Hypothalamic NR3C1 DNA methylation in rats exposed to prenatal stress.

BACKGROUND: Human and animal studies have indicated that maternal prenatal stress (PS) has molecular and behavioral effects during pregnancy and early life. The present study aimed to evaluate the epigenetic changes of the NR3C1 gene involved in the HPA axis in the hypothalamic tissues of rats exposed to PS induced by chronic unpredictable mild stress (CUMS). Behavioral and molecular effects of these changes on the next generation were also assessed. METHODS AND RESULTS: CUMS protocol was used to generate stress in pregnant Wistar rats. To determine the effects of stress on anhedonia and movement, sucrose preference test, forced swimming test, and open field test were performed. Following these behavioral experiments, bisulfite sequencing PCR for DNA methylation levels of the NR3C1 gene, RT-qPCR for mRNA levels, and Western blot techniques for protein analysis were used in the hypothalamic tissue of sacrificed rats. Depression-like behaviors were evident in the behavioral tests of stress-exposed mothers and pups. In PS-exposed pups, hypothalamic NR3C1 promoter methylation was higher, and NR3C1 mRNA levels and NR3C1 protein levels were lower compared with controls, regardless of sex. CONCLUSION: Our results confirm the relationship between PS and epigenetic changes of HPA axis-related genes and show that NR3C1 gene methylation status in pups is sensitive to PS during pregnancy. Environmental maternal stress may have transgenerational effects that are potentially associated with adverse outcomes in the pups.

Determination of miRNA expression profile in patients with prostate cancer and benign prostate hyperplasia.

BACKGROUND: It is a known fact that the role of microRNAs (miRNA) has a very important place in cancer development and progression. miRNAs target a significant part of pathways as well as genes. This study aimed to compare the differential expression profiles of miRNAs in prostate cancer and benign prostatic hyperplasia patients. METHODS: Peripheral blood mononuclear cells (PBMCs) and tissue samples were collected from prostate cancer (PCa) (n: 20) and benign prostatic hyperplasia (BPH) (n: 20) patients. Total RNA isolation was performed. As a result of the RNA concentration and purity measurement, each patient group was pooled, and the miRNAs profiles comparison was performed with the Affymetrix Microarray System. RESULTS: In tissue samples, 37 different expressed miRNAs were identified in PCa patients compared to BPH patients. In PBMCs samples, 27 different expressed miRNAs were identified in PCa patients compared to benign prostatic hyperplasia patients. As a result of the comparison of tissue and PBMCs samples, it was determined that down regülated hsa-miR-494-3p, hsa-miR-3128, hsa-miR-8084 were common miRNAs. 3 (HIF1A, NHS,INSL4) targets identified for hsa-miR-494-3p, 2 (HIF1A, AVRP1A) for hsa-miR-3128, 3 (AVRP1A, NHS, INSL4) for hsa-miR-8084. DISCUSSION: Our results suggested that determined common hsa-miR-494-3p, hsa-miR-3128, hsa-miR-8084 and their target HIF1A, AVRP1A, NHS, INSL4 may play a crucial role in therapeutic and early diagnostic strategies for prostate cancer. The present study may represent the first in-depth analysis of PBMCs and tissue samples miRNA profiles.

Lysozyme gene treatment in testosterone induced benign prostate hyperplasia rat model and comparasion of its' effectiveness with botulinum toxin injection.

OBJECTIVES: To compare the effects and histopathological changes of botulinum neurotoxin type A and lysozyme gene injections into prostate tissue within a testosterone induced benign prostate hyperplasia rat model. MATERIALS AND METHODS: 40 male Wistar rats were randomized into four Groups. Group-1: Control, Group-2: Testosterone replacement, Group-3: Testosterone+botulinum neurotoxin type A, Group-4: Testosterone+plazmid DNA/liposome complex. RESULTS: Estimated prostate volume of the testosterone injected Groups were higher than the control (p <0.05). Actual prostate weight of the testosterone injected Groups was higher than the control Group (p <0.05). Testosterone undecanoate increased the prostate weight by 39%. Botulinum neurotoxin type A treatment led to an estimated prostate volume and actual prostate weights decreased up to 32.5% in rats leading to prostate apoptosis. Lysozyme gene treatment led to an estimated prostate volume and actual prostate weights decrease up to 38.7%. CONCLUSION: Lysozyme gene and botulinum neurotoxin type A treatments for prostate volume decreasing effect have been verified in the present study that could be anew modality of treatment in prostatic benign hyperplasia that needs to be verified in large randomized human experimental studies.

