Mrna and Mirna Profiles in the Nucleus Accumbens Are Associated with Psychological Stress-Induced Susceptible and Resilient Mice

Pharmacology, Biochemistry and Behavior 199 (2020) 173062

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Pharmacology, Biochemistry and Behavior

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mRNA and miRNA profiles in the nucleus accumbens are associated with psychological stress-induced susceptible and resilient mice

Yanjun Lu a, Jiuyong Yang a, Jinyan Sun a, Wei Lu a,*, Jin-Hui Wang a,b,c,** a Qingdao University, School of Pharmacy, Qingdao, Shandong 266021, China b University of Chinese Academy of Sciences, Beijing 100049, China c Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

ARTICLE INFO ABSTRACT

Keywords: Background: Stress may be one of the main causes of fear and anxiety. Previous studies have shown that the Psychological stress nucleus accumbens is involved in emotional responses. However, in the nucleus accumbens, the mRNA and Susceptibility miRNA profiles of stress susceptibility and resilience of psychological stress still need to be studied. Resilience Materials and methods: In this study, by observing the conspecific being attacked, the witness group experienced Anxiety psychological stress. After five days of psychological stress, the fear memory of mice was measured by social Nucleus accumbens interaction test, and the degree of anxiety was measured by elevated plus maze. mRNA and miRNA profilesin the nucleus accumbens tissue of control, susceptible and resilient mice were established by high-throughput sequencing. Results: In susceptible mice versus resilient mice, the Differentially expressed genes (DEGs) may be related to psychological stress-induced susceptibility. DEGs enriched in Cell adhesion molecules, Neuroactive ligand- receptor interaction, Gap junction, PI3K-Akt, VEGF, Jak-STAT, Ras, and Chemokine pathways were up- regulated. DEGs enriched in cGMP-PKG, B cell receptor, and NOD-like receptor pathways were down- regu lated. The sequencing results of mRNAs and miRNAs were verified by qRT-PCR and dual luciferase reporter assay. Conclusion: The imbalance of different synapses and pathways in the nucleus accumbens may be related to susceptibility and resilience caused by psychological stress.

1. Introduction nucleus accumbens also mediates a variety of stress responses by interacting with the limbic, cognitive, and motor circuits (Azogu and Stress is essential in the occurrence and expression of many mental Plamondon, 2017; Floresco, 2015). The changes of molecules in the illnesses. Stressful events in life are often associated with fear (Izquierdo nucleus accumbens may regulate various stress responses by changing et al., 2016; Makkar et al., 2010; Maren and Holmes, 2016). Excessive the function of neurons and then affecting the circuit. Structural ele stress is the cause of many mental illnesses, such as phobia, depression, ments and functions in neuronal circuits, including the nucleus accum generalized anxiety and PTSD (Coutellier and Usdin, 2011; Desmedt bens, are related to reward and fear memory. Cellular processes and et al., 2015; Dunsmoor and Paz, 2015; Orsini and Maren, 2012). Due to signaling molecules in the nucleus accumbens may be associated with their genetic within-strain variability, individuals respond differently to anxiety(Bosch-Bouju et al., 2016). In recent years, miRNAs have been stress exposure; some are susceptible to stress exposure, while some are used in the research of mental disorders such as anxiety and major capable of actively coping with stressors, being stress resilient (Khan depression. Some miRNAs play a crucial role in the stress response of et al., 2020). However, the molecular mechanism that mediates these animals (Haramati et al., 2011; Issler et al., 2014). Therefore, the stress resistance is still unclear. comprehensive study on mRNA and miRNA in nucleus accumbens is The nucleus accumbens is the linchpin structure of the reward cir more helpful to solve the comprehensive molecular problems, which is cuit, which is involved in the regulation of emotional response, and the related to fear memory and anxiety caused by stress.

* Correspondence to: W. Lu, Qingdao University, School of Pharmacy, 38 Dengzhou road, Qingdao, Shandong 266021, China. ** Correspondence to: J.-H. Wang, University of Chinese Academy of Sciences, Institute of Biophysics, 15 Datun Road, Chaoyang District, Beijing 100101, China. E-mail addresses: [email protected] (W. Lu), [email protected] (J.-H. Wang). https://doi.org/10.1016/j.pbb.2020.173062 Received 25 September 2020; Received in revised form 15 October 2020; Accepted 18 October 2020 Available online 22 October 2020 0091-3057/© 2020 Elsevier Inc. All rights reserved. Y. Lu et al. Pharmacology, Biochemistry and Behavior 199 (2020) 173062

Fig. 1. Experimental process of psychological stress. (a) After seven days of acclimatization, the C57 mice were screened by EPM test and social interaction test before psychological stress. The witness mice were subjected to psychological stress for five days. EPM test and social interaction test were performed again. The nucleus accumbens were removed for transcriptome sequencing. (b) C57 mouse was placed in the side with CD1, and the mice in the witness group were placed on the other side of the cage. The witness group watched the same species under attack, and thus experienced psychological stress. (c) Social avoidance detection is performed in an interactive social area (50*50 cm), with 10 * 10 cm transparent and perforated small cages on the edges. The area around the small cage is definedas the communication area.

The psychological and pathological effects of stress on specific in dividuals are significantlydifferent (Krishnan, 2014; Russo et al., 2012). After psychological stress, mice can be divided into susceptible and resilient based on their different effects on physical and mental states. Stress can induce a range of physiological responses (including nervous, endocrine and immune systems) and may be harmful in some cases. Susceptible mice showed fear, anxiety and other symptoms, while resilient mice did not show these symptoms(Berton et al., 2006; Krishnan et al., 2007). Therefore, it is necessary to study the mRNA and miRNA profiles related to susceptibility and resilience induced by psy chological stress, as it remains to be systemically studied. To study the relationship between the molecules of the nucleus accumbens and psychological stress response, our strategy is shown below. These mice were divided into control and witness groups. The control group were raised normally without any treatment. And the witness group experienced psychological stress by observing that conspecific mice being attacked. After psychological stress, the mice were tested for fear memory and anxiety-like behavior. According to the test results, the witness group were further divided into susceptible and resilient groups. After the behavior test, the nucleus accumbens tissues of the mice were taken out and their mRNA and miRNA profiles were sequenced. Through the combined comparison and analysis of mRNA and miRNA, we hoped to find molecular and pathways of susceptility and resilience caused by psychological stress in the nucleus accumbens, and then develop new treatment methods and strategies.

2. Materials and methods

2.1. Mice Fig. 2. Social avoidance test of mice after psychological stress treatment. Control n = 12, resilient n = 16, susceptible n = 12. (a) shows the social C57BL/6J mice and CD-1 mice were purchased from Beijing life behavior tracks of control, susceptible and resilient mice on day6 with and without CD1. (b) shows the ratio (%) of the stay time of the three groups of River Experimental Animal Technology Co., Ltd. and Qingdao Daren mice in the communication area on day 0 and day6 when there was a CD1 fortune Animal Technology Co., Ltd. The experimental protocols were attacker in the small container and no CD1 attacker. Two-way ANOVA was used approved by the Animal Use and Care Committee of Qingdao University. for the comparisons among control, resilient and susceptible mice, while four The mice were fed with free access to water and food. The ambient ◦ asterisks show p < 0.0001, two asterisks show p < 0.01, one asterisk show p temperature was 22 ± 2 C. Relative humidity was 55 ± 5%. The < 0.05.

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Fig. 3. Test of EPM in mice after psychological stress treatment. Control n = 12, resilient n = 16, susceptible n = 12. (a) shows the susceptible, resilient, and control mice tracks on the EPM before (day 0) and after (day 6) psychological stress. (b) shows the time (%) of the control, resilient and susceptible mice in the open zone on day 0 and day 6. Two-way ANOVA was used for the comparisons among control, resilient and susceptible mice, while one asterisk show p < 0.05.

circadian rhythm was 12 h in the dark (07:00–19:00), 12 h in the light 2.3. Psychological stress (19:00–07:00). The resident-intruder paradigm was used as the social pressure for 2.2. Timeline of the experimental process this experiment(Golden et al., 2011; Montagud-Romero et al., 2018), psychological stress in social interactions may be more close to real life Concerning Golden ‘s protocol, 15-week-old male CD1 mice were than stress caused by electrical stimulations. During the adaptation used as attackers(Golden et al., 2011). Based on previous research, male period, male CD1 mice were placed in a room that took up half of a 5-week-old C57BL/6 J mice were used as subjects. When male C57 mice normal cage (29 × 17.5 × 12.5 cm). A neighboring room (29 × 17.5 × enter the residence of CD1 mice, CD1 mice are more aggressive(Ham 12.5 cm) was separated by a transparent partition. After adaptation mels et al., 2015). Mice were allowed to have a seven-day adaptation period, the attacked C57 mice were placed in the same cage as the CD1 period before the screening (Fig. 1a). At the end of the acclimation mice, and the mice in the witness group were placed on the other side of period, EPM (see below) and social interaction test (see below) were the cage. The witness group experienced psychological stress by used to detect anxiety and fear in mice. Mice with a ratio higher than 0.5 observing that conspecific mice being attacked and bitten by CD1 in the social interaction test and an open arm time ratio higher than (Fig. 1b). Psychological stress was performed twice a day for five days. +2% in the EPM test were selected (Du et al., 2019), these mice were The duration of psychological stress is the time that it takes for CD1 mice considered to have no innate social avoidance and anxiety-like behavior. to attack the beaten C57 mice 5 times, and the time was about 5–10 min These mice were randomly divided into control and witness groups. (Sun et al., 2019). Before psychological stress, CD1’s aggressive After the adaptation period, mice in the witness group underwent psy behavior was tested. chological stress experiments for 5 days (see below). Then the anxiety state and social avoidance of the control and witness groups were tested 2.4. Elevated plus maze (EPM) detection again by EPM and social interaction test. After the behavioral test, ac cording to the behavioral results of the mice, the mice in the witness EPM tests were used to evaluating anxiety-like behavior in mice. group were divided into susceptible group and resilient group. After 24 h EPM is composed of two closed arms (30 × 5 × 15.25 cm), two opposite of behavioral test, the nucleus accumbens tissues on both sides of the × × ◦ open arms (30 5 cm), and a central area (5 5 cm), 40 cm above the mice were collected and stored at 80 C. Control mice were treated in ground. Record the tracks and time of the mice entering each arm within the same way as witness mice, the genes that may be changed by 5 min. The standard for detecting anxiety is the percentage of time that behavioral tests were excluded. the mice stayed in the open arms to the total time. The shorter the time stayed in the open arm, the more anxious the mice were. Prior to psy chological stress, mice with a residence time ratio of less than 2% in the

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Table 1 Table 1 (continued ) Signaling pathways identified by KEGG based on DEGs data of NAc in suscep KEGG Term Count Genes tible versus control. Entry

