Σφακιανάκης Αλέξανδρος
ΩτοΡινοΛαρυγγολόγος
Αναπαύσεως 5 Άγιος Νικόλαος
Κρήτη 72100
00302841026182
00306932607174
alsfakia@gmail.com

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Τρίτη 10 Αυγούστου 2021

MicroRNA-449a inhibits cell proliferation and migration by regulating mutant p53 in MDA-MB-468 cells

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Exp Ther Med. 2021 Sep;22(3):1020. doi: 10.3892/etm.2021.10452. Epub 2021 Jul 15.

ABSTRACT

The present study aimed to investigate the role of microRNA (miR)-449a in the proliferation, migration and apoptosis of MDA-MB-468 breast cancer cells and examine the association between miR-449a and mutant p53 in these cells. Cell proliferation, migration and invasion were examined using a crystal violet staining assay, wound healing scratch assay and Transwell assay, respectively. The expression level of miR-449a and p53 was detected by reverse transcription-quantitative PCR or western blotting. The results indicated that knockdown of mutant p53 suppressed the proliferation and migration of MDA-MB-468 cells by inhibiting the PI3K/AKT/mTOR signaling pathway. In addition, miR-449a suppressed proliferation and migration via downregulation of mutant p53 expression in MDA-MB-468 cells. Therefore, miR-449a may function as a tumor suppressor by regula ting p53 expression in breast cancer cells, which may have potential implications in the treatment of patients with triple-negative breast cancer carrying mutant p53.

PMID:34373706 | PMC:PMC8343910 | DOI:10.3892/etm.2021.10452

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iTRAQ-based quantitative proteomics analysis of the hepatoprotective effect of melatonin on ANIT-induced cholestasis in rats

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Exp Ther Med. 2021 Sep;22(3):1014. doi: 10.3892/etm.2021.10446. Epub 2021 Jul 15.

ABSTRACT

The therapeutic effects of melatonin on cholestatic liver injury have received widespread attention recently. The aim of the present study was to investigate the mechanisms of the anti-cholestatic effects of melatonin against α-naphthyl isothiocyanate (ANIT)-induced liver injury in rats and to screen for potential biomarkers of cholestasis through isobaric tags for relative and absolute quantitation (iTRAQ) proteomics. Rats orally received melatonin (100 mg/kg body weight) or an equivalent volume of 0.25% carboxymethyl cellulose sodium salt 12 h after intraperitoneal injection of ANIT (75 mg/kg) and were subsequently sacrificed at 36 h after injection. Liver biochemical indices were determined and liver tissue samples were stained using hematoxylin-eosin staining, followed by iTRAQ quantitative proteomics to identify potential underlying therape utic mechanisms and biomarkers. The results suggested that the expression levels of alanine transaminase, aspartate aminotransferase, total bilirubin and direct bilirubin were reduced in the rats treated with melatonin. Histopathological observation indicated that melatonin was effective in the treatment of ANIT-induced cholestasis. iTRAQ proteomics results suggested that melatonin-mediated reduction in ANIT-induced cholestasis may be associated with enhanced antioxidant function and relieving abnormal fatty acid metabolism. According to pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes, the major metabolic pathways for the metabolism of melatonin are fatty acid degradation, the peroxisome proliferator-activated receptor signaling pathway, fatty acid metabolism, chemical carcinogenesis, carbon metabolism, pyruvate metabolism, fatty acid biosynthesis and retinol metabolism, as well as drug metabolism via cytochrome P450. Malate dehydrogenase 1 and glutathi one S-transferase Yb-3 may serve as potential targets in the treatment of ANIT-induced cholestasis with melatonin.

PMID:34373700 | PMC:PMC8343461 | DOI:10.3892/etm.2021.10446

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Molecular diagnosis of McArdle disease using whole-exome sequencing

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Exp Ther Med. 2021 Sep;22(3):1029. doi: 10.3892/etm.2021.10461. Epub 2021 Jul 18.

