Patients and study approval
This study was approved by the ethics committee at Renmin Hospital of Wuhan University (approval ID: WDRY2021-K041) and all individuals provided written informed consent. MDD patients were recruited from the Department of Psychiatry at our hospital. All participants were graded on the Hamilton Depression Scale (HAMD; 24 items)
CUS exposure
Adult male ICR mice (6 weeks of age) were purchased from Beijing Vital River Laboratory Animal Technology (China) and housed at 23 ± 2℃ on a 12-h light/dark cycle with standard food and water ad libitum. After adaption to the environment for 1-week, chronic unpredictable stress (CUS) was performed to induce depressive-like behaviors. The stressors included the following: (1) a forced swim at 40℃ for 4 min; (2) a forced swim at 4℃ for 2 min; (3) the presence of moistened sawdust in the cage for 24 h; (4) tail-squeezing for 1 min; (5) crowding for 2 h; (6) food or water deprivation for 24 h. Further details are described in our previous report [13].
Lentivirus microinjection
A TUG1 short hairpin RNA (shRNA) lentivirus (1.5 mL, 109 viral genomes/mL; MineBio Life Sciences Ltd., Shanghai, China) was microinjected bilaterally into the hippocampus of each experimental mouse, as described previously [14].
Behavioral tests
(1) The sucrose preference test (SPT): Mice were habituated to drinking water and 1% sucrose solution for 3 days prior to testing. After one day of drinking only water, animals were then given access for 24 h to one bottle of water and one bottle of sucrose. The volumes of the two fluids were recorded at the beginning and ending of the tests, and the sucrose preference (%) was calculated as the ratio of sucrose intake to the total volume of fluid intake.
(2) The forced swim test (FST): Animals were placed in a large glass container (height: 30 cm; diameter: 18 cm) containing a water depth of 20 cm at 23–25℃. Immobility time was recorded for 4 min after habituating for 2 min. Immobility was defined as moving only to remain afloat.
(3) The tail suspension test (TST): Mice were suspended by the tail, and immobility time was recorded for 4 min after habituating for 2 min. Immobility time in the TST was defined as no movements other than by the whiskers or respiration.
High-throughput screening analysis
Eight blood samples were collected from four healthy individuals and four MDD patients. Differential genes were then identified by RNA-sequencing and high-throughput screening analysis (Baikede Biotechnology Co. Ltd., Wuhan, China).
Cell cultures
Primary mouse neurons were harvested from mice (P1 to P3 in age). The hippocampi were then removed and digested in 0.25% trypsin at 37℃ for 30 min. After centrifugation for 5 min at 1500 rpm and 4℃, the dissociated cells were resuspended in DMEM/F-12 (50:50) medium supplemented with N2 and 10% FBS. Next, cells were cultured in poly-lysine-coated culture flasks for 4 h. The supernatant was recovered and replaced by a fresh neural basal medium supplemented with B27; 10 µM of cytosine arabinoside was added into the medium to repress the growth of astrocytes.
Plasmids and transfection
TUG1, DUSP14, and GPX4 overexpression plasmids and the siRNA-TUG1 plasmid were provided by Baikede Biotechnology Co. Ltd (Wuhan, China). The oligomer for the TUG1 siRNA was as follows: 5'-GGGUUUUUUGUUUUGUUUUUU-3'. Neurons (1 × 105) were transfected with overexpression plasmids (1.0 µg) or siRNA plasmid (1.0 µM) and 1µL of lipofectamine 2000 (Invitrogen, USA) in 500 µL of medium for 24 h.
Cell viability assays
Neurons (1× 104 cells/well) were grown in 96-well plates for 24 h and transfected with the indicated plasmid for 24 h followed by the indicated drug treatment. The supernatant was removed from each well and replaced by 100 µL of fresh medium containing 10 µL of CCK-8 solution (Sangon Biotech, Shanghai, China); this was then incubated for 2 h. Next, cell absorbance was detected at 450 nm by a microplate reader (BIO-RAD, USA).
Measurement of ROS levels
ROS levels were measured with a ROS Assay Kit (Beyotime Biotechnology, Shanghai, China) in accordance with the manufacturer's directions. In brief, neurons were recovered and suspended in diluted DCFH-DA and then incubated at 37℃ for 20 min. Neurons were washed three times in a serum-free medium; then, fluorescence excitation/emission (488/525 nm) was determined by a microplate reader (BIO-RAD, USA).
Detection of MDA and GSH levels
The concentrations of MDA and GSH in hippocampal tissues and neurons were detected by a Lipid Peroxidation Assay Kit (Beyotime Biotechnology) and a GSH Assay Kit (Beyotime Biotechnology) in accordance with the manufacturer's directions.
