Circ_005077 accelerates myocardial lipotoxicity induced by high-fat diet via CyPA/p47PHOX mediated ferroptosis.

The long-term high-fat diet (HFD) can cause myocardial lipotoxicity, which is characterized pathologically by myocardial hypertrophy, fibrosis, and remodeling and clinically by cardiac dysfunction and heart failure in patients with obesity and diabetes. Circular RNAs (circRNAs), a novel class of noncoding RNA characterized by a ring formation through covalent bonds, play a critical role in various cardiovascular diseases. However, few studies have been conducted to investigate the role and mechanism of circRNA in myocardial lipotoxicity. Here, we found that circ_005077, formed by exon 2-4 of Crmp1, was significantly upregulated in the myocardium of an HFD-fed rat. Furthermore, we identified circ_005077 as a novel ferroptosis-related regulator that plays a role in palmitic acid (PA) and HFD-induced myocardial lipotoxicity in vitro and in vivo. Mechanically, circ_005077 interacted with Cyclophilin A (CyPA) and inhibited its degradation via the ubiquitination proteasome system (UBS), thus promoting the interaction between CyPA and p47phox to enhance the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase responsible for ROS generation, subsequently inducing ferroptosis. Therefore, our results provide new insights into the mechanisms of myocardial lipotoxicity, potentially leading to the identification of a novel therapeutic target for the treatment of myocardial lipotoxicity in the future.

Melatonin attenuates dental pulp stem cells senescence due to vitro expansion via inhibiting MMP3.

OBJECTIVE: We aimed to identify the crucial genes involved in dental pulp stem cell (DPSC) senescence and evaluate the impact of melatonin on DPSC senescence. METHODS: Western blotting, SA-ß-Gal staining and ALP staining were used to evaluate the senescence and differentiation potential of DPSCs. The optimal concentration of melatonin was determined using the CCK-8 assay. Differentially expressed genes (DEGs) involved in DPSC senescence were obtained via bioinformatics analysis, followed by RT-qPCR. Gain- and loss-of-function studies were conducted to explore the role of MMP3 in DPSC in vitro expansion and in response to melatonin. GSEA was employed to analyse MMP3-related pathways in cellular senescence. RESULTS: Treatment with 0.1 µM melatonin attenuated cellular senescence and differentiation potential suppression in DPSCs due to long-term in vitro expansion. MMP3 was a crucial gene in senescence, as confirmed by bioinformatics analysis, RT-qPCR and Western blotting. Furthermore, gain- and loss-of-function studies revealed that MMP3 played a regulatory role in cellular senescence. Rescue assays showed that overexpression of MMP3 reversed the effect of melatonin on senescence. GSEA revealed that the MMP3-dependent anti-senescence effect of melatonin was associated with the IL6-JAK-STAT3, TNF-α-Signalling-VIA-NF-κB, COMPLEMENT, NOTCH Signalling and PI3K-AKT-mTOR pathways. CONCLUSION: Melatonin attenuated DPSC senescence caused by long-term expansion by inhibiting MMP3.

miR-141-3p regulates saturated fatty acid-induced cardiomyocyte apoptosis through Notch1/PTEN/AKT pathway via targeting PSEN1.

It has been reported that miR-141-3p levels are markedly upregulated in the cardiomyocytes of obese rats induced by a high-fat diet. However, the role of miR-141-3p in myocardial lipotoxicity remains elusive. In the present study, the role of miR-141-3p in lipotoxic injury of H9c2 cells induced by palmitic acid (PA) and its possible mechanisms were assessed. The results indicated that miR-141-3p was significantly upregulated in PA-induced cardiomyocytes. miR-141-3p inhibitor enhanced the cell viability, reduced the release of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), and troponin I (CTN-I), decreased cell apoptosis rate, and repressed the activation of mitochondrial apoptosis pathway in PA-treated H9c2, whereas treatment with miR-141-3p mimics resulted in the opposite effects. Mechanistically, it was further revealed that miR-141-3p could specifically bind to presenilin 1 (PSEN1) 3'UTR, and upregulating miR-141-3p levels reduced the expression of PSEN1, thereby inhibiting the activation of the Notch1/PTEN/AKT pathway. Additionally, inhibition of Notch1/AKT signaling pathway by its inhibitor could abrogate the effect of miR-141-3p on mitochondrial-mediated apoptosis induced by PA. In conclusion, the present study demonstrates that miR-141-3p regulates saturated fatty acid-induced cardiomyocyte apoptosis through Notch1/PTEN/AKT pathway via targeting PSEN1, which gains a new insight into the mechanisms of myocardial lipotoxic injury.

