Each site was injected with 2 μL AAV.

Two weeks later, 5 mg/mL 6-hydroxydopamine (6-OHDA) (Sigma-Aldrich, St. Louis, MO, USA) was injected into the medial forebrain bundle (from bregma: AP = −4.3 mm, ML = −1.6 mm, and DV = −8.4 mm from skull surface) and the substantia nigra pars compacta (from bregma: AP = −4.8 mm, ML = −1.7 mm, and DV = −8.0 mm from the skull surface) to achieve a full nigrostriatal lesion. Each site received 2 μL with an injection rate of 0.5 μL/min. The sham lesion group received the same dose of saline instead of 6-OHDA. Other operations were the same as the above AAV vector injection process.

To assess the successful establishment of a PD model, rats were injected subcutaneously with 0.5 mg/kg apomorphine (Sigma-Aldrich) 3 weeks after the 6-OHDA-induced lesion, with the turning behavior recorded. Rats displaying more than 7 full contralateral turns/min during the 30 min period after the injection of apomorphine were selected for L-DOPA administration.

Rats received a single daily intraperitoneal injection of L-DOPA (12 mg/kg; Sigma-Aldrich) combined with benserazide (6 mg/kg; Sigma-Aldrich). The sham LID group rats received single daily injections of the same volume of saline.

Three batches of animal experiments were conducted. The first batch was divided into the 0 week (PD) group (n = 4), 2 weeks group (n = 4), 4 weeks group (n = 4), 6 weeks group (n = 5), 8 weeks group (n = 4), and 10 weeks group (n = 5) according to the duration of L-DOPA treatments. The second batch included the LncRNA + LID group (n = 15), sham + LID group (n = 11), sham + PD group (n = 5), and sham group (n = 6). The LID group was injected with empty vector containing a fluorescein label. The third batch included the empty vector of the NONRATT023402.2/ShNGFR (sham) group (n = 7), AAV-NONRATT023402.2 and the empty vector of the ShNGFR (LncRNA) group (n = 5), AAV-ShNGFR and the empty vector of the NONRATT023402.2 (ShNGFR) group (n = 8), and the AAV-NONRATT023402.2/ShNGFR (LncRNA + ShNGFR) group (n = 8). The fourth batch included the empty vector of the rno-miR-3065-5p (sham) group and the AAV-rno-miR-3065-5p inhibitor (microRNA) group (n = 7).

Behavioral testing

The cylinder test was used to evaluate forepaw use contralateral to the lesion side and to assess the success of modeling [39, 40]. Each rat was placed into a transparent cylinder (20 cm diameter and 30 cm height) and recorded for 5 min. The number of weight-bearing contacts made on the cylinder wall with the left, right, or both forepaws was recorded. Contralateral forepaw use was calculated according to the following equation: [(the number of contralateral forepaw movements)/(total number of forepaw movements) + (1/2) both forepaw movements] ×100% [41].

To assess dyskinesia behavior, rats were observed for AIM as described previously [9, 42]. In brief, the rats were monitored for 1 min every 30 min over a period of 3 h, immediately after L-DOPA administration for signs of axial, forelimb, orolingual dyskinesia, and locomotive activities (Fig. 1A). The scale of each sign was rated from 0 to 4 based on duration and severity. Total AIM scores were calculated as the sum of the score per observation point.

Primary striatal neuronal cultures

Rat striatum tissues were extracted from fetal SD rats (embryonic day 17) under sterile conditions. The striatum tissues were digested for 10 min in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Scientific, MA, United States) containing 0.02% papain at 37°C and the tissues were gently triturated for 15–20 times, followed with 8 min centrifugation. Then cells were plated onto flasks or 6-wells plates precoated with poly-D-lysine (Sigma-Aldrich, St. Louis, United States) at a density of 5 × 105 cells/ml. The plating medium included DMEM, 10% fetal bovine serum (FBS), 5% horse serum, 1 mM L-glutamine. After 4 h, discard the plating medium and use neurobasal medium (Thermo Scientific) supplemented with 2% B27 (Thermo Scientific). On the 7th day, cells were used for processing and experiments.