KEGG Term Count Genes rectifying channel, subfamily J, Entry member 6) ↑, Mas1 (MAS1 oncogene) ↑, Ptgs2 (prostaglandin- ↑ Adra1d (adrenergic receptor, endoperoxide synthase 2) , alpha 1d) ↑, Cckbr Kcnj3 (potassium inwardly- (cholecystokinin B receptor) ↑, rectifying channel, subfamily J, ↑ Chrna10 (cholinergic receptor, member3) , ↓ Kcnj6 (potassium inwardly- nicotinic, alpha polypeptide 10) , mmu04728 Dopaminergic synapse 3 Chrna5 (cholinergic receptor, rectifying channel, subfamily J, ↑ nicotinic, alpha polypeptide 5) ↑, member 6) , Chrnb3 (cholinergic receptor, Mapk11 (mitogen-activated ↑ nicotinic, beta polypeptide 3) ↑, protein kinase 11) , Grm2(glutamate receptor, Grm2 (glutamate receptor, Neuroactive ligand- ↑ mmu04080 15 metabotropic 2) ↑, Grm8 metabotropic 2) , receptor interaction mmu05030 Cocaine addiction 2 (glutamate receptor, metabotropic Bdnf (brain derived neurotrophic ↑ 8) ↑, Hcrtr1(hypocretin (orexin) factor) , receptor 1) ↑, Nmbr(neuromedin B Kcnj3 (potassium inwardly- receptor) ↑, Prlhr (prolactin rectifying channel, subfamily J, ↑ releasing hormone receptor) ↓, member 3) , Oxytocin signaling Kcnj6 (potassium inwardly- Rxfp1 (relaxin/insulin-like family mmu04921 3 peptide receptor 1) ↑, Sstr2 pathway rectifying channel, subfamily J, ↑ (somatostatin receptor 2) ↑, Sstr3 member 6) , (somatostatin receptor 3) ↑, Trhr Ptgs2 (prostaglandin- ↑ (thyrotropin releasing hormone endoperoxide synthase 2) , ↓ receptor) ↓, Fzd5 (rizzled class receptor 5) , Fzd5 (frizzled class receptor 5) ↓, Wnt10a (wingless-type MMTV Wnt10a (wingless-type MMTV Hippo signaling integration site family, member mmu04390 3 ↑ integration site family, member pathway 10A) , Signaling pathways 10A) ↑, Trp73 (transformation related ↓ regulating Otx1 (orthodenticle homeobox 1) protein 73) , mmu04550 5 pluripotency of stem ↑, Mapk11 (mitogen-activated VEGF signaling protein kinase 11) ↑, cells Esrrb (estrogen related receptor, mmu04370 2 beta) ↑, pathway Ptgs2 (prostaglandin- ↑ Mapk11 (mitogen-activated endoperoxide synthase 2) , ↓ protein kinase 11) ↑, Cdh15 (cadherin 15) , Lbp (lipopolysaccharide binding Cell adhesion H2-DMb1 (histocompatibility 2, mmu04514 3 ↓ protein) ↓, molecules (CAMs) class II, locus Mb1) , NF-kappa B signaling ↑ mmu04064 3 Blnk (B cell linker) ↑, Ntng2 (netrin G2) , pathway Ptgs2 (prostaglandin- Fxyd2 (FXYD domain-containing ↓ endoperoxide synthase 2) ↑, ion transport regulator 2) , Adra1d (adrenergic receptor, Adra1d (adrenergic receptor, ↑ cGMP-PKG signaling alpha 1d) ↑, alpha 1d) , mmu04022 3 Cckbr (cholecystokinin B receptor) pathway Kcnmb1 (potassium large Calcium signaling ↑, conductance calcium-activated mmu04020 4 pathway Trhr (thyrotropin releasing channel, subfamily M, beta ↓ hormone receptor) ↓, member 1) , Il12a (interleukin 12a) ↑, Tnnc1 (troponin C, cardiac/slow RIG-I-like receptor skeletal) ↑, mmu04622 2 Mapk11 (mitogen-activated signaling pathway ↑ Lbp (lipopolysaccharide binding protein kinase 11) , protein) ↓, Fxyd2 (FXYD domain-containing Toll-like receptor ↑ ion transport regulator 2) ↓, mmu04620 3 Il12a (interleukin 12a) , cAMP signaling signaling pathway mmu04024 3 Sstr2 (somatostatin receptor 2) ↑, Mapk11 (mitogen-activated pathway protein kinase 11) ↑, Bdnf (brain derived neurotrophic ↑ Grm2 (glutamate receptor, factor) , metabotropic 2) ↑, Kcnj3 (potassium inwardly- Grm8 (glutamate receptor, rectifying channel, subfamily J, ↑ member 3) ↑, mmu04724 Glutamatergic synapse 3 metabotropic 8) , mmu05032 Morphine addiction 2 Kcnj3 (potassium inwardly- Kcnj6 (potassium inwardly- rectifying channel, subfamily J, rectifying channel, subfamily J, ↑ member 3) ↑, member 6) , ↑ Bdnf (brain derived neurotrophic Fgf5 (fibroblastgrowth factor 5) , ↑ Rap1 signaling Met (met proto-oncogene) ↑, factor) , mmu04015 3 Neurotrophin Mapk11 (mitogen-activated pathway Mapk11 (mitogen-activated mmu04722 3 ↑ signaling pathway protein kinase 11) ↑, protein kinase 11) , Trp73 (transformation related Kcnj3 (potassium inwardly- protein 73) ↓, rectifying channel, subfamily J, ↑ Estrogen signaling member 3) ↑, Met (met proto-oncogene) , mmu04915 2 Ntng2 (netrin G2) ↑, pathway Kcnj6 (potassium inwardly- mmu04360 Axon guidance 3 Robo3 (roundabout guidance rectifying channel, subfamily J, ↑ receptor 3) ↑, member 6) , Kcnj3 (potassium inwardly- Mapk11 (mitogen-activated ↑ rectifying channel, subfamily J, protein kinase 11) , mmu04726 Serotonergic synapse 3 mmu04668 TNF signaling pathway 2 member 3) ↑, Ptgs2 (prostaglandin- ↑ Kcnj6 (potassium inwardly- endoperoxide synthase 2) , mmu04010 3 Fgf5 (fibroblastgrowth factor 5) ↑, (continued on next page)

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Table 1 (continued ) Table 2 Signaling pathways identified by KEGG based on DEGs data of NAc in suscep KEGG Term Count Genes Entry tible versus resilient.

Bdnf (brain derived neurotrophic KEGG Term count Genes MAPK signaling factor) ↑, Entry pathway Mapk11 (mitogen-activated Col5a2 (collagen, type V, alpha 2) ↑ protein kinase 11) , ↑, Hkdc1 (hexokinase domain PI3K-Akt signaling Kdr (kinase insert domain protein Insulin signaling containing 1) ↑, mmu04151 4 ↑ mmu04910 2 pathway receptor) , pathway Pygl (liver glycogen Tnxb (tenascin XB) ↑, ↓ phosphorylase) , Vwf (Von Willebrand factor) ↑, ↓ Fzd5 (frizzled class receptor 5) , Kdr (kinase insert domain protein Wnt10a (wingless-type MMTV VEGF signaling receptor) ↑, mmu04310 Wnt signaling pathway 2 mmu04370 2 integration site family, member pathway Pla2g4e (phospholipase A2, group ↑ 10A) , IVE) ↑, Col6a5 (collagen, type VI, alpha Crh (corticotropin releasing ↓ PI3K-Akt signaling 5) , hormone) ↓, mmu04151 3 ↑ mmu04730 Long-term depression 2 pathway Fgf5 (fibroblastgrowth factor 5) , Pla2g4e (phospholipase A2, group ↑ Met (met proto-oncogene) , IVE) ↑, ↑ Fgf5 (fibroblastgrowth factor 5) , Il11ra2 (interleukin 11 receptor, mmu04014 Ras signaling pathway 2 ↑ Jak-STAT signaling Met (met proto-oncogene) , mmu04630 2 alpha chain 2) ↑, pathway FoxO signaling Mapk11 (mitogen-activated Gm2002 (predicted gene 2002) ↑, mmu04068 1 ↑ pathway protein kinase 11) , H2-Q8 (histocompatibility 2, Q B cell receptor ↑ Cell adhesion region locus 8) ↑, mmu04662 1 Blnk (B cell linker) , mmu04514 2 signaling pathway molecules (CAMs) H2-T10 (histocompatibility 2, T Glucagon signaling Pygl (liver glycogen region locus 10) ↑ mmu04922 1 ↓ pathway phosphorylase) , Fxyd2 (FXYD domain-containing cAMP signaling GnRH signaling Mapk11 (mitogen-activated mmu04024 2 ion transport regulator 2) ↓, mmu04912 1 ↑ pathway pathway protein kinase 11) , Sstr5 (somatostatin receptor 5) ↑, Trp73 (transformation related Kdr (kinase insert domain protein mmu04115 p53 signaling pathway 1 ↓ protein 73) , receptor) ↑, Jak-STAT signaling mmu04014 Ras signaling pathway 2 mmu04630 1 Il12a (interleukin 12a) ↑, Pla2g4e (phospholipase A2, group pathway IVE) ↑, HIF-1 signaling Hkdc1 (hexokinase domain Chrnb3 (cholinergic receptor, mmu04066 1 ↑ Neuroactive ligand- pathway containing 1) , mmu04080 2 nicotinic, beta polypeptide 3) ↑, receptor interaction T cell receptor Mapk11 (mitogen-activated Sstr5 (somatostatin receptor 5) ↑, mmu04660 1 ↑ signaling pathway protein kinase 11) , cGMP-PKG signaling Fxyd2 (FXYD domain-containing mmu04022 1 Fc epsilon RI signaling Mapk11 (mitogen-activated pathway ion transport regulator 2) ↓, mmu04664 1 ↑ pathway protein kinase 11) , Rap1 signaling Kdr (kinase insert domain protein mmu04015 1 Thyroid hormone Fxyd2 (FXYD domain-containing pathway receptor) ↑, mmu04919 1 ↓ signaling pathway ion transport regulator 2) , B cell receptor mmu04662 1 Cd72 (CD72 antigen) ↓, Prolactin signaling Mapk11 (mitogen-activated signaling pathway mmu04917 1 ↑ pathway protein kinase 11) , NOD-like receptor Trip6 (thyroid hormone receptor mmu04621 1 NOD-like receptor Mapk11 (mitogen-activated signaling pathway interactor 6) ↓, mmu04621 1 ↑ signaling pathway protein kinase 11) , Pla2g4e (phospholipase A2, group mmu04724 Glutamatergic synapse 1 Kcnj6 (potassium inwardly- IVE) ↑, mmu04727 GABAergic synapse 1 rectifying channel, subfamily J, nsulin signaling Fxyd2 (FXYD domain-containing ↑ mmu04910 1 member 6) , pathway ion transport regulator 2) ↓, Fc epsilon RI signaling Pla2g4e (phospholipase A2, group Note: ↑ indicates up-regulation in the tissue of Nac from susceptible versus mmu04664 1 pathway IVE) ↑, control mice, whereas ↓ represents down-regulation. mmu04540 Gap junction 1 Tuba1c (tubulin, alpha 1C) ↑, Pla2g4e (phospholipase A2, group mmu04726 Serotonergic synapse 1 IVE) ↑, Thyroid hormone Fxyd2 (FXYD domain-containing mmu04919 1 signaling pathway ion transport regulator 2) ↓, Oxytocin signaling Pla2g4e (phospholipase A2, group mmu04921 1 open arm were deleted, these mice were considered to have natural pathway IVE) ↑, anxiety-like behavior. GnRH signaling Pla2g4e (phospholipase A2, group mmu04912 1 pathway IVE) ↑, Thyroid hormone Fxyd2 (FXYD domain-containing 2.5. Social interaction test mmu04918 1 synthesis ion transport regulator 2) ↓, Chemokine signaling Ccl9 (chemokine (C–C motif) mmu04062 1 To test whether C57 mice are afraid of CD1 mice, we used the social pathway ligand 9) ↑, interaction test. The mice were tested in social interaction cages. In a 50 MAPK signaling Pla2g4e (phospholipase A2, group mmu04010 1 × 50 cm open field,placed a 10 × 10 cm square transparent small hole pathway IVE) ↑, box (for CD1 mice) so that C57 mice could observe CD1 mice (Fig. 1c). Note: ↑ indicates up-regulation in the tissue of Nac from susceptible versus The 5 cm area around the small cage was definedas the communication resilient mice, whereas ↓ represents down-regulation. area. The ratios of the time C57 stayed in the communication area with a CD1 mice to the time C57 stayed in the communication area without a CD1 mice were recorded, the ratio reflectswhether C57 mice have social avoidance behaviors on CD1 mice. The avoidance behavior of C57 on 2.6. Susceptible and resilient categories detection CD1 mice is manifested by avoiding activities near the container with CD1 and staying in the communication area for less time. Prior to psy After these behavior tests, the mice in the witness group were divided chological stress, mice with a social interaction test ratio below 0.5 were into susceptible and resilient. Compared with the initial screening re deleted, suggesting that they had an innate fear of CD1 mice. sults, the mice whose rate of stay in the communication area (in the social interaction test) decreased by more than 20% and less than 1 were