DOCK8 Serves as a Prognostic Biomarker and Is Related to Immune Infiltration in Patients With HPV Positive Head and Neck Squamous Cell Carcinoma.

PURPOSE: Dedicator of cytokinesis 8 (DOCK8) was reported to have a vital link to immunoregulation. However, the mechanisms by which it drives immune infiltration in cancer remain uncertain. We tried to assess the role of DOCK8 in patients with cancer, especially human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC). METHODS: Data on the expression and survival of DOCK8 in patients with various cancers were analyzed using the Oncomine and TIMER databases. The TIMER database assessed the relationship of DOCK8 with immune infiltration levels and various markers of multiple immune cells. Gene set enrichment analysis revealed tumor-associated biological processes related to DOCK8. ENCODE database was used to explore relevant transcription factors of DOCK8, and a PPI network was constructed using GENEMINIA. The expression and survival role of DOCK8 was confirmed in patients from independent GEO datasets. RESULTS: We determined that DOCK8 expression was upregulated or downregulated in various cancers unlike in healthy tissues. A high expression of DOCK8 was significantly correlated with a favorable prognosis in HPV-positive HNSCC and lung adenocarcinoma (LUAD). Furthermore, multivariate Cox regression analysis revealed that DOCK8 was an independent prognostic factor of HPV-positive HNSCC. Additionally, elevated DOCK8 expression was positively correlated with multiple immune cell infiltration levels and immune marker expression associated with particular immune cell subsets. Also, 14 pathways involved in immune activities and carcinogenesis, 22 potential TFs, and co-expression proteins of DOCK8 indicated DOCK8 to be related to tumor-associated biological processes. Ultimately, we verified that DOCK8 is upregulated and confers a favorable overall survival and progression-free survival status in patients with HPV-positive HNSCC. CONCLUSION: These results elucidate that high expression of DOCK8 indicates a favorable prognosis in patients with HPV-positive HNSCC as well as increased microenvironmental immune infiltration levels. It would provide new insights into the prognosis predicting and clinical regimen decision making in patients with HPV-positive HNSCC.

MiR-30e-5p is sponged by Kcnq1ot1 and represses Angiotensin II-induced hypertrophic phenotypes in cardiomyocytes by targeting ADAM9.

Prolonged cardiac hypertrophy, a pathological compensatory response of the heart, finally leads to heart failure. Numerous studies have illustrated the vital roles of non-coding RNAs (ncRNAs) in cardiac hypertrophy. Here, we probed into the role and probable mechanism of microRNA-30e-5p (miR-30e-5p) in Angiotensin II (Ang-II)-stimulated hypertrophic cardiomyocytes. Intriguingly, the expression of hypertrophic markers, cell surface area and protein/DNA ratio were all reduced in Ang-II-induced hypertrophic cardiomyocytes when miR-30e-5p expression was augmented. Then, ADAM9 was screened out as the target of miR-30e-5p and ADAM9 overexpression rescued the effect of miR-30e-5p upregulation in Ang-II-treated cardiomyocytes. Moreover, we identified Kcnq1ot1 as the upstream of miR-30e-5p/ADAM9 axis and verified that Kcnq1ot1 aggrandized ADAM9 expression in Ang-II-treated cardiomyocytes through absorbing miR-30e-5p. Furthermore, rescue assays confirmed that ADAM9 up-regulation abrogated the repressive effect of Kcnq1ot1 depletion on Ang-II-induced cardiac hypertrophy. In conclusion, Kcnq1ot1 sequestered miR-30e-5p to release ADAM9 to facilitate cardiac hypertrophy, indicating that Kcnq1ot1 might be used as a potentially therapeutic target for cardiac hypertrophy.

Down-regulation of GAS5 ameliorates myocardial ischaemia/reperfusion injury via the miR-335/ROCK1/AKT/GSK-3ß axis.

