Up-regulation of miR-138 inhibits hypoxia-induced cardiomyocyte apoptosis via down-regulating lipocalin-2 expression

Abstract

Hypoxia-induced cardiomyocyte apoptosis contributes significantly to the development of numerous cardiac diseases, such as ischemic heart disease, heart failure, etc. Promoting cell survival by inhibiting apoptosis is one of the available strategies to attenuate cardiac dysfunction caused by cardiomyocyte loss. Previous studies have been demonstrated that miR-138 and lipocalin-2 (Lcn2) play important roles in cardiomyocyte apoptosis and survival. We presently determined whether Lcn2 is a target gene of miR-138 involved in hypoxia-induced cardiomyocyte apoptosis. Firstly, mimics of miR-138 were transfected into HL-1 cells to investigate its effect on cell apoptosis. Using 3-(4,5-dimethyl-thiazol-2-y1) 2,5-diphenyl tetrazolium bromide (MTT) and Annexin V-FITC/PI flow cytometer assays, over-expression of miR-138 significantly enhanced the cell growth and significantly attenuated the cell apoptosis in hypoxic conditions. Dual-luciferase reporter gene and western blot results confirmed Lcn2 was a direct target of miR-138. Then, the recombinant plasmid, pcDNA3.1/Lcn2 was transfected into the HL-1 cells that over-expressed miR-138. We further observed that the over-expression of Lcn2 diminished the protection of miR-138 over-expression from hypoxia-induced cell survival and apoptosis. In conclusion, our study demonstrated that up-regulation of miR-138 inhibits the hypoxia-induced cardiomyocyte apoptosis via down-regulating the pro-apoptotic gene expression of Lcn2.

Keywords: apoptosis; cardiomyocyte; hypoxia; lipocalin-2; miR-138.

Figure 1

Expression of miR-138 in hypoxic cardiomyocytes. qRT-PCR revealed that the expression of miR-138 was significantly decreased in the hypoxic HL-1 cells. HL-1 cells were cultured in 1% O₂ and 5% CO₂. *P < 0.05.

Figure 2

Effect of miR-138 over-expression on cell growth and apoptosis in hypoxic cardiomyocytes. (a) The over-expression of miR-138 in miR-138 upon mimetic transfection was validated using qRT-PCR. HL-1 cells transfected with empty plasmid were used as a negative control (NC). *P < 0.05. (b) After transfected with miR-138 mimics, HL-1 cells were cultured in 1% O₂ and 5% CO₂ (hypoxia) for different duration, and cell survival curve was measured by MTT. *P < 0.05. (c) Exposed to hypoxia for 48 h, cell apoptosis was tested by Annexin V-FITC/PI flow cytometry, and the proportion of apoptosis cells was measured. *P < 0.05. (d) Cells treated with miR-138 mimics versus cells treated with miR-NC. MTT: 3-(4,5-dimethyl-thiazol-2-y1) 2,5-diphenyl tetrazolium bromide. (A color version of this figure is available in the online journal)

Figure 3

Lcn2 was target gene of miR-138. The mRNA (a) and protein (b) expressions of Lcn2 in hypoxic cardiomyocytes. (c) Sequence alignment of miR-138 and 3'UTR of Lcn2 using TargetScan algorithm. (d) HL-1 cells were co-transfected with miR-138 mimics and a luciferase reporter containing a fragment of the Lcn2 3'UTR harboring either the miR-138 binding site (Lcn2-3′UTR-WT) or a mutant (Lcn2-3′UTR-MUT). The assays showed that luciferase activity in the Lcn2-3′UTR-WT group was significantly decreased compared to the luciferase activity of the mutant groups. (e) Western blot detection of Lcn2 expression in HL-1 cells treated with miR-138-overexpression or NC constructs. *P < 0.05. GADPH: glyceraldehyde 3-phosphate dehydrogenase; NC: negative control.

Figure 4

Validation of the role of Lcn2 in the over-expression of miR-138-influenced cell growth and apoptosis of hypoxic cardiomyocytes. (a) The Lcn2 levels were analyzed by western blot. Lcn2 was high expressed in miR-138 + Lcn2 cells. (b) The miR138 + Lcn2 cells/miR138 + vector cells were cultured in 1% O₂ and 5% CO₂ (hypoxia) for different duration, and cell survival curve was measured by MTT. *P < 0.05. (c) Exposed to hypoxia for 48 h, cell apoptosis was tested by Annexin V-FITC/PI flow cytometry, and proportion of apoptosis cells was measured. *P < 0.05. (d) miR138 + Lcn2 cells versus miR-138 + vector cells. GADPH: glyceraldehyde 3-phosphate dehydrogenase; MTT: 3-(4,5-dimethyl-thiazol-2-y1) 2,5-diphenyl tetrazolium bromide. (A color version of this figure is available in the online journal)