Profiling of Long Non-coding RNAs and mRNAs by RNA-Sequencing in the Hippocampi of Adult Mice Following Propofol Sedation

Jun Fan, Quan Zhou, Yan Li, Xiuling Song, Jijie Hu, Zaisheng Qin, Jing Tang, Tao Tao, Jun Fan, Quan Zhou, Yan Li, Xiuling Song, Jijie Hu, Zaisheng Qin, Jing Tang, Tao Tao

Abstract

Propofol is a frequently used intravenous anesthetic agent. The impairment caused by propofol on the neural system, especially the hippocampus, has been widely reported. However, the molecular mechanism underlying the effects of propofol on learning and memory functions in the hippocampus is still unclear. In the present study we performed lncRNA and mRNA analysis in the hippocampi of adult mice, after propofol sedation, through RNA-Sequencing (RNA-Seq). A total of 146 differentially expressed lncRNAs and 1103 mRNAs were identified. Bioinformatics analysis, including gene ontology (GO) analysis, pathway analysis and network analysis, were done for the identified dysregulated genes. Pathway analysis indicated that the FoxO signaling pathway played an important role in the effects of propofol on the hippocampus. Finally, four lncRNAs and three proteins were selected from the FoxO-related network for further validation. The up-regulation of lncE230001N04Rik and the down-regulation of lncRP23-430H21.1 and lncB230206L02Rik showed the same fold change tendencies but changes in Gm26532 were not statistically significant in the RNA-Seq results, following propofol sedation. The FoxO pathway-related proteins, PI3K and AKT, are up-regulated in propofol-exposed group. FoxO3a is down-regulated at both mRNA and protein levels. Our study reveals that propofol sedation can influence the expression of lncRNAs and mRNAs in the hippocampus, and bioinformatics analysis have identified key biological processes and pathways associated with propofol sedation. Cumulatively, our results provide a framework for further study on the role of lncRNAs in propofol-induced or -related neurotoxicity, particularly with regards to hippocampus-related dysfunction.

Keywords: RNA-sequencing; hippocampus; long non-coding RNA; neurotoxicity; propofol.

Figures

Figure 1
Figure 1
Experimental design and behavioral readouts of MWM tests. (A). Schematic illustration of the experimental design; (B) The latency of mice to reach the platform, in the two groups. Two-way ANOVA showed that propofol-induced anesthesia increases the escape latency of MWM, when compared with the control group. (*P < 0.05), the Bonferroni test shows that the propofol-treated mice have a longer escape latency compared to that of the mice receiving control treatment, from day 1 to day 5 (#P < 0.05 vs. control group at each day). Results are presented as mean ± standard error (n = 8, *P < 0.05); (C) Number of platform crossings in the probe trial. Results are presented as median and interquartile range (n = 8, *P < 0.05); (D) Representative searching swimming paths of two mice in the probe tests. (Left: Control group, Right: Propofol group).
Figure 2
Figure 2
The comprehensive evaluation and screening of candidate lncRNAs. (A). The main workflow for screening provisional lncRNAs; (B). The box plots of FPKM distribution in different groups, showing no significant differences between the control and propofol groups; (C). The Venn diagram of the coding potential of screened transcripts using CNCI, CPC, PFAM, and phyloCSF, 9587 transcripts were predicted to have no coding potential by all of the four methods; (D) The full length of mRNAs is longer than lncRNAs; (E) The violet plots of expression levels of lncRNAs and mRNAs.
Figure 3
Figure 3
Transcriptome profile of RNA-Seq data distinguishing control and propofol groups. (A) A volcano plot of differentially expressed lncRNAs and mRNAs between control and propofol groups; (B) Unsupervised hierarchical clustering of the expression profiles of differentially expressed lncRNAs in the propofol group, compared with the control group; (C) Unsupervised hierarchical clustering of the expression profiles of differentially expressed mRNA in the propofol group compared with the control group.
Figure 4
Figure 4
The top 10 enrichment scores in gene ontology (GO) enrichment analysis on target genes of differentially expressed lncRNAs. (A) Analysis of the up-regulated lncRNAs; (B) Analysis of the down-regulated lncRNAs. Red bars represent biological process terms; Green bars represent cell component terms; Blue bars represent molecular function terms.
Figure 5
Figure 5
The KEGG pathway enrichment analysis on target genes of differentially expressed lncRNAs. (A,B) The top 10 enrichment scores in the KEGG pathway analysis of the target genes of the up-regulated (A) and down-regulated (B) lncRNAs; (C,D) The network of most enriched pathways of the up-regulated (C) and down-regulated (D) lncRNAs and related genes.
Figure 6
Figure 6
The top 10 enrichment scores in gene ontology (GO) enrichment analysis of differentially expressed mRNAs. (A) Analysis of the up-regulated mRNAs; (B) Analysis of the down-regulated mRNAs; Red bars represent biological process terms; Green bars represent cell component terms; Blue bars represent molecular function terms.
Figure 7
Figure 7
The KEGG pathway enrichment analysis on differentially expressed mRNAs. The top 10 enrichment scores in the KEGG pathway analysis of the up-regulated mRNAs (A) and top 3 down-regulated mRNAs (B).
Figure 8
Figure 8
The visualization of the lncRNA/FoxO gene co-expression network in FoxO pathways. The circular nodes represent the FoxO-genes, and the diamond nodes represent the FoxO-lncRNAs. The black lines show connections between lncRNAs and their target mRNAs; the purple line shows integration of protein-protein interactions and the thicker lines represent the larger combined score.
Figure 9
Figure 9
Validation of selected lncRNAs and proteins in the FoxO pathway. (A) Three of the qRT-PCR-validated lncRNAs (E230001N04Rik, RP23-430H21.1, B230206L02Rik and Gm26532) showed the same fold change patterns as those in the RNA-Seq results. The differences in Gm26532 were not statistically significant. (B) Quantitative analysis of selected proteins in the FoxO pathway that are differentially expressed in the Prop group vs. Con group. The PI3K and AKT are significantly up-regulated, but FoxO3a is decreasing in the Prop group. Data was normalized to the house keeping gene U6 (lncRNA) or GAPDH (mRNA), *P < 0.05.

