Study population and sample collection

Fifty-eight patients with SZ were selected from local hospital. Inclusion criteria: (1) the diagnosis of SZ followed the diagnostic criteria stipulated in the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders IV (DSM-5) [16]; (2) complete clinical data of patients; (3) normal intelligence; (4) patients who did not take antipsychotic drugs before their first enrollment; (5) no history of mental illness or family history. Exclusion criteria: (1) patients with brain trauma; (2) patients with dysfunction of heart, liver, kidney, and other important organs; (3) accompanied by other mental disorders; (4) patients with alcohol dependence or drug abuse. Another 57 healthy subjects were selected as control group. The implementation of this study was approved by the Ethics Committee of Shenzhen Polytechnic University and conforms to the relevant provisions of the Declaration of Helsinki. All volunteers and their families were informed and signed written informed consent.

General information was collected from all volunteers, including gender, age, body mass index (BMI), family history. On the next day after inclusion, 5 mL of fasting venous blood was collected and centrifuged at 4000r/min at 4 ℃ for 10 min. The upper serum was taken and stored in a −80 ℃ refrigerator for later use.

Determination of serum miR-15a-5p expression level

Total RNA was extracted from serum using TRIzol reagent, and cDNA was obtained by reverse transcription reaction using RNA as a template according to PrimeScript RT reagent kit instructions. RT-qPCR was carried out according to the instructions of the real-time fluorescence quantitative PCR kit. The reaction system was 20 μL, including 10 μL of SYBR Premix, 1 μL of cDNA, 8 μL of sterile purified water, and 0.5 μL of upstream and downstream primers. The cycle parameters of PCR amplification are as follows: pre-denaturation at 90 ℃ for 10 min (1 cycle), followed by 40 cycles of denaturation at 94 ℃ for 30 s, annealing at 60 ℃ for 30 s, and extension at 70 ℃ for 30 s. Each reaction was repeated for three times. U6 was used as the internal reference gene, and the relative expression level of serum miR-15a-5p was expressed by (RQ) = 2−△△Ct method.

Psychopathology data collection

The symptoms of psychopathology were assessed with the Positive and Negative Symptom Scale (PANSS) for SZ [17]. PANSS consisted of 7 positive symptoms, 7 negative symptoms and 16 general psychopathological symptoms. In this study, PANSS items P1, P3, P5, and G9 were used to estimate positive symptoms; N1, N2, N3, N4, N6, and G7 items were used to evaluate negative symptoms; P2, N5, and G11 items were used to evaluate cognitive function; P4, P7, G8, and G14 items were used to estimate arousal; and G2, G3, and G6 items were used to evaluate depressive mood.

Animals and grouping

Twenty-five male SD rats with a body weight of (55 ± 5) g were raised in a cage alone and were given light from 7:00 to 19:00 every day to allow them to drink and eat freely. The rats began the experiment after a week of environmental adaptation. The rats were randomly divided into 5 groups, with 5 rats in each group. The blank group, also known as the sham group, received intraperitoneal injections of sodium chloride (0.9%, m/v) every day for 2 weeks. The animal models of SZ are usually constructed by intraperitoneally injection of MK-801. Rats were intraperitoneally injected with MK-801 (0.2 mg/kg) at 10:00 a.m. every day for 2 weeks to establish SZ model. In addition, the SZ model rats were divided into miR-15a-5p mimic group (SZ + 2 μM miR-15a-5p mimic), miR-15a-5p inhibitor group (SZ + 2 μM miR-15a-5p inhibitor) and miR-negative control group (SZ + 2 μM miR-NC). The above mimics, inhibitors, and negative controls of miR-15a-5p were administered by intraperitoneal injection.

Morris water maze test

Morris water maze test was used to evaluate the cognitive function of SZ rats. The water maze is 210 cm in diameter, 51 cm in height, 10 cm in platform diameter, and 19–22 ℃ in water temperature. The water chamber was divided into four quadrants, and a platform was placed inside the chamber. The platform is placed at 1 cm below the third quadrant. The rats were first trained for 5 days, four times a day. The rats were placed at a random position in the water maze, allowing the rats to find the platform within 60 s and rest on the platform for 10 s. If the rats did not find the platform within 60 s, they were artificially placed on the platform for 10 s, and the escape latency of rats was recorded. Subsequently, the platform was removed, and the rats were placed in the first quadrant, and the times and time of passing through the original platform position in the third quadrant were recorded.

Open field test

The open field box (100 × 100 × 50cm3) was divided into 9 areas. Put the camera directly above the box and connect it to the computer. Adjust the parameters to start recording. The rats were placed in the center of the box with their backs to the experimenter, and they were allowed to explore in the box for 10 min. Motor behavior in rats was quantified as total distance between spontaneous activity and rest time. In addition, the tank should be cleaned with 70% ethanol between the two tests to remove odors of other animals.

Bioinformatics analysis

To further understand the biological function of miR-15a-5p, we tried to study the downstream target genes of miR-15a-5p by bioinformatics methods. We used TargetScan, miRDB and Martarbase databases to predict the target genes of miR-15a-5p and obtained the target genes supported by these three databases by constructing Venn diagram. Subsequently, we performed Gene ontology (GO) analysis on these target genes to determine the molecular functions (MF), cellular components (CC), and biological processes (BP) of these genes. Further, the signal pathways of potential enrichment of these target genes were briefly analyzed through Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis.

Data analysis

SPSS 17.0 statistical software was used for statistical analysis. Quantitative data were expressed as mean ± standard deviation, and the comparison between the two groups was made by independent sample t test. Qualitative data were represented by n, and Chi-square test was chosen for comparison between the two groups. Pearson correlation coefficient analysis was used to analyze the correlation between clinical indicators and serum miR-15a-5p; Logistic regression was used to evaluate the risk factors for the occurrence of SZ; SZ was taken as the dependent variable (Assignment: SZ = 1, non-SZ = 0), and age, gender (Assignment: female = 1, male = 0), education level (Assignment: > 9 years = 1, ≤ 9 years = 0), marital status (Assignment: unmarried = 1, married = 0) and miR-15a-5p level were taken as independent variables. The diagnostic value of serum miR-15a-5p in SZ was analyzed by the receiver operator characteristic curve. P < 0.05 was considered a significant difference.