miR-200a-5p regulates myocardial necroptosis induced by Se deficiency via targeting RNF11

Abstract

Necroptosis has been discovered as a new paradigm of cell death and may play a key role in heart disease and selenium (Se) deficiency. Hence, we detected the specific microRNA (miRNA) in response to Se-deficient heart using microRNAome analysis. For high-throughput sequencing using Se-deficient chicken cardiac tissue, we selected miR-200a-5p and its target gene ring finger protein 11 (RNF11) based on differential expression in cardiac tissue and confirmed the relationship between miR-200a-5p and RNF11 by dual luciferase reporter assay and real-time quantitative PCR (qRT-PCR) in cardiomyocytes. We further explored the function of miR-200a-5p and observed that overexpression of miR-200a-5p spark the receptor interacting serine/threonine kinase 3 (RIP3)-dependent necroptosis in vivo and in vitro. To understand whether miR-200a-5p and RNF11 are involved in the RIP3-dependent necroptosis pathway, we presumed that oxidative stress, inflammation response and the mitogen-activated protein kinase (MAPK) pathway might trigger necroptosis. Interestingly, necroptosis trigger, z-VAD-fmk, failed to induce necroptosis but enhanced cell survival against necrosis in cardiomyocytes with knockdown of miR-200a-5p. Our present study provides a new insight that the modulation of miR-200a-5p and its target gene might block necroptosis in the heart, revealing a novel myocardial necrosis regulation model in heart disease.

Keywords: Cardiomyocytes; Necroptosis; RNF11; Selenium; miR-200a-5p.

Graphical abstract

Fig. 1

HE staining for myocardial tissues in the control group and Se-deficient group. Histopathological analysis of the heart in the control group and Se-deficient group.

Fig. 2

Different miRNA and mRNA expression profiles and qRT-PCR detectionin vivoandin vitroand luciferase activity assay. (A) Heat map with intuitive reflection of the features of expression profiles changes in the control group and Se-deficient group. (B) Myocardial tissue miRNA levels of miR-200a-5p in the control group and Se-deficient group. Bars represent the mean ± SD of 30 individuals. Bars represent the mean ± SD of triplicate cell cultures. *p < 0.05 by Student's t-test. (C) To investigate the most appropriate transfection concentration in cardiomyocytes model with transfecting different concentrations of miR-200a-5p-mimic, miR-200a-5p-inhibitor, mimic negative control and inhibitor negative control for 24 h. miRNA levels of miR-200a-5p was determined by RT-PCR. The optimum concentration of miR-200a-5p-mimic and miR-200a-5p-inhibitor used in this paper were always 100 nM and 50 nM, respectively. Bars that do not share the same letters are significantly different (p < 0.05) from each other. The data were expressed as the mean ± SD of triplicate cell cultures. (D) The mRNA levels of RNF11 in the cardiomyocytes transfected with mimic, inhibitor and control cardiomyocytes. Bars that do not share the same letters are significantly different (p < 0.05) from each other. The data were expressed as the mean ± SD of triplicate cell cultures. (E) The mRNA and protein levels of RNF11 in the control myocardial tissue and Se-deficient myocardial tissue. Bars represent the mean ± SD of 30 individuals. Bars represent the mean ± SD of triplicate cell cultures. *p < 0.05 by Student's t-test. (F–G) Correlation between RNF11 and miR-200a-5p in luciferase reporter gene assay results. pMIR-RNF11-WT plasmids was mutated in the miRNA target sites, and designated as pMIR-RNF11-Mut. miR-200a-5p-mimic inhibits RNF11-WT expression but not mutant RNF11-MUT expression. Bars that do not share the same letters are significantly different (p < 0.05) from each other. The data were expressed as the mean ± SD of triplicate cell cultures.

