Fluoxetine ameliorates depressive symptoms by regulating lncRNA expression in the mouse hippocampus
摘要: 抑郁症是一种与衰老相关的精神障碍疾病，可导致患者发病率和死亡率的增加。目前临床上具有显著抑郁症状的老年抑郁症患者的发病率也在逐年增加。最近的研究表明，在抑郁症发病期间，大脑中长链非编码RNA（lncRNA）表达的改变会影响神经发育，并对抑郁症具有调节功能。但是大多数lncRNA的功能尚未被研究。在本研究中，我们利用慢性不可预知温和应激（CUMS）抑郁小鼠模型，用抗抑郁药氟西汀进行6周干预后，分析了在抑郁样行为小鼠模型以及氟西汀干预后的CUMS小鼠海马组织中lncRNA的表达水平的差异。结果发现，与正常小鼠相比，CUMS诱导的小鼠海马组织中共有282个lncRNA差异表达，其中134个lncRNA在CUMS抑郁样小鼠中表达上调，148个lncRNA在CUMS抑郁样小鼠中表达下调）（P<0.05）。在氟西汀干预后，与CUMS抑郁样小鼠相比，我们发现370个lncRNA呈现差异表达。我们进一步对这些差异表达的lncRNA进行GO功能分析发现，差异表达的lncRNA与蛋白结合，氧结合和转运活性之间存在关联。通过基因和基因组百科全书（KEGG）信号通路分析表明，失调的lncRNA可能参与了炎症反应途径。氟西汀可通过调节海马中lncRNA的表达来有效缓解CUMS诱导的小鼠抑郁症状。本文的发现为老年抑郁症发生的潜在机制提供了有价值的见解。Abstract: Depression is a prevalent mental disorder that is associated with aging and contributes to increased mortality and morbidity. The overall prevalence of geriatric depression with clinically significant symptoms is currently on the rise. Recent studies have demonstrated that altered expressions of long non-coding RNAs (lncRNAs) in the brain affect neurodevelopment and manifest modulating functions during the depression. However, most lncRNAs have not yet been studied. Herein, we analyzed the transcriptome of dysregulated lncRNAs to reveal their expressions in a mouse model exhibiting depressive-like behaviors, as well as their corresponding response following antidepressant fluoxetine treatment. A chronic unpredictable mild stress (CUMS) mouse model was applied. A six-week fluoxetine intervention in CUMS-induced mice attenuated depressive-like behaviors. In addition, differential expression analysis of lncRNAs was performed following RNA-sequencing. A total of 282 lncRNAs (134 up-regulated and 148 down-regulated) were differentially expressed in CUMS-induced mice relative to non-stressed counterparts (P<0.05). Moreover, 370 differentially expressed lncRNAs were identified in CUMS-induced mice after fluoxetine intervention. Gene Ontology (GO) analyses showed an association between significantly dysregulated lncRNAs and protein binding, oxygen binding, and transport activity, while the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that these dysregulated lncRNAs might be involved in inflammatory response pathways. Fluoxetine effectively ameliorated the symptoms of depression in CUMS-induced mice by regulating the expression of lncRNAs in the hippocampus. The findings herein provide valuable insights into the potential mechanism underlying depression in elderly people.
Figure 1. Behavioral assessments and the schematic diagram of the indcution process of depressive-like hehaviors in mice
In the SPT assessment (A), mice exposed to CUMS displayed decreased sucrose preference when compared to NOR mice; these effects were ameliorated after fluoxetine treatment. In the FST (B) and TST (C) assessments, immobility time was longer in the CUMS group than in the FLU and the NOR groups. * P-value<0.001. D: Schematic diagram showing the induction process of depressive-like behaviors in mice. Changes in the expression of 5-HT (E) and BDNF (F) in samples from CUMS, FLU, and NOR groups (n=4 per group). A significant decrease in 5-HT concentration was observed in the CUMS group compared to the NOR group. 5-HT concentration was increased after fluoxetine treatment (E). BDNF concentration increased after fluoxetine treatment in the FLU group compared to the CUMS group, in which it was decreased (F). Data indicate mean±SEM. *P-value<0.05. NOR: Normal control group; CUMS: CUMS induced depression group; FLU: Fluoxetine-treated group; SPT: Sucrose preference test; FST: Forced swim test; TST: Tail suspension test.
