Circulating "LncPPARδ" From Monocytes as a Novel Biomarker for Coronary Artery Diseases

Yue Cai, Yujia Yang, Xiongwen Chen, Duofeng He, Xiaoqun Zhang, Xiulan Wen, Jiayong Hu, Chunjiang Fu, Dongfeng Qiu, Pedro A Jose, Chunyu Zeng, Lin Zhou, Yue Cai, Yujia Yang, Xiongwen Chen, Duofeng He, Xiaoqun Zhang, Xiulan Wen, Jiayong Hu, Chunjiang Fu, Dongfeng Qiu, Pedro A Jose, Chunyu Zeng, Lin Zhou

Abstract

To investigate long noncoding RNA NONHSAT112178 (LncPPARδ) as a biomarker for coronary artery disease (CAD) in peripheral blood monocyte cells, RT-qPCR was performed to validate the microarray results, receiver operating characteristic curve was applied to study the potential of LncPPARδ as a biomarker. Diagnostic models from LncPPARδ alone or combination of risk factors were constructed by Fisher criteria. The expression of genes neighboring the LncPPARδ gene was examined with RT-qPCR in THP-1 cell line treated with LncPPARδ siRNA. Using a diagnostic model by Fisher criteria, the consideration of risk factors increased the optimal sensitivity from 70.00% to 82.00% and decreased the specificity from 94.00% to 78.00%. The consideration of risk factors also increased area under the receiver operating characteristic curve from 0.727 to 0.785 (P = 0.001), from 0.712 to 0.768 (P = 0.01), and from 0.769 to 0.835 (P = 0.07), in the original, training, and test sets, respectively. Finally, we found that the expression of peroxisome proliferator-activated receptor δ (PPARδ), Adipose Differentiation-Related Protein (ADRP), and Angiopoietin-like 4 (ANGPTL4) were affected by LncPPARδ silencing.Our present study indicated that LncPPARδ, especially combined with risk factors, can be a good biomarker for CAD. LncPPARδ regulates the expression of neighboring protein-coding genes, PPARδ and its direct target genes ADRP and ANGPTL4.

Trial registration: ClinicalTrials.gov NCT01629225.

Conflict of interest statement

The authors have no conflicts of interest to disclose.

Figures

FIGURE 1
FIGURE 1
Heat map of lncRNA expression from microarray analysis of combined circulating monocyte samples of patients with CAD and control subjects (n = 3, PBMCs from 5 patients were pooled as 1 sample), respectively. The expression of lncRNA is hierarchically clustered on the y-axis, and CAD or control monocyte samples are hierarchically clustered on the x-axis. The relative lncRNA expression is depicted according to the color scale shown on the left. Red indicates up-regulation; green, down-regulation. “P1M-P3M” indicates CAD samples; “N1M-N3M,” control samples (A). Scatter plot of lncRNA expression in test samples versus normal samples. X-axis depicts data values of normal samples, Y-axis depicts data values of test samples. Dots located above the upper green line and below the under green line represent fold change ≥2.0. “Test” indicates CAD samples; “Normal,” control samples (B). CAD = coronary artery heart disease, PBMC = peripheral blood monocyte.
FIGURE 2
FIGURE 2
Expression levels and ROC curve analyses of LncPPARδ. (A) Expression of LncPPARδ in the PBMCs from CAD patients and control subjects determined by qRT-PCR (∗P < 0.001, vs Control, n = 20). (B) ROC curve analyses of LncPPARδ for diagnosis of CAD in pilot samples. AUC = area under the ROC curve, CAD = coronary artery heart disease, PBMC = peripheral blood monocyte, ROC = receiver operating characteristic.
FIGURE 3
FIGURE 3
Diagnostic value of LncPPARδ with or without combination of other risk factors on CAD. Original set included 211 CAD patients and 171 control subjects (A); training set included 161 CAD patients and 121 control subjects (B); and test set included 50 CAD patients and 50 control subjects (C), respectively. CAD = coronary artery heart disease.
FIGURE 4
FIGURE 4
Diagnostic specificity of LncPPARδ in PBMCs for CAD. LncPPARδ levels were checked in CAD and other cardiovascular diseases, including arrhythmia (n = 8), valvular disease (n = 8), dilated cardiomyopathy (n = 6), hyperlipidemia (n = 8), hypertension (n = 8), type 2 diabetes mellitus (n = 12), abdominal aortic aneurysm (n = 2), and viral myocarditis (n = 7). Horizontal lines indicate the median. CAD = coronary artery heart disease, PBMC = peripheral blood monocyte.
FIGURE 5
FIGURE 5
Stability of LncPPARδ in PBMC exposed at varying time periods at room temperature (A) and different freeze-thaw cycles (B). Expression of LncPPARδ in the PBMCs was determined by qRT-PCR (∗P < 0.001, vs control, n = 12). PBMC = peripheral blood monocyte.
FIGURE 6
FIGURE 6
Effects of LncPPARδ on mRNA expressions of PPARδ, ADRP, and ANGPTL4 in THP-1 cells after transfection of LncPPARδ siRNA. Knock-down efficiency of LncPPARδ siRNA determined by qRT-PCR (A). The effects of LncPPARδ silencing on PPARδ, ADRP, and ANGPTL4 expression determined by qRT-PCR at 24 h after siRNA treatment (B–D). PPARδ = peroxisome proliferator-activated receptor δ. ∗P < 0.001, compared with control, n = 6.

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