ABSTRACT

Whole-exome sequencing (WES) analysis has been used recently as a diagnostic tool for finding molecular defects. In the present study, researchers attempted to analyze molecular defects through WES in a 13-year-old female patient who had not been diagnosed through a conventional genetic approach. DNA was extracted and subjected to WES analysis to identify the genetic defect. A total of 106,728 exons and splicing variants were selected, and synonymous single nucleotide variants (SNVs) and general single nucleotide polymorphisms (SNPs) were filtered out. Finally, nonsynonymous SNVs (c.C415T and c.C389T) of the PYGM gene were identified in nine compound heterozygous mutations. PYGM encodes myophosphorylase and degrades glycogen in the muscle to supply energy to muscle cells. The present study revealed that the patient's father had a c. C389T mutation and the mother had a c.C415T mutation, resulting in A130V and R139W missense mutations, respectively. To the best of our knowledge, the A130V variant in PYGM has not been reported in the common variant databases. All variations of the patient's family detected using WES were verified by Sanger sequencing. Because the patient had compound heterozygous mutations in the PYGM gene, the patient was presumed to exhibit markedly decreased muscle phosphorylase activity. To assess the function of myophosphorylase, an ischemic forearm exercise test was performed. The blood ammonia level sharply increased and the lactate level maintained a flat curve shape similar to the typical pattern of McArdle disease. Therefore, the diagnosis of the patient was confirmed to be McArdle disease, a glycogen storage disease. Through WES analysis, accurate and early diagnosis could be made in the present study. This report describes a novel compound heterozygous mutation of the PYGM gene in a Korean patient.

PMID:34373715 | PMC:PMC8343624 | DOI:10.3892/etm.2021.10461

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Morphine pretreatment protects against cerebral ischemic injury via a cPKCγ-mediated anti-apoptosis pathway

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Exp Ther Med. 2021 Sep;22(3):1016. doi: 10.3892/etm.2021.10448. Epub 2021 Jul 15.

ABSTRACT

It has been reported that morphine pretreatment (MP) can exert neuroprotective effects, and that protein kinase C (PKC) participates in the initiation and development of ischemic/hypoxic preconditioning in the brain. However, it remains unknown whether PKC is involved in MP-induced neuroprotection. The aim of the present study, which included in vivo and in vitro experiments, was to determine whether the conventional γ isoform of PKC (cPKCγ) was involved in the protective effects of MP against cerebral ischemic injury. The present study included an in vivo experiment using a mouse model of middle cerebral artery occlusion and an in vitro experiment using neuroblastoma N2a cells with oxygen-glucose deprivation (OGD). Furthermore, a cPKCγ antagonist, Go6983, was used to determine the involvement of cPKCγ in the prote ctive effects of MP against cerebral ischemic injury. In the in vivo experiment, neurological deficits, ischemic infarct volume, neural cell damage, apoptosis and caspase-3 activation were evaluated. In the in vitro experiment, flow cytometry was used to determine the activation of caspase-3 in N2a cells with OGD. It was found that MP protected against cerebral ischemic injury. However, intracerebroventricular injection of the cPKCγ antagonist before MP attenuated the neuroprotective effect of MP and increased the activation of cleaved caspase-3. These findings suggested that MP may provide protection against cerebral ischemic injury via a cPKCγ-mediated anti-apoptosis pathway.

PMID:34373702 | PMC:PMC8343874 | DOI:10.3892/etm.2021.10448

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Weighted gene co-expression network analysis of the association between upregulated AMD1, EN1 and VGLL1 and the progression and poor prognosis of breast cancer

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Exp Ther Med. 2021 Sep;22(3):1030. doi: 10.3892/etm.2021.10462. Epub 2021 Jul 18.