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Table 3 Table 3 (continued ) Signaling pathways identified by KEGG based on DEGs data of NAc in resilient KEGG Term Count Genes versus control. Entry

KEGG Term Count Genes 1S subunit) ↑, Entry Ptger2 (prostaglandin E receptor 2 ↑ LOC547349 (MHC class I family (subtype EP2)) , ↓ member) ↓, Cdh15(cadherin 15) Sstr5 (somatostatin receptor 5) , ↓, Cldn23 (claudin 23) ↓, H2-Q1 Bdnf (brain derived neurotrophic ↑ (histocompatibility 2, Q region factor) , locus 1) ↓, H2-Q7 Bdnf (brain derived neurotrophic ↑ (histocompatibility 2, Q region factor) , Cell adhesion locus 7) ↓, H2-Q8 Neurotrophin Trp73 (transformation related mmu04514 9 mmu04722 3 ↓ molecules (CAMs) (histocompatibility 2, Q region signaling pathway protein 73) , locus 8) ↓, H2-T10 Mapk11 (mitogen-activated ↑ (histocompatibility 2, T region protein kinase 11) , locus 10) ↓, H2-Bl Ptger2 (prostaglandin E receptor 2 ↓ (subtype EP2)) ↑, (histocompatibility 2,blastocyst) , Inflammatory Mapk11 (mitogen-activated H2-DMb1(histocompatibility 2, mmu04750 mediator regulation of 3 ↓ protein kinase 11) ↑, class II, locus Mb1) , TRP channels Mas1(MAS1 oncogene) ↑, Adra1b Pla2g4e (phospholipase A2, group ↓ (adrenergic receptor, alpha 1b) ↑, IVE) , Adra1d (adrenergic receptor, Cacna1s (calcium channel, alpha 1d) ↑, Grm8 (glutamate voltage-dependent, L type, alpha ↑ receptor, metabotropic 8) ↑, Nmbr 1S subunit) , (neuromedin B receptor) ↑, Ptger2 mmu04726 Serotonergic synapse 3 Pla2g4e (phospholipase A2, group ↑ Neuroactive ligand- (prostaglandin E receptor 2 IVE) , mmu04080 10 receptor interaction (subtype EP2)) ↑, P2rx3 Ptgs2 (prostaglandin- ↑ (purinergic receptor P2X, ligand- endoperoxide synthase 2) , gated ion channel,3) ↑, Sstr3 Cacna1s (calcium channel, (somatostatin receptor 3) ↑, Sstr5 voltage-dependent, L type, alpha ↓ 1S subunit) ↑, (somatostatin receptor 5) , Trhr Oxytocin signaling (thyrotropin releasing hormone mmu04921 3 Pla2g4e (phospholipase A2, group pathway ↓ receptor) ↓, IVE) , Adra1b (adrenergic receptor, Ptgs2 (prostaglandin- ↑ alpha 1b) ↑, Adra1d (adrenergic endoperoxide synthase 2) , receptor, alpha 1d) ↑, Cacna1s Wnt10a (wingless-type MMTV (calcium channel, voltage- integration site family, member Calcium signaling ↑ mmu04020 5 dependent, L type, alpha 1S 10A) , pathway ↑ Hippo signaling Wnt6 (wingless-type MMTV subunit) , P2rx3(purinergic mmu04390 3 receptor P2X, ligand-gated ion pathway integration site family, member 6) ↑ channel, 3) ↑, Trhr (thyrotropin , releasing hormone receptor) ↓， Trp73 (transformation related ↓ Cacna1s (calcium channel, protein 73) , voltage-dependent, L type, alpha Adra1b (adrenergic receptor, ↑ 1S subunit) ↑, alpha 1b) , ↑ Adra1d (adrenergic receptor, Fgf3 (fibroblastgrowth factor 3) , cGMP-PKG signaling mmu04022 3 alpha1d) ↑, Bdnf (brain derived neurotrophic pathway MAPK signaling factor) ↑, Cacna1s (calcium channel, mmu04010 6 pathway Dusp4 (dual specificity voltage-dependent, L type, alpha ↑ phosphatase 4) ↑, 1S subunit) , Pla2g4e (phospholipase A2, group Col4a3 (collagen, type IV, alpha ↓ IVE) ↑, 3) , Col5a2 (collagen, type V, ↓ Mapk11 (mitogen-activated PI3K-Akt signaling alpha 2) , Col6a5 (collagen, type mmu04151 5 ↓ protein kinase 11) ↑, pathway VI, alpha 5) , Fgf3 (fibroblast ↑ Wnt10a (wingless-type MMTV growth factor 3) , Lamb3 ↓ integration site family, member (laminin, beta 3) , ↓ 10A) ↑, Tuba1c (tubulin, alpha 1C) , mmu04540 Gap junction 2 ↑ Signaling pathways Wnt6 (wingless-type MMTV Tuba8 (tubulin, alpha 8) , regulating integration site family, member 6) Lbp (lipopolysaccharide binding mmu04550 4 ↑ NF-kappa B signaling protein) ↓, pluripotency of stem , mmu04064 2 cells Otx1 (orthodenticle homeobox 1) pathway Ptgs2 (prostaglandin- ↑ ↑, endoperoxide synthase 2) , Mapk11 (mitogen-activated Lbp (lipopolysaccharide binding ↑ Toll-like receptor protein) ↓, protein kinase 11) , mmu04620 2 Cacna1s (calcium channel, signaling pathway Mapk11(mitogen-activated ↑ voltage-dependent, L type, alpha protein kinase 11) , 1S subunit) ↑, Grm8 (glutamate receptor, GnRH signaling ↑ mmu04912 3 Mapk11 (mitogen-activated metabotropic 8) , pathway mmu04724 Glutamatergic synapse 2 protein kinase 11) ↑, Pla2g4e (phospholipase A2, group ↓ Pla2g4e (phospholipase A2, group IVE) , IVE) ↑, Wnt10a (wingless-type MMTV Cd247 (CD247 antigen) ↓, integration site family, member ↑ T cell receptor Itk (IL2 inducible T cell kinase) ↓, 10A) , mmu04660 3 mmu04310 Wnt signaling pathway 2 signaling pathway Mapk11 (mitogen-activated Wnt6 (wingless-type MMTV protein kinase 11) ↑, integration site family, member 6) ↑ cAMP signaling Cacna1s (calcium channel, , mmu04024 4 Rap1 signaling pathway voltage-dependent, L type, alpha mmu04015 2 Fgf3 (fibroblastgrowth factor 3) ↑, pathway (continued on next page)

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Table 3 (continued ) resilient mice were used for the following research. KEGG Term Count Genes Entry 2.7. RNA purification from the nucleus accumbens tissue Mapk11 (mitogen-activated protein kinase 11) ↑, After the behavioral tests were completed, mice were anesthetized ↑ ◦ Fgf3 (fibroblastgrowth factor 3) , by using Isoflurane,then perfused with 4 C saline and decapitated, the mmu04014 Ras signaling pathway 2 Pla2g4e (phospholipase A2, group IVE) ↓, nucleus accumbens tissues were separated on ice-cold glass slide. The Cacna1s (calcium channel, total RNAs were obtained by TRIzol reagent (Life Technologies, Carls mmu05010 Alzheimer’s disease 1 voltage-dependent, L type, alpha bad, CA, USA) as previously described (Du et al., 2019). RNA samples 1S subunit) ↑, were sent to Beijing Genomics Institute (BGI) for high-throughput Cacna1s (calcium channel, sequencing. mmu04727 GABAergic synapse 1 voltage-dependent, L type, alpha 1S subunit) ↑, NOD-like receptor Mapk11 (mitogen-activated mmu04621 1 2.8. Bioinformatics analysis for mRNA and miRNA signaling pathway protein kinase 11) ↑, Mapk11 (mitogen-activated mmu04728 Dopaminergic synapse 1 protein kinase 11) ↑, The analysis followed previous research reports(Shen et al., 2019). Cacna1s (calcium channel, Fragments per kilobase per million (FPKM) and Differentially expressed mmu04725 Cholinergic synapse 1 voltage-dependent, L type, alpha genes (DEGs) between the two groups were screened out via the DESeq2 1S subunit) ↑, package. Genes in low expression levels (FPKM<0.5) were removed. The B cell receptor mmu04662 1 Cd72 (CD72 antigen) ↑, signaling pathway threshold to identify DEGs was that the fold change was more than 2 times (|log2(fold)| > 1) and the P-value was less than 0.05. Canonical Note: ↑ indicates up-regulation in the tissue of Nac from resilient versus control pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) mice, whereas ↓ represents down-regulation. database were used in the enrichment analyses of DEGs. To obtain miRNA count, high-quality clean reads ranging in 18–25 nt = definedas susceptible (n 12). The witness mice whose ratio decreased were matched to the known miRNA precursor of corresponding species by below 20% or increased and the ratio above 1 were defined as in miRBase. The expression of miRNAs that meet the following criteria is = ’ resilient (n 16) (Golden et al., 2011). Those that didn t meet the above considered to be recognized miRNAs: During the matching process, = two criteria were definedas atypical mice (n 3). Only susceptible and miRNAs can be consistent with miRNA precursor in miRBase without

Fig. 4. The validation of differentially expressed mRNAs in the nucleus accumbens from control (n = 3) versus susceptible (n = 3) mice. Four asterisks show p < 0.0001, three asterisks show p < 0.001, two asterisks show p < 0.01, one asterisk show p < 0.05, in which two-sample t-test was used for the comparisons between control mice versus susceptible mice.