Growth arrest-specific transcript 5 (GAS5), along non-coding RNA (LncRNA), is highly expressed in hypoxia/reoxygenation (H/R)-cardiomyocytes and promotes H/R-induced apoptosis. In this study, we determined whether down-regulation of GAS5 ameliorates myocardial ischaemia/reperfusion (I/R) injury and further explored its mechanism. GAS5 expression in cardiomyocytes and rats was knockdown by transfected or injected with GAS5-specific small interfering RNA or adeno-associated virus delivering small hairpin RNAs, respectively. The effects of GAS5 knockdown on myocardial I/R injury were detected by CCK-8, myocardial enzyme test, flow cytometry, TTC and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining. qRT-PCR and luciferase reporter assay were carried out to analyse the relationship between GAS5 and miR-335. The regulation of GAS5 on Rho-associated protein kinase 1 (ROCK1) expression, the activation of PI3K/AKT/GSK-3ß pathway and mitochondrial permeability transition pore (mPTP) opening was further evaluated. The results indicated that GAS5 knockdown enhanced the viability, decreased apoptosis and reduced the levels of lactate dehydrogenase and creatine kinase-MB in H/R-treatment cardiomyocytes. Meanwhile, down-regulation of GAS5 limited myocardial infarct size and reduced apoptosis in I/R-heart. GAS5 was found to bind to miR-335 and displayed a reciprocal inhibition between them. Furthermore, GAS5 knockdown repressed ROCK1 expression, activated PI3K/AKT, thereby leading to inhibition of GSK-3ß and mPTP opening. These suppressions were abrogated by miR-335 inhibitor treatment. Taken together, our results demonstrated that down-regulation of GAS5 ameliorates myocardial I/R injury via the miR-335/ROCK1/AKT/GSK-3ß axis. Our findings suggested that GAS5 may be a new therapeutic target for the prevention of myocardial I/R injury.

Brazilin prevents against myocardial ischemia-reperfusion injury through the modulation of Nrf2 via the PKC signaling pathway.

BACKGROUND: Brazilin, a major ingredient of Caesalpinia sappan L., possesses multiple pharmaceutical activities, although whether or not brazilin exerts any protective effect on myocardial ischemia-reperfusion injury (MIRI) has not yet been reported. The present study determined the cardioprotective effects of brazilin, and elucidated the role of nuclear factor E2-associated factor 2 (Nrf2) in this process. METHODS: Following treatment with brazilin, H9c2 cells were subjected to 6 h of hypoxia/3 h of reoxygenation. CCK-8 assay and flow cytometry were employed to detect cell viability and apoptosis, respectively. Furthermore, after brazilin treatment, isolated rat hearts underwent 30 min of ischemia, followed by 90 min of reperfusion. Triphenyltetrazolium chloride (TTC) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining were performed to measure myocardial infarct size and apoptosis, respectively. The changes in the levels of proteins were detected by western blotting. RESULTS: Brazilin treatment dose-dependently led to a significant enhancement in cell viability, a reduction in myocardial infarct size, and a decrease in release of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH). Moreover, brazilin also remarkably inhibited apoptosis and led to various improvements in cardiac function. Additionally, brazilin treatment caused a marked alleviation of oxidative stress, as evidenced by the fact that brazilin reduced the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), while enhancing the activities of superoxide dismutase (SOD) and glutathione peroxidase (GXH-Px). Mechanistically, it was found that brazilin induced Nrf2 nuclear translocation, with a concomitant upregulation of both heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase (NQO1) expression. Furthermore, the phosphorylation level and transcriptional activity of Nrf2 were enhanced by brazilin, although these enhancements were abrogated by treatment with a protein kinase C (PKC) inhibitor. Finally, it was observed that the protective effects of brazilin could be negated through inhibition of Nrf2, which suggested that the cardioprotection afforded by brazilin was Nrf2-dependent. CONCLUSIONS: Taken together, our results have demonstrated that brazilin may afford protection against MIRI through the activation of Nrf2 via the PKC signaling pathway. These results may lay the foundation for the further use of brazilin in the prevention of MIRI in clinical practice.

Erratum to "Astaxanthin Attenuates Hypertensive Vascular Remodeling by Protecting Vascular Smooth Muscle Cells from Oxidative Stress-Induced Mitochondrial Dysfunction".

[This corrects the article DOI: 10.1155/2020/4629189.].

miRNA-27a Transcription Activated by c-Fos Regulates Myocardial Ischemia-Reperfusion Injury by Targeting ATAD3a.