ABSTRACT

Breast cancer is the most prevalent malignancy among females, but the molecular mechanisms involved in its pathogenesis and progression have remained to be fully elucidated. The aim of the present study was to identify novel potential therapeutic targets for breast cancer. The dataset GSE76275 was downloaded from the Gene Expression Omnibus database and weighted gene co-expression network analysis (WGCNA) was performed to identify hub genes. Furthermore, the dataset GSE25055, containing gene expression data and clinical information, was downloaded to validate the expression and survival association of these hub genes. In addition, the datasets GSE25065 and GSE42568 were used to validate the association between hub gene expression levels and clinical features. Immunohistochemistry (IHC), reverse transcription-quantitative PCR, as well as prolifera tion, migration, invasion and apoptosis assays, were used to verify gene expression and function. A total of 4,052 genes were selected for WGCNA and 18 modules were established; the red module was identified as the key module, as it had a strong positive correlation with the tumor grade. Survival analyses of hub genes [S-adenosylmethionine decarboxylase proenzyme (AMD1), homeobox protein engrailed-1 (EN1) and vestigial-like protein (VGLL1)] indicated that higher levels of gene expression were associated with poor prognosis of patients with breast cancer. This association was based on survival analysis of GSE25055 using the Kaplan-Meier plotter tool. Expression validation revealed that the upregulation of hub genes was associated with advanced tumor grade and malignant molecular subtype (basal-like). IHC results from the Human Protein Atlas also demonstrated that protein expression levels of the hub genes were higher in tumor tissues compared with those in adjacent normal tissues. Fu rthermore, the expression levels of AMD1, EN1 and VGLL1 were strongly correlated with each other. These results demonstrated that AMD1 is highly expressed in breast cancer tissues and cells and AMD1 knockdown decreased the proliferation and metastatic potential, while increasing apoptosis of breast cancer cells. These results suggested that AMD1, EN1 and VGLL1 are likely to contribute to breast cancer progression and unfavorable prognosis.

PMID:34373716 | PMC:PMC8343771 | DOI:10.3892/etm.2021.10462

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Comparison of the differentiation abilities of bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells toward nucleus pulposus-like cells in three-dimensional culture

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Exp Ther Med. 2021 Sep;22(3):1018. doi: 10.3892/etm.2021.10450. Epub 2021 Jul 15.

ABSTRACT

Nucleus pulposus cell (NPC) transplantation can be a potential therapeutic approach for intervertebral disc degeneration (IDD). However, low cell viability has restricted the therapeutic capacity of NPCs, and sources of natural NPCs are limited. Bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived mesenchymal stem cells (ADSCs) can be differentiated toward NPC-like cells. However, it is unknown whether there are differences in the abilities of these two cell types to differentiate into NPC-like cells, or which cell type exhibits the best differentiation ability. The present study compared the abilities of BMSCs and ADSCs to differentiate toward NPC-like cells with or without a 3D culture system to lay a foundation for stem cell transplantation therapy for IDD. BMSCs were isolated from the rat whole bone marrow cell using the rep eated adherent culture method. ADSCs were isolated from rat adipose tissues in the subcutaneous inguinal region using the enzyme digestion method. Cells were identified using flow cytometry. Cell viability was assessed via Cell Counting Kit-8 assays, and reverse transcription-quantitative PCR and western blotting were carried out to evaluate the expression of NPC markers and chondrocyte-specific genes. Glycosaminoglycans (GAGs) and proteoglycans were examined via Alcian blue and safranin O staining, respectively. ADSCs in 3D culture displayed the highest cell proliferative ability, compared with the 2D culture system and BMSC culture. In addition, ADSCs in 3D culture exhibited increased GAG and proteoglycan synthesis than BMSCs. Compared with BMSCs in 3D culture, ADSCs in 3D culture exhibited higher mRNA and protein expression of NPC marker genes (hypoxia-inducible factor 1-α, glucose transporter 1) and chondrocyte-specific genes (Sox-9, aggrecan and type II collagen). The present findings indicated that ADSCs exhibited a better ability to differentiate into NPC-like cells in 3D culture compared with BMSCs, which may be of value for the regeneration of intervertebral discs using cell transplantation therapy.