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Fig. 5. The validation of differentially expressed mRNAs in the nucleus accumbens from resilient (n = 3) versus susceptible (n = 3) mice. Four asterisks show p < 0.0001, three asterisks show p < 0.001, two asterisks show p < 0.01, one asterisk show p < 0.05, in which two-sample t-test was used for the comparisons between control mice versus resilient mice.

mismatched markers, and at least 16 nt of mature miRNA markers in SYBR qPCR Master Mix (Vazyme, Q711-02, Nanjing, China). Table S1 miRBase overlap allowable offsets can then be counted. For unannotated lists the primers used by qRT-PCR to verify mRNA. The relative remaining reads, the Miredp package was used to predict new miRNAs expression levels of mRNAs were normalized to a reference gene, with potential stem-loop structure(Friedlander et al., 2012). To correct GAPDH. The relative expression levels of miRNAs were normalized to biased results for low expression, miRNAs with reading counts less than U6 small nucleolar RNA. 5 were removed in differential expression analysis. DESeq2 package was used to compare miRNAs expression. The cri terion for identifying the differential expression of miRNAs was that the 2.11. Dual luciferase reporter assay fold change was more than 2 times and the Q-value was less than 0.001. The targeted gene sequence containing the targeted site was ampli fiedby 2 × Phanta Max Master Mix (Vazyme, P515-01, Nanjing, China). 2.9. Integrated mRNA/miRNA network analysis The products were digested by XhoI/NotI and ligated with the luciferase vector psiCHECKTM-2. Dual luciferase reporter assay was based on Integrating differentially expressed miRNAs and mRNAs to identify previous reports(Du et al., 2019). The activities of fireflyluciferase and potential target genes regulated by miRNAs. The criterion was that the Renilla luciferase were detected by Dual-Glo® Luciferase Assay System miRNAs and mRNAs should be reversed simultaneously, and the miR (Promega, Cat. E2920, USA). NAs should predict mRNAs by RNAhybrid, Targetscan, and miRanda. Cytoscape software (San Diego, CA USA) was used to make the inter active networks between miRNAs and their target mRNAs. 2.12. Statistical analysis

2.10. Validations of miRNA and mRNA by quantitative real-time PCR All data were expressed as mean ± SEM. The unpaired student t-test was used for qRT-PCR and dual luciferase reporter assay. Two-way For mRNAs, cDNAs were synthesized by HiScript III RT SuperMix for ANOVA with post-hoc comparison by Bonferroni’s multiple compari qPCR kit (Vazyme, R323-01, Nanjing, China). For miRNAs, cDNAs were sons test was used to compare the data from behavioral tests among synthesized using the Mir-X miRNA First-Strand Synthesis Kit (Clontech, groups. The relationship between miRNA and its target prediction was 638315, CA, USA). The cDNAs were amplifiedby the ChamQ Universal evaluated by the Pearson correlation coefficient.

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Fig. 6. The validation of differentially expressed mRNAs in the nucleus accumbens from control (n = 3) versus resilient (n = 3) mice. Four asterisks show p < 0.0001, three asterisks show p < 0.001, two asterisks show p < 0.01, one asterisk show p < 0.05, in which two-sample t-test was used for the comparisons between resilient mice versus susceptible mice.

3. Results In order to detect anxiety-like behavior in mice, we used the elevated plus maze. Fig. 3a shows the susceptible, resilient, and control mice 3.1. Psychological stress-induced fear memory and anxiety-like behaviors tracks on the EPM before (day 0) and after (day 6) psychological stress. Fig. 3b shows the percentage of time spent on the open arm on day 0 and According to the criteria that mice have normal social activities and day6 as a percentage of time spent on the total arm. Percentages of time anxiety levels, 20% of the mice were excluded, leaving about 80% of the staying in open arms for control, resilient and susceptible at day 0 are mice. To test whether C57 mice are afraid of CD1 mice, we used the 13.43 ± 1.48%, 13.15 ± 1.45%, and 13.80 ± 1.62%. There is no sta social interaction test. Fig. 2a shows the social behavior tracks of con tistical difference between the three groups (control versus resilient, p > trol, susceptible and resilient mice on day6 with and without CD1. The 0.99, t = 0.1332; control versus susceptible, p > 0.99, t = 0.5384; fewer the tracks of C57 mice in the communication area, the greater the resilient versus susceptible, p > 0.99, t = 0.4424, Total df = 74, two-way fear of CD1 in C57 mice. After psychological stress, when there were ANOVA). Percentages of time staying in open arms for control, resilient CD1 mice, the behavioral tracks of the mice in the susceptible group in and susceptible at day 6 are 13.12 ± 1.71%, 12.54 ± 1.02%, and 7.22 ± the communication area were significantly reduced than without CD1. 1.64%, respectively. There were statistical differences among groups at Fig. 2b shows the ratio of the dwell time of the three groups of mice in day 6 (control versus susceptible, p = 0.0292, t = 2.654; resilient versus the communication area on day 0 and day 6. As can be seen from the susceptible, p = 0.0375, t = 2.560 Total df = 74, two-way ANOVA). The figure,the ratio of mice in the three groups was similar on day 0, with no time ratios in the control group are 13.43 ± 1.48% on day 0 and 13.12 ± statistical difference (control versus resilient, p > 0.99, t = 0.1788; 1.71% on day 6 (p > 0.99, n = 12). The time ratios in the resilient group control versus susceptible, p > 0.99, t = 0.5950; resilient versus sus are13.15 ± 1.45% on day 0 and 12.54 ± 1.02% on day 6 (p > 0.99, n = ceptible, p > 0.99, t = 0.4573, Total df = 74, two-way ANOVA). There 16). The susceptible group was 13.80 ± 1.62% on day 0 and 7.22 ± were statistical differences among the three groups on day6 (control 1.64% on day 6 (p = 0.0028, n = 12). These results indicate that mice in versus resilient, p = 0.0002, t = 4.266; control versus susceptible, p = the susceptible group show greater anxiety after experiencing psycho 0.0025, t = 3.482; resilient versus susceptible, p < 0.0001, t = 7.988, logical stress than mice in the control and resilient groups. Total df = 74, two-way ANOVA). The susceptible group was 1.16 ± 0.05 ± = = on day 0 and 0.74 0.04 on day 6 (p 0.0013, n 12), the resilient 3.2. mRNA differential expression in the nucleus accumbens among ± ± < group was 1.12 0.05 on day 0 and 1.61 0.10 on day 6 (p 0.0001, n control, resilient and susceptible mice = 16), the control mice were 1.10 ± 0.07 on day 0 and 1.14 ± 0.11 on > = day 0 (p 0.9999, n 12). These data indicated that after experiencing High-throughput sequencing was used to sequence mRNA and psychological stress, the mice in the susceptible group showed fear- miRNA from the nucleus accumbens of the control (n = 3), resilient (n = avoidance behaviors to the CD1 mice, while the mice in the resilient 3), and susceptible (n = 3) groups. The overall qualities of RNA- group did not show the fear of CD1. Sequencing were shown in the supplementary data (Table S2 and

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Table 4 Table 5 The changed miRNAs predict target mRNAs in susceptible versus control. The changed mRNAs predict miRNAs in susceptible versus control.

