LncRNA‐mRNA competing endogenous RNA network depicts transcriptional regulation in ischaemia reperfusion injury

Abstract The study aimed to investigate time‐course transcriptomes in myocardial ischaemia reperfusion injury (IRI) via RNA‐Seq. Transcriptomes of 10 samples derived from patients with acute ST‐segment elevation myocardial infarction (ASTEMI) who were assigned to percutaneous coronary intervention (PCI), were sequenced at the time of 0 (before PCI), 2, 12, 24 and 72 hours after PCI, respectively. Using the genefilter package in r, wgcna and stem, different expression lncRNA (DEL) and mRNA (DEM) were analysed. Out of 756 mRNAs and 206 lncRNAs shared by enrolled patients, 135 RNAs were screened to be significantly associated with the IRI. Furthermore, combined with lncRNA‐mRNA, lncRNA‐miRNA and miRNA‐mRNA network, 51 RNAs and 131 relationship pairs were ascertained in the competing endogenous RNAs (ceRNA) network. Among these nodes, SH2D3C and GTF2H4 were significantly enriched in cellular response to stress and their interaction module were isolated from functional ceRNA network. Subsequently, their critical role was confirmed via down‐regulation of SH2D3C and GTF2H4 expression in vitro model. These results identified that lncRNA‐mRNA ceRNA network, associated significantly with IRI, functioned as critical regulative pivotal roles after PCI‐AMI, and SH2D3C and GTF2H4 may be the most responsive transcriptional regulator in the early‐phase of IRI.


| MATERIAL S AND ME THODS
In the current study, PAXgene Blood RNA tubes (BD, San Jose, CA, USA) were used to store the peripheral blood samples of patients To obtain genes associated with IRI, 206 DELs and 756 DEMs were subjected to further analyses. When co-efficient R 2 = 0.9, power = 6 was obtained and utilized for the CV calculation ( Figure 1A). The cluster tree was constructed with thresholds of gene number ≥ 20 and CutHeight = 0.95, according to the criteria of hybrid dynamic shear tree ( Figure 1B). Then, the co-efficient between model and IRI was around 0.8 and the significance between whole RNAs and IRI at detected points was 0.0099 ( Figure 1C, Table   S1); with P < 0.05, 6 of time-point significantly associated models were identified using STEM, including 48, 32, 66, 39, 44 and 54 RNAs respectively ( Figure 1D).
About 18 997 lncRNA-mRNA pairs and 105 188 RNA expression associated pairs were combined to select the overlapped lncRNA-mRNA pairs; 42 pairs were selected to construct the lncRNA-mRNA regulatory network (Figure 2A). According to miRcode and starBase databases, the lncRNA-miRNA and miRNA-mRNA regulatory networks were combined to construct the ceRNA regulatory network ( Figure 2B). Furthermore, functional enrichment analyses of mRNA involved in ceRNA network were deeply analysed. Only SH2D3C and GTF2H4 were found to significantly (P < 0.05) enrich in GO_BP: GO:0033554 ~ cellular response to stress ( Figure 2C). The module contained 16 nodes: two lncRNAs, two mRNAs, 12 miRNAs, and formed 22 edges: two lncRNA-mRNA pairs, six lncRNA-miRNA pairs and 14 miRNA-mRNA pairs. The peak was found at 12 hours after PCI ( Figure 2D). Especially, both SH2D3C and RP11-203J24.9 presented negative correlations with IRI and could be targeted by hsa-miR-15a and hsa-miR-214; GTF2H4 and LINC00243 presented positive correlations with IRI and could be regulated by hsa-miR-10a, hsa-miR-122 and hsa-miR-150. To demonstrate the critical roles of SH2D3C and GTF2H4 in IRI, we did experiments in in vitro model (hypoxia/reoxygenation injury) to provide further evidence by down-regulating SH2D3C and GTF2H4 expression.
The results showed that the viability of cardiac microvascular endothelial cells was significantly reduced in the process of hypoxia/ reoxygenation injury and in the presence of si-SH2D3C and si-GTF2H4 ( Figure 2E-G).

| CON CLUS ION
Our results identified that lncRNA-mRNA ceRNA network, associated significantly with IRI, functioned as critical regulative pivotal roles after PCI-AMI and SH2D3C and GTF2H4 could F I G U R E 1 Identification of ischaemia reperfusion injury (IRI)-associated RNAs and models. A, Result of power value selection. X axis represented the power value and Y axis represented the square of log(k) and log(p(k)). The red line represented the standard line that indicated square value = 0.9. B, Clustering of IRI-associated RNAs. C, IRI-associated modules. D, Clustering of RNAs varied with time tendency. Different squares indicated clusters. Number in top left corner represented the gene number, and in the left bottom represented the clustered P value. Table: models and RNAs associated with IRI be the most responsive transcriptional regulators in the earlyphase of IRI.

CO N FLI C T S O F I NTE R E S T S
The authors declare that there is no conflict of interests regarding the publication of this paper.