Silencing TTTY15 mitigates hypoxia-induced mitochondrial energy metabolism dysfunction and cardiomyocytes apoptosis via TTTY15/let-7i-5p and TLR3/NF-κB pathways

doi:10.1016/j.cellsig.2020.109779

Cellular Signalling

Volume 76, December 2020, 109779

https://doi.org/10.1016/j.cellsig.2020.109779 Get rights and content

Highlights

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TTTY15 was highly induced in response to hypoxia stress.
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Knockdown of TTTY15 suppressed apoptosis and mitochondrial damage in hypoxia-induced AC16 cells.
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TTTY15 served as a miRNA sponge for let-7i-5p.
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TTTY15/let-7i-5p axis participated in hypoxia injury in cardiomyocytes through TLR3/NF-κB signaling pathway.

Abstract

Noncoding RNAs are interweaved in pathological processes in myocardial ischemia (MI), such as long noncoding RNA (lncRNA) and microRNAs (miRNAs). The aim of this study was to figure out the role of Testis-specific transcript Y-linked 15 (TTTY15) and let-7i-5p in cell model of MI in cardiomyocytes. Hypoxia-induced cell injury was assessed by Cell counting kit 8 assay, flow cytometry, commercial kits and western blotting. As a result, hypoxia stress induced inhibition on cell proliferation, glucose uptake, and ATP production, and promotion on apoptosis, lactate dehydrogenase (LDH) release, and lactic acid production in human cardiomyocyte AC16 cells. During hypoxia injury, expression of TTTY15 and let-7i-5p was measured by real-time quantitative polymerase chain reaction, and TTTY15 was upregulated, accompanied with let-7i-5p downregulation. Functionally, either silencing TTTY15 or overexpressing let-7i-5p could attenuate hypoxia-induced apoptosis and mitochondrial energy metabolism dysfunction in AC16 cells. Moreover, there was an interaction between TTTY15 and let-7i-5p via target binding, as evidenced by dual-luciferase reporter assay and RNA immunoprecipitation assay. Knockdown of let-7i-5p could counteract the protective role of TTTY15 deletion in hypoxic AC16 cells. Meanwhile, toll-like receptor 3 (TLR3)/nuclear factor-kappa B (NF-κB) signaling was validated by western blotting. Expression of TLR3, tumor necrosis factor receptor-associated factor 6 (TRAF6) and phosphorylated p65 was promoted in hypoxic AC16 cells, which was abrogated by TTTY15 silencing along with let-7i-5p upregulation. Collectively, TTTY15 knockdown protects cardiomyocytes against hypoxia-induced apoptosis and mitochondrial energy metabolism dysfunction in vitro through let-7i-5p/TLR3/NF-κB pathway to suppress.

Section snippets

List of abbreviations

TTTY15	Testis-specific transcript Y-linked 15
TLR3	toll-like receptor 3
TRAF6	receptor-associated factor 6
MI	Myocardial infarction
CVDs	Cardiovascular diseases
LDH	lactate dehydrogenase
ATP	adenosine triphosphate

Cell culture and hypoxia treatment

The human adult ventricular cardiomyocyte AC16 cells (SCC109) were obtained from Merck (Darmstadt, Germany), and cultured in Dulbecco's modified Eagle's medium: Nutrient mixture F-12 (DMEM/F12; Gibco, Gaithersburg, MD, USA). Normally, the AC16 cells were incubated in an aseptic condition of 95% air and 5% CO₂ in 37 °C. AC16 cells in 90% confluence were transferred in an environment of 94% N₂, 5% CO₂ and 1% O₂ in 37 °C for 12 h (hypoxia treatment) or a condition of 95% air and 5% CO₂ in 37 °C

Hypoxia induced apoptosis and mitochondrial energy metabolism dysfunction in human cardiomyocyte AC16 cells

Sustained exposure to hypoxia could induce cell injury. And to confirm hypoxia injury in cardiomyocytes, AC16 cells were disposed to 1% oxygen condition instead of normal 21% oxygen for 12 h. A series of functional experiments were following performed. CCK-8 showed a cell viability inhibition (approximate 50%, P < 0.05) after hypoxia treatment, accompanied with declined Cyclin D1 expression (Fig. 1A and G), suggesting cell proliferation was inhibited in response to hypoxia. Flow cytometry

Discussion

There were quite a few of lncRNAs that had been involved in hypoxia-induced myocardial injuries in cardiomyocyte fibrosis [23], apoptosis and autophagy [24], migration and invasion [25], Ca homeostasis and mitochondrial fission [26,27]. Little research had been focused on the role of lncRNA in mitochondrial energy metabolism in cardiomyocytes. Here, we investigated the dysregulation of TTTY15 and its role in hypoxia-induced human cardiomyocyte AC16 cells proliferation, apoptosis and glucose

Funding

None.

Authors' contributions

Han Zhang and Xiufang Zou designed the study, analyzed the data and wrote the manuscript. Feng Liu analyzed the data and performed the experiments.

Declaration of Competing Interest

The authors declare that they have no financial conflicts of interest.

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¹: These authors contributed equally to this work.

View full text

Silencing TTTY15 mitigates hypoxia-induced mitochondrial energy metabolism dysfunction and cardiomyocytes apoptosis via TTTY15/let-7i-5p and TLR3/NF-κB pathways

Highlights

Abstract

Section snippets

List of abbreviations

Cell culture and hypoxia treatment

Hypoxia induced apoptosis and mitochondrial energy metabolism dysfunction in human cardiomyocyte AC16 cells

Discussion

Funding

Authors' contributions

Declaration of Competing Interest

Biochim. Biophys. Acta Mol. basis Dis.

Mol. Med. Rep.

Life Sci.

Gene

Biochim. Biophys. Acta

Mol. Ther. Nucleic Acids

Biomed. Pharmacother.

Eur. Urol.

J. Biol. Chem.

Cardiovascular risk in chronic obstructive pulmonary disease

Respirology

Myocardial apoptosis in heart disease: does the emperor have clothes?

Basic Res. Cardiol.

Mitochondrial quality control in cardiac cells: mechanisms and role in cardiac cell injury and disease

J. Cell. Physiol.

The independence of and associations among apoptosis, autophagy, and necrosis

Signal Transduct. Target Ther.

Circulating noncoding RNAs as biomarkers of cardiovascular disease and injury

Circ. Res.

Regulatory non-coding RNAs in acute myocardial infarction

J. Cell. Mol. Med.

Long noncoding RNAs in patients with acute myocardial infarction

Circ. Res.

Identification of microRNAs as potential cellular monocytic biomarkers in the early phase of myocardial infarction: a pilot study

Sci. Rep.

miR-27a-5p attenuates hypoxia-induced rat cardiomyocyte injury by inhibiting Atg7

Int. J. Mol. Sci.

Long noncoding RNA MIAT: a potential role in the diagnosis and mediation of acute myocardial infarction

Mol. Med. Rep.