Astragaloside IV inhibits oxidized low‑density lipoprotein‑induced endothelial damage via upregulation of miR‑140‑3p

Abstract

Oxidized low‑density lipoprotein (ox‑LDL)‑mediated endothelial cell injury has an important role in the vascular complications of type 2 diabetes. Astragaloside IV (ASV) is an active component of Radix Astragali, which has been demonstrated to exert protective effects against endothelial damage. The present study explored whether microRNAs (miRNAs) are involved in mediating the protective effects of ASV on ox‑LDL‑induced damage in human umbilical vein endothelial cells (HUVECs). RNA sequencing and reverse transcription‑quantitative PCR analyses revealed that ox‑LDL treatment significantly downregulated miR‑140‑3p expression in HUVECs. miR‑140‑3p overexpression promoted cell proliferation and inhibited apoptosis in ox‑LDL‑induced HUVECs. However, inhibition of miR‑140‑3p expression could reverse the effects of ASV on ox‑LDL‑induced HUVECs and reactivate ASV‑inhibited PI3K/Akt signaling in ox‑LDL‑induced HUVECs. In addition, Krüppel‑like factor 4 (KLF4) was identified as a target of miR‑140‑3p in ox‑LDL‑treated HUVECs. Subsequent experiments revealed that KLF4 overexpression partially counteracted the protective effects of miR‑140‑3p or ASV treatment in ox‑LDL‑induced HUVECs. Taken together, the current findings demonstrated that the protective effects of ASV on HUVECs were dependent on miR‑140‑3p upregulation and subsequent inhibition of KLF4 expression, which in turn suppressed the PI3K/Akt signaling pathway. The present results shed light to the molecular mechanism by which ASV alleviated ox‑LDL‑induced endothelial cell damage.

Figure 1

miR-140-3p overexpression promotes proliferation and inhibits apoptosis in ox-LDL induced HUVECs. (A) Volcano plot showing the differential expression of all miRNAs following RNA sequencing analysis. (B) Heat map and clustering analysis identified 120 significantly dysregulated miRNAs, including 60 downregulated miRNAs and 60 upregulated miRNAs at fold change >2 (P<0.05). Red indicates upregulation and green indicates downregulation. (C) miR-140-3p levels were analyzed by RT-qPCR in HUVECs treated with 100 µg/ml ox-LDL for 0, 6, 12 and 24 h (n=3). (D) miR-140-3p levels were analyzed by RT-qPCR following transfection of HUVECs with miR-NC or miR-140-3p mimics (n=3). (E) Cell proliferation of HUVECs was evaluated using Cell Counting Kit-8 agent, following overexpression of miR-140-3p with or without ox-LDL treatment (n=3). (F) Representative plots and (G) quantification of flow cytometry analysis to detect cell apoptosis in HUVECs. Three independent experiments were conducted. Data are presented as the mean ± standard deviation of at least three experiments. ^#P<0.05 vs. control; ^*P<0.05 vs. ox-LDL+miR-NC. ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vein endothelial cells; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control.

Figure 2

ASV protects HUVECs from ox-LDL-induced damage by upregulating miR-140-3p expression. (A) miR-140-3p levels were analyzed by reverse transcription-quantitative PCR in HUVECs with or without ASV treatment (0, 20, 50, 100 µM for 24 h; n=3). ^**P<0.01 vs. blank; ^#P<0.05 and ^##P<0.01 vs. ox-LDL alone. (B) miR-140-3p levels in HUVECs transfected with miR-140-3p inhibitors or inhibitor control (n=3). ^**P<0.01 vs. inhibitor control. (C) Cell proliferation was evaluated using the Cell Counting Kit-8 agent in HUVECs transfected with miR-140-3p inhibitors or inhibitor control under ASV treatment (n=3). ^**P<0.01 vs. control; ^##P<0.01 vs. ox-LDL alone; ^&&P<0.01 vs. ox-LDL+ASV+inhibitor control. (D) Representative plots and (E) quantification of flow cytometry analysis to detect cell apoptosis of ox-LDL-induced HUVECs following ASV treatment and/or miR-140-3p downregulation. ^**P<0.01 vs. control; ^##P<0.01 vs. ox-LDL alone; ^&&P<0.01 vs. ox-LDL+ASV+inhibitor control. (F) Representative blots and (G) quantification of PI3K and (H) Akt phosphorylation levels in ox-LDL-induced HUVECs following ASV treatment and/or miR-140-3p downregulation. ^**P<0.01 vs. control; ^##P<0.01 vs. ox-LDL alone; ^&&P<0.01 vs. ox-LDL+ASV+inhibitor control. ASV, astragaloside IV; HUVECs, human umbilical vein endothelial cells; ox-LDL, oxidized low-density lipoprotein; RT-qPCR, reverse transcription-quantitative PCR.

