Expression of miR-16 in different parts of rats with depression and its association with serotonin transporter
Abstract Objective To investigate the expression of miR-16 in different parts of the nervous system of chronic unpredictable mild stress depression model rats and its association with serotonin (5-HT) transporter. Methods There were 10 rats in the control group and the chronic unpredictable mild stress depression model group. The rats in the model group received 21 days of unpredictable mild stress stimulation, and the control rats were normally reared. The cerebrospinal fluid was collected from the rats of both groups after anesthesia, and miR-16 was measured. Then the rats were killed by decapitation, the prefrontal cortex, the nucleus raphe nucleus and the hippocampus were isolated, and the miR-16 and 5-HT transporter proteins were determined. Results The relative expression of miR - 16 in the cerebrospinal fluid and the nucleus raphe was lower in the model group than in the control group. The miR - 16 in the nucleus was negatively correlated with the 5 - HT amine transporter. The miR - 16 and 5 in the prefrontal cortex and hippocampus were negative. - There was no significant difference in the HT transporter compared with the control group (P > 0.05). Conclusion The mesothelial nucleus miR - 16 may be involved in the pathological process of depression by regulating the expression of 5 - HT transporter. The cerebrospinal fluid miR - 16 may also be associated with depression. Key words chronic unpredictable mild stress depression model; miR-16;5-HT; transporter The World Health Organization has announced that depression has become the world's fourth-largest disease and is expected to become the second-largest disease after coronary heart disease by 2020. Depression has a high prevalence rate (10% to 25% for women with a lifetime prevalence, 5% to 12% for men), high recurrence rate (30% in 1 year, 70% in 5 years), and high suicide rate (10%) ～ 25%) is one of the global public health problems, which brings great spiritual loss and huge economic burden to individuals, families and society. However, its etiology and pathogenesis are still unclear, which makes it extremely difficult to diagnose and treat the disease. MicroRNAs (miRNAs) are a hotspot in epigenetic research in recent years. They are a class of evolutionarily conserved, 18-25 nucleotide non-coding RNA molecules that pass through the 3' untranslated region of the mRNA of the target gene (3' - Untranslated region (UTR) binding, which causes degradation of mRNA or inhibition of translation, thereby regulating protein expression and plays an important role in the development and development of biological development and disease. Existing studies have revealed that miR-16 may be associated with depression, and its mechanism of involvement in depression may be that it uses the serotonin transporter (SERT) gene as a target gene in brain tissue. Participate in the regulation of SERT translation level, thus affecting the function of 5-HT neurotransmitter in brain tissue. However, it has not yet been determined which parts of the nervous system are involved in the onset of depression. In this study, we established a model of chronic unpredictable mild stress depression in rats, and compared the differences of miR-16 in different parts (cerebrospinal fluid, prefrontal cortex, hippocampus, and nucleus) between the depression model group and the control group, and analyzed miR-16 and SERT. The correlation of protein is to explore the role of miR-16 in depression and its mechanism, and to provide a reference for understanding the pathogenesis of depression and finding suitable biomarkers. Materials and Methods 1. Laboratory animals and groupings: 20 clean female SD rats, purchased from Zhejiang Academy of Medical Sciences, laboratory animal certificate number: SCXK (Zhejiang) 2008 - 0033, weighing about 150g, randomly divided into control group (10) and depression model Group (10). The temperature of the breeding room is 23 ± 1 °C and the relative humidity is 55% ± 5%. 2. Establishment of a depression model: Chronic unpredictable mild stress (CUMS) model rats were randomized to receive one of the following stimuli: 4 °C ice bath for 5 min, water for 24 h, day and night fall, tail suspension 10 min 45 ° C high temperature 5 min, fasting 48 h, rat cage tilt 24 h, wet litter 24 h, tail 1 min, restraint stress 2 h, for 3 weeks. The rats in the control group were bright (8: 00 to 20: 00) and 12 hours dark (20: 00 to 8: 00) every day for 12 hours, and they were given free access to drinking water. 