miRNAs The predicted target miRNAs The predicted target Geng symbol The predicted Geng The predicted mRNAs that match DEGs in mRNAs that match DEGs miRNAs that match Symbol miRNAs that match transcriptome * in transcriptome * DEGs in DEGs in transcriptome * transcriptome * let-7c-2- Bdnf↑, Wisp1↑, Chrna5↑, miR- Scube1↑, Galnt9↑, Espnl↑, 3p↓ Mical2↑, 3473b↓ Fezf2↑, Mical2c↑, 1190005I06Rik↓ miR-490-5p↑ Lipg↑ miR-499-5p↓ miR- Cyp26b1↑, Il12a↑, miR-451a↑miR-34b- miR- Pkp1↑, Bdnf↑, Medag↑, Baiap3↓ Crhbp↑ miR-27a-5p↓ 700- Adcyap1↑, Bmp3↑, Igfn1↑, 5p↑ 376a-3p↓ Chst8↑, Cd300lg↑, 5p↓ Arhgap25↑, miR-486a-3p↓, miR- Rspo2↑, Sstr3↑, Grm2↑, Scube1↑, Nr4a2↑, Dnaaf3↓ miR-34b-5p↑ Cwh43↑ 182-5p↓, miR-199b- miR- Nrn1↑, Met↑, Krt80↑, miR-377- Tmem200a↑, Mapk11↑, 3p↓ 10a- Arhgap25↑, Kcnj3↑, Mas1↑, 5p↓ Espnl↑, Rnf223↑, Rtn4rl2↑, Dsc3↓ let-7a-1-3p↑ Mapk11↑ miR-377-5p↓ 5p↓ Cd300lg↑, Mical2↑, Enkur↓ miR-490-5p↑ Mas1↑ miR-10a-5p↓ miR- Bdnf↑, Alas2↑, Otop2↑, Cbln2↑, Espnl↑, Cwh43↑, miR-671–3p↓, miR- miR- Fam216b↓ miR-297c-5p↑ Medag↑ 133a- Bmp3↑, Synj2↑, Rtn4rl2↑, Fhod3↑, St8sia5↑, 376a-3p↓ 486a-3p↓ 3p↓ Npr3↑, Trib1↑, Cd300lg↑, Fzd5↓ miR-490-5p↑ Met↑ miR-10a-5p↓ miR- Grm8↑, Adcyap1↑, miR-29b-3p↓, let-7c- Bdnf↑, Kcnj6↑, Cwh43↑, miR-499- 182- Fam19a1↑, Sstr2↑, Gfra2↑, 2-3p↓, miR-505-3p↓, Igfn1↑, Fhod3↑, 5p↓ Lrrc43↓ miR-490-5p↑ Mical2↑ 5p↓ Lipg↑, Kcnj6↑, Mroh5↑, miR-377-5p↓, miR- miR- Serinc2↑, Alas2↑, Grm2↑, Cbln2↑, Bdnf↑, Wisp1↑, 339-5p↓, miR-3473b↓ miR- 199a- St6galnac4↑, Chst8↑, Aldh1a7↑, Synj2↑, miR-451a↑, miR- 497a-5p↓ Lrrcc1↓ Mroh5↑ miR-499-5p↓ 3p↓ 9130024F11Rik↑, Npr3↑, Arhgap25↑, 451a↑, miR-429-3p↑, miR- Serpinb8↑, Bdnf↑, miR-96-5p↓, miR- Wisp1↑, Chrnb3↑, Synj2↑, miR- 199a- Cyp26b1↑, Necab3↑, 200c-3p↓, miR-700- Igfn1↑, Adgra1↑, Hs3st2↑, 486b-5p↓ Musk↓ miR-297c-5p↑ Cyp26b1↑ 5p↓ Espnl↑, Igfn1↑, Ntng2↑, 5p↓, miR-486b-5p↓, miR- Cpne9↑, Kcnk4↑, Hcrtr1↑, miR-339-5p↓, miR- Gm11762↑, Otx1↑, Rtn4r↑, 199b- Synj2↑, Cwh43↑, Npr3↑, Neil3↓ miR-429-3p↑ Necab3↑ miR-486b-5p↓ 7044-3p↓ 3110035E14Rik↑, 3p↓ Ntng2↑, let-7c-2-3p↓, miR- Togaram2↓ let-7a-1-3p↑ Chrna5↑ Scube1↑, Satb2↑, Kcng4↑, 107-5p↓ miR-1a- Nptx1↑, Cckbr↑, Npr3↑, miR- Serinc2↑, Gfra2↑, Tcap↑, Trp73↓ miR-669f-5p↑ Npas1↑ miR-298-3p↓ 3p↓ Arhgap25↑, Trib1↑, 7056-3p↓ Esrrb↑, Trhde↑, miR-497a-5p↓, miR- Cd300lg↑, 505-3p↓, miR-1a-3p↓, Ttc16↓ miR-451a↑ Npr3↑ miR- miR-199b-3p↓, miR- Ovol2↑, Cyp26b1↑, Krt80↑, miR-764- 200c- Serpinb8↑, Espnl↑, Trhde↑, 133a-3p↓ 9130024F11Rik↑, 3p↓ 3p↓ miR-219a-5p↓, miR- Ttc21a↓ miR-669f-5p↑ Nptx1↑ miR- 1a-3p↓ Cbln2↑, Bdnf↑, C1ql3↑, miR- Pkp1↑, Medag↑, Tmem40↑, 206- Ttll6↓ let-7a-1-3p↑ Nr4a2↑ miR-377-5p↓ Synj2↑, Arhgap25↑, 671–3p↓ Synj2↑, Klhl30↑, 3p↓ Ubxn10↓ miR-451a↑ Nrn1↑ miR-10a-5p↓ miR- Cyp26b1↑, Grm8↑, Wisp1↑, let-7a-1-3p↑, miR- miR-486b-5p↓, miR- Bdnf↑, Adcyap1↑, Nptx1↑, miR-96- Upb1↓ Ntng2↑ 219a- Tmem252↑, Arhgap25↑, 34b-5p↑ 199b-3p↓ Arhgap25↑, 5p↓ 5p↓ Trib1↑, Casq1↓ miR-451a↑ Otop2↑ miR-133a-3p↓ miR- miR-7044-3p↓, miR- Crhbp↑, Wisp1↑, Aldh1a7↑, miR- 3110035E14Rik↑ miR-7044-3p↓ Otx1↑ 27a- Musk↓, Fam216b↓, 3544-3p↓ Cd300lg↑, 297c-5p↑ 5p↓ miR-200c-3p↓, miR- 9130024F11Rik↑ Ovol2↑ miR-200c-3p↓ Cyp26b1↑, Serinc2↑, 199a-3p↓ miR- Trabd2b↑, Kcnj6↑, Espnl↑, miR- Ttc16↓, Casq1↓, Ubxn10↓, miR-700-5p↓, miR- 339- miR-671–3p↓, miR- Igfn1↑, Arhgap25↑, St8sia5↑, 451a↑ Baiap3↓, Lrrcc1 ↓, Adcyap1↑ 107-5p↓, miR-219a- Pkp1↑ 5p↓ 376a-3p↓ Mical2↑, 5p↓, miR-499-5p↓ Hkdc1↑, Kcnk4↑, Rtn4r↑, miR-298-3p↓, miR- miR- Adgra1↑ miR-199a-5p↓ Rnf223↑ Gfra2↑, Tmem40↑, Npas1↑, miR-490- Fzd5↓, Lrrc43↓, Enkur↓, 377-5p↓ 298- Rnf223↑, Arhgap25↑, 5p↑ 1190005I06Rik↓, miR-199a-3p↓, miR- 3p↓ Alas2↑ Rspo2↑ miR-10a-5p↓ Fhod3↑, 133a-3p↓ miR- miR-497a-5p↓, miR- Sstr3↑, Scube1↑, Kcng4↑, miR-429- Aldh1a7↑ Rspo3↑ miR-298-5p↓ 298- Neil3↓Lrrcc1↓ 27a-5p↓ Tmem252↑, Rspo3↑, Trib1↑, 3p↑ 5p↓ miR-298-3p↓miR- Anxa1↑ miR-3083-3p↓ Rtn4r↑ miR- 7044-3p↓ miR- 29b- Grm2↑, Kcnj6↑, Mical2↑, Trp73↓, Ttc21a↓, miR-298-3p↓, miR- 669f-5p↑ 3p↓ 96-5p↓, miR-497a- miR- 5p↓, miR-700-5p↓, Sstr2↑, Synj2↑, Anxa1↑, miR-34b- 3083- Upb1↓, Baiap3↓, Dnaaf3↓, miR-3544-3p↓, miR- miR-377-5p↓, miR- Trhde↑, Klhl14↑, 5p↑ Arhgap25↑ Rtn4rl2↑ 3p↓ 219a-5p↓, miR-10a- 133a-3p↓ 5p↓, miR-339-5p↓, ↑ Note: indicates up-regulation in the tissue of Nac from susceptible versus miR-1a-3p↓, miR- control mice, whereas↓ represents down-regulation. 206-3p↓, miR-497a-5p↓, let-7c- ↓ ↓ Table S3). The clean small RNA readings in each library range from 10 to 2-3p , miR-182-5p , miR-219a-5p↓, miR- Bdnf↑ Satb2↑ miR-1a-3p↓ 44 nucleotides, of which the richest length is 22 nucleotides (Fig. S4). 376a-3p↓, miR-486b- High-quality clean reading segments with more than 18 nucleotides 5p↓, miR-133a-3p↓, were mapped to the mice genome. Genome-matching readings were miR-206-3p↓ divided into different categories of small RNA based on their biogenesis miR-700-5p↓, miR- miR-298-5p↓, miR- Bmp3↑ 107-5p↓, miR-133a- Scube1↑ 377-5p↓, miR- and annotation (Fig. S5). High-throughput sequencing was performed 3p↓, 3473b↓, miR-1a-3p↓ on the mRNAs in the nucleus accumbens, and the FPKM value was miR-7056-3p↓, miR- calculated by the DESeq2 package, the low-expression genes were C1ql3↑ miR-206-3p↓ Serinc2↑ 339-5p↓, miR-199a- removed (FPKM <0.5). 3p↓ The differentially expressed mRNAs and miRNAs were screened (continued on next page)

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Table 5 (continued ) susceptible and resilient mice, 117 differentially expressed mRNAs were Geng symbol The predicted Geng The predicted screened out, in which 51 mRNAs were down-regulated and 66 mRNAs miRNAs that match Symbol miRNAs that match were up-regulated in susceptible mice (Table S6). Between resilient and DEGs in DEGs in control mice, 269 differentially expressed mRNAs were screened out, in transcriptome * transcriptome * which 96 mRNAs were up-regulated and 173 mRNAs were down- miR-764-3p↓, miR- regulated in resilient mice (Table S8). Table S5, Table S7 and Table S9 Car10↑ miR-505-3p↓ Serpinb8↑ 107-5p↓, miR-486b- list the pathways identifiedby KEGG based on DEGs between susceptible ↓ 5p and control mice, susceptible and resilient mice, resilient mice and miR-486a-3p↓, miR- Cbln2↑ 497a-5p↓, miR-206- Smoc2↑ miR-3544-3p↓ control mice, some of the neural-related pathways are listed in 3p↓, Tables 1–3. Among the genes related to synapses, the top half of the miR-505-3p↓, miR- miR-3083-3p↓, miR- Cckbr↑ Sstr2↑ table shows genes encoding synapse elements, while the bottom half 1a-3p↓, 499-5p↓ shows genes encoding pathways that regulate synapses. Among genes miR-486a-3p↓, miR- related to pathway, and the top half of the table shows genes encoding 27a-5p↓, miR-376a- miR-298-5p↓, miR- Cd300lg↑ 3p↓, miR-10a-5p↓, Sstr3↑ molecules that constitute the pathway, the bottom half shows genes 10a-5p↓ miR-10a-5p↓, miR-1a- encoding molecules that regulate the pathway. 3p↓, miR-497a-5p↓, miR- Chst8↑ St6galnac4↑ miR-199a-3p↓ 3.3. qRT-PCR validates mRNA results 376a-3p↓, miR-486a-3p↓, miR- Cpne9↑ miR-199b-3p↓ St8sia5↑ 339-5p↓ To verify the data of mRNA sequencing analysis, we performed qRT- miR-3083-3p↓, miR- PCR from the nucleus accumbens tissue. Compared with control mice, 3083-3p↓, miR-497a- the expression of Trhr and Dnaaf3 decreased in susceptible mice, while 5p↓, miR-671–3p↓, Galnt9↑ miR-3473b↓ Synj2↑ the expression of Chrna5, Sstr2, Bdnf, Chrnb3, Adra1d, Cckbr, Kcnj3 and miR-199b-3p↓, miR- 133a-3p↓, miR-199a- Kcnj6 increased (Fig. 4). Compared with the resilient group, Baiap3 5p↓, miR-206-3p↓, expression was reduced in susceptible mice, while Zfp729a, Tuba1c, miR-298-3p↓, miR- Sstr5, Col5a2, Chrnb3, Kdr, H2-T10 expression was increased (Fig. 5). ↑ ↓ ↑ ↓ Gfra2 7056-3p , miR-505- Tcap miR-7056-3p Compared with the mice in the control group, the expression of Trhr, 3p↓, miR-499-5p↓, Gm11762↑ miR-7044-3p↓ Tmem200a↑ miR-377-5p↓ Sstr5, and Tas1r1 in the resilient group decreased, while the expressions miR-29b-3p↓, miR- of Grm8, Adra1b, Adra1d, Cacan1s, and Otx1 increased (Fig. 6). The qRT- miR-298-5p↓, miR- Grm2↑ 10a-5p↓, miR-199a- Tmem252↑ 96-5p↓ PCR analysis results are consistent with the mRNA sequencing results, 3p↓ which can validate our research. miR-96-5p↓, miR- miR-298-3p↓, miR- Grm8↑ Tmem40↑ The expression levels of mRNAs could be affected by miRNAs. The 499-5p↓ 671–3p↓ Hcrtr1↑ miR-199b-3p↓ Tnnc1↑ miR-107-5p↓ bindings of miRNAs with their dicers can degrade mRNAs and weaken Hkdc1↑ miR-298-3p↓ Chrnb3↑ miR-199a-5p↓ the translations of mRNAs (Afonso-Grunz and Muller, 2015; Dwivedi, Hs3st2↑ miR-199a-5p↓ Trabd2b↑ miR-339-5p↓ 2014). If the expressions of mRNAs decrease, the corresponding ex miR-182-5p↓, miR- miR-3083-3p↓, miR- pressions of miRNAs will be up-regulated, or vice versa. We analyzed the 700-5p↓, miR-486b- Igfn1↑ Trhde↑ 7056-3p↓, miR-764- 5p↓, miR-339-5p↓, miRNA expression profiles in the nucleus accumbens. 3p↓, miR-505-3p↓ miR-199a-5p↓, miR-298-5p↓, miR- 3.4. miRNA differential expression in the nucleus accumbens among 96-5p↓, miR-505-3p↓, Il12a↑ miR-700-5p↓ Trib1↑ control, resilient and susceptible mice miR-1a-3p↓, miR- 133a-3p↓ miR-298-5p↓, miR- In susceptible versus control mice, 40 differentially expressed miR Kcng4↑ Esrrb↑ miR-7056-3p↓ 1a-3p↓ NAs were screened out, of which 7 miRNAs were up-regulated and 33 ↓ ↑ ↓ ↑ miR-505-3p , miR- miRNAs were down-regulated (Table S10). In susceptible versus resil Kcnj3 miR-10a-5p Fam19a1 ↓ 499-5p ient mice, 41 differentially expressed miRNAs were screened out, of miR-486a-3p↓, miR- miR-29b-3p↓, miR- 764-3p↓, miR-377- which 26 miRNAs were up-regulated and 15 miRNAs were down- 182-5p↓, miR-505- Kcnj6↑ Espnl↑ 5p↓, miR-486b-5p↓, regulated (Table S11). In resilient versus control mice, 42 differen 3p↓, miR-339-5p↓, miR-339-5p↓, miR- miR-499-5p↓ tially expressed miRNAs were screened out, of which 15 miRNAs were 3473b↓ up-regulated and 27 miRNAs were down-regulated (Table S12). Ac miR-298-3p↓, miR- Kcnk4↑ Fap↑ miR-3544-3p↓ cording to the three databases, the target mRNAs of miRNAs were pre 199b-3p↓ Klhl14↑ miR-3083-3p↓ Fezf2↑ miR-3473b↓ dicted and matched with the mRNA measured by mRNA sequencing. miR-298-3p↓, miR- Tables 4, 6, 8 respectively shows that the changed miRNAs predict target Klhl30↑ miR-671–3p↓ Fhod3↑ 486a-3p↓, miR-182- mRNAs between susceptible and control mice, between susceptible and ↓ ↓ 5p , miR-107-5p , resilient mice, between resilient and control mice. Tables 5, 7, 9 miR-96-5p↓, miR- miR-200c-3p↓, miR- 497a-5p↓, let-7c-2- respectively shows the changed mRNAs and their corresponding miR Krt80↑ Wisp1↑ 10a-5p↓ 3p↓, miR-27a-5p↓, NAs between susceptible and control mice, between susceptible and miR-199a-5p↓, resilient mice, between resilient and control mice. The interactive net Note: ↑ indicates up-regulation in the tissue of Nac from susceptible versus works of miRNAs and corresponding mRNAs between two groups were control mice, whereas ↓ represents down-regulation. constructed (Figs. S1-S3). To further validate the miRNAs sequencing results, we selected 15 miRNAs to perform qRT-PCR (Fig. 7). The qRT-PCR results were consistent with the miRNAs sequencing results, which further validated according to the criteria described in the materials and methods. Be our study. tween susceptible and control mice, 227 differentially expressed mRNAs were screened out, in which 98 mRNAs were down-regulated and 129 mRNAs were up-regulated in susceptible mice (Table S4). Between

