Identification of the Potential Key Long Non-coding RNAs in Aged Mice With Postoperative Cognitive Dysfunction

Abstract

Postoperative cognitive dysfunction (POCD) is a significant complication of surgery, particularly in elderly patients. Emerging researches showed that long non-coding RNA (lncRNA) may play a vital role in the pathogenesis of POCD. Here we aimed to identify potential key lncRNAs involved in the development of POCD. LncRNA and mRNA expression profiles in hippocampal tissues from POCD and control mice were analyzed by microarray assay. Gene ontology (GO) and KEGG pathway enrichment analyses were conducted to probe the functions of dysregulated genes. Then, important factors of the mainly affected biological processes were measured in the hippocampus. Correlated coding-non-coding co-expression (CNC) networks were constructed. Finally, the potential key pairs of lncRNA and target mRNA implicated in POCD were probed. Our data showed that 868 differentially expressed lncRNAs and 690 differentially expressed mRNAs were identified in total. GO and KEGG analyses indicated that the differentially expressed genes were mainly associated with inflammatory and apoptotic signaling pathways. Surgery-induced inflammatory cytokines and apoptosis were significantly increased in hippocampal tissues of aged mice. In CNC network analysis, we found that LncRNA uc009qbj.1 was positively correlated with apoptosis-associated gene Vrk2 level. LncRNA ENSMUST00000174338 correlated positively with expression of the inflammation and apoptosis-associated gene Smad7. LncRNA NONMMUT00000123687 mediated gene expression by binding the inflammation-regulated transcription factor Meis2. Our results suggested that these potential key lncRNAs and mRNAs may play a crucial role in the development of POCD through mediating neuronal inflammation or apoptosis.

Keywords: aging; apoptosis; inflammation; long non-coding RNA; microarray; postoperative cognitive dysfunction.

FIGURE 1

Surgical trauma induces anxiety-like behavior in aged mice. The time in the central area was taken as measures of anxiety and exploratory behavior in the open-field test. (A) Mean velocity. (B) Total travel distance. (C) Rearing activity. (D) Entries into the center. (E) % central area. Data are presented as means ± SEM; ^*P < 0.05, vs. Control, Student’s t-test, N = 10 for all analyses.

FIGURE 2

Unilateral nephrectomy impairs associative cognitive function on day 3 post-surgery. (A) Fear conditioning test (FCT) design. (B) FCT workflow. Blue arrows indicate sound stimuli and red arrows indicate electric shock stimuli. (C) Baseline freezing levels. (D,E) Freezing levels in the contextual test on day 1 and day 3 post-surgery. (F,G) Freezing levels in the cued tone test on day 1 and day 3 post-surgery. Data are presented as means ± SEM; ^∗∗∗ P < 0.001, vs. Control, Student’s t-test, N = 12 for all analyses.

FIGURE 3

LncRNA and mRNA expression profiles. (A) The scatterplots of lncRNA expression profiles. (B) The scatterplots of mRNA expression profiles. The scatterplots of lncRNA and mRNA were used to assess the lncRNA and mRNA expression variations between the surgery group and the control group. The green lines represent the fold change (the default value is 2 times). (C) Subgroup analysis of differentially expressed lncRNAs was based on their gene mapping and the relationships between adjacent protein-encoding genes. (D) Relative expression of five lncRNAs. (E) Relative expression of five mRNAs. Data are presented as means ± SEM; ^*P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, vs. Control, Student’s t-test, N = 10 for all analyses.

FIGURE 4

KEGG pathway enrichment analysis is a functional analysis for mRNAs. The P-value (EASE-score, Fisher-P-value or Hypergeometric-P-value) indicates the significance of the pathway correlated to the conditions. The lower the P-value, the more significant the pathway is (The recommended P-value cut-off is 0.05). (A) Pathways involved in up-regulated transcripts. (B) Pathways involved in down-regulated transcripts.

FIGURE 5

Expression of inflammatory cytokines and neuronal apoptosis were significantly enhanced in hippocampal tissue after surgery. Hippocampal tissues were harvested on day 3 after surgery from the two groups. (A–E) mRNA expression of CXCL1 (A), CXCL2 (B), CCL2 (C), TNF-α (D) and IL-1β (E). Proinflammatory cytokine expression was measured by qRT-PCR. (F) Representative images of TUNEL assay in the CA1 of the hippocampus. Scale bar, 20 μm. (G) Quantitative analysis of TUNEL-positive cells. Red arrows point to representative TUNEL-positive cells. The cells were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI). (H) Protein levels of cleaved caspase-3. (I) Quantification of protein levels of cleaved caspase-3. Data are presented as means ± SEM; ^∗∗P < 0.01, ^∗∗∗ P < 0.001, vs. Control, Student’s t-test, N = 6 for all analyses.

FIGURE 6

Co-expression networks of third lncRNAs with associated mRNAs. Co-expressed lncRNA-mRNA pairs were identified through strict screening criteria (correlation coefficients > 0.95 or < –0.95, P < 0.01). The differentially expressed lncRNAs were predicted to function by regulating the closely related mRNA. Red circles represent lncRNAs, and green circles denote mRNAs. Solid and dotted lines represent positive correlation and negative correlation, respectively.