References

    1. Bernard D., Prasanth K. V., Tripathi V., Colasse S., Nakamura T., Xuan Z., et al. . (2010). A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression. EMBO J. 29, 3082–3093. 10.1038/emboj.2010.199
    1. Briggs J. A., Wolvetang E. J., Mattick J. S., Rinn J. L., Barry G. (2015). Mechanisms of long Non-coding RNAs in mammalian nervous system development, plasticity, disease, and evolution. Neuron 88, 861–877. 10.1016/j.neuron.2015.09.045
    1. Bunch H. (2018). Gene regulation of mammalian long non-coding RNA. Mol. Genet. Genomics 293, 1–15. 10.1007/s00438-017-1370-9
    1. Cabili M. N., Trapnell C., Goff L., Koziol M., Tazon-Vega B., Regev A., et al. . (2011). Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev. 25, 1915–1927. 10.1101/gad.17446611
    1. Chen B., Deng X., Wang B., Liu H. (2016). Etanercept, an inhibitor of TNF-a, prevents propofol-induced neurotoxicity in the developing brain. Int. J. Dev. Neurosci. 55, 91–100. 10.1016/j.ijdevneu.2016.10.002
    1. Deng X., Chen B., Wang B., Zhang J., Liu H. (2017). TNF-alpha mediates the intrinsic and extrinsic pathway in Propofol-Induced neuronal apoptosis via PI3K/Akt signaling pathway in rat prefrontal cortical neurons. Neurotox. Res. 32, 409–419. 10.1007/s12640-017-9751-8
    1. Derrien T., Johnson R., Bussotti G., Tanzer A., Djebali S., Tilgner H., et al. . (2012). The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res. 22, 1775–1789. 10.1101/gr.132159.111
    1. Erasso D. M., Camporesi E. M., Mangar D., Saporta S. (2013). Effects of isoflurane or propofol on postnatal hippocampal neurogenesis in young and aged rats. Brain Res. 1530, 1–12. 10.1016/j.brainres.2013.07.035
    1. Fan J., Zhou Q., Qin Z., Tao T. (2016). Effect of propofol on microRNA expression in rat primary embryonic neural stem cells. BMC Anesthesiol. 16:95. 10.1186/s12871-016-0259-1
    1. Feng T., Shao F., Wu Q., Zhang X., Xu D., Qian K., et al. . (2016). miR-124 downregulation leads to breast cancer progression via LncRNA-MALAT1 regulation and CDK4/E2F1 signal activation. Oncotarget 7, 16205–16216. 10.18632/oncotarget.7578
    1. Gong W., Zheng J., Liu X., Liu Y., Guo J., Gao Y., et al. . (2017). Knockdown of long Non-Coding RNA KCNQ1OT1 restrained glioma cells' malignancy by activating miR-370/CCNE2 axis. Front. Cell. Neurosci. 11:84. 10.3389/fncel.2017.00084
    1. Guttman M., Amit I., Garber M., French C., Lin M. F., Feldser D., et al. . (2009). Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 458, 223–227. 10.1038/nature07672
    1. Guttman M., Garber M., Levin J. Z., Donaghey J., Robinson J., Adiconis X., et al. . (2010). Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nat. Biotechnol. 28, 503–U166. 10.1038/nbt.1633
    1. Hoekman M. F., Jacobs F. M., Smidt M. P., Burbach J. P. (2006). Spatial and temporal expression of FoxO transcription factors in the developing and adult murine brain. Gene Expr. Patterns 6, 134–140. 10.1016/j.modgep.2005.07.003
    1. Hsiao H. T., Wu H., Huang P. C., Tsai Y. C., Liu Y. C. (2016). The effect of propofol and sevoflurane on antioxidants and proinflammatory cytokines in a porcine ischemia-reperfusion model. Acta Anaesthesiol. Taiwan 54, 6–10. 10.1016/j.aat.2015.11.002