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Table 6 The changed miRNAs predict target mRNAs in susceptible versus resilient.

miRNAs The predicted target mRNAs that match DEGs in transcriptome * miRNAs The predicted target mRNAs that match DEGs in transcriptome *

miR-203-3p↓ Gal3st2b↑Lpp↑ miR-193a-3p↓ Cd300lf↑Myl1↑ miR-212-5p↓ Tmem181c-ps↑LOC100861615↑ miR-199a-5p↓ Bspry↑Chrnb3↑Csprs↑ miR-3065-5p↓ Lpp↑Chrnb3↑Col5a2↑ miR-199b-3p↓ Gpr15↑Lpp↑Abhd1↑ miR-339-5p↓ Tnxb↑ miR-1b-5p↓ Chrnb3↑ miR-341–3p↓ Gpr15↑Eno1b↑ miR-200b-3p↓ Lpp↑Adm↑Dsp↑Mms22l↑ miR-673-5p↓ Angptl7↑Vwf↑ miR-122-5p↑ 3110062M04Rik↓Cd72↓ miR-700-5p↓ Il11ra2↑ miR-132-5p↑ Hyal1↓ miR-7080-3p↓ Pla2g4e↑Col5a2↑ miR-184-3p↑ Fyb2↓Catsperd↓ miR-7662–3p↓ Lpp↑Adm↑Cd300lf↑Tmem252↑Sstr5↑ miR-211-5p↑ Col13a1↓Ptchd4↓Lrrcc1↓ miR-96-5p↓ Dnah6↑Tmem252↑Tmod4↑ miR-34b-5p↑ Ggnbp1↓Baiap3↓Vmn2r30↓ miR-200c-3p↓ Bspry↑ miR-34c-3p↑ Endou↓Etnppl↓ miR-124-3p↓ Lpp↑ miR-34c-5p↑ Baiap3↓Lrrcc1↓ miR-1251-5p↓ Tmem181c-ps↑Gm2002↑Angptl7↑Tnxb↑ miR-375-3p↑ Musk↓ miR-133a-3p↓ Zfp729a↑Alas2↑BC021767↑ miR-378d↑ Cd72↓ miR-135a-5p↓ Cd300lf↑Zfp729a↑ miR-669f-5p↑ Cd72↓ miR-135b-3p↓ Ccdc78↑ miR-744-5p↑ Tshz1↓Lsmem1↓Tm6sf2↓ miR-183-5p↓ Morf4l1b↑Rsc1a1↑Spink8↑Col5a2↑

Note: ↑ indicates up-regulation in the tissue of Nac from susceptible versus resilient mice, whereas ↓ represents down-regulation.

Table 7 The changed mRNAs predict miRNAs in susceptible versus resilient.

mRNA The predicted miRNAs that match DEGs in mRNA The predicted miRNAs that match DEGs in transcriptome * transcriptome *

3110062M04Rik↓ miR-122-5p↑ Gpr15↑ miR-199b-3p↓miR-341–3p↓ Baiap3↓ miR-34b-5p↑miR-34c-5p↑ Dnah6↑ miR-96-5p↓ Tshz1↓ miR-744-5p↑ Il11ra2↑ miR-700-5p↓ Vmn2r30↓ miR-34b-5p↑ LOC100861615↑ miR-212-5p↓ miR-124-3p↓miR-199b-3p↓miR-200b-3p↓miR-203-3p↓miR-3065-5p↓miR- Tm6sf2↓ miR-744-5p↑ Lpp↑ 7662–3p↓ Ptchd4↓ miR-211-5p↑ Col5a2↑ miR-183-5p↓miR-3065-5p↓miR-7080-3p↓ Musk↓ miR-375-3p↑ Csprs↑ miR-199a-5p↓ Lsmem1↓ miR-744-5p↑ Mms22l↑ miR-200b-3p↓ Catsperd↓ miR-184-3p↑ Morf4l1b↑ miR-183-5p↓ Lrrcc1↓ miR-211-5p↑miR-34c-5p↑ Chrnb3↑ miR-199a-5p↓miR-1b-5p↓miR-3065-5p↓ Hyal1↓ miR-132-5p↑ Myl1↑ miR-193a-3p↓ Endou↓ miR-34c-3p↑ Pla2g4e↑ miR-7080-3p↓ Etnppl↓ miR-34c-3p↑ Cd300lf↑ miR-135a-5p↓miR-193a-3p↓miR-7662–3p↓ Col13a1↓ miR-211-5p↑ Rsc1a1↑ miR-183-5p↓ Fyb2↓ miR-184-3p↑ Spink8↑ miR-183-5p↓ Ggnbp1↓ miR-34b-5p↑ Sstr5↑ miR-7662–3p↓ Abhd1↑ miR-199b-3p↓ Ccdc78↑ miR-135b-3p↓ Adm↑ miR-200b-3p↓miR-7662–3p↓ Tmem181c-ps↑ miR-1251-5p↓miR-212-5p↓ Alas2↑ miR-133a-3p↓ Tmem252↑ miR-7662–3p↓miR-96-5p↓ Angptl7↑ miR-1251-5p↓miR-673-5p↓ Tmod4↑ miR-96-5p↓ Zfp729a↑ miR-133a-3p↓miR-135a-5p↓ Tnxb↑ miR-1251-5p↓miR-339-5p↓ Eno1b↑ miR-341–3p↓ BC021767↑ miR-133a-3p↓ Gal3st2b↑ miR-203-3p↓ Bspry↑ miR-199a-5p↓miR-200c-3p↓ Dsp↑ miR-200b-3p↓ Vwf↑ miR-673-5p↓ Gm2002↑ miR-1251-5p↓

Note: ↑ indicates up-regulation in the tissue of Nac from susceptible versus resilient mice, whereas ↓ represents down-regulation.

3.5. Verification of targeting relationship between miR-497a-5p, miR- however, no statistical difference was found in the mutant type (Fig. 8b/ 199a-5p, miR-29b-3p and Bdnf, Chrnb3, Kcnj6 mRNA d/f). These results indicate that Bdnf, Chrnb3, Kcnj6 mRNA are targets of miR-497a-5p, miR-199a-5p, miR-29b-3p, which are consistent with our To verify the relationship between miRNAs and predicted target bioinformatics analysis in miRNA target gene prediction. mRNAs, qRT-PCR and dual luciferase reporter assays to detect whether miR-497a-5p, miR-199a-5p, and miR-29b-3p were targeted to Bdnf, 4. Discussion Chrnb3, and Kcnj6, respectively. According to qRT-PCR analysis, we found a negative correlation between miR-497a-5p and Bdnf (Fig. 8a), By high-throughput sequencing of mRNA and miRNA, we quantita miR-199a-5p and Chrnb3 (Fig. 8c), miR-29b-3p and Kcnj6 (Fig. 8e). In tively analyzed the nucleus accumbens brain regions of control, resil dual luciferase reporter assay, we constructed wild-type and mutant ient, and susceptible mice. In susceptible and resilient mice, up- vectors, respectively. miR-497a-5p, miR-199a-5p, and miR-29b-3p regulated genes in the susceptible mice related to synaptic elements mimics could significantly reduce the relative activities of wild-type include Pla2g4e, Chrnb3, Sstr5, encode Glutamatergic synapse and dual luciferase reporter plasmids for Bdnf, Chrnb3, and Kcnj6 mRNA, Neuroactive ligand-receptor interaction. The down-regulated mainly

12 Y. Lu et al. Pharmacology, Biochemistry and Behavior 199 (2020) 173062

Table 9 The changed mRNAs predict miRNAs in resilient versus control.