MicroRNA-27a (miR-27a) has been implicated in myocardial ischemia-reperfusion injury (MIRI), but the underlying mechanism is not well understood. This study is aimed at determining the role of miR-27a in MIRI and at investigating upstream molecules that regulate miR-27a expression and its downstream target genes. miR-27a expression was significantly upregulated in myocardia exposed to ischemia/reperfusion (I/R) and cardiomyocytes exposed to hypoxia/reoxygenation (H/R). c-Fos could regulate miR-27a expression by binding to its promoter region. Moreover, overexpression of miR-27a led to a decrease in cell viability, an increase in LDH and CK-MB secretion, and an increase in apoptosis rates. In contrast, suppression of miR-27a expression resulted in the opposite effects. ATPase family AAA-domain-containing protein 3A (ATAD3a) was identified as a target of miR-27a. miR-27a regulated the translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus and H/R-induced apoptosis via the regulation of ATAD3a. It was found that inhibiting miR-27a in vivo by injecting a miR-27a sponge could ameliorate MIRI in an isolated rat heart model. In conclusion, our study demonstrated that c-Fos functions as an upstream regulator of miR-27a and that miR-27a regulates the translocation of AIF from the mitochondria to the nucleus by targeting ATAD3a, thereby contributing to MIRI. These findings provide new insight into the role of the c-Fos/miR-27a/ATAD3a axis in MIRI.

The lncRNA Malat1 regulates microvascular function after myocardial infarction in mice via miR-26b-5p/Mfn1 axis-mediated mitochondrial dynamics.

RATIONALE: Myocardial infarction (MI) is a leading cause of cardiovascular mortality globally. The improvement of microvascular function is critical for cardiac repair after MI. Evidence now points to long non-coding RNAs (lncRNAs) as key regulators of cardiac remodelling processes. The lncRNA Malat1 is involved in the development and progression of multiple cardiac diseases. Studies have shown that Malat1 is closely related to the regulation of endothelial cell regeneration. However, the potential molecular mechanisms of Malat1 in repairing cardiac microvascular dysfunction after MI remain unreported. METHODS AND RESULTS: The present study found that Malat1 is upregulated in the border zone of infarction in mouse hearts, as well as in isolated cardiac microvascular endothelial cells (CMECs). Targeted knockdown of Malat1 in endothelial cells exacerbated oxidative stress, attenuated angiogenesis and microvascular perfusion, and as a result decreased cardiac function in MI mice. Further studies showed that silencing Malat1 obviously inhibited CMEC proliferation, migration and tube formation, which was at least in part attributed to disturbed mitochondrial dynamics and activation of the mitochondrial apoptosis pathway. Moreover, bioinformatic analyses, luciferase assays and pull-down assays indicated that Malat1 acted as a competing endogenous RNA (ceRNA) for miR-26b-5p and formed a signalling axis with Mfn1 to regulate mitochondrial dynamics and endothelial functions. Overexpression of Mfn1 markedly reversed the microvascular dysfunction and CMEC injuries that were aggravated by silencing Malat1 via inhibition of excessive mitochondrial fragments and mitochondria-dependent apoptosis. CONCLUSIONS: The present study elucidated the functions and mechanisms of Malat1 in cardiac microcirculation repair after MI. The underlying mechanisms of the effects of Malat1 could be attributed to its blocking effects on miR-26b-5p/Mfn1 pathway-mediated mitochondrial dynamics and apoptosis.

microRNA Expression Profiles in Myocardium of High-Fat Diet-Induced Obesity Rat.

PURPOSE: A high-fat diet (HFD) can lead to cardiac dysfunction, hypertrophy, and fibrosis. This study aimed to explore microRNA expression profiles in the myocardium of HFD-induced obesity rat. MATERIALS AND METHODS: Wistar rats were randomly divided into two groups, and fed with normal chow diet (NCD) or HFD for 20 weeks. Cardiac function was evaluated by echocardiography. Left ventricular myocardium was harvested to assess the extent of myocardial morphology alteration. MicroRNA expression was analyzed using Agilent miRNA microarray and quantitative real-time PCR (qRT-PCR) was used to validate the microarray data. The mirdbV6 database was used to forecast the miRNA target genes. The role of microRNAs in palmitate-induced cardiac hypertrophy and fibrosis in primary neonatal rat cardiomyocytes was evaluated by loss- and gain-of-function experiments. RESULTS: Significant changes in cardiac function, hypertrophy, fibrosis, and apoptosis were found in HFD rats as compared with NCD rats. miR-141-3p and miR-144-3p were also significantly upregulated in the myocardium of HFD-induced obesity rat. A series of genes involved in essential biological processes, including anatomical structure development and metabolic process, was targeted by these two miRNAs. These target genes were also implicated in signaling pathways involved in the PI3K-Akt signaling pathway, Wnt signaling pathway, autophagy, and protein processing in the endoplasmic reticulum. Inhibition of miR-141 or overexpression of miR-144 attenuated palmitate-induced cardiac hypertrophy and fibrosis. In contrast, overexpression of miR-141 or inhibition of miR-144 aggravated palmitate-induced cardiac hypertrophy and fibrosis. CONCLUSION: This study identifies that miR-141 and miR-144 are candidate miRNAs associated with the development of HFD-induced cardiac dysfunction and structure alteration.