    1. Hwangbo D. S., Gersham B., Tu M. P., Palmer M., Tatar M. (2004). Drosophila dFOXO controls lifespan and regulates insulin signalling in brain and fat body. Nature 429, 562–566. 10.1038/nature02549
    1. Jandura A., Krause H. M. (2017). The new RNA world: growing evidence for long noncoding rna functionality. Trends Genet. 33, 665–676. 10.1016/j.tig.2017.08.002
    1. Ji P., Diederichs S., Wang W., Böing S., Metzger R., Schneider P. M., et al. . (2003). MALAT-1, a novel noncoding RNA, and thymosin beta4 predict metastasis and survival in early-stage non-small cell lung cancer. Oncogene 22, 8031–8041. 10.1038/sj.onc.1206928
    1. Jiang Q., Wang Y., Shi X. (2017). Propofol inhibits neurogenesis of rat neural stem cells by upregulating MicroRNA-141-3p. Stem Cells Dev. 26, 189–196. 10.1089/scd.2016.0257
    1. Kennedy L. M., Pham S. C., Grishok A. (2013). Nonautonomous regulation of neuronal migration by insulin signaling, DAF-16/FOXO, and PAK-1. Cell Rep. 4, 996–1009. 10.1016/j.celrep.2013.07.045
    1. Kong L., Zhang Y., Ye Z. Q., Liu X. Q., Zhao S. Q., Wei L., et al. . (2007). CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Res. 35, W345–W349. 10.1093/nar/gkm391
    1. Krzisch M., Sultan S., Sandell J., Demeter K., Vutskits L., Toni N. (2013). Propofol anesthesia impairs the maturation and survival of adult-born hippocampal neurons. Anesthesiology 118, 602–610. 10.1097/ALN.0b013e3182815948
    1. Langfelder P., Horvath S. (2008). WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 9:599. 10.1186/1471-2105-9-559
    1. Li Y., Wu Y., Li R., Wang C., Jia N., Zhao C., et al. . (2015). Propofol regulates the surface expression of GABAA receptors: implications in synaptic inhibition. Anesth. Analg. 121, 1176–1183. 10.1213/ANE.0000000000000884
    1. Lin M. F., Jungreis I., Kellis M. (2011). PhyloCSF: a comparative genomics method to distinguish protein coding and non-coding regions. Bioinformatics 27, I275–I282. 10.1093/bioinformatics/btr209
    1. Liu Y., Yan Y., Inagaki Y., Logan S., Bosnjak Z. J., Bai X. (2017). Insufficient Astrocyte-Derived Brain-Derived neurotrophic factor contributes to Propofol-Induced neuron death through Akt/Glycogen synthase kinase 3beta/Mitochondrial fission pathway. Anesth. Analg. 125, 241–254. 10.1213/ANE.0000000000002137
    1. Lv H. R. (2017). lncRNA-Map2k4 sequesters miR-199a to promote FGF1 expression and spinal cord neuron growth. Biochem. Biophys. Res. Commun. 490, 948–954. 10.1016/j.bbrc.2017.06.145
    1. Medema R. H., Kops G. J., Bos J. L., Burgering B. M. (2000). AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 404, 782–787. 10.1038/35008115
    1. Moseley H., Shankar K. B., Kumar Y., Hallsworth R., Krishnan A. (1988). Propofol: a new intravenous anaesthetic. West Indian Med. J. 37, 229–231.
    1. Nakae J., Kitamura T., Silver D. L., Accili D. (2001). The forkhead transcription factor FoxO1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression. J. Clin. Invest. 108, 1359–1367. 10.1172/JCI200112876
    1. Nie Y., Lu Y. X., Lv L. H. (2015). Effect of propofol on generation of inflammatory mediator of monocytes. Asian Pac. J. Trop. Med. 8, 964–970. 10.1016/j.apjtm.2015.10.008