Figure 2. Transcriptome-wide identification and characterization of lncRNAs
A: A total of 8 281 lncRNAs were identified using the CNCI, CPC2, Pfam Scan, and PhyloCSF tools. B: Identified lncRNAs were mainly classified as lincRNA, anti-sense lncRNA, and intronic lncRNA. C: Conservation comparison of the identified lncRNAs, mRNAs, and predicted novel lncRNAs. D, E: The exon number (D) and length (E) of lncRNAs identified were comparatively shorter than mRNA transcripts. F: Length of ORF in most identified lncRNAs was less than 300 bp.
Figure 3. Differentially expressed lncRNAs (DElnRNAs) comparison between CUMS vs. NOR groups and FLU vs. CUMS groups
A, B: Volcano map of 282 and 370 differentially regulated lncRNAs based on a group-wise comparison of CUMS vs. NOR groups (A), and FLU vs. CUMS groups (B), respectively (P-value<0.05). C: Venn map showing 95 commonly expressed lncRNAs identified in a comparison between FLU vs. CUMS groups and CUMS vs. NOR groups (P-value<0.05). D: Three lncRNAs (LNC_004212, LNC_001203, and ENSMUST00000148687.1) with contrasting expression patterns between FLU and CUMS groups vs. CUMS and NOR groups based on RNA-seq analysis (Q-value<0.05).
Figure 4. Chromosomal distribution and hierarchical clustering analysis of DElnRNAs in CUMS, FLU and NOR mice
A: Distribution of DElnRNAs on all chromosomes in CUMS vs. NOR groups. Among the 282 DElnRNAs, the 134 up-regulated and 148 down-regulated lncRNAs were enriched on chromosome 1, 2, 7, 9, 10, 11 and 2, 4, 5, 8, 10, 12, 15, respectively. B: Distribution of DElnRNAs on all chromosomes in FLU vs. CUMS groups. After fluoxetine treatment, the 236 up-regulated and 134 down-regulated lncRNAs mapped to chromosomes 4, 6, 10, 11, 17 and 2, 7, 9, 11, 17, respectively. P-value<0.05. C: Hierarchical clustering showing that lncRNAs differentially expressed (P-value<0.05) in FLU vs. CUMS and CUMS vs. NOR groups were clustered into four main categories. DElnRNAs in the same category may participate in similar processes resulting in depressive-like behaviors. Samples from the same group were clustered together, indicating minimal variation in samples from the same group.
Figure 5. GO analysis of putative target genes
A: GO analysis of mRNAs co-localized with DElnRNAs between FLU and CUMS mice. A total of 154 co-localized differentially expressed transcripts were found to be enriched with the GO terms of organismal development, intracellular part, organelle, and macromolecular complex binding when comparing FLU and CUMS groups. B: GO analysis of mRNAs co-expressed with DElnRNAs between FLU and CUMS mice. A total of 679 co-expressed transcripts indicated an association with the GO terms of response to stimulus, intracellular part, organelle or cytoplasm of the cell, and protein binding. C, D: GO annotation of mRNA targets co-localized with 48 DElnRNAs which were up-regulated in CUMS-induced mice compared to NOR mice, and down-regulated in FLU treated compared to CUMS mice. These 48 lncRNA transcripts were predicted to be associated with oxygen binding and transport activity. E: Validation of lncRNA expressions in the hippocampus of CUMS, FLU, and NOR mice by qRT-PCR. Six of eight randomly selected lncRNAs were up-regulated, whereas Tmem134 and LNC_004971 were down-regulated in CUMS mice compared to NOR controls. Up-regulated lncRNAs in CUMS mice compared to NOR controls were down-regulated after fluoxetine treatment, thereby validating the RNA-seq results.
