MicroRNA expression in the hippocampal CA1 region under deep hypothermic circulatory arrest

Abstract

Using deep hypothermic circulatory arrest, thoracic aorta diseases and complex heart diseases can be subjected to corrective procedures. However, mechanisms underlying brain protection during deep hypothermic circulatory arrest are unclear. After piglet models underwent 60 minutes of deep hypothermic circulatory arrest at 14°C, expression of microRNAs (miRNAs) was analyzed in the hippocampus by microarray. Subsequently, TargetScan 6.2, RNA22 v2.0, miRWalk 2.0, and miRanda were used to predict potential targets, and gene ontology enrichment analysis was carried out to identify functional pathways involved. Quantitative reverse transcription-polymerase chain reaction was conducted to verify miRNA changes. Deep hypothermic circulatory arrest altered the expression of 35 miRNAs. Twenty-two miRNAs were significantly downregulated and thirteen miRNAs were significantly upregulated in the hippocampus after deep hypothermic circulatory arrest. Six out of eight targets among the differentially expressed miRNAs were enriched for neuronal projection (cyclin dependent kinase, CDK16 and SLC1A2), central nervous system development (FOXO3, TYRO3, and SLC1A2), ion transmembrane transporter activity (ATP2B2 and SLC1A2), and interleukin-6 receptor binding (IL6R) - these are the key functional pathways involved in cerebral protection during deep hypothermic circulatory arrest. Quantitative reverse transcription-polymerase chain reaction confirmed the results of microarray analysis. Our experimental results illustrate a new role for transcriptional regulation in deep hypothermic circulatory arrest, and provide significant insight for the development of miRNAs to treat brain injuries. All procedures were approved by the Animal Care Committee of Xuanwu Hospital, Capital Medical University, China on March 1, 2017 (approval No. XW-INI-AD2017-0112).

Keywords: bioinformatics; cerebral protection; deep hypothermic circulatory arrest; gene ontology enrichment analysis; hippocampus; microRNA; microarray; nerve regeneration; neural regeneration; post-transcriptional expression.

Figure 1

Manipulation procedure for deep hypothermic circulatory arrest (DHCA) with cardiopulmonary bypass (CPB). min: Minutes; Preop.: pre-operation.

Figure 2

Hierarchical clustering of differentially expressed miRNAs in the hippocampus of sham and DHCA-treated piglets. Each row represents one miRNA with significantly different expression between sham and DHCA groups (P < 0.01). Each column represents a biological replicate; in each panel, the left three columns represent sham piglets, while the right three represent DHCA-treated piglets. Colors represent the expression of each miRNA: red, upregulation; green, downregulation. DHCA: Deep hypothermic circulatory arrest.

Figure 3

Density plots of Pearson correlation coefficients of predicted miRNA-mRNA pairs. (A–C) For each miRNA that was significantly expressed, the correlation between expression of miRNA and predicted target mRNAs was evaluated in six samples by calculating the Pearson correlation coefficient. This was repeated for all predicted target mRNAs of the corresponding miRNA; the distribution of these coefficients (red line) is shown in the density plot. The control curve (blue line) was used to illustrate the distribution of an equal number of coefficients, whereby each coefficient was obtained from the correlation of miRNA expression and expression of a randomly non-predicted mRNA. Compared with the control curve, a left shift of the red curve indicates that predicted mRNA targets are significantly more inversely correlated with that miRNA. Only the identified miRNAs exhibiting a significant left shift (Student’s t-test P < 0.05) are shown: miR-194 (A), miR-23a^* (B), and miR-27a^* (C). The X axis represents r, the Pearson correlation coefficient.

Figure 4

Network of inverse correlation between each differentially expressed miRNA and its putative mRNA targets. Red represents differentially expressed miRNA. Green represents inversely correlated mRNA targets of each miRNA. Yellow represents anti-correlated mRNAs shared among differentially expressed miRNAs.

Figure 5

Select miRNA-mRNA pairs with strong inverse correlation. (A) miR-194-SLC1A2 (r = –0.79, P = 0.034); (B) miR-122-FOXO3 (r = –0.84, P = 0.018); (C) miR-122-SMU1 (r = –0.64, P = 0.12); (D) miR-10b-IL6R (r = –0.72, P = 0.066); (E) miR-10b-FOXO3 (r = –0.74, P = 0.05); (F) miR-10b-SMU1 (r = –0.73, P = 0.06).

Figure 6

Inverse correlation of each differentially expressed miRNA with expression of its target mRNA. (A) miR-23a^*-IPPK (r = –0.75, P = 0.05); (B) miR-23a^*-TYRO3 (r = –0.67, P = 0.099); (C) miR-23a^*-CDK16 (r = –0.74, P = 0.05); (D) miR-23a^*-SLC1A2 (r = –0.62, P = 0.13); (E) miR-27a^*-ATP2B2 (r = –0.60, P = 0.15); (F) miR-27a^*-TYRO3 (r = –0.66, P = 0.11); (G) miR-27a^*-IPPK (r = –0.67, P = 0.0994); (H) miR-27a^*-SLC1A2 (r = –0.86, P = 0.013); (I) miR-23a^*-ATP2B2 (r = –0.78, P = 0.039); (J) miR-27a^*-CDK16 (r = –0.69, P = 0.089); (K) miR-135a^*-IL6R (r = –0.78, P = 0.04). IPPK: Inositol-pentakisphosphate 2-kinase; CDK: cyclin dependent kinase.

Figure 7

Validation of miRNA microarray data by quantitative reverse transcription-polymerase chain reaction. Relative expression of six miRNAs was normalized to the expression of an internal control (U6) using 2^–ΔΔCT to calculate the difference in expression. P values were calculated using a two-sided Student’s t-test. *P < 0.05, **P < 0.01, vs. sham group. Temporal expression profiles of selected regulated miRNAs as determined by quantitative reverse transcription-polymerase chain reaction analysis of hippocampal RNA. miRNAs, such as miR-194, -200c, -10b, and -122, were selected from the panel of downregulated miRNAs; and miR-27a^* and -23a^* were selected from the panel of upregulated miRNAs to confirm the findings of microarray and bioinformatic analyses. Data represent changes in threshold cycle between DHCA and sham groups (ΔCt = Ct_sham−Ct_DHCA; n = 3). DHCA: Deep hypothermic circulatory arrest.

Figure 8

Functional enrichment analysis of 255 inversely correlated target mRNAs of selected miRNAs. Red vertical line represents Bonferroni correlated P = 0.05.