MiRNA‐199a‐5p targets WNT2 to regulate depression through the CREB/BDNF signaling in hippocampal neuron

Abstract Introduction This study mainly investigated the role of miR‐199a‐5p in depression. Methods qRT‐PCR and western blotting were employed to detect the expressions of miR‐199a‐5p, CREB and BDNF. Sucrose preference test, forced swimming test, and tail suspension test were performed to evaluate depression‐related symptoms. MTT assays and flow cytometry were used to examine the cell reproduction and apoptotic cells of hippocampal neuron. Results The data demonstrated that the expression levels of miR‐199a‐5p in the cerebrospinal fluids and serums of depression patient and the hippocampus of chronic unpredictable mild stress (CUMS) mouse were significantly increased. However, the expressions of WNT2, p‐CREB, and BDNF were inhibited. In addition, miR‐199a‐5p‐inhibitor enhanced sucrose preferences of CUMS mouse and decreased immobile time in sucrose preference test and forced swimming test. Knockdown of WNT2 attenuated the effects of miR‐199a‐5p‐inhibitor on cell reproduction and apoptotic cells of hippocampal neuron and the expression of WNT2, p‐CREB, and BDNF. Conclusion MiR‐199a‐5p can target WNT2 to enhance the development of depression through regulation of the CREB/BDNF signaling. Trial registration: JNU‐Hos‐49284.

of specific miRNAs may contribute to various human diseases, including cancer, Alzheimer's disease, and Parkinson's disease (Martins et al., 2011). Studies have identified miRNAs that are involved in depression. For example, miR-1202 was reported to be a primatespecific and brain-enriched miRNA involved in major depression and antidepressant treatment (Lopez et al., 2014). MiR-199a-5p plays a suppressive role in tumor cell reproduction (Raimondi et al., 2014;H. Yi et al., 2013). This study was carried out to investigate the role of miR-199a-5p in the pathophysiology of depression.
The WNT pathway actively participates in embryogenesis, development of nervous system, and adult hippocampal neurogenesis (Huelsken & Behrens, 2002). The WNT signaling pathway was also demonstrated to play essential roles in the treatment of major depression. It was reported that WNT and its downstream factors were involved in the pathophysiology of depression (Voleti & Duman, 2012). In addition, elevated temporal cortex CREB concentrations in major depression were observed (Dowlatshahi et al., 1998). Another study reported that inhibited function of the CREB1/BDNF/NTRK2 pathway had a negative impact on the risk mechanism of depression (Juhasz et al., 2011). In this study, interactions among miR-199a-5p, the WNT pathway, and the CREB1/BDNF axis in the development of depression were investigated.

| Patients information and sample preparation
This study recruited MDD patient with standards of 1) Patients had major depressions diagnosed by Chinese Classification and diagnostic criteria of Mental Disorders, 3rd edition; 2) Patients had Hamilton Depression Scale scores >= 24; Patient were excluded if they had: 1) Primary organic diseases, mental illnesses, drug use, or history of bipolar disorders. The control group had no mental/neurological illnesses. This study was approved by the Ethics Committee of The second affiliated hospital of Baotou medical college. All participants signed the written informed consent.
Cerebrospinal fluid (CSF, 4 ml) was collected by lumbar puncture in 3 male MDD patients and 4 female MDD patients aged at 31.26 ± 8.05 years old, as well as 3 male control participants and 4 female participants aged at 32.38 ± 8.56 years old. Fasting venous blood samples (4 ml) were obtained in another 20 MDD patients (9 males and 11 females, aged at 34.84 ± 9.59 years old), and 20 healthy controls (10 males and 10 females, aged at 35.12 ± 10.06 years old).
After collection, CSF and blood samples were centrifuged at 4°C for 10 min and then stored at −80°C.

| Animal information
Adult male Kunming mice were purchased from the Vital River Laboratory Animal Technology Co, Ltd (Beijing, China). Mice were kept at 12 hr/12 hr day/night cycle and with free access to food and drink.
Twelve mice were grouped into chronic unpredictable mild stress (CUMS) and control (n = 6) groups. Twenty-four mice were grouped into the control, CUMS, CUMS + NC-inhibitor, and CUMS + miR-199a-5p-inhibitor (n = 6) groups. Another 18 mice were grouped into CUMS, CUMS + fluoxetine, and CUMS + fluoxetine +miR-199a-5p mimics (n = 6) groups. and the control (Shanghai, China) were intracerebroventricularly administered to mice with 0.5 nmol/mouse (L.-T. Yi et al., 2014). The injection was carried out one time per week for 3 weeks. The fluoxetine group had 10 mg/kg fluoxetine by intraperitoneal injections one time per day for 2 weeks. For euthanasia, animals were deeply anesthetized with sodium pentobarbital (30 mg/kg body weight, Sigma Chemical Co.) through intraperitoneal injection. Mice were finally sacrificed by cervical dislocation. All animal experiments were performed following the Institutional Animal Care Committee of The second affiliated hospital of Baotou medical college.

