Protective effect of microRNA‑381 against inflammatory damage of endothelial cells during coronary heart disease by targeting CXCR4

Abstract

Coronary heart disease (CHD) is the leading cause of human morbidity and mortality worldwide. MicroRNA (miRNA) profiling is an innovative method of identifying biomarkers for many diseases and may be a powerful tool in the diagnosis and treatment of CHD. The present study aimed to analyze the effects of miRNA (miR)‑381 on the inflammatory damage of endothelial cells during CHD. A total of 21 patients with CHD and 21 healthy control patients were enrolled in this study. Reverse transcription‑quantitative PCR, western blotting and immunofluorescence assays were conducted to examine the expression levels of miR‑381, C‑X‑C chemokine receptor type 4 (CXCR4), Bcl‑2, Bax, Cleaved‑Caspases‑3 and ‑9, p38, ERK1/2 and JNK. Cell Counting Kit‑8, EdU and flow cytometry experiments were performed to evaluate cell proliferation and apoptosis. An ELISA was adopted to determine the expressions of inflammatory factors (interleukins‑8, ‑6 and ‑1β, and tumor necrosis factor‑α). In addition, a dual‑luciferase reporter assay was used to determine the relationship between miR‑381 and CXCR4. Decreased miR‑381 expression and increased CXCR4 expression in the plasma were observed in the CHD group compared with the normal group, which indicated a negative relationship between miR‑381 and CXCR4. Overexpression of miR‑381 significantly promoted the proliferation and inhibited the apoptosis of oxidized low‑density lipoprotein (OX‑LDL)‑induced human umbilical vein endothelial cells (HUVECs) through mitogen‑activated protein kinase pathway by targeting and inhibiting CXCR4. Furthermore, overexpression of miR‑381 reduced the release of inflammatory factors in OX‑LDL‑induced HUVECs. By contrast, reduced expression of miR‑381 exerted the opposite effects, which were subsequently reversed by silencing CXCR4 expression. Results from the present study indicated that miR‑381 was a CHD‑related factor that may serve as a potential molecular target for CHD treatment.

Keywords: microrna-381; coronary heart disease; c-X-c chemokine receptor type 4; endothelial cells; inflammation.

Figure 1.

Relative expression levels of miR-381 and CXCR4 in the plasma of patients in the normal and CHD groups. (A) RT-qPCR assay was performed to evaluate the expression of miR-381 in in the plasma of the normal and CHD groups. (B) RT-qPCR assay was performed to evaluate the expression of CXCR4 in the plasma of the normal and CHD groups. *P<0.05 vs. Normal. CHD, coronary heart disease; CXCR4, C-X-C chemokine receptor type 4; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR.

Figure 2.

CXCR4 is a target gene of miR-381. (A) RT-qPCR assay was performed to evaluate the expression of miR-381 to confirm the successful transfection of miR-381 mimics or miR-381 inhibitors. (B) Laser scanning confocal microscopy was employed to detect the transfection by observing green fluorescence protein (magnification, ×200). (C) RT-qPCR was performed to evaluate the levels of CXCR4 in cells transfected with siRNA-CXCR4. (D) TargetScan was used to predict the target genes of miR-381; a putative miR-381 binding site was identified in the 3′UTR of CXCR4 (highlighted in red in the MUT sequence). (E) To determine if CXCR4 was a direct target of miR-381, a dual-luciferase reporter assay was performed. (F) Western blotting and (G) RT-qPCR were used to test the protein and mRNA expression levels, respectively, of CXCR4 in cells transfected with miR-381 mimics or miR-381 inhibitors. (H) RT-qPCR assay determination of the expression of miR-381 in cells transfected with siRNA-CXCR4. *P<0.05 vs. the corresponding NC group. CXCR4, C-X-C chemokine receptor type 4; miR, microRNA; MUT, mutant; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA; UTR, untranslated region; WT, wild-type.

Figure 3.

Expressions of miR-381 and CXCR4 in OX-LDL-induced HUVECs. RT-qPCR assay tested the expression levels of (A) miR-381 and (B) CXCR4. (C) Western blotting and (D) immunofluorescence assays were performed to analyze the protein expression level of CXCR4 in OX-LDL-induced HUVECs (magnification, ×200). *P<0.05 vs. Blank. CXCR4, C-X-C chemokine receptor type 4; HUVECs, human umbilical vein endothelial cells; OX-LDL, oxidized low-density lipoprotein; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR.

Figure 4.

Effects of miR-381 on OX-LDL-induced cell proliferation. (A and B) Cell Counting Kit-8 assay was performed to detect the proliferation of OX-LDL-induced HUVECs. *P<0.05 vs. Blank or miR-381 inhibitiors. (C) EdU assay was performed to detect the proliferation of OX-LDL-induced HUVECs (magnification, ×200). *P<0.05. CXCR4, C-X-C chemokine receptor type 4; HUVECs, human umbilical vein endothelial cells; miR, microRNA; NC, negative control; OX-LDL, oxidized low-density lipoprotein; siRNA, small interfering RNA.

Figure 5.

Effects of miR-381 on inflammatory cytokine release in OX-LDL-induced cells. ELISA was used to detect the levels of IL-8, IL-6, IL-1β and TNF-α in the cell supernatant. *P<0.05. CXCR4, C-X-C chemokine receptor type 4; IL, interleukin; miR, microRNA; NC, negative control; OX-LDL, oxidized low-density lipoprotein; siRNA, small interfering RNA; TNF, tumor necrosis factor.

Figure 6.

Effects of miR-381 on cell apoptosis of OX-LDL-induced HUVECs. (A) Flow cytometry was used to evaluate the apoptosis of OX-LDL-induced HUVECs transfected with miR-381 mimics, mimics NC, inhibitors, inhibitors NC, siRNA CXCR4, siRNA NC or miR-381 inhibitors + siRNA CXCR4. *P<0.05 vs. NC. (B) Western blotting assay was performed to evaluate the protein expression levels of apoptotic-related proteins, including Bcl-2, Bax, Cleaved-Caspase-3 and Cleaved-Caspase-9. *P<0.05 vs. blank. CXCR4, C-X-C chemokine receptor type 4; HUVECs, human umbilical vein endothelial cells; miR, microRNA; NC, negative control; OX-LDL, oxidized low-density lipoprotein; siRNA, small interfering RNA.

Figure 7.

Effects of miR-381 on MAPK signaling pathway. Western blotting was conducted to determine the protein expression levels of MAPK signaling pathway-related proteins (p-p38, p38, p-ERK, ERK, p-JNK and JNK) in OX-LDL-induced HUVECs transfected with miR-381 mimics, mimics NC, inhibitors, inhibitors NC, siRNA CXCR4, siRNA NC or miR-381 inhibitors + siRNA CXCR4. *P<0.05 vs. Blank. CXCR4, C-X-C chemokine receptor type 4; ERK, extracellular signal-regulated kinase; HUVECs, human umbilical vein endothelial cells; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; miR, microRNA; NC, negative control; OX-LDL, oxidized low-density lipoprotein; p-, phosphorylated; siRNA, small interfering RNA.