    1. Paik J. H., Ding Z., Narurkar R., Ramkissoon S., Muller F., Kamoun W. S., et al. . (2009). FoxOs cooperatively regulate diverse pathways governing neural stem cell homeostasis. Cell Stem Cell 5, 540–553. 10.1016/j.stem.2009.09.013
    1. Punta M., Coggill P. C., Eberhardt R. Y., Mistry J., Tate J., Boursnell C., et al. . (2012). The Pfam protein families database. Nucleic Acids Res. 40, D290–D301. 10.1093/nar/gkr1065
    1. Qiao H., Li Y., Xu Z., Li W., Fu Z., Wang Y., et al. . (2017). Propofol affects neurodegeneration and neurogenesis by regulation of autophagy via effects on intracellular calcium homeostasis. Anesthesiology 127, 490–501. 10.1097/ALN.0000000000001730
    1. Rafalski V. A., Brunet A. (2011). Energy metabolism in adult neural stem cell fate. Prog. Neurobiol. 93, 182–203. 10.1016/j.pneurobio.2010.10.007
    1. Ramos A. D., Andersen R. E., Liu S. J., Nowakowski T. J., Hong S. J., Gertz C., et al. . (2015). The long noncoding RNA Pnky regulates neuronal differentiation of embryonic and postnatal neural stem cells. Cell Stem Cell 16, 439–447. 10.1016/j.stem.2015.02.007
    1. Ren K., Xu R., Huang J., Zhao J., Shi W. (2017). Knockdown of long non-coding RNA KCNQ1OT1 depressed chemoresistance to paclitaxel in lung adenocarcinoma. Cancer Chemother. Pharmacol. 80, 243–250. 10.1007/s00280-017-3356-z
    1. Rinn J. L., Chang H. Y. (2012). Genome regulation by long noncoding RNAs. Annu. Rev. Biochem. 81, 145–166. 10.1146/annurev-biochem-051410-092902
    1. Samir A., Gandreti N., Madhere M., Khan A., Brown M., Loomba V. (2015). Anti-inflammatory effects of propofol during cardiopulmonary bypass: a pilot study. Ann. Card. Anaesth. 18, 495–501. 10.4103/0971-9784.166451
    1. Song C., Zhang J., Liu Y., Pan H., Qi H. P., Cao Y. G., et al. . (2016). Construction and analysis of cardiac hypertrophy-associated lncRNA-mRNA network based on competitive endogenous RNA reveal functional lncRNAs in cardiac hypertrophy. Oncotarget 7, 10827–10840. 10.18632/oncotarget.7312
    1. Spéder P., Liu J., Brand A. H. (2011). Nutrient control of neural stem cells. Curr. Opin. Cell Biol. 23, 724–729. 10.1016/j.ceb.2011.08.004
    1. Sun L., Luo H., Bu D., Zhao G., Yu K., Zhang C., et al. . (2013). Utilizing sequence intrinsic composition to classify protein-coding and long non-coding transcripts. Nucleic Acids Res. 41:e166. 10.1093/nar/gkt646
    1. Sun W. C., Liang Z. D., Pei L. (2015). Propofol-induced rno-miR-665 targets BCL2L1 and influences apoptosis in rodent developing hippocampal astrocytes. Neurotoxicology 51, 87–95. 10.1016/j.neuro.2015.08.001
    1. Sun W., Yang Y., Xu C., Guo J. (2017). Regulatory mechanisms of long noncoding RNAs on gene expression in cancers. Cancer Genet. 216–217, 105–110. 10.1016/j.cancergen.2017.06.003
    1. Szklarczyk D., Morris J. H., Cook H., Kuhn M., Wyder S., Simonovic M., et al. . (2017). The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res. 45, D362–D368. 10.1093/nar/gkw937
    1. Tan M. C., Widagdo J., Chau Y. Q., Zhu T., Wong J. J., Cheung A., et al. . (2017). The Activity-Induced long Non-Coding RNA Meg3 modulates AMPA receptor surface expression in primary cortical neurons. Front. Cell. Neurosci. 11:124. 10.3389/fncel.2017.00124