| Sucrose preference test
After CUMS, mice were deprived of water after 8 p.m. The next day, mice had 1% sucrose with 1 hr/day for 3 d from 8 to 9 a.m. On the test day, sucrose preference test was carried out after 8 hr of water deprivation. After that, mice had free access to drink 1% sucrose or water for 1 hr. The test last for 2 d, followed by switching of drink bottles. After the test, the weight loss of bottles was considered as liquid consumptions.

| Forced swimming test
A glass beaker was filled with 18 cm of water at 22°C, and mice were forced to swim in it for 6 min. The immobility duration of mice was measured by double-blinded observers, as the time that mice didn't move or had little movement. After forced swimming test, mice were cleaned and put back into the cage.

| Tail suspension test
Mice were stuck by rubber at 1 cm to tail tip and suspended from top for six min. Immobility durations that mice were utterly motionless were calculated by double-blinded observers.

| Hippocampus
Mice were sacrificed after CUMS. The hippocampus was taken, frozen, and stored at −80°C. ELISA kits were used to detect the concentrations of corticosterone (CORT) in mouse serum.

| QRT-PCR
Total RNAs were isolated from tissues and cells using Trizol reagent (Invitrogen). cDNA samples were synthesized with reversetranscription and used as template for qRT-PCR. U6 was used as the internal control. The expression levels were determined using 2 −ΔΔCt method. The primer sequences were listed in Table 1.

| Western blotting
Hippocampus homogenate was lysed, centrifuged, and separated via SDS-PAGE. Total proteins were transferred to PVDF membrane (Amersham). The membrane was blocked in 5% skim milk at 25°C for 1 hr, and then treated with anti-WNT2, anti-CREB, anti-p-CREB, anti-BDNF, and antiβ-actin (Santa Cruz Biotechnology, US) at 4°C for overnight. Then, the membrane was treated with horseradish peroxidase-labeled secondary antibody (Invitrogen, US).
Chemiluminescence kit was used to visualize the signals (Biobyt, UK).

| Cell culture and transfections
C57BL/6J newborn (P0) mice were obtained from Animal Center of The second affiliated hospital of Baotou medical college.
Hippocampal neuron was taken from newborn mice and incubated in neurobasal medium with L-glutamine and B27 (Life Technologies).

| MTT assays
Hippocampal neuron was seeded in 96-well plates in medium with 20 μl 5 mg/ml MTT and maintained at 37°C with 5% CO 2 for 4 hr.
Next, the supernatant was removed and 150 μl DMSO was added.
ELISA was used to measure the signals at 490 nm.

| Flow cytometry
The hippocampal neuron was centrifuged at 600 rpm for 2 min.
The supernatant was removed and 10 μl Annexin V-FITC and 5 μl Propidium iodide were added for incubation at 4°C for 30 min in dark.
The apoptotic cells were detected using a flow cytometer (Coulter).

| Cell transfections
HEK293 cells were obtained from Gefan Biotechnology, China and maintained in DMEM with 10% FBS, 100 mg/L penicillin, and 100 mg/L streptomycin. Cells were cultured at 37°C with 5% CO 2 .
Luciferase activities were measured using Dual-luciferase reporter assay (Promega, US).

| Statistical analysis
SPSS 17.0 was used for data analysis. The data were expressed as means ± standard deviation (SD). For each analysis, the data sets were firstly analyzed for their normality (Shapiro-Wilk test) and equal variance (modified Levene test) assumptions prior to the use of parametric statistical methods. Then, differences between 2 groups were evaluated by Student's t test, and difference in multiple groups was evaluated by one-way analysis of variance (ANOVA) followed by post hoc analyses test if data conformed to normality and homogeneity of variance. p < .05 was considered statistically significant.