Western Blotting
Western blotting was performed in accordance with our previous research [15]. In brief, proteins from hippocampal tissues and neurons were extracted by RIPA lysis buffer (Thermo Fisher Scientific, USA) supplemented with protease inhibitor. After centrifugation at 12000 rpm for 12 min at 4℃, the protein concentration in the supernatant was detected using the bicinchoninic acid assay (Beyotime Biotechnology). Samples of supernatant were fully mixed with loading buffer and boiled at 95℃ for 7 min. Then, 40 µg of protein from each sample was loaded on an SDS-PAGE gel and separated by electrophoresis; then, separated proteins were transferred onto a nitrocellulose membrane. Protein was blocked by 5% non-fat milk for 70 min. Membranes were then incubated overnight at 4℃ with primary antibodies against Acsl4 (Abcam, UK, #ab155282), Aifm2 (CUSABIO, China, #CSB-PA874794LA01HU), GPX4 (ab125066), DUSP14 (Abnova, China, #H00011266-PW1), and β-actin (ab5694). The following morning, the membranes were incubated with an appropriate HRP-labeled secondary antibody for 90 min at room temperature. Finally, the gray value of each separated protein was measured by a chemiluminescence detection system (Thermo Scientific, USA).
Real-time PCR
Total RNA from hippocampal tissues and neurons was extracted with a Trizol RNA extraction kit (Thermo Fisher Scientific, USA). Total RNA was extracted from serum samples with an extraction kit (Qiagen, Germany) in accordance with the manufacturer’s directions. 200 ng RNA was then used for reverse transcription. Then, cDNAs were used as templates for real-time PCR with HiScript III RT SuperMix (Vazyme Biotech, USA) and SYBR green master mix (Vazyme Biotech). β-actin was used as an internal control. The primers for the target genes are shown in Table 1.
Table 1
Primer sequences for target genes
Gene | Primer sequence |
TUG1 | Forward: 5′-CAAGAAACAGCAACACCAGAAG-3′ |
| Reverse: 5′-TAAGGTCCCCATTCAAGTCAGT-3′ |
Acsl4 | Forward: 5′-CCACACTTATGGCCGCTGTT-3′ |
| Reverse: 5′-GGGCGTCATAGCCTTTCTTG-3′ |
Aifm2 | Forward: 5′-CAAGATCAACAGCTCCGCCTACC-3′ |
| Reverse: 5′-CGTCGGCACAGTCACCAATGG-3′ |
GPX4 | Forward: 5′-CCGCCTTTGCCGCCTAC-3′ |
| Reverse: 5′-TTTACTTCGGTCTTGCCTCACT-3′ |
DUSP14 | Forward: 5′-GGACTCTTGAGGAAGAAGGAGAC-3′ |
| Reverse: 5′-TCAACGTCACACTTCATGATGGA-3′ |
β-actin | Forward: 5′-ACTGCCGCATCCTCTTCCT-3′ |
| Reverse: 5′-TCAACGTCACACTTCATGATGGA-3′ |
RNA immunoprecipitation (RIP)
Fresh hippocampo were rinsed three times and lysed in a glass homogenizer with ice-cold PBS. Following centrifugation for 5 min at 1500 rpm and 4℃, the cell pellets were recovered and resuspended in an equal volume of RIP lysis buffer (Thermo Fisher Scientific). RIP experiments were performed with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) in accordance with the manufacturer's instructions. In brief, lysates were incubated with DUSP14 antibody (#H00011266-PW1) or IgG antibody (Abcam, ab172730) overnight at 4℃. The beads were then added and incubated for 6 h at 4 ℃. Then, we washed and resuspended the beads in TRIzol. Finally, RNA levels were measured by real-time PCR.
Co-immunoprecipitation
The procedures for this experiment were performed in accordance with our previous report [15]. In brief, cell lysates were centrifuged and the supernatants were collected and incubated overnight at 4℃ with 1 µg of anti-Ub antibody (Abcam, ab209263). Then, the complex was precipitated by Protein A agarose beads (Roche, Mannheim, Germany). After washing the beads, the conjugated protein was recovered by Laemmli sample buffer and boiled at 95℃ for 7 min. Western blotting was then used to detect the expression levels of DUSP14 protein.
Statistical analyses
Statistical analyses were performed with SPSS version 20.0 (IBM, Chicago, IL, USA). Data are shown as mean ± SD. Receiver operating characteristic (AUC) curve analysis was performed to analyze the diagnosis value of TUG1 for MDD. Spearman’s correlation was performed to analyze the association between TUG1 levels and HAMD-24 scores in MDD patients. Comparisons between the two groups were performed with a t-test. P < 0.05 was regarded as statistically significant.