Figure 3

miR-140-3p upregulation promotes cell proliferation and inhibits cell apoptosis by targeting KLF4 in ox-LDL-induced HUVECs. (A) The predicted binding sites between miR-140-3p and KLF4, by using the bioinformatics tool Starbase v3.0. (B) Relative luciferase activity in HUVECs transfected with miR-140-3p mimic or miR-NC together with luciferase reporter constructs containing either the wide-type or mutant-type 3′ untranslated region of KLF4. ^##P<0.01 vs. miR-NC. (C) Relative mRNA expression levels of KLF4 in HUVECs stimulated with 100 µg/ml ox-LDL for 0, 6, 12, and 24 h (n=3). ^*P<0.05 and ^**P<0.01 vs. control. (D) Relative mRNA expression levels of KLF4 and (E) KLF4 protein expression levels in ox-LDL-induced HUVECs with or without miR-140-3p overexpression (n=3). ^**P<0.01 vs. control; ^#P<0.05 vs. ox-LDL+miR-NC. (F) Relative mRNA expression levels of KLF4 in HUVECs transfected with pcDNA (empty vector control) or pcDNA-KLF4 at 24 h post-transfection (n=3). ^**P<0.01 vs. control. (G) Relative protein expression levels of KLF4 in HUVECs transfected with pcDNA or pcDNA-KLF4 at 24 h post-transfection (n=3). ^**P<0.01 vs. control. (H) Cell proliferation was evaluated using Cell Counting Kit-8 agent in HUVECs transfected with miR-140-3p mimics or pcDNA-KLF4 (n=3). ^**P<0.01 vs. control; ^#P<0.05 vs. ox-LDL alone; and ^&P<0.05 vs. ox-LDL+miR-140-3p mimics. (I) Representative plots and (J) quantification of flow cytometry analysis to detect cell apoptosis in HUVECs. Data are presented as the mean ± standard deviation of at least three independent experiments. ^**P<0.01 vs. control; ^#P<0.05 vs. ox-LDL alone; and ^&P<0.05 vs. ox-LDL+miR-140-3p mimics. KLF4, Krüppel-like factor 4; ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vein endothelial cells; NC, negative control.

Figure 3

miR-140-3p upregulation promotes cell proliferation and inhibits cell apoptosis by targeting KLF4 in ox-LDL-induced HUVECs. (A) The predicted binding sites between miR-140-3p and KLF4, by using the bioinformatics tool Starbase v3.0. (B) Relative luciferase activity in HUVECs transfected with miR-140-3p mimic or miR-NC together with luciferase reporter constructs containing either the wide-type or mutant-type 3′ untranslated region of KLF4. ^##P<0.01 vs. miR-NC. (C) Relative mRNA expression levels of KLF4 in HUVECs stimulated with 100 µg/ml ox-LDL for 0, 6, 12, and 24 h (n=3). ^*P<0.05 and ^**P<0.01 vs. control. (D) Relative mRNA expression levels of KLF4 and (E) KLF4 protein expression levels in ox-LDL-induced HUVECs with or without miR-140-3p overexpression (n=3). ^**P<0.01 vs. control; ^#P<0.05 vs. ox-LDL+miR-NC. (F) Relative mRNA expression levels of KLF4 in HUVECs transfected with pcDNA (empty vector control) or pcDNA-KLF4 at 24 h post-transfection (n=3). ^**P<0.01 vs. control. (G) Relative protein expression levels of KLF4 in HUVECs transfected with pcDNA or pcDNA-KLF4 at 24 h post-transfection (n=3). ^**P<0.01 vs. control. (H) Cell proliferation was evaluated using Cell Counting Kit-8 agent in HUVECs transfected with miR-140-3p mimics or pcDNA-KLF4 (n=3). ^**P<0.01 vs. control; ^#P<0.05 vs. ox-LDL alone; and ^&P<0.05 vs. ox-LDL+miR-140-3p mimics. (I) Representative plots and (J) quantification of flow cytometry analysis to detect cell apoptosis in HUVECs. Data are presented as the mean ± standard deviation of at least three independent experiments. ^**P<0.01 vs. control; ^#P<0.05 vs. ox-LDL alone; and ^&P<0.05 vs. ox-LDL+miR-140-3p mimics. KLF4, Krüppel-like factor 4; ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vein endothelial cells; NC, negative control.

Figure 4

KLF4 overexpression reverses the protective effects of ASV on ox-LDL-induced HUVECs. (A) Cell viability was assessed by Cell Counting Kit-8 assay in ox-LDL-induced HUVECs following ASV treatment and/or KLF4 overexpression (n=3). (B) Representative plots and (C) quantification of flow cytometry analysis to detect cell apoptosis in ox-LDL-induced HUVECs following ASV treatment and/or KLF4 overexpression (n=3). (D) Representative blots and (E) quantification of PI3K and (F) Akt phosphorylation levels in ox-LDL-induced HUVECs following ASV treatment and/or KLF4 overexpression. Data are presented as the mean ± standard deviation of at least three independent experiments. ^**P<0.01 vs. control; ^##P<0.01 vs. ox-LDL alone; ^&&P<0.01 vs. ox-LDL+ASV+vector. KLF4, Krüppel-like factor 4; ASV, astragaloside IV; ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vein endothelial cells.