Knockdown of lncRNA ENST00000609755.1 Confers Protection Against Early oxLDL-Induced Coronary Heart Disease

Abstract

Background: This study investigated the association between long non-coding RNAs (lncRNAs) and coronary heart disease (CHD) and further elucidated the potential biological roles of lncRNAs in CHD pathogenesis. Methods: A case-control study (590 patients and 590 controls) was conducted from February 2017 and March 2019 in Fuzhou, China. Environmental factors were investigated using questionnaires and physical examinations. Five representative lncRNAs were screened using lncRNA microarray (peripheral blood in 5 cases and 5 controls) and further verified by quantitative real-time polymerase chain reaction (peripheral blood leukocyte in 100 cases and 100 controls). Oxidized low-density lipoprotein (oxLDL) was used to induce a human coronary artery endothelial cell (HCAECs) injury model, and loss of function was used to elucidate the role of lncRNA ENST00000609755.1 (lnc-MICALL2-2) in oxLDL-induced HCAECs injury. Results: A total of 320 lncRNAs were found dysregulated in CHD patients (fold change> 2, p < 0.05). The results of a discovery microarray, population verification and HCAEC experiments suggested the lnc-MICALL2-2 is upregulated in CHD subjects and in an oxLDL-induced HCAECs injury model. Conversely, lnc-MICALL2-2 inhibition in vitro attenuated the effects of oxLDL on HCAECs morphology, proliferation, and apoptosis. Conclusion: Elevated expression of lnc-MICALL2-2 is an independent risk factor for CHD, and knockdown subsequently confers protection against early pathological processes of oxLDL-induced CHD.

Keywords: coronary heart disease; environmental factors; human coronary artery endothelial cells; long non-coding RNA; oxidized low-density lipoprotein.

Figure 1

Differentially expressed lncRNAs in CHD vs. non-CHD subjects. (A) Heat map analysis from 10 samples in the CHD subjects (A1, A2, A3, A4, and A5) and non-CHD subjects (B1, B2, B3, B4, and B5) revealed different lncRNA expression profiles. The red color indicates relatively up-regulated lncRNAs, and the green color represents relatively down-regulated lncRNAs; the black portions indicate no significant difference. (B,C) Olcano plot and scatter plot analysis of differentially expressed lncRNAs in CHD and non-CHD subjects. Red or green points represent up-regulated or down-regulated lncRNAs (fold change >2.0, p < 0.05), respectively.

Figure 2

Comparison of the expression levels of lncRNAs between the CHD and non-CHD subjects. Five differentially expressed lncRNAs were validated by qRT-PCR. Data are expressed as means ± SD, (n = 30–100/group), *p < 0.05. Student t-test was used for all comparisons.

Figure 3

The expression levels of differentially expressed lncRNAs in HCAECs. (A) Purification identification of HCAECs by vWF. vWF factor (red) and cellular localization (blue) double staining ( ×200 times) of HCAECs. (B) Cell viabilities were compared after culturing at different concentrations of oxLDL for 24, 48, and 72 h (n = 3/group). (C) Comparison of the expression levels of differentially expressed lncRNAs after cell culture at different concentrations of oxLDL for 24, 48, and 72 h (n = 3/group). Values are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 vs. normal HCAECs; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. 50 μg/ml oxLDL; ^&&p < 0.01, ^&&&p < 0.001 vs. 100 μg/ml oxLDL in panels b and c. Data are expressed as means ± SD. one-way ANOVA was used for all comparisons.

Figure 4

The function of knockdown of lnc-MICALL2-2 in oxLDL-induced HCAECs injury. (A) Lnc-MICALL2-2 interference fragment screening. *p < 0.05, **p < 0.01, ***p < 0.001 vs. normal HCAECs; ^##p < 0.01 vs. HCAECs treated with siRNA-1; ^&p < 0.05 vs. HCAECs treated with siRNA-2 (n = 3/group). (B) Relative expression of lnc-MICALL2-2 in normal HCAECs, HCAECs treated with siRNA, oxLDL, and oxLDL + siRNA. (C) HCAECs morphology of different treatment groups under electron microscopic observation (n = 3/group). (D) LDH activity in HCAECs culture medium of different treatment groups (n = 3/group). (E) HCAECs cell proliferation of different treatment groups activity by Cell Counting Kit-8 detection (n = 9/group). (F,G) HCAECs apoptosis of different treatment groups by flow cytometry (n = 3/group). Values are expressed as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 in (B,D,E,G). One-way ANOVA was used for all comparisons.