Animal grouping and treatment
In this study, 20 Wistar male rats were acquired from the Center of Laboratory Animals at our institute. All rats were about 8 weeks in age and weighed 250 to 300 g. The animal protocols of this study have been approved by the Institutional Animal Ethics Committee. After 7 days of adaptation, the rats were divided into 3 groups, i.e., 1. SHAM group; 2. SIA group; and 3. SIA + EA group. All rats were placed in individual cages at 22 ± 1 °C during the experiment. The establishment of the SIA model and corresponding EA treatment will be described below.
Establishment Of An Sia Rat Model
Scopolamine hydrobromide (Sigma-Aldrich, St. Louis, MO) was dissolved in 0.9% saline and then injected into rats via intraperitoneal injection at a dosage of 3 mg/kg. The injection was carried out once a day for seven days consecutively. The rats in the SHAM group were given the injection of 0.9% saline at the same dosage and the same frequency. After the 7 days of consecutive injection were finished, all rats were euthanized by CO2 suffocation to collect their brain tissues for subsequent analyses.
The treatment with EA was carried out immediately after the SIA rats gained full conscience after the induction of anesthesia. To carry out the EA treatment, the site of acupuncture, Renzhong, which is located at the junction beneath the nasal septum along the cleft lip midline, as well as the site of Neiguan, which is located at the junction between the flexor carpi radialis and palmaris longus, were subjected to electro stimulation delivered via a 0.25 mm × 30 mm single use needle. During the EA procedure, the frequency of electro stimulation was set to 2 Hz, while the intensity of the electro stimulation was set to 3.0 mA. The EA stimulation was applied for 1 min in 12 h intervals until the rats were sacrificed.
Novel Object Recognition (nor) Test
The NOR test was carried out to assess recognition memory according to a procedure described in (Singh and Thakur, 2014).
Rna Isolation And Real-time Pcr
Samples of collected tissues and cultured cells were pulverized and then lysed using a QIAzol lysis buffer (Qiagen, Valencia, CA). Then, total RNA content was isolated from each sample by using a miRNeasy mini assay kit (Qiagen, Valencia, CA) according to manual instructions. Then, 2 µg of RNA in each sample was reversely transcribed into cDNA by using a Superscript II assay kit for reverse transcription (Invitrogen, Carlsbad, CA). Finally, real time PCR was carried out by using a Prism 7900 HT real time PCR system (Applied Biosystems, Foster City, CA) in conjunction with a SYBR green master mix (Applied Biosystems, Foster City, CA) following the instructions provided by the manufacturer. The relative expression of rno-miR-183-5p, rno-miR-34c-3p, rno-miR-210-3p, rno-miR-758-5p, rno-miR-568, rno-miR-196c-3p, and SIN3A mRNA was quantified using the 2−ΔΔCT method.
Cell Culture And Transfection
SH-SY5H cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Ham's F-12 medium added with 5% fetal bovine serum, 100 µg/ml streptomycin, as well as 100 U/ml penicillin. The cell culture was carried out in a 37 °C humidified incubator containing 95% air and 5% CO2. When cells were 80% confluent, they were transfected with one of 6 candidate miRNAs, i.e., rno-miR-183-5p (A), rno-miR-34c-3p (B), rno-miR-210-3p (C), rno-miR-758-5p (D), rno-miR-568 (E), or rno-miR-196c-3p (F), for 48 h before the mRNA and protein expression of SIN3A was detected. The transfection was carried out using Lipofectamine 2000 purchased from Invitrogen (Carlsbad, CA) following a routine transfection protocol.
Vector Construction, Mutagenesis And Luciferase Assay
To clarify the regulatory relationship between SIN3A and rno-miR-183-5p, rno-miR-34c-3p, rno-miR-210-3p, rno-miR-758-5p, rno-miR-568 or rno-miR-196c-3p, the 3‘UTR of SIN3A containing the binding sites for above miRNAs were respectively cloned into different pcDNA luciferase vectors (Promega, Madison, WI) to produce vectors for wide type SIN3A 3‘UTR. Then, site-directed mutagenesis was carried out using a Quick Change II site-directed mutagenesis assay kit (Stratagene, San Diego, CA) to generate mutant 3‘UTR of SIN3A harboring the mutated binding sites for above miRNAs, respectively, which were also cloned into different pcDNA luciferase vectors to produce vectors for mutant SIN3A 3‘UTR. In the next step, SH-SY5H cells were co-transfected with each of rno-miR-183-5p, rno-miR-34c-3p, rno-miR-210-3p, rno-miR-758-5p, rno-miR-568 or rno-miR-196c-3p in conjunction with wild type or mutant 3‘UTR of SIN3A using Lipofectamine 2000. At 48 h post transfection, the luciferase activity of transfected cells was assayed using a Bright Glo luciferase kit (Promega, Madison, WI) following the instructions provided by the manufacturer.
Western Blot Analyses
Samples of collected tissues and cultured cells were prepared into a homogenate and centrifuged to collect the protein supernatant. Then, after quantifying protein concentrations by using a BCA assay kit (Pierce Biotechnology, Rockford, IL), an appropriate amount of sample protein was resolved by 10% denaturing SDS-PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA), which were then blocked at room temperature for 1 h by using an Odyssey blocking reagent (Li-Cor, Lincoln, NE), incubated overnight in a 4 oC fridge with anti-SIN3A primary antibodies, and further incubation with appropriate secondary antibodies. The relative protein expression was analyzed by using an Odyssey imaging system (Li-Cor, Lincoln, NE).
Samples of collected rat brain tissues were sliced into 4 µm sections, fixed using PBS containing 4% paraformaldehyde, and quenched with 3% H2O2 to remove the activity of endogenous peroxidase. Then, after being blocked for 1 h in 5% BSA, the sections were treated overnight in a 4 °C fridge with anti-SIN3A primary antibodies (1:4000 dilution, Abcam, Cambridge, MA). After washing and further incubation with biotin-labeled secondary antibodies (1:1000 dilution, Abcam, Cambridge, MA), the slides were incubated with HRP-tagged avidin/streptavidin and counterstained with DAPI before the positive expression of SIN3A was analyzed underneath a microscope.
All experimental results were shown in means ± SD. The comparison of different groups was carried out using Student’s t test. A difference was deemed statistically significant if its probability was < 0.05, i.e., P < 0.05. All statistical analyses were done using SPSS 21.0 (IBM, Chicago, IL) and Prism 6.0 (GraphPad, San Diego, CA).