3. Assessment of Depressive Behavior: Depression-like behavior was assessed using a sucrose consumption test and a market test. (1) Sugar consumption experiment: 1 time before and after modeling, completed in 3 days. Rats were kept in single cage on the first day, and 2 bottles of 1% sucrose water were given. On the second day, 1 bottle was taken. Sucrose water is exchanged for tap water for sucrose water preference training. On the third day, after the rats were banned for 23 hours, 1 bottle of 1% sucrose water and 1 bottle of tap water were given, and the consumption of sucrose water and tap water within 1 hour was calculated to determine the sugar water preference rate. Sugar water preference rate (%) = sugar consumption / (sugar consumption + tap water consumption) × 100%. (2) Market experiment: Containers with length, width and height of 100cm, 100cm and 40cm respectively (provided by Shanghai Xinsoft), the bottom is divided into 25 squares of equal area, the inner wall is black, a camera is placed directly above the container, which will be large The rats were placed in the center and the walking score and vertical score within 3 minutes were recorded (walking score: 1 paw of the rat entered 1 grid 1 point; vertical score: 1 point of the rat's front paw off the ground or climbing the bucket wall 1 time) . The open field test was carried out at 18: 00 to 19: 00, in a quiet, dark environment, and the rat feces were cleaned each time to remove any possible taste. A total of two open field tests were performed before and after modeling. 4. Cerebrospinal fluid, prefrontal cortex, hippocampus, and central nucleus of the nucleus: After the depression behavioral index was measured, the head was fixed on the directional instrument after anesthesia. Cut the head and neck, disinfect, and cut a longitudinal incision along the longitudinal axis with a scalpel (about 2 cm). Use the scissors to bluntly separate the dorsal muscles of the neck. In order to avoid bleeding, the muscles attached to the bone at the deepest level are scraped off with a scalpel back, exposing the occipital foramen, and the cerebrospinal fluid is directly extracted from the occipital foramen. The rats were killed by decapitation, and the prefrontal cortex, hippocampus and nucleus were rapidly separated on ice according to the stereotactic map of rat brain of Bao Xinmin et al. 5. Western blot analysis of SERT protein: Mem - PER Eukaryotic Membrane Protein Extraction Rea-gent Kit ( Thermo Fisher Scientific, USA) was used to extract membrane proteins from the prefrontal cortex, hippocampus, and nucleus. The Bradford method quantifies proteins. The procedure for determining the SERT protein by Western blot was carried out according to the article by Huff et al. Anti-SERT primary antibody (1:250 dilution), anti-beta-actin primary antibody (1:250 dilution) and horseradish peroxidase-labeled secondary antibody (1:5000 dilution) were purchased from Santa Cruz, Canada. SERT protein bands were analyzed using the Chemi DocTM XRS + (Bio- rad USA) system's own software and corrected with β-actin. 6. Real-time PCR assay for miR - 16: by miRcutemiRNA Extraction and isolation kit (Beijing Tiangen Biochemical Technology Co., Ltd.) Extract total RNA from cerebrospinal fluid, prefrontal cortex, hippocampus and mesothelial nucleus, and measure its concentration and purity with nanodrop (thermo scientific company); take 2μg RNA for reverse transcription. The c DNA was used for the next real-time quantitative fluorescent PCR amplification. U6 is the internal reference calibration gene, primer sequence: 5' - ACGCAAATTCGT-GAAGCGTTCCAT - 3', miR-16 primer sequence: 5' - TAGCAG-CACGTAAATTGGCG - 3'. The downstream primer is a universal primer [Beijing Tiangen Biochemical Technology Co., Ltd. miRcute miRNA fluorescence quantitative detection kit (SYBRGreen) comes with]. Real-time quantitative PCR amplification procedures are: pre-denaturation 94 ° C 2 min; PCR reaction: 94 ° C 20s, 60 ° C 34s 40 cycles, real-time PCR instrument purchased from ABI company stepone plus, using the instrument software to automatically analyze the data to obtain the cycle threshold (Ct value) of each sample, and obtain the miR of each sample by the standard curve method. - 16 content, the resulting data is corrected with the corresponding U6. 7. Statistical methods: Using SPSS 19. 0 Statistical software analysis data, independent sample t test was used for comparison between groups, and Pearson correlation analysis was used for correlation analysis. Data are expressed as mean ± standard deviation ( x ± s). Take P < 0. 