Animal grouping and treatment
In this study, 25 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, 24 rats were randomly selected and divided into 4 groups with 6 rats in each group, i.e., 1. SHAM group; 2. SIA group; 3. SIA + EA group; and 4. EA group. Comparisons were made among the SHAM group, SIA group and SIA+EA group to study the effect of EA on SIA rats. And comparisons were also made between the SHAM group and EA group to identify potential miRNAs that responded to EA treatment. 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 injections 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 consciousness after the induction of anesthesia. According to previous publications , the acupuncture site of Renzhong located at the junction beneath the nasal septum along the cleft lip midline, as well as the Neiguan site located at the junction between the ﬂexor carpi radialis and palmaris longus, were subjected to electrostimulation 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 on the rats immediately after full recovery from anesthesia until the rats were sacrificed.
Measurement of rat model memory consolidation
The Novel object recognition (NOR) test was carried out to assess recognition memory according to a procedure described elsewhere (Singh and Thakur, 2014). In brief, we used objects of similar sizes with different shapes in the experiment. For the habituation process, each rat was placed in the box for 5 min on two successive days before returning to the home cage. On the third day, two similar objects were placed and rats were allowed to interact with the objects for 5 min and before returning to the home cage. After 48 h of training, we replaced one object with a novel object before re-introducing the rats to interact with the objects. The objects and box were wiped by 70 % ethanol after each trial to remove odor of previous mice and the positions of the objects were similar during training and test. The time spent with novel objects was calculated as TNovel/(TNovel + TFamiliar) X 100. Specially, TNovel refers to time spent with novel object and TFamiliar refers to time spent with familiar object. And rearing, NCD and locomotive activity were also evaluated (Matsumoto J, 2014; Hanania T, 2004) and involved in the statistical analysis as potential confounding factors.
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 were 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 an SYBR green master mix (Applied Biosystems, Foster City, CA) following the instructions provided by the manufacturer. The relative expression of rno-miR-183-5p (Primer-F: 5’- TATGGCACTGGTAGAATTCAC-3’; Primer-R: 5’-GAACATGTCTGCGTATCTC-3’), rno-miR-34c-3p (Primer-F: 5’-AGGCAGTGTAGTTAGCTGATT-3’; Primer-F: 5’- GAACATGTCTGCGTATCTC-3’), rno-miR-210-3p (Primer-F: 5’-AGCCACTGCCCACAGCAC-3’; Primer-R: 5’-GAACATGTCTGCGTATCTC-3’), rno-miR-758-5p (Primer-F: 5’-TGGTTGACCAGAGAGCAC-3’; Primer-R: 5’- GAACATGTCTGCGTATCTC-3’), rno-miR-568 (Primer-F: 5’-ATGTATAAATGTATACACAC-3’; Primer-R: 5’-GAACATGTCTGCGTATCTC-3’), rno-miR-196c-3p (Primer-F: 5’- AGGTAGTTTCGTGTTGTTG-3’; Primer-R: 5’-GAACATGTCTGCGTATCTC-3’), Arc (Primer-F: 5’-TATTCAGGCTGGGTCCTGTC-3’; Primer-R: 5’-TGGAGCAGCTTATCCAGAGG-3’), Egr1 (Primer-F: 5’-AGCGAACAACCCTATGAGCA-3’; Primer-R: 5’-TCGTTTGCTGGGATAACTC-3’ ), Homer1 (Primer-F: 5’-GAAGTCGCAGGAGAAGATG-3’; Primer-R:5’-TGATTGCTGAATTGAATGTGTACC-3’), Narp (Primer-F: 5’-GTTCTGGGGAGTTCAAGGCA-3’; Primer-R: 5’-AGGAAGTGGCTCAGGCATCT-3’) and SIN3A mRNA (Primer-F: 5’-CAGAATGACACCAAGGTCCTGAG-3’; Primer-R: 5’- CATACGCAAGTGAGAGGTGTGG-3’) 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 the cells were 80% confluent, they were transfected with one of 6 candidate miRNAs, i.e., rno-miR-183-5p, rno-miR-34c-3p, rno-miR-210-3p, rno-miR-758-5p, rno-miR-568, or rno-miR-196c-3p, 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 sequences of SIN3A containing the binding sites for the above miRNAs were respectively cloned into different pcDNA luciferase vectors (Promega, Madison, WI) to produce vectors for wide type SIN3A 3‘UTRs. 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 sequences of SIN3A harboring the mutated binding sites for above miRNAs, respectively, and the mutant 3‘UTR sequences were also cloned into different pcDNA luciferase vectors to produce vectors for mutant SIN3A 3‘UTRs. 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 analysis
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 (ab129087, 1:1000 dilution, Abcam, Cambridge, MA), anti-Arc primary antibodies (ab18950, 1:1000 dilution, Abcam, Cambridge, MA), anti-Egr1 primary antibodies (ab133695, 1:1000 dilution, Abcam, Cambridge, MA), anti-Homer1 primary antibodies (ab184955, 1:1000 dilution, Abcam, Cambridge, MA) and anti-Narp primary antibodies (ab191563, 1:1000 dilution, Abcam, Cambridge, MA) and further incubated with HRP-conjugated secondary antibodies (ab6721, 1:2000 dilution, Abcam, Cambridge, MA). The relative protein expression was analyzed using an Odyssey imaging system (Li-Cor, Lincoln, NE).
Samples of collected rat hippocampus 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 (ab129087, 1:100 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 horseradish peroxidase (HRP)-tagged avidin/streptavidin and counter-stained with 4',6-diamidino-2-phenylindole (DAPI) before the positive expression of SIN3A was analyzed underneath a microscope.
All experimental results were shown in mean ± SD. The comparison of different groups was carried out using Student’s t-test (for inter-group comparison) and one-way ANOVA (for multi-group comparison). 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). Each experiment was repeated 3 times.