PMID:34373704 | PMC:PMC8343765 | DOI:10.3892/etm.2021.10450

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Therapeutic hypothermia effect on asphyxial cardiac arrest-induced renal ischemia/reperfusion injury via change of Nrf2/HO-1 levels

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Exp Ther Med. 2021 Sep;22(3):1031. doi: 10.3892/etm.2021.10463. Epub 2021 Jul 18.

ABSTRACT

The present study aimed to investigate the renoprotective effect of therapeutic hypothermia (TH) on renal ischemia-reperfusion injury (RI/RI) induced by asphyxial cardiac arrest (CA) in rats. A total of 48 male rats were randomly divided into five groups: i) Sham (n=6); ii) Normothermia + CA (Normo.) (n=14); iii) Normo. and 2 h of TH after return of spontaneous circulation (ROSC) (n=12); iv) Normo. and 4 h of TH after ROSC (n=9); and v) Normo. and 6 h of TH after ROSC (n=7). All rats except the Sham group underwent asphyxia CA and were sacrificed 1 day after ROSC. The survival rate increased from 42.8% in the Normo. group to 50, 66.6 and 85.7% in the groups with 2, 4 and 6 h of TH after CA, respectively. TH attenuated the histopathological changes of the renal tissues following ROSC and the levels of blood urea nitrogen, serum creatinine and malo ndialdehyde in renal tissues. On immunohistochemistry, the relative optical density of nuclear erythroid-related factor-2 (Nrf2) and heme oxygenase (HO-1) expression in renal tissues increased in the Normo. group compared with that in the Sham group and exhibited further significant increases at 6 h of TH after ROSC. In conclusion, TH attenuated renal injury and increased the expression of Nrf2 and HO-1 in a TH treatment time-dependent manner.

PMID:34373717 | PMC:PMC8343472 | DOI:10.3892/etm.2021.10463

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Entheseal involvement in a group of psoriatic arthritis patients: An ultrasonographic study

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Exp Ther Med. 2021 Sep;22(3):1044. doi: 10.3892/etm.2021.10476. Epub 2021 Jul 21.

ABSTRACT

Psoriatic arthritis (PsA) is an inflammatory potentially destructive disease that requires early diagnosis and therapeutic approach. Its main pathogenic event and the condition's hallmark is considered to be enthesitis. Clinical examination of the enthesis can be a challenge in the clinical practice; thus, ultrasonography (US) has emerged as an indispensable imaging tool for evaluating both structural and inflammatory changes of this structure. In the present study, we aimed to analyze the type and frequency of entheseal involvement in PsA patients by US examination, performing a retrospective study on 41 patients diagnosed with PsA. Ultrasonographically confirmed enthesitis, identified according to Outcome Measures in Rheumatology group (OMERACT, initially Outcome Measures in Rheumatoid Arthritis Clinical Trials) definitions, was present in 26 o f the included patients, Achilles enthesis being the most common site involved. The prevalence of tendon structure abnormalities and the presence of entesophytes underlines the importance of chronic inflammation on entheseal sites. US examination has proven to be a reliable imaging method, with significant and continuous improvement, which is clearly a requisite part for current understanding and diagnosis of enthesitis and more than this, for the patient follow-up algorithm.

PMID:34373730 | PMC:PMC8343913 | DOI:10.3892/etm.2021.10476

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Genome-wide methylation analysis reveals differentially methylated CpG sites and altered expression of heart development-associated genes in fetuses with cardiac defects

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Exp Ther Med. 2021 Sep;22(3):1032. doi: 10.3892/etm.2021.10464. Epub 2021 Jul 18.