Table 1. qRT-PCR primers used for validation of lncRNA expression identified from RNA-seq
lncRNA transcript ID Transcript symbol name Forward primer sequence (5'–3') Reverse primer sequence (5'–3') PCR product size (bp) LNC_007274 – CTTGGTCAGAAGCATCTGGA AAGAACAGGCTTCGAGAACG 273 ENSMUST00000148687.1 Gm16638 ATCCAGCAGACAGCACTATG GTTGCCTCTGTGTTCAGAAG 278 LNC_001203 – CACAGTTGCTTCTAAGCCAG ATACAGAGAGCGCAGCATTC 259 LNC_004971 – TGGTGACATTCTTCAGCTCC ATTGCAGAAGAGGCCGATAG 145 ENSMUST00000139987.8 Tmem134 GATCTTCATCTACTGCGCTG CAGGTGTAGGTTGGAGGAAT 159 ENSMUST00000182520.1 Gm26917 AATGGTGCTACCGGTCATTC ACACCTCTCTTATCCGCTCT 194 ENSMUST00000152663.7 4933431E20Rik GACTCAGATCCATCCGTTCA CAGTGTCTGGACCTGTTCAT 280 ENSMUST00000174808.1 Malat1 CCTTCCTGTGTGGCAAGAAT CTGCAAGCACAACTTGAGGT 223 –: Not available.
Table 2. DElnRNAs between CUMS-induced mice and normal controls (adjusted P-value <0.05)
LncRNA transcript_id CUMS/NOR fold change (log2 ratio) P-value Q-value LNC_001649 –13.699 2 8.93E–05 0.029 085 LNC_002578 –13.348 5 0.000102 0.031 665 LNC_007352 –10.429 3 3.50E–05 0.014 861 LNC_001657 –9.480 77 6.31E–05 0.022 996 LNC_007186 –8.750 24 8.35E–07 0.000 832 LNC_004212 –8.231 64 3.01E–06 0.002 228 LNC_003004 –7.265 23 0.00018 0.045 952 LNC_002630 –7.001 84 0.000174 0.044 864 LNC_003171 –5.972 24 0.000118 0.034 934 LNC_001871 3.741 192 5.21E–05 0.020 057 LNC_002576 5.643 55 6.22E–06 0.004 041 LNC_005505 7.062 69 0.000196 0.048 071 LNC_000648 7.296 817 9.84E–05 0.030 782 LNC_002056 7.889 765 1.61E–05 0.008 33 ENSMUST00000139987.8 7.965 873 0.000105 0.032 184 LNC_001203 8.904 771 3.63E–05 0.015 174 ENSMUST00000148687.1 8.997 071 1.36E–05 0.007 63 LNC_003699 9.458 857 5.65E–05 0.021 329 LNC_007164 10.498 72 1.48E–05 0.007 939
Table 3. DELnRNAs between fluoxetine-treated CUMS-induced mice and CUMS-induced mice (adjusted P-value <0.05)
LncRNA transcript_id FLU/CUMS fold change (log2 ratio) P-value Q-value LNC_000804 –12.589 2 7.49E–16 2.95E–12 LNC_004168 –11.758 1 2.41E–12 5.29E–09 LNC_007274 –10.935 3 4.54E–06 0.001 971 LNC_005854 –10.426 8 1.12E–05 0.003 987 ENSMUST00000148687.1 –9.016 46 1.63E–05 0.005 382 LNC_004971 –8.843 16 0.000 204 0.036 612 LNC_004518 –7.637 87 7.27E–05 0.017 453 LNC_001203 –7.069 03 0.000 151 0.029 595 LNC_001138 3.050 837 3.00E–08 3.39E–05 LNC_004347 3.307 226 3.81E–06 0.001 737 ENSMUST00000182520.1 5.833 658 1.36E–23 2.54E–19 LNC_007241 6.265 049 4.08E–05 0.011 235 LNC_003126 7.047 628 0.000 264 0.043 714 LNC_002186 7.188 645 0.000 182 0.034 032 LNC_003822 7.450 247 0.000 115 0.02 4392 LNC_007358 7.570 922 0.000 133 0.027 026 LNC_004212 7.790 258 4.21E–05 0.011 393 LNC_003821 8.000 836 1.37E–05 0.004 665 LNC_000872 8.030 34 7.94E–06 0.00 313 LNC_001392 8.045 196 0.000 21 0.037 484 LNC_001105 8.478 254 3.56E–11 7.19E–08 LNC_000238 8.580 248 0.000 309 0.048 759 LNC_002941 8.760 41 0.000 251 0.042 275 LNC_007536 9.109 974 0.000 196 0.035 421 ENSMUST00000123998.1 10.423 95 1.09E–05 0.003 939 LNC_003023 11.197 15 2.82E–06 0.001 406 LNC_000806 13.241 01 4.17E–05 0.011 379 ENSMUST00000174808.1 16.987 89 3.93E–05 0.010 935
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ZR-2020-294 Supplementary Tables and Figures.pdf