Synergic Effects of Doxorubicin and Melatonin on Apoptosis and Mitochondrial Oxidative Stress in MCF-7 Breast Cancer Cells: Involvement of TRPV1 Channels.

Transient receptor transient receptor potential vanilloid 1 (TRPV1) is a Ca(2+)-permeable channel gated by oxidative stress and capsaicin (CAP) and modulated by melatonin (MEL) and capsazepine (CPZ). A combination of doxorubicin (DOX) and MEL may offer a potential therapy for breast cancer by exerting antitumor and anti-apoptotic effects and modulating Ca(2+) influx and TRPV1 activity. We aimed to investigate the effects of MEL and DOX on the oxidative toxicity of MCF-7 human breast cancer cells, in addition to the activity of the TRPV1 channel and apoptosis. The MCF-7 cells were divided into the following six treatment groups: control, incubated with MEL (0.3 mM), incubated with 0.5 µM DOX, incubated with 1 µM DOX, incubated with MEL + 0.5 µM DOX, or incubated with MEL + 1 µM DOX. The intracellular free Ca(2+) concentration was higher in the DOX groups than in the control, and the concentration was decreased by MEL. The intracellular free Ca(2+) concentration was further increased by treatment with the TRPV1 channel activator CAP (0.01 mM), and it was decreased by the CPZ (0.1 mM). The intracellular production of reactive oxygen species, mitochondrial membrane depolarization, apoptosis level, procaspase 9 and PARP activities, and caspase 3 and caspase 9 activities were higher in the DOX and MEL groups than in the control. Apoptosis and the activity of caspase 9 were further increased in the DOX plus MEL groups. Taken together, the findings indicate that MEL supported the effects of DOX by activation of TRPV1 and apoptosis, as well as by inducing MCF-7 cell death. As the apoptosis and caspase activity of cancer cells increase because of their elevated metabolism, MEL may be useful in supporting their apoptotic capacity.

Standardizing urethral stricture models in rats: a comprehensive study on histomorphologic and molecular approach.

PURPOSE: To create a reproducible and standardized urethral stricture model in rats, evaluating both histomorphologic findings and gene expression data. In studies involving experimental animals, more standardization is needed for the creation of a urethral stricture model. METHODS: Sixteen male rats were randomized into two groups. The Sham group (n:8) underwent only a penoscrotal incision, while the stricture group (n:8) had their urethras exposed through a penoscrotal incision, followed by electrocauterization to the corpus spongiosum. On the 15th day, blood and urethral tissues were harvested for histologic and molecular analyses. Histomorphologic, immunohistochemical, and reverse transcription polymerase chain reaction analyses were performed. RESULTS: The stricture group exhibited more severe and intense spongiofibrosis, inflammation, epithelial desquamation, and congestion in vascular structures compared to the controls (p < 0.05). The urethral tissue in the stricture group showed an increased ratio of inflammation parameters, including Collagen 1A1, Collagen 3A1, elastin, Transforming growth factor ß1, α Smooth muscle actin, Platelet-derived growth factor α, and Platelet-derived growth factor ß. Transforming growth factor ß1, Platelet-derived growth factor α, and Platelet-derived growth factor ß each correlated highly with the other six parameters (r > 0.60, p < 0.05). CONCLUSION: Developing electrocoagulation-induced urethral stricture in rats is a simple, reliable, inexpensive, and reproducible. Reporting histologic data with qualitative and semi-quantitative scoring will enhance data standardization, aiding reader understanding and analysis. Transforming growth factor ß and Platelet-derived growth factor play key roles in fibrosis during stricture development. Incorporating these cytokines in urethral stricture animal model studies can demonstrate successful stenosis creation.