Geng Symbol The predicted Geng Symbol The predicted miRNAs that match miRNAs that DEGs in match DEGs in transcriptome * transcriptome *

* miR-27a-5p↑miR- 4930481A15Rik↓ Alkbh3↑ miR-7070-3p↓ 378d↑ 6430573F11Rik↓ miR-467b-5p↑ Arhgap33os↑ miR-7070-3p↓ miR-133b-3p↑miR- Adcyap1↓ 499-5p↑miR-499- Armc3↑ miR-1251-5p↓ transcriptome 5p↑ in Aldh1a1↓ miR-744-5p↑ Asf1b↑ miR-96-5p↓ miR-135b- DEGs Aldh1a2↓ miR-378d↑ Ccdc78↑ 3p↓miR-451a↓ miR-135b- match Aspg↓ miR-10a-5p↑ Cd247↑ 3p↓miR-183- ↓ ↓

that 5p miR-183-5p let-7a-1-3p↑miR- 1247-5p↑miR-

mRNAs 133a-3p↑miR-206- Bdnf↓ 3p↑miR-34b- Cdh15↑ miR-363-3p↓

target 3p↑miR-34c- 3p↑miR-375- 3p↑miR-532-5p↑ let-7a-1-3p↑miR- miR-363-3p↓miR- predicted Cacna1s↓ Cldn23↑ 10a-5p↑ 451a↓

The Adcyap1 ↓ Col10a1 Qrfpr St6galnac4 Lsr Rspo3 Pkib Qrfpr ↓ Pkib St6galnac4 ↓ Fgl2 Serinc2 Igfn1 ↓ Gm10037 Scube1 ↓ Pappa2 Misp Fosl2 Bdnf ↓ Cbln2 Slc5a5 4930481A15Rik ↓ Cd72 ↓ Igfn1 Qrfpr Nwd2 Mapk11 Serinc2 Hbb-bs ↓ Bdnf Nmbr Nptx2 Cbln2 Fosl2 Bdnf ↓ Scube1 Plcxd2 Endou Gpr150 Tmem215 Dusp4 ↓ Sstr3 Plcxd2 Pdzrn3 ↓ Otx1 Bdnf ↓ Etnk2 Grm8 Serinc2 Cd72 ↓ Igfn1 Scube1 Aldh1a2 Sstr3 Tmem132c 4930481A15Rik Pkib Slc5a5 Dusp4 ↓ 6430573F11Rik Cpne9 Klhl14 Cbln2 ↓ Nptxr Egln3 Adcyap1 ↓ Cd72 Fam19a1 Lipg Cnn1 Nptxr Grm8 Ngb Tmem215 Fosl2 Bdnf ↓ Myoc Gadl1 Pkib Scube1 ↓ Myoc Cbln2 Pappa2 Klhl14 Fosl2 Scube1 ↓ Lsr Aldh1a1 Fosl2 Slc13a4 ↓ Nptxr miR-206-3p↑miR- miR-183-5p↓miR- Cbln2↓ 34b-3p↑miR-493- Col5a2↑ 3065-5p↓ 3p↑miR-539-3p↑ miR-122-5p↑miR- Cd72↓ 296-3p↑miR- Col6a5↑ miR-3074-5p↓ 378d↑miR-499-5p↑ let-7a-1-3p↑miR-

miRNAs miR-133b-3p ↑ miR-153-3p ↑ miR-199a-3p ↑ miR-199a-5p ↑ miR-1a-3p ↑ miR-206-3p ↑ miR-27a-5p ↑ miR-296-3p ↑ miR-34b-3p ↑ miR-34c-3p ↑ miR-34c-5p miR-3547-3p ↑ miR-375-3p ↑ miR-378d ↑ miR-467b-5p ↑ miR-493-3p ↑ miR-499-5p ↑ miR-532-5p ↑ miR-539-3p ↑ miR-744-5p ↑ miR-764-3p ↑ Cnn1↓ Colq↑ miR-135b-3p↓ 499-5p↑ miR-124-3p↓miR- 182-5p↓miR- Col10a1↓ miR-133b-3p↑ Crocc2↑ 3074-5p↓miR- 7070-3p↓ down-regulation.\. Cpne9↓ miR-467b-5p↑ D330041H03Rik↑ miR-363-3p↓ Dkkl1↓ miR-10a-5p↑ Dnah11↑ miR-3065-5p↓ miR-34c-5p↑miR- Dusp4↓ Dnah6↑ miR-96-5p↓ 467b-5p↑ represents miR-1251- 5p↓miR-182- Efcab6↓ miR-10b-5p↑ Dnah8↑ 5p↓miR-3065-

whereas ↓ 5p↓ Egln3↓ miR-493-3p↑ Dynlt1f↑ miR-212-5p↓ miR-363-3p↓miR- mice, 451a↓miR-

* Endou↓ miR-34c-3p↑ Ect2↑ 451a↓miR-673- ↓ ↓ control 5p miR-673-5p miR-122-5p↑miR- Etnk2↓ Eno1b↑ miR-341–3p↓ 375-3p↑ versus Fam19a1↓ miR-499-5p↑ Fam216b↑ miR-1251-5p↓ transcriptome control. miR-451a↓miR- in Fgl2↓ miR-199a-3p↑ Fbln7↑ 673-5p↓

resilient let-7a-1-3p↑miR- DEGs versus 1a-3p↑miR-34b-

from Fosl2↓ 3p↑miR-499- Fndc1↑ miR-363-3p↓ match 5p↑miR-539- resilient Nac

that 3p↑miR-744-5p↑ in of Gadl1↓ miR-532-5p↑ Folr1↑ miR-341–3p↓ Gm10037↓ miR-199a-5p↑ Foxj1↑ miR-124-3p↓ mRNAs

tissue ↓ ↓ ↑ ↑ miR-183-5p miR- mRNAs Gpnmb let-7a-1-3p Gbp11 363-3p↓ the

target Gpr150↓ miR-34c-3p↑ Gpr15↑ miR-341–3p↓ in

target miR-375-3p↑miR- Grm8↓ H2-DMb1↑ miR-183-5p↓ 499-5p↑ ↓ ↑ ↑ ↓

predicted Hbb-bs miR-34b-3p H2-Q1 let-7i-3p

predict Igfbp2↓ miR-10a-5p↑ Iqca↑ miR-212-5p↓ The Vax1 ↑ H2-Q1 Prss56 Slc26a5 ↑ Rd3 Itk Lpp Trp73 Foxj1 Crocc2 Tmem254c ↑ Parp3 Pipox Aire Rd3 Dnah8 Slc14a2 Tmem181c-ps Mki67 Lrrc43 Fam216b Ttc21a Armc3 Colq ↑ Cd247 Ccdc78 Ak7 Rd3 ↑ Lrp2bp Dnah8 Trp73 Crocc2 Col5a2 ↑ Rsc1a1 Majin Gbp11 H2-DMb1 Cd247 Tmprss4 Spink8 Morf4l1b Togaram2 ↑ Dynlt1f Aire Iqca Xaf1 Tmem181c-ps LOC100861615 Col5a2 ↑ Dnah8 Dnah11 Slc14a2 Lpp Rd3 ↑ Col6a5 Rsph1 Crocc2 Gpr15 ↑ Tas1r1 Eno1b Folr1 Tmprss4 Cdh15 ↑ Fndc1 Cldn23 Parp3 Sfi1 Gbp11 Ect2 Tas1r1 D330041H03Rik Lrrc43 Ttll2 Khdrbs2 ↑ Cldn23 Ect2 Fbln7 Lpp Ccdc78 Pif1 Ect2 ↑ Fbln7 Kif2c Adam21 Spef2 Mia Aire ↑ Zfp729a Alkbh3 Mns1 Itk Crocc2 Arhgap33os Pipox ↑ Asf1b Itk Tmod4 Pif1 Dnah6 Bdnf ↓ Ptgs2 Ptger2 Myoc Gpnmb Cnn1 Nptxr Klhl14 Cacna1s Pkib Fosl2 Nrn1 ↓ Mas1 Sstr3 Dkkl1 Igfbp2 Cacna1s Rspo2 Aspg Medag ↓ Efcab6 Cd72 ↓ Etnk2 St6galnac4 Nwd2 Myoc Otx1 Serinc2 Bdnf ↓ Lsr Ngb Serinc2 Bdnf ↓ ↓ ↑ miR-124-3p miR- up-regulation ↓ miR-199a-5p miR- ↑ ↓ Igfn1 ↑ ↑ Itk 7070-3p miR-96- 296-3p miR-378d ↓ miRNAs 5p let-7a-1-3p↑miR- ↓ ↑ ↑ ↓

indicates Klhl14 467b-5p miR-539- Khdrbs2 miR-451a 8 ↑ 3p↑ changed let-7i-3p ↓ miR-124-3p ↓ miR-1251-5p ↓ miR-135b-3p ↓ miR-182-5p ↓ miR-183-5p ↓ miR-212-5p ↓ miR-3065-5p ↓ miR-3074-5p ↓ miR-341 – 3p ↓ miR-363-3p ↓ miR-451a ↓ miR-673-5p ↓ miR-7070-3p ↓ miR-96-5p ↓ let-7a-1-3p ↑ miR-10a-5p ↑ miR-10b-5p ↑ miR-122-5p ↑ miR-1247-5p ↑ miR-133a-3p ↑ miRNAs (continued on next page) Table The Note:

13 Y. Lu et al. Pharmacology, Biochemistry and Behavior 199 (2020) 173062

Table 9 (continued ) Table 9 (continued )

Geng Symbol The predicted Geng Symbol The predicted Geng Symbol The predicted Geng Symbol The predicted miRNAs that match miRNAs that miRNAs that match miRNAs that DEGs in match DEGs in DEGs in match DEGs in transcriptome * transcriptome * transcriptome * transcriptome *