RNA interference

On the basis of the manufacturer’s protocol, cells were transfected with siRNA using LipofectamineTM 3000 reagent (Thermo Scientific). The knockdown efficiency was determined by RT-qPCR 24 h after transfection. siRNA sequences are as follows (Table 1).

RNA extraction and sequencing

Rats were deeply anesthetized with isoflurane and decapitated. The striatum was separated using a microdissection procedure. Total RNA was isolated from the right striatum of rats using a TransZol Up Plus RNA Kit (Cat#ER501-01; TransGen Biotech, Beijing, China) following the manufacture’s instructions, and checked for an RNA integrity number to inspect RNA integrity using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US). Qualified total RNA was further purified by a RNAClean XP Kit (Cat# A63987; Beckman Coulter, Brea, CA, USA) and RNase-Free DNase Set (Cat#79254; Qiagen, Hilden, Germany).

The cluster was generated by cBot with the library diluted to 10 pM and then sequenced on an Illumina HiSeq X-ten system (Illumina, San Diego, CA, USA). Library construction and sequencing were performed by Shanghai Biotechnology (Shanghai, China). Raw reads were preprocessed by filtering-out rRNA reads, sequencing adapters, short-fragment reads, and other low quality reads. HISAT2 (version 2.0.4) [43] was used to map the clean reads to the Rnor6.0 reference genome. To quantify the mRNAs, their expressions were determined as fragments per kilobase of transcript per million mapped reads using StingTie (version 1.3.1) [44]. Differentially expressed genes (DEGs) were identified using the edgeR package [45] with a threshold log2fold-change (FC) > 1.2 and P < 0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms were annotated to the DEGs.

Prediction of target miRNAs and target genes, and gene network construction

The potential target miRNAs of NONRATT023402.2 were predicted using the miRDB database [46], and the target genes of miRNAs were predicted using the TargetScan database [47]. The predicted target genes were intersected with upregulated DEGs to narrow the scope of the study.

The lncRNA-miRNA-mRNA ceRNA network was constructed based on the relationships between lncRNAs, using predicted target miRNAs and target mRNAs in Cytoscape (version 3.9.1). Among the target genes, the protein-protein interaction (PPI) network was obtained from the STRING database with a confidence > 0.2, with Cytoscape used for visualization. The module of the PPI network was analyzed using the MCODE plug [48].

Dual luciferase reporter assays

The interactions between NONRATT023402.2 and rno-miR-3065-5p, rno-miR-3065-5p, NGFR, and c-Fos, and the promotor region of NONRATT023402.2 were verified using the dual-luciferase reporter assay. The luciferase reporter plasmids, pMIR-REPORT and pGL4.74 (Syngenbio, Beijing, China), encoding firefly luciferase (hluc+) and Renilla luciferase (hRluc), respectively, were used for all assays. The sequences of NONRATT023402.2, rno-miR-3065-5p, NGFR, and the promotor region of NONRATT023402.2 were obtained from the National Center for Biotechnology Information, and their binding sites were predicted using RNAhybrid (version 2.2).

Wild-type and mutant sequence fragments of NONRATT023402.2 and NGFR were inserted into their respective plasmids. HEK293FT cells were seeded into 96-well plates, and 100 μL of transfection solution containing 0.5 µL Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) and 25 ng reporter plasmids with 50 nM rno-miR-3065-5p mimic or 50 nM mimic control were added, respectively. The cells were then washed and harvested 24 h after transfection. Luciferase activities were measured using the Dual Luciferase Reporter Assay Kit (Promega, Milwaukee, WI, USA) and Centro XS (Berthold Technologies, Bad Wildbad, Germany). No mutant group was used in the assay of the c-Fos and NONRATT023402.2 promoters. The experiments were then repeated six times.