05 is statistically significant. Discuss In this study, we established a chronic unpredictable mild stress rat depression model to study the expression of miR - 16 in different parts of the rat nervous system and its association with SERT, so as to further clarify the pathophysiological process of miR - 16 involved in depression. . In vertebrates, miRNAs in brain tissue are much more abundant than other tissues, suggesting that miRNAs may play an important role in brain function. Some researchers have found that miRNAs in cerebrospinal fluid may be associated with mental illness. For example, in 2014, Muller et al. showed that miR-146a in cerebrospinal fluid of patients with Alzheimer's disease is lower than that of healthy people. But so far, there have been no reports of miR-16 levels in cerebrospinal fluid in depression. Therefore, the cerebrospinal fluid of the depression model rats was collected in this experiment, and the miR-16 assay was performed, and the level was found to be lower than that of the control rats. The results need to be further confirmed to determine the feasibility of cerebrospinal fluid miR-16 as a biomarker of depression. This study also measured miR-16 in the prefrontal cortex, hippocampus, and nucleus of the brain. Some researchers have found that some miRNAs in the prefrontal cortex of suicidal patients have abnormalities, such as miR-494 and miR-335, which are lower than healthy controls. However, no reports of miR-16 in the prefrontal cortex of depression have been reported. The level of miR-16 in the prefrontal cortex of rats in this model shows that the level of miR-16 in the prefrontal cortex of the model group is compared with the control group. The difference was not statistically significant, and the expression of SERT protein in this part was not significantly different from the control group, indicating that the prefrontal cortex miR-16 may not be involved in the pathological process of depression. In recent years, the study of hippocampal miR-16 and depression has attracted the attention of some researchers. For example, Zhang Yi et al found that hippocampal miR-16 is higher than normal rats in the model of maternal deprivation of rats. The authors believe that hippocampal miR-16 is involved in the pathological process of depression-like behavior induced by maternal deprivation. In another study, the antidepressant fluoxetine reduced miR-16 in mouse hippocampus. If miR-16 was neutralized using synthetic anti-miR-16, anti-miR-16 showed antidepressant Like a role. However, in the study of Bai et al, the authors used a chronic unpredictable mild stress to establish a depression model, and no changes were found in hippocampal miR-16 of model rats. Based on the inconsistency of hippocampal miR-16 in the results of different depression models, and the differences in hippocampal SERT proteins in Bai et al., this study also analyzed hippocampal miR-16 in model rats, and also analyzed the differences in hippocampal SERT. It was shown that miR-16 was not elevated in the hippocampus of model rats. This conclusion is consistent with the paper published by Bai et al., and the expression of SERT protein in this part is not statistically different from the control group. The above results indicate that hippocampal miR-16 does not participate in pathological processes associated with chronic unpredictable mild stress depression, suggesting that the mechanisms of depression induced by different stress may be different. The central nucleus accumulates 5 - HT neurons, and its main function is to produce the transmitter 5-HT, which is of great significance in the study of the relationship between miR-16 and depression. Baudry et al. injected fluoxetine (1 μmol/L, 2 μl/min) directly into the nucleus of the nucleus for 3 days and found that miR - 16 was elevated in this area. At 5 times, the expression of SERT was reduced by a factor of 2; direct injection of miR-16 (1 μl, 2 μmol/L) into this region also observed a decrease in SERT expression; however, when fluoxetine and anti-resistant When miR-16 was injected together, SERT expression was unaffected, and it was thus seen that fluoxetine was passed. miR-16 regulates the expression of SERT protein. However, in the animal model of depression, the difference between the mesial nucleus miR-16 and the control has not been reported. This experiment showed that the miR-16 of the nuclei in the chronic unpredictable mild stress depression model was lower than that of the control group, but the SERT protein level was higher than that of the control group, and there was a significant negative correlation between the two. The author believes that the low miR-16 status of the nucleus can lead to the overexpression of SERT protein, which affects the function of the 5-HT transmitter system and thus participates in the depression process caused by chronic unpredictable mild stress. In conclusion, this study explored miR-16 by measuring miR-16 levels in different parts of the nervous system. Participate in possible parts of depression and its mechanisms. The results showed that there were abnormalities in cerebrospinal fluid and mesothelial nucleus miR-16 in chronic unpredictable mild stress depression rats. MiR-16 may be involved in the pathogenesis of depression by regulating SERT protein expression. 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