Lipg↓ miR-499-5p↑ Kif2c↑ miR-673-5p↓ miR-1251-5p↓miR- miR-1247-5p↑miR- 212-5p↓miR-7070- Lsr↓ 133b-3p↑miR-744- LOC100861615↑ miR-212-5p↓ 3p↓ 5p↑ Ak7↑ miR-135b-3p↓ Zfp729a↑ miR-7070-3p↓ miR-124-3p↓miR- ↑ Mapk11↓ miR-296-3p↑ Lpp↑ 3065-5p↓miR- Note: indicates up-regulation in the tissue of Nac from resilient versus control 451a↓ mice, whereas ↓ represents down-regulation. Mas1↓ miR-10a-5p↑ Lrp2bp↑ miR-182-5p↓ miR-1251- included Fxyd2, encodes Thyroid hormone synthesis. In terms of signal 5p↓miR-363- Medag↓ miR-10b-5p↑ Lrrc43↑ 3p↓miR-363- transduction pathways, down-regulated genes in the nucleus accumbens 3p↓miR-363-3p↓ of mice in the susceptible group include Fxyd2, Cd72 and Trip6, encode Misp↓ miR-1a-3p↑ Majin↑ miR-183-5p↓ cAMP, cGMP-PKG, B cell receptor, and NOD-like receptor pathway. Up- let-7a-1-3p↑miR- regulated genes include Col5a2, Kdr, Tnxb, Vwf, Pla2g4e, Il11ra2, Myoc↓ 122-5p↑miR-532- Mia↑ miR-673-5p↓ Gm2002, Sstr5, Ccl9, encode PI3K-Akt, VEGF, Jak-STAT, cAMP, Ras, and 5p↑miR-539-3p↑ miR-1247-5p↑miR- Chemokine pathway. The DEGs may be related to psychological stress- Ngb↓ Mki67↑ miR-1251-5p↓ 499-5p↑ induced susceptibility. Nmbr↓ miR-34b-3p↑ Mns1↑ miR-7070-3p↓ We found that some pathways were significantly enriched and ↓ ↑ ↑ ↓ Nptx2 miR-34b-3p Morf4l1b miR-183-5p related to nerve system through the KEGG analysis. For example, the let-7a-1-3p↑miR- miR-1251- Nptxr↓ 493-3p↑miR-499- Parp3↑ DEGs enriched in Neuroactive ligand-receptor interaction pathway were 5p↓miR-363-3p↓ 5p↑miR-764-3p↑ associated with brain diseases such as Parkinson’s disease and so on miR-451a↓miR- (Kong et al., 2015; Su et al., 2009; Wei et al., 2020). In this pathway, Nrn1↓ miR-10a-5p↑ Pif1↑ 96-5p↓ Chrnb3, Prlhr, Trhr, Cckbr, Rxfp1 and others are closely related to the miR-122-5p↑miR- miR-1251- Nwd2↓ Pipox↑ nerve system(Cordero-Erausquin et al., 2000; Kourtian et al., 2017). 296-3p↑ 5p↓miR-96-5p↓ miR-122-5p↑miR- Genes in Cell adhesion molecules (CAMs) pathway play an important Otx1↓ Prss56↑ let-7i-3p↓ 3547-3p↑ role in neurite growth and regeneration, myelin regeneration and miR-124-3p↓miR- neuronal differentiation (Chooi and Chew, 2019). During nerve devel miR-1a-3p↑miR- 1251-5p↓miR- Pappa2↓ Rd3↑ opment, CAMs is involved in cell migration, axon guidance, target 539-3p↑ 182-5p↓miR- 3074-5p↓ recognition and synaptic formation (Zhang et al., 2008). We have also Pdzrn3↓ miR-3547-3p↑ Rsc1a1↑ miR-183-5p↓ found some pathways with significant enrichment. Although there are let-7a-1-3p↑miR- few studies showed the relationship between these pathways and ↑ ↓ 133b-3p miR-153- ↑ ↓ neuroscience, DEGs in these pathways have also been found in our Pkib ↑ ↑ Rsph1 miR-3074-5p 3p miR-378d miR- sequencing results. For example, Met gene is enriched in Malaria 532-5p↑ miR-34c-3p↑miR- pathway, which is significantly enriched, is also enriched in Axon Plcxd2↓ Sfi1↑ miR-363-3p↓ 34c-5p↑ guidance pathway. At the same time, we also found that although there miR-1251- is no significant difference in some pathways, the pathways and their ↓ ↑ ↑ ↓ Ptger2 let-7a-1-3p Slc14a2 5p miR-3065- enriched genes are more neurologically related. For example, Neuro 5p↓ Ptgs2↓ let-7a-1-3p↑ Slc26a5↑ miR-124-3p↓ trophin signaling pathway is thought to be related to stress and fear ` miR-133b-3p↑miR- memory (Regue-Guyon et al., 2018), cGMP-PKG signaling pathway is Qrfpr↓ 153-3p↑miR-296- Spef2↑ miR-673-5p↓ related to depression and cognitive deficits (Zhou et al., 2017). 3p↑ We focused on the analysis of susceptible and resilient mRNA and ↓ ↑ ↑ ↓ Rspo2 miR-10a-5p Spink8 miR-183-5p miRNA expression profiles in the nucleus accumbens, which helps to miR- Rspo3↓ miR-133b-3p↑ Tas1r1↑ 341–3p↓miR- discover pathways related to susceptibility and resilience. Research data 363-3p↓ shows that, except for genes in some pathways that are all up-regulated miR-1a-3p↑miR- or all down-regulated. In some pathways, some genes are down- 34c-3p↑miR- miR-1251- Scube1↓ Tmem181c-ps↑ regulated, while other genes in this pathway are up-regulated. The 378d↑miR-539- 5p↓miR-212-5p↓ 3p↑miR-744-5p↑ imbalance of gene expression in these pathways may be related to sus miR-122-5p↑miR- ceptibility and resilience. In addition to the imbalance between genes in 1247-5p↑miR- a single pathway, the up-regulated and down-regulated of multiple Serinc2↓ Tmem254c↑ miR-1251-5p↓ 199a-3p↑miR-296- pathways can also make the entire molecular network in the nucleus ↑ ↑ 3p miR-375-3p accumbens unbalanced. It further could lead to abnormal neuronal and Slc13a4↓ miR-764-3p↑ Tmod4↑ miR-96-5p↓ miR-206-3p↑miR- miR-183-5p↓miR- synaptic functions in the nucleus accumbens, which may lead to stress Slc5a5↓ Tmprss4↑ 378d↑ 341–3p↓ susceptibility or resilience. miR-10a-5p↑miR- Sstr3↓ Togaram2↑ miR-212-5p↓ By analyzing the genes of the two groups of susceptible versus con 34c-5p↑miR-378d↑ trol and resilient versus control, we found that compared with the mice miR-122-5p↑miR- in the control group, there were genes in the same direction of change in 133b-3p↑miR- miR-124-3p↓miR- St6galnac4↓ Trp73↑ 133b-3p↑miR-199a- 182-5p↓ susceptible and resilient mice. Then we speculated that these genes and 3p↑ their translations protein are associated with psychological stress Tmem132c↓ miR-378d↑ Ttc21a↑ miR-1251-5p↓ treatment. This hypothesis is based on the fact that both the susceptible miR-34c-3p↑miR- Tmem215↓ Ttll2↑ miR-363-3p↓ group and the resilient group are experienced psychological stress, but 499-5p↑ Adam21↑ miR-673-5p↓ Vax1↑ let-7i-3p↓ their responses to psychological stress are different. By comparing sus Aire↑ Xaf1↑ miR-212-5p↓ ceptible versus control, resilient versus control with two groups of overlapping altered mRNAs, we found that the genes Adra1d, Bdnf,

14 Y. Lu et al. Pharmacology, Biochemistry and Behavior 199 (2020) 173062

Fig. 7. The validation of differentially expressed miRNA in the nucleus accumbens. (a). susceptible mice versus control mice. (b). susceptible mice versus resilient mice. (c). resilient mice versus control mice. ****p < 0.0001, ***p < 0.001, **p < 0.01, * p < 0.05, in which two-sample t-test was used for the comparisons.

Mapk11, Sstr3, Wnt10a, and Grm8 had higher expression levels in sus The channel mediates the slow inhibition of currents, which play an ceptible and resilient than in the control group. The expression levels of important role in regulating resting potential, neuronal excitability and genes Dnali1, Lbp, Pipox, Trhr, Trp73 and so on in the control group were neurotransmitter release (Li et al., 2019). Kcnj6 plays a role in neuro higher than those in the susceptible and resilient group. These over degenerative diseases such as depression, reward and Down syndrome lapping genes may be related to psychological stress. These changed (Cooper et al., 2012). Chrnb3 is involved in memory and attention, sleep genes are involved in the Glutamatergic synapse, Calcium, Neuro and wakefulness, reward and pain, etc. (Cordero-Erausquin et al., 2000). trophin, Hippo, NF-kappa B pathway and so on. Mice in the susceptible Although no studies have found its role in stress susceptibility and and resilient groups have different performances, but the two groups of resilience, the overexpression of Chrnb3 is related to the susceptibility to mice have experienced psychological stress, and the genes of the two cocaine and alcohol addiction(Haller et al., 2014). groups of mice have the same direction of change when compared with We focus on analyzing the molecular expression profiles of mRNA the control group of mice, which reminds us that psychological stress and miRNA in the nucleus accumbens of mice. The nucleus accumbens is can also cause changes in certain genes. a significantstructure to regulate the emotional response. Some genes in In order to verify the research results, we completed high-throughput the nucleus accumbens may be discoveries related to stress susceptibility sequencing of mRNA and miRNA, the qRT-PCR of some mRNAs and and resilience. For example, some molecules related to adrenergic, miRNAs, and the interaction between mRNA and miRNA. qRT-PCR glutamic, cholinergic, dopaminergic and serotoninergic synapses, and validated the accuracy of the sequencing results of mRNAs and miR molecules related to intracellular cAMP and calcium pathways. There NAs (Figs. 4–7). Analysis of the dual-luciferase reporter assay confirmed fore, it is necessary to study the molecular expression profiles of the the interactions between miR-497a-5p, miR-199a-5p, miR-29b-3p and nucleus accumbens in susceptible and resilient groups, which can help to Bdnf, Chrnb3, Kcnj6, respectively (Fig. 8). understand the role of the nucleus accumbens in susceptibility and Studies have shown that Bdnf (brain-derived neurotrophic factor) is resilience, and can find new targets for the treatment of psychological associated with the susceptibility to social failure in mice. Bdnf can stress disorders. promote fear learning and avoidance behavioral responses to the stress of chronic social failure (Rosenhauer et al., 2019). The expression of CRediT authorship contribution statement Bdnf in nucleus accumbens of mice susceptible to social defeat was significantly up-regulated. (Dong et al., 2017; Wook Koo et al., 2016). Yanjun Lu, Jiuyong Yang, Jinyan Sun contributed to experiments and Importantly, the down-regulation of Bdnf reversed the social avoidance data analysis. Wei Lu and Jin-Hui Wang contributed to concept, project of susceptible mice (Labont´e et al., 2019). Kcnj6 encodes inward rectifier design and paper writing. potassium channels activated by G protein (GIRK2) (Honda et al., 2018).

15 Y. Lu et al. Pharmacology, Biochemistry and Behavior 199 (2020) 173062

Fig. 8. The miRNAs targeted mRNAs were validated by qRT-PCR and Luciferase reporter assay (n = 3). Correlation between miRNAs and its prediction target expression by qRT-PCR in the nucleus accumbens tissue. (a). the correlation between Chrnb3 and miR- 199a-5p (r = 0.9124; p = 0.0030). (b). miR-199a- 5p and Chrnb3 were validated by dual-luciferase re porter assay. (c). The correlation between Bdnf and miR-497a-5p (r = 0.9176; p = 0.0026). (d). miR- 497a-5p and Bdnf were validated by dual-luciferase reporter assay. (e). The correlation between Kcnj6 and miR-29b-3p (r = 0.9883; p < 0.0001). (f). miR- 29b-3p and Kcnj6 were validated by dual-luciferase reporter assay.

Declaration of competing interest Appendix A. Supplementary data

All authors declare no competing interest. All authors have read and Supplementary data to this article can be found online at https://doi. approved the final version of the manuscript. org/10.1016/j.pbb.2020.173062.

Acknowledgement References

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