The expression profile analysis and functional prediction of lncRNAs in peripheral blood mononuclear cells in maintenance hemodialysis patients developing heart failure

Abstract

Heart failure (HF) is the leading cause of death in patients with maintenance hemodialysis (MHD). Biomarkers has an important guiding role in the early diagnosis, risk stratification, and prognostic assessment of HF. Increasing studies have indicated that long non-coding RNAs (lncRNAs) have played an indispensable role in the regulatory network of HF. This study was aiming to explore the expression profiles of lncRNAs in patients treated with MHD developing heart failure. Peripheral blood mononuclear cells were isolated from 4 hemodialysis patients with reduced ejection fraction (HFrEF) and 4 hemodialysis patients with preserved ejection fraction (HFpEF), respectively. The expression profile analysis of lncRNAs was performed by using Illumina Novaseq 6000 sequencer. Quantitative real time polymerase chain reaction (qRT-PCR) was used to verify the expression of representative differentially expressed lncRNAs. Based on lncRNA-miRNA-mRNA-KEGG network analysis, the potential role of candidate lncRNAs and their association with the severity of HF were further evaluated. In total, 1,429 differentially expressed lncRNAs were found between patients with HFrEF and patients with HFpEF, of which 613 were up-regulated and 816 were down-regulated (P < 0.05). Five candidate lncRNAs were screened out by a series of bioinformatic analyses. After being compared with miRBase, ENST00000561762, one of the 5 candidates, was considered the most likely lncRNA to be serving as a precursor for miRNA. Nine predicted target genes were found by further lncRNA-miRNA-mRNA-KEGG network analysis, and among which ITGB5 was enriched in the actin dynamics signaling pathway. In another cohort of hemodialysis patients, the expression of lncRNA ENST00000561762 was verified by qRT-PCR. Further analysis revealed that there was a strong correlation between left ventricular ejection fraction and ENST00000561762, proBNP, and 6-minute walk distance, respectively. LncRNAs expression profile was remarkably different in hemodialysis patients with HFrEF compared to those with HFpEF. Among which, lncRNA ENST00000561762 was considered as a promising biomarker for patients with HFrEF as it was predicted to be a miRNA precursor to regulate the actin dynamics signaling pathway.

Keywords: HF; MHD; PBMC; lncRNAs.

Fig. 1

Expression level of lncRNA in each sample. For each sample, lncRNAs were analyzed using 3D PCA plot (A) and correlation coefficient matrix (B). These 6503 lncRNAs are common to the results obtained from the four databases (C). Test, group of hemodialysis patients with reduced ejection fraction (HFrEF). Control, group of hemodialysis patients with preserved ejection fraction (HFpEF).

Fig. 2

The differentially expressed lncRNAs between HFrEF and HFpEF groups. (A) Volcano map of differentially expressed lncRNAs. (B) Cluster heat maps of differentially expressed lncRNAs. (C) Bar chart of differentially expressed lncRNAs. Test, group of hemodialysis patients with reduced ejection fraction (HFrEF). Control, group of hemodialysis patients with preserved ejection fraction (HFpEF).

Fig. 3

Functional enrichment analysis of lncRNAs. (A) GO analysis. (B) KEGG pathways. Test, group of hemodialysis patients with reduced ejection fraction (HFrEF). Control, group of hemodialysis patients with preserved ejection fraction (HFpEF).

Fig. 4

Prediction of miRNA precursors and its target gene. (A) The result of lncRNA-miRNA-mRNA interaction network. (B) LncRNA-miRNA-mRNA network was constructed for lncRNA ENST00000561762. The miRNA was shown as green triangle and the target gene (mRNA) was shown as red rectangle.

Fig. 5

Correlation analysis between lncRNA ENST00000561762 and heart failure related. Clinical indexes. To further clarify the confidence of lncRNA expression profile, the expression level of lncRNA ENST00000561762 was detected in 74 patients using qRT-PCR. The 74 patients were divided into group I (LVEF < 40%), group II (LVEF41-50%), and group III (LVEF > 51%). (A) Differential expression of lncRNA ENST00000561762 among groups I, II, and III. (B–E) Differences in relative levels of ESV (B), LV (C), pro-BNP (D), and LA (E) among groups I, II, and III. (F–H) The correlation analysis between LVEF and lncRNA ENST00000561762 (F), pro-BNP and LVEF(G), as well as 6MWD and LVEF (H). *, P < 0.05, **, P < 0.01, ***, P < 0.005, ****, P < 0.001. LVEF, left ventricular ejection fraction. ESV, left ventricular systolic volume. LV, left ventricular diameter. pro-BNP, B-type natriuretic peptide. LA, left atrial diameter. 6MWD, 6-minute walk test.