Fig. 3

Different mRNA expression of TNF-α-related genes in vivo and in vitro. (A) The mRNA expression of TAB, TAK1, RIP2, TNF-α, NF-κb and RNF11 in the control myocardial tissue and Se-deficient myocardial tissue. Bars represent the mean ± SD of 30 individuals. *p < 0.05 by Student's t-test. (B) The mRNA levels of TAB, TAK1, RIP2, TNF-α, NF-κb and RNF11 in cardiomyocytes transfected with mimic and inhibitor and control cardiomyocytes. Bars represent the mean ± SD of triplicate cell cultures. Bars that do not share the same letters are significantly different (p < 0.05) from each other.

Fig. 4

miR-200a-mediated modulation of RIP3-mediated necroptosis. (A) The ultrastructural changes of cardiomyocytes transfected with mimic, inhibitor and control cardiomyocytes. (B) The mRNA expression of TNF-α, MLKL, RIP1, RIP3, Caspase8 and FADD in the control myocardial tissue and Se-deficient myocardial tissue. Bars represent the mean ± SD of 30 individuals. *p < 0.05 by Student's t-test. (C) The mRNA levels of TNF-α, MLKL, RIP1, RIP3, Caspase8 and FADD in cardiomyocytes transfected with mimic and inhibitor and control cardiomyocytes. Bars represent the mean ± SD of triplicate cell cultures. Bars that do not share the same letters are significantly different (p < 0.05) from each other. (D) Detection results of staining with the AO/EB of cardiomyocytes. The normal cells are stained green. The early apoptotic cells are stained bright green, and later apoptotic cells are stained orange. The necrotic cells are stained red. (E) Cardiomyocytes were transfected with mimic and inhibitor for 24 h. Cell viabilities and necrosis rate were measured by the fluorescence intensity of Annexin V-FITC and PI to identify healthy (double negative), early apoptotic (Annexin V positive), and necrosis (double-positive) cell populations after treatment of z-VAD-fmk (added TNF-α) with/without BHA or Nec-1.

Fig. 5

Protein levels of necroptosis-relatedin vivoandin vitro. The protein levels of RNF11, MLKL, RIP1, RIP3 and Caspase8 were detected in vivo and in vitro. Cardiomyocytes were treated with z-VAD-fmk (added TNF-α) with/without Nec-1 after transfected with mimic and inhibitor. Bars represent the mean ± SD. Bars that do not share the same letters are significantly different (p < 0.05) from each other. *p < 0.05 by Student's t-test.

Fig. 6

miR-200a-5p-mediated modulation of redox states. (A) ROS generation was performed by immunofluorescence using DCFH-DA (green fluorescence, 5 mM) in cells. Cardiomyocytes were treated with z-VAD-fmk (added TNF-α) with/without Nec-1 after transfected with mimic and inhibitor. Cardiomyocytes were visualized using fluorescence microscopy. (B) Oxidative stress markers of H₂O_2, GSH, SOD and T-AOC contents were measured in vivo and in vitro. Cardiomyocytes were treated with z-VAD-fmk (added TNF-α) with/without BHA or Nec-1 after transfected with mimic and inhibitor. Bars represent the mean ± SD. Bars that do not share the same letters are significantly different (p < 0.05) from each other. *p < 0.05 by Student's t-test.

Fig. 7

miR-200a-5p-mediated modulation of MAPKs activation. (A-C) The mRNA levels of ERK, JNK and p38 were detected in vivo and vitro. Cardiomyocytes were treated with z-VAD-fmk (added TNF-α) with/without BHA or Nec-1 after transfected with mimic and inhibitor. Bars represent the mean ± SD. Bars that do not share the same letters are significantly different (p < 0.05) from each other. *p < 0.05 by Student's t-test. (D-F) The protein levels of phosphorylation ERK, phosphorylation JNK, phosphorylation p38, total ERK, total JNK and total p38 were detected in vivo and in vitro. Cardiomyocytes were treated with z-VAD-fmk (added TNF-α) with/without BHA or Nec-1 after transfected with mimic and inhibitor. Bars represent the mean ± SD. Bars that do not share the same letters are significantly different (p < 0.05) from each other. *p < 0.05 by Student's t-test.