    1. Trapnell C., Roberts A., Goff L., Pertea G., Kim D., Kelley D. R., et al. . (2012). Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578. 10.1038/nprot.2012.016
    1. Twaroski D. M., Yan Y., Olson J. M., Bosnjak Z. J., Bai X. (2014). Down-regulation of MicroRNA-21 is involved in the Propofol-induced neurotoxicity observed in human stem Cell-derived neurons. Anesthesiology 121, 786–800. 10.1097/ALN.0000000000000345
    1. Twaroski D. M., Yan Y., Zaja I., Clark E., Bosnjak Z. J., Bai X. (2015). Altered mitochondrial dynamics contributes to Propofol-induced cell death in human stem Cell-derived neurons. Anesthesiology 123, 1067–1083. 10.1097/ALN.0000000000000857
    1. Ucar M., Ozgül U., Polat A., Toprak H. I., Erdogan M. A., Aydogan M. S., et al. . (2015). Comparison of antioxidant effects of isoflurane and propofol in patients undergoing donor hepatectomy. Transplant. Proc. 47, 469–472. 10.1016/j.transproceed.2014.11.043
    1. Velly L. J., Guillet B. A., Masmejean F. M., Nieoullon A. L., Bruder N. J., Gouin F. M., et al. . (2003). Neuroprotective effects of propofol in a model of ischemic cortical cell cultures: role of glutamate and its transporters. Anesthesiology 99, 368–375. 10.1097/00000542-200308000-00018
    1. Vutskits L., Gascon E., Tassonyi E., Kiss J. Z. (2005). Clinically relevant concentrations of propofol but not midazolam alter in vitro dendritic development of isolated gamma-aminobutyric acid-positive interneurons. Anesthesiology 102, 970–976. 10.1097/00000542-200505000-00016
    1. Wang J., Ma R., Ma W., Chen J., Yang J., Xi Y., et al. . (2016). LncDisease: a sequence based bioinformatics tool for predicting lncRNA-disease associations. Nucleic Acids Res. 44:e90. 10.1093/nar/gkw093
    1. Wang W., Lu R., Feng D. Y., Liang L. R., Liu B., Zhang H. (2015). Inhibition of microglial activation contributes to propofol-induced protection against post-cardiac arrest brain injury in rats. J. Neurochem. 134, 892–903. 10.1111/jnc.13179
    1. Yan Y., Qiao S., Kikuchi C., Zaja I., Logan S., Jiang C., et al. . (2017). Propofol induces apoptosis of neurons but not astrocytes, oligodendrocytes, or neural stem cells in the neonatal mouse hippocampus. Brain Sci. 7:130. 10.3390/brainsci7100130
    1. Zhang D., Zhou X. H., Zhang J., Zhou Y. X., Ying J., Wu G. Q., et al. . (2015). Propofol promotes cell apoptosis via inhibiting HOTAIR mediated mTOR pathway in cervical cancer. Biochem. Biophys. Res. Commun. 468, 561–567. 10.1016/j.bbrc.2015.10.129
    1. Zhao J., Li L., Peng L. (2015). MAPK1 up-regulates the expression of MALAT1 to promote the proliferation of cardiomyocytes through PI3K/AKT signaling pathway. Int. J. Clin. Exp. Pathol. 8, 15947–15953.
    1. Zhong L., Luo F., Zhao W., Feng Y., Wu L., Lin J., et al. . (2016). Propofol exposure during late stages of pregnancy impairs learning and memory in rat offspring via the BDNF-TrkB signalling pathway. J. Cell. Mol. Med. 20, 1920–1931. 10.1111/jcmm.12884

Source: PubMed

Szponzorok és közreműködők

Egészségi állapot

Kábítószer-beavatkozások

3
Iratkozz fel