Comparison Analysis of Dysregulated LncRNA Profile in Mouse Plasma and Liver after Hepatic Ischemia/Reperfusion Injury

Abstract

Long noncoding RNAs (LncRNAs) have been believed to be the major transcripts in various tissues and organs, and may play important roles in regulation of many biological processes. The current study determined the LncRNA profile in mouse plasma after liver ischemia/reperfusion injury (IRI) using microarray technology. Microarray assays revealed that 64 LncRNAs were upregulated, and 244 LncRNAs were downregulated in the plasma of liver IRI mouse. Among these dysregulated plasma LncRNAs, 59-61% were intergenic, 22-25% were antisense overlap, 8-12% were sense overlap and 6-7% were bidirectional. Ten dysregulated plasma LncRNAs were validated by quantitative PCR assays, confirming the accuracy of microarray analysis result. Comparison analysis between dysregulated plasma and liver LncRNA profile after liver IRI revealed that among the 308 dysregulated plasma LncRNAs, 245 LncRNAs were present in the liver, but remained unchanged. In contrast, among the 98 dysregulated liver LncRNAs after IRI, only 19 were present in the plasma, but remained unchanged. LncRNA AK139328 had been previously reported to be upregulated in the liver after IRI, and silencing of hepatic AK139328 ameliorated liver IRI. Both microarray and RT-PCR analyses failed to detect the presence of AK139328 in mouse plasma. In summary, the current study compared the difference between dysregulated LncRNA profile in mouse plasma and liver after liver IRI, and suggested that a group of dysregulated plasma LncRNAs have the potential of becoming novel biomarkers for evaluation of ischemic liver injury.

Fig 1. Genomic loci of the plasma LncRNAs in mouse chromosomes.

The loci of LncRNAs in mouse chromosomes has been indicated in blue. The increased and decreased LncRNAs had been marked in red and green, respectively. The bars represent the folds of LncRNAs expression change. The numbers and symbols represent chromosome numbers. Please refer to GEO accession number GSE60726 for the detailed information of all plasma LncRNAs.

Fig 2. Dysregulated LncRNA profile in the plasma of I/R-treated mouse.

Dysregulated LncRNAs in mouse plasma were identified by microarray assays as described in the methodology section. The threshold for up-regulation was fold change≥1.5 and for down-regulation, fold change≤0.7. Increased LncRNAs was presented in panel A, whereas decreased LncRNAs presented in panel B. The detailed information of the dysregulated LncRNAs was referred to S2 and S3 Tables.

Fig 3. Quantitative assays of increased LncRNAs in I/R-treated mouse plasma.

A-E) Five upregulated LncRNAs with most meaningful elevation in expression fold picked up by microarray assays were further analyzed and confirmed by quantitative RT-PCR assay. In panel A-D, the data were normalized to sham group. In Panel E, the data were normalized to I/R group because the signal of LncRNA Ak013346 in real time RT-PCR assays was too weak in sham group. The representative gel image had been provided for analysis of each LncRNA. I/R, ischemia/reperfusion; Sham, plasma of sham mice; I/R, plasma of I/R-treated mice. N = 5, *P<0.05 versus sham group.

Fig 4. Quantitative assays of decreased LncRNAs in I/R-treated mouse plasma.

A-E) Five downregulated LncRNAs with most meaningful decrease in expression fold picked up by microarray assays were further analyzed and confirmed by quantitative RT-PCR assay. In all panels, the data were normalized to sham group. The representative gel image had been provided for analysis of each LncRNA. I/R, ischemia/reperfusion; Sham, plasma of sham mice; I/R, plasma of I/R-treated mice. N = 5, *P<0.05 versus sham group.

Fig 5. The distribution of LncRNAs between mouse liver and plasma.

This figure had been made based on the data presented in Table 1, Table 2, S1 Table and LncRNA profile in the plasma and liver published in our previous study [12]. Up, upregulated LncRNAs in plasma or liver; Down, downregulated LncRNAs in plasma or liver; the numbers in the figure represent the numbers of LncRNAs present in plasma or liver. Total LncRNAs in liver: 20073 [12]; total LncRNAs in plasma: 10206; total LncRNAs present in both liver and plasma:9196.

Fig 6. AK139328 is not present in mouse plasma.

A) The distribution of validated plasma LncRNAs in mouse livers. B) AK139328 is not present in mouse plasma. RT-PCR assay was performed to detect the existence of LncRNAs in mouse plasma and livers. The gel images shown here were the representatives of 3 independent experiments.