Quantitative real-time PCR

Total RNA was extracted using the Total RNA Extraction Kit (DNase I) (Cat#GPQ1801; GenePool) and reverse transcription was performed using the lncRNA cDNA Synthesis Kit (Cat#GPQ1806; GenePool) according to the manufacturer’s instructions. The qRT-PCR was performed in 20 μL reaction tubes containing 10 μL FastSYBR Mixture, 0.4 μL of each primer (10 μM), 2 μL cDNA template, and 7.2 μL dH2O using a BIOER LineGene 9600Plus instrument (Bioer Technology, Hangzhou, China) under the following conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 60 s. The forward and reverse primer sequences are listed in Table 2. The relative expression levels were calculated using the 2−ΔΔCT method after normalization with the reference control.

Table 2 Primers used in analysis of genes expression by qRT-PCR.

Western blot analysis

Western blot analysis was performed as previously described [9] using the following primary antibodies: TH antibody (ab112, 1:200), c-Fos antibody (ab7963, 1:500), p75 NGF receptor antibody (ab52987, 1:1000), and PI3 kinase p85 alpha (phosphor Y607) antibody (ab182651, 1:500) (all from Abcam, Cambridge, MA, USA); PI3 kinase p85 (19H8) antibody (#4257; 1:500), AKT antibody (#9272; 1:1000), and phospho-Akt (Ser473) antibody (#9271, 1:1000) (all from Cell Signaling Technology, Danvers, MA, USA). GAPDH antibody (ab181602; 1:3000; Abcam) was used for the loading control. Protein band density was quantified using Quantity One software (version 4.6.2, Bio-Rad, Hercules, CA, USA).

Immunofluorescence (IF)

Rats were anesthetized and transcardially perfused with 0.9% saline solution followed by 4% cold paraformaldehyde (PFA). Harvested rat brain tissues were fixed in 4% PFA and embedded in paraffin. The specimens (4 μm thick) were dried, washed, permeabilized, blocked in 5% goat serum, and incubated overnight at 4 °C with tyrosine hydroxylase (TH) antibody (ab112; 1:700), c-Fos antibody (ab7963; 1:50), or p75 NGF receptor antibody (ab52987; 1:100) (all from Abcam) and then incubated with the appropriate fluorochrome-conjugated secondary antibodies. Sections were mounted with medium containing diamidino-2-phenylindole (DAPI; to stain nuclei) (Vector Laboratories, Burlingame, CA, USA). The images were analyzed using Pannoramic Viewer software (3D HISTECH, Budapest, Hungary).

RNA fluorescence in situ hybridization (FISH)

FISH was performed to detect the subcellular location of NONRATT023402.2. Brain sections were digested in a pepsin solution, fixed in formaldehyde, and dehydrated by gradient ethanol solutions. The sections were then incubated with a digoxin (DIG)-labeled probe (5′-DIG-AGTAACGCTGAGTCTCGTGAGTCTGGTTCCAT-DIG-3′), followed by incubation with a DyLight 594-conjugated IgG fraction (ab96873; Abcam) coupled with a monoclonal mouse DIG antibody (ab116590; Abcam;). Nuclei were then counterstained with DAPI.

Statistical analysis

Statistical analyses were performed using SPSS statistical software for Windows, version 19.0.0 (SPSS, Chicago, IL, USA) and Prism 9.0.0 (GraphPad, La Jolla, CA, USA) software. Prior to significance testing, normal distribution and homogeneity of variances were confirmed by Shapiro-Wilk test and Brown-Forsythe testing. Data were compared using Student’s t-test (two groups) or by one-way analysis of variance followed by an appropriate multiple comparisons test (more than two groups). Data are expressed as the mean ± SEM. An alpha level of P < 0.05 was employed for significance testing.



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