| The expression levels of miR-199a-5p were significantly elevated in the serum of CSF and MDD patient
The expression of miR-139-5p and miR-199a-5p were detected in CSF and serum of MDD patient. It showed that the expression levels of miR-139-5p (t test, t (4) = −8.015, p = .001) and miR-199a-5p (t test,

| WNT2 was a direct target of MIR-199a-5p
The possible binding between miR-199a-5p and WNT2 was pre-

| Silencing of WNT2 attenuated the effect of MIR-199a-5p-inhibitor on cell reproduction and apoptosis of hippocampal neuron
As shown in Figure 5a, the miR-199a-5p-inhibitor could enhance cell re-  reported . In this study, the expression of candidate miRNAs in CSF and serum in MDD patient were investigated. It was found that the expression levels of miR-139-5p and miR-199a-5p

| D ISCUSS I ON
were elevated in CSF and MDD serum, and the increase in the expression levels of miR-199a-5p were more obvious compared with the control patient. To the best of our knowledge, we are the first to discover that miR-199a-5p was up-regulated in depressive samples.
It was reported that the expression levels of miR-132 were significantly increased in patients with depression (Fang et al., 2018).
CUMS mice were shown to have increased depression-like behaviors and reduced hippocampal expression levels of miR-124 (Higuchi et al., 2016). In our study, the expression levels of miR-199a-5p in CUMS mice were elevated compared to that in the control mouse.
The expression levels of WNT2, p-CREB, and BDNF in CUMS mice were reduced. In addition, we also observed a considerable reduction of sucrose preference in CUMS. The concentrations of CORT in CUMS serum were enhanced. Similar to previous studies, we found that the expression levels of miR-199a-5p were substantially elevated in the hippocampus of CUMS mice.
MiRNAs were identified to involve in numeric biological functions, including developmental transitions (La Torre et al., 2013), neuronal patterning (Kosik, 2006), cell apoptosis (Cheng et al., 2005), and fat metabolism (P. Xu et al., 2003). miR-199a-5p was reported to be involved in the promotion of cell reproduction in autosomal dominant polycystic kidney diseases (L. Sun, Zhu, et al., 2015). However, in our study, we noticed that overexpression of miR-199a-5p suppressed cell reproduction and enhanced apoptosis of hippocampal neuron.
It was reported that miR-199a-5p could target the WNT2 signaling pathway and regulate cell reproduction of smooth muscle cells (Gheinani et al., 2015). Similarly, we also found possible binding between miR-199a-5p and WNT2. The expression levels of WNT2-WT were significantly reduced in cells with the overexpression of miR-199a-5p, while the activities of WNT2-WT were considerably elevated in cells transfected with miR-199a-5p-inhibitor. In addition, the expression of WNT2, p-CREB, and BDNF in hippocampal neuron transfected by miR-199a-5p mimics were suppressed. Knockdown of WNT2 attenuated the effects of miR-199a-5p-inhibitor on cell reproduction and apoptosis of hippocampal neuron. These data further confirmed that WNT2 was a direct target of miR-199a-5p.
MiR-124 was reported to be the most abundant in the brain, and its dysregulation has been related to neurodegeneration, neuroimmune disorder, and CNS stress . In this study, we found that miR-199a-5p could act as a novel marker for major depression-related brain neuronal regulations. Our data showed that F I G U R E 5 Silencing of WNT2 attenuated the effect of miR-199a-5p-inhibitor on cell reproduction and apoptosis of hippocampal neuron. Hippocampal neuron transfected with NC-inhibitor, miR-199a-5p-inhibitor, miR-199a-5p-inhibitor + si-NC, or miR-199a-5p-inhibitor + si- miR-199a-5p-inhibitor changed the effect of CUMS on SP, as well as the immobility duration in the forced swimming test and tail suspension test. Moreover, miR-199a-5p-inhibitor reversed the increasing of the expression levels of CORT and miR-199a-5p, and the decreasing of the expression levels of WNT2, p-CREB, and BDNF resulted from

CUMS.
Fluoxetine could increase the activity of the ERK-CREB signal system and alleviates the depressive-like behaviors in rats exposed to chronic forced swim stress (Qi et al., 2008). In this study, we also found that fluoxetine can elevate the SP of CUMS mice and decrease the immobility duration in the forced swimming test and tail suspension test. However, overexpression of miR-199a-5p relieved the effect.
In addition, the decreasing in the expression levels of CORT and miR-199a-5p, and the increasing in the expression levels of WNT2, p-CREB, and BDNF resulted from fluoxetine, were all reversed by a miR-199a-5p mimic. It indicated that miR-199a-5p had the opposite function of fluoxetine on CUMS mice.

| CON CLUS I ONS
MiR-199a-5p can target WNT2 to enhance the developments of depression by regulating the CREB/BDNF signaling.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/brb3.2107.

DATA AVA I L A B I L I T Y S TAT E M E N T
The analyzed data sets generated during the study are available from the corresponding author on reasonable request.