ABSTRACT

DNA methylation, as an epigenetic mechanism, has a vital role in heart development. An increasing number of studies have investigated aberrant DNA methylation in pediatric or adult heart samples from patients with congenital heart defects (CHD). Placenta tissue, umbilical cord blood, or newborn blood have also been used to detect DNA methylation biomarkers for CHD. However, few studies have compared the methylation levels in fetal heart tissue with cardiac defects with that in normal controls. The present study conducted an integrative whole-genome and CpG site-specific DNA methylation analysis of fetal heart samples from 17 isolated cardiac defect cases, 14 non-isolated cardiac defect cases, and 22 controls with normal hearts, using methylated DNA immunoprecipitation microarray and MassARRAY EpiTYPER assays. Expression of genes adjacent to diffe rentially methylated regions (DMRs) was measured by RT-qPCR and western blot analysis. The results revealed that fetuses with cardiac defects presented global hypomethylation. Genomic analysis of DMRs revealed that a proportion of DMRs were located in exons (12.4%), distal intergenic regions (11.14%), and introns (8.97%). Only 55.7% of DMRs were observed at promoter regions. Functional enrichment analysis for genes adjacent to these DMRs revealed that hypomethylated genes were involved in embryonic heart tube morphogenesis and immune-related regulation functions. Intergenic hypermethylation of EGFR and solute carrier family 19 member 1 (SLC19A1), and intragenic hypomethylation of NOTCH1 were validated in fetal heart tissues with cardiac defects. Only SLC19A1 expression was significantly decreased at the mRNA level, while EGFR, NOTCH1, and SLC19A1 expression were all significantly decreased at the protein level. In conclusion, the present study demonstrated that fetal cardiac defects may be associated with alterations in regional and single CpG site methylation outside of promoter regions, resulting in differentiated expression of corresponding genes associated with heart development. These results present new insights into the epigenetic mechanisms underlying abnormal heart development.

PMID:34373718 | PMC:PMC8343574 | DOI:10.3892/etm.2021.1046 4

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Differential expression of long non-coding RNAs SRA, HCG22 and MHRT in children with Kawasaki disease

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Exp Ther Med. 2021 Sep;22(3):1022. doi: 10.3892/etm.2021.10454. Epub 2021 Jul 15.

ABSTRACT

Kawasaki disease (KD) is an acute, self-limited inflammatory illness during childhood that may lead to thrombosis in the coronary arteries (CA). The major aims of the present study were to estimate the serum levels of long non-coding RNAs (lncRNAs) and the metabolic profiles of patients with KD. A total of 40 specimens were obtained from pediatric patients (40 specimens before and 40 specimens after treatment) who were diagnosed with KD (n=40). The controls comprised healthy children without KD (n=40). The serum levels of lncRNAs steroid receptor RNA activator (SRA), human leukocyte antigen complex group 22 (HCG22) and myosin heavy chain-associated RNA transcript (MHRT) were determined using reverse transcription-quantitative PCR. Subsequently, the correlation between the expression levels of lncRNAs and biochemical parameters of patients was ass essed. Receiver operating characteristic curves were constructed to determine the diagnostic value of the lncRNAs. The results indicated that the serum levels of lncRNAs SRA and HCG22 were higher in patients with acute KD compared with those in healthy controls. B-type natriuretic peptide (BNP) and C-reactive protein were positively correlated with HCG22 in patients with acute KD, while total cholesterol and low-density lipoprotein were negatively correlated with HCG22 in patients with acute KD. The lncRNA MHRT was significantly upregulated in convalescent KD compared with acute KD following intravenous immunoglobulin therapy. In patients with convalescent KD, creatine kinase was positively correlated with MHRT, while BNP and adenosine deaminase were negatively correlated with MHRT. In conclusion, to the best of our knowledge, the present study was the first to identify that the serum levels of lncRNAs SRA and HCG22 in patients with acute KD were higher compared with those in contro l subjects. MHRT levels in patients with convalescent KD were higher than those in the acute phase. LncRNAs SRA and HCG22 may have crucial roles in KD and are potential novel diagnostic biomarkers for KD. LncRNA MHRT may be considered a novel biomarker for predicting the clinical prognosis of patients with KD.

PMID:34373708 | PMC:PMC8343885 | DOI:10.3892/etm.2021.104 54

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