Astaxanthin Attenuates Hypertensive Vascular Remodeling by Protecting Vascular Smooth Muscle Cells from Oxidative Stress-Induced Mitochondrial Dysfunction.

Oxidative stress aggravates mitochondrial injuries and accelerates the proliferation of vascular smooth muscle cells (VSMCs), which are important mechanisms contributing to vascular remodeling in hypertension. We put forward the hypothesis that Astaxanthin (ATX), known to possess strong features of antioxidant, could attenuate vascular remodeling by inhibiting VSMC proliferation and improving mitochondrial function. The potential effects of ATX were tested on spontaneously hypertensive rats (SHRs) and cultured VSMCs that injured by angiotensin II (Ang II). The results showed that ATX lowered blood pressure, reduced aortic wall thickness and fibrosis, and decreased the level of reactive oxygen species (ROS) and H2O2 in tunica media. Moreover, ATX decreased the expression of proliferating cell nuclear antigen (PCNA) and ki67 in aortic VSMCs. In vitro, ATX mitigated VSMC proliferation and migration, decreased the level of cellular ROS, and balanced the activities of ROS-related enzymes including NADPH oxidase, xanthine oxidase, and superoxide dismutase (SOD). Besides, ATX mitigated Ca2+ overload, the overproduction of mitochondrial ROS (mtROS), mitochondrial dysfunction, mitochondrial fission, and Drp1 phosphorylation at Ser616. In addition, ATX enhanced mitophagy and mitochondrial biosynthesis by increasing the expression of PINK, parkin, mtDNA, mitochondrial transcription factor A (Tfam), and PGC-1α. The present study indicated that ATX could efficiently treat vascular remodeling through restraining VSMC proliferation and restoring mitochondrial function. Inhibiting mitochondrial fission by decreasing the phosphorylation of Drp1 and stimulating mitochondrial autophagy and biosynthesis via increasing the expression of PINK, parkin, Tfam, and PGC-1α may be part of its underlying mechanisms.

PHLDA1 is a new therapeutic target of oxidative stress and ischemia reperfusion-induced myocardial injury.

AIM: Oxidative stress plays an important role in myocardial ischemia-reperfusion injury. Pleckstrin homology-like domain, family A, member 1 (PHLDA1) was first identified in apoptosis induced by T cell receptor activation, and was shown to play a different role in different cell types and under different stimuli. The role and mechanism of PHLDA1 in oxidative stress-induced cardiomyocyte injury and cardiac ischemia-reperfusion were therefore determined. MAIN METHODS: Cell viability and apoptotic rate were measured by Cell Counting Kit-8 and flow cytometry, respectively. Mitochondrial membrane potential was measured using JC-1 test kit. Reactive oxygen species (ROS) production was detected using ROS kit. HE staining was used to detect histological morphology, 2,3,5-triphenyltetrazolium chloride staining to detect infarct size, terminal deoxynucleotidyl transferase dUTP nick end labeling staining to detect the apoptotic rate, and immunohistochemistry and western blot analysis to detect protein expression. The binding of PHLDA1 to Bcl-2 associated X (Bax) was detected by immunoprecipitation. KEY FINDINGS: The results indicated that PHLDA1 is highly expressed in oxidative stress-induced cardiomyocyte and myocardial ischemia-reperfusion injuries. PHLDA1 overexpression in cardiomyocytes promoted oxidative stress-induced cardiomyocyte injury. At the same time, PHLDA1 knockdown improved oxidative stress-induced cardiomyocyte and myocardial ischemia-reperfusion injuries. In addition, PHLDA1 binds to Bax and the interaction is enhanced under H2O2 stimulation. SIGNIFICANCE: The present results indicated that PHLDA1 interacts with Bax to participate in oxidative stress-induced cardiomyocyte injury and myocardial ischemia reperfusion injury.