Protective effect and mechanism of miR-194-5p inhibitor on myocardial injury in mice with type 2 diabetic cardiomyopathy

doi:10.11958/20260039

Abstract

Abstract:

Objective To investigate the protective effect and its potential mechanism of miR-194-5p inhibitor (miR-194-5p antagomir) on myocardial injury in mice with type 2 diabetic cardiomyopathy (DCM). Methods Sixty C57BL/6 mice were randomly divided into the normal control (NC) group, the DCM group, the DCM+antagomir control group and the DCM+miR-194-5p antagomir group (n=15). The DCM model was established by high-fat diet combined with intraperitoneal injection of streptozotocin. After successful model establishment, mice in the DCM+miR-194-5p antagomir group were injected via the tail vein with miR-194-5p antagomir dissolved in PBS at a dose of 10 mg/kg, while mice in the control group were injected with the same dose of antagomir control dissolved in PBS, twice a week for 4 weeks. Twelve weeks after the intervention, cardiac function was evaluated by echocardiography. Myocardial cell cross-sectional area was detected by wheat germ agglutinin (WGA) staining. Inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α in myocardial tissue were determined by enzyme-linked immunosorbent assay (ELISA). The expression levels of miR-194-5p and dual-specificity phosphatase 9 (DUSP9) mRNA were detected by real-time fluorescence quantitative PCR (RT-qPCR). Myocardial mitochondrial ultrastructure was observed by transmission electron microscopy. The target of miR-194-5p was predicted by bioinformatics methods, and pathway enrichment analysis was performed. The dual-luciferase reporter gene assay was used to verify the targeted regulation of DUSP9 by miR-194-5p. The expression levels of DUSP9 protein and mitogen-activated protein kinase (MAPK) pathway-related molecules were detected by Western blot assay. Results Compared with the NC group, left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were decreased in the DCM group and the DCM+antagomir control group, and myocardial cell cross-sectional area, apoptosis rate, levels of inflammatory factors IL-6 and TNF-α, phosphorylated ERK (p-ERK) and phosphorylated p38 (p-p38) increased (P<0.05). The expression of miR-194-5p mRNA was up-regulated, while the mRNA and protein expression of DUSP9 were down-regulated (P<0.05), and the myocardial mitochondrial ultrastructure was severely damaged. Compared with the DCM group and the DCM+antagomir control group, LVEF and LVFS were increased, and myocardial cell cross-sectional area, apoptosis rate, inflammatory factor levels, p-ERK and p-p38 were decreased in the DCM+miR-194-5p antagomir group (P<0.05). The expression of miR-194-5p mRNA was down-regulated, while the mRNA and protein expression of DUSP9 were up-regulated (P<0.05), and the myocardial mitochondrial ultrastructural injury was alleviated. Bioinformatics prediction indicated that DUSP9 was a potential target of miR-194-5p, and the MAPK signaling pathway was the most significantly enriched pathway among the target genes. The dual-luciferase reporter assay confirmed that miR-194-5p could directly target the 3'-UTR region of DUSP9. Conclusion Inhibition of miR-194-5p attenuates myocardial injury in DCM mice, likely by targeting DUSP9 for upregulation, thereby suppressing the MAPK signaling pathway.

Key words: diabetic cardiomyopathies, microRNAs, dual-specificity phosphatases, p38 mitogen-activated protein kinases, apoptosis, extracellular signal-regulated kinase, miR-194-5p

CLC Number:

R542.2

Figures/Tables 13

References 20

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基因名称	引物序列（5'→3'）	产物大小/bp
miR-194-5p	上游：CGCTGTAACAGCAACTCCAT	75
	下游：GTGCAGGGTCCGAGGT	75
U6	上游：AGCCGAAACAACCAGAGCAC	95
	下游：CAGGTAGGCGATGGTGAAGG	95
DUSP9	上游：GTGTGAGGTGCCTACTTGCTC	201
	下游：GCTCAGTCTTCTGTCCTTGGA	201
β-actin	上游：CATGTACGTTGCTATCCAGGC	181
	下游：CTCCTTAATGTCACGCACGAT	181

基因名称	引物序列（5'→3'）	产物大小/bp
miR-194-5p	上游：CGCTGTAACAGCAACTCCAT	75
	下游：GTGCAGGGTCCGAGGT	75
U6	上游：AGCCGAAACAACCAGAGCAC	95
	下游：CAGGTAGGCGATGGTGAAGG	95
DUSP9	上游：GTGTGAGGTGCCTACTTGCTC	201
	下游：GCTCAGTCTTCTGTCCTTGGA	201
β-actin	上游：CATGTACGTTGCTATCCAGGC	181
	下游：CTCCTTAATGTCACGCACGAT	181

组别	LVEF/%		LVFS/%
NC组	73.20±4.34		37.93±2.98
DCM组	56.33±1.97^a		22.67±2.16^a
DCM+antagomir control组	55.79±3.27^a		23.56±3.02^a
DCM+miR-194-5p antagomir组	61.80±2.12^bc		31.29±1.89^bc
F	103.400^＊＊		113.500^＊＊
组别		LVEDD/mm		LVESD/mm
NC组		3.45±0.09		2.51±0.12
DCM组		4.47±0.15^a		3.41±0.09^a
DCM+antagomir control组		4.52±0.24^a		3.49±0.13^a
DCM+miR-194-5p antagomir组		3.98±0.13^bc		3.01±0.11^bc
F		142.700^＊＊		234.700^＊＊

组别	LVEF/%		LVFS/%
NC组	73.20±4.34		37.93±2.98
DCM组	56.33±1.97^a		22.67±2.16^a
DCM+antagomir control组	55.79±3.27^a		23.56±3.02^a
DCM+miR-194-5p antagomir组	61.80±2.12^bc		31.29±1.89^bc
F	103.400^＊＊		113.500^＊＊
组别		LVEDD/mm		LVESD/mm
NC组		3.45±0.09		2.51±0.12
DCM组		4.47±0.15^a		3.41±0.09^a
DCM+antagomir control组		4.52±0.24^a		3.49±0.13^a
DCM+miR-194-5p antagomir组		3.98±0.13^bc		3.01±0.11^bc
F		142.700^＊＊		234.700^＊＊

组别	IL-6	TNF-α
NC组	20.47±1.98	9.10±1.21
DCM组	41.38±4.82^a	30.14±3.99^a
DCM+antagomir control组	43.89±3.97^a	29.41±4.02^a
DCM+miR-194-5p antagomir组	28.42±3.41^bc	18.12±2.43^bc
F	134.100^＊＊	153.200^＊＊