miRNA-193b-5p Suppresses Pancreatic Cancer Cell Proliferation, Invasion, Epithelial Mesenchymal Transition, and Tumor Growth by Inhibiting eEF2K.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer deaths in the US due to the lack of effective targeted therapeutics and extremely poor prognosis. OBJECTIVE: The study aims to investigate the role of miR-193b and related signaling mechanisms in PDAC cell proliferation, invasion, and tumor growth. METHODS: Using PDAC cell lines, we performed cell viability, colony formation, in vitro wound healing, and matrigel invasion assays following transfection with miR-193b mimic or control-miR. To identify potential downstream targets of miR-193b, we utilized miRNA-target prediction algorithms and investigated the regulation of eukaryotic elongation factor-2 kinase (eEF2K) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways and mediators of epithelial mesenchymal transition (EMT). The role of miR-193b in PDAC tumorigenesis was evaluated in in vivo tumor growth of Panc-1 xenograft model in nude mice. RESULTS: We found that miR-193b is under expressed in PDAC cells compared to corresponding normal pancreatic epithelial cells and demonstrated that ectopic expression of miR-193b reduced cell proliferation, migration, invasion, and EMT through downregulation of eEF2K signaling in PDAC cells. miR-193b expression led to increased expression of E-Cadherin and Claudin-1 while decreasing Snail and TCF8/ZEB1 expressions via eEF2K and MAPK/ERK axis. In vivo systemic injection of miR-193b using lipid-nanoparticles twice a week reduced tumor growth of Panc-1 xenografts and eEF2K expression in nude mice. CONCLUSIONS: Our findings suggest that miR-193b expression suppresses PDAC cell proliferation, migration, invasion, and EMT through inhibition of eEF2K/MAPK-ERK oncogenic axis and that miR-193b-based RNA therapy might be an effective therapeutic strategy to control the growth of PDAC.

Cytogenetic abnormalities detected in patients with non-obstructive azoospermia and severe oligozoospermia.

PURPOSE: To find the frequency and types of major chromosomal abnormalities with nonobstructive azoospermia and severe oligozoospermia to give appropriate genetic counseling before assisted reproduction techniques in Isparta (South of Turkey), and to investigate the general characteristics in this infertile male population. METHODS AND PATIENTS: A total of 115 infertile males (92 were azoospermic, 23 severe oligospermic) were studied for the cytogenetic evaluation prior to use of assisted reproduction techniques. Also, 60 fertile males as a control group were studied. Karyotyping was performed on peripheral blood lymphocytes according to the standard methods. Levels of luteinising hormone, follicle-stimulating hormone (FSH), testosterone and prolactin were obtained and a testicular sonography examination was conducted. RESULTS: The total prevalence of chromosomal abnormalities was found to be 4.3% (5/115), including 4 patients with Klinefelter's Syndrome and 1 patient with gonadal dysgenesis (46XX). All of them were azoospermic males, corresponding to a frequency of 5.4% (5/92 patients). Oligozoospermic males and control males had no chromosomal abnormalities. There was a significant difference in serum FSH, LH, mean testicular volume and smoking when comparing patients (both azoospermic and oligozoospermic) and control groups (p<0.05). Also, there was a significant difference in serum FSH, LH and mean testicular volume when compared with azoospermic and oligozoospermic patients (p<0.05). CONCLUSIONS: The occurrence of chromosomal abnormalities among infertile males strongly suggests the need for routine genetic testing and counseling prior to the employment of assisted reproduction techniques.

Cytogenetic finding of breast cancer cases and in their first-degree relatives.

PURPOSE: The aim of this study was to evaluate and compare the rate of sister chromatid exchange (SCE), the occurrence of micronuclei, and the lymphocyte proliferation rate index (PRI) in patients with breast cancer, their first-degree relatives, and healthy volunteers. METHODS: We analyzed the frequency of SCE and micronuclei, and the PRI in the peripheral blood lymphocytes of 30 women with breast cancer, 22 of their female family members, and 20 age-matched healthy female volunteers. RESULTS: SCE occurred significantly more often in the lymphocytes of breast cancer patients (10.84±0.4 per metaphase), compared with their first-degree relatives (7.45±0.54) and controls (5.94±0.2) (p<0.001 for both). The mean SCE frequency was not statistically different between first-degree relatives and controls (p=0.071). Similarly, micronuclei occurred at a significantly higher rate in breast cancer patients (9.6±0.72), and in their first-degree relatives (7±0.64), compared to controls (3.85±0.4) (p<0.001 and p=0.001, respectively). There was also a significant difference between the occurrence of micronuclei in patients compared to their family members (p=0.021). The PRI was significantly lower in patients (1.61±0.1), compared with both their first-degree relatives (1.75±0.1), and controls (1.74±0.1) (p=0.001 and p=0.002, respectively). CONCLUSION: Increased SCE and the occurrence of micronuclei, as well as a reduced PRI are associated with breast cancer. Furthermore, increased SCE and the frequency of micronuclei in a first-degree relative suggest that they exhibit greater genetic instability than women of the same age.