Introduction
Brain ischemia is a major cause of death and neurologic disability in many countries. It is a frequent clinical condition of difficult therapeutic solution mainly because its ...physiopathological studies are embarrassed by factors like the diversity of the anatomical location of the lesions, its etiology and clinical signs and symptoms. The alcoholism is a health problemin many societies,larger studies are needed on effects of alcohol associated with cerebral ischemia. It is known that neurons can die few days after stroke and some researchers suggest that apoptosis is the main physiopathological mechanism involved in delayed neuronal death.
Objectives
To evaluate the histopathological changes, morphometry, expression of apoptosis‐related proteins CASPASE3 and BCL2, serum expression of miR‐21 after experimental cerebral ischemia followed by reperfusion for 48 hours, with or without a model of chronic alcoholism.
Material and Methods
We used 50 Wistar rats, divided into 5 experimental groups: Control: only anesthetic procedure; Sham: complete simulation of the surgical procedure; Ischemia (I): focal cerebral ischemia for 90 minutes + reperfusion for 48 hours; Alcoholic(A): received daily absolute ethyl alcohol diluted to 20% in water for four weeks, and, Alcoholic and Ischemic (I + A): the same treatment in group A and I. The brain samples collected were processed for histopathological analysis (by light microscopy and transmission electron microscopy), morphometric (counting of neurons in the striatum), both by the technique of luxol fast blue, and immunohistochemistry (for protein expression of CASPASE3 and BCL2). The blood of the ventral tail artery was collected for analysis of expression of miR‐21 by qPCR.
Results
We observed histopathological changes in animals with a higher degree of ischemic and ischemic groups associated with alcoholism in three areas analyzed: swelling of neuronal cytoplasm, foamy nucleus, neural cells with pyknotic nuclei, interstitial edema and foci of inflammatory infiltrate. The neuronal loss was higher in the medial striatum of animals in groups I and I+A. The protein expression of CASPASE3 was higher than the expression of BCL2, especially in groups I and I + A for the two proteins. The expression of CASPASE3 was higher in the region corresponding to the penumbra. Histopathology revealed that the ischemic focus had a greater number of pyknotic nuclei suggestive of necrosis.
Conclusions
The histopathologic and morphometric changes observed mainly in the ischemic focus were correlated with the expression of CASPASE3, the greater the area of penumbra, and were more severe in the ischemic animals when associated with chronic alcoholism. The expression of BCL2 was slightly higher where histopathological changes and expression of CASPASE3 were less evident. The serum expression of miR‐21 was not indicative of ischemic and/or associated with chronic alcoholism.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Introduction
Glioblastomas (GBM) are the most common malignant primary tumors in the brain, and show high mortality rate. Despite the current advances in the therapy, the GBMs are extremely resistant ...to ionizing radiation and chemotherapy, and the number of relapse is high. Studies bring this eminent tumorigenic potential due to the presence of a subpopulation of neoplastic cells with characteristics of stem cells, called cancer stem cells (CSCs). In gliomas, the isolation of tumor stem cells have been done by antigenic markers and observing the culture conditions of normal neural stem cells in vitro. That is, the floating proliferation of tumor cells when cultured, are analogous to neurospheres derived from normal neural stem cells in defined culture conditions. It is believed that CSCs are responsible for the restoration of the tumor and the low efficiency of the treatment, as these cells demonstrate malignant properties as tumorigenesis, chemoresistance and radioresistance. The practical implication of this finding is that no current therapy is able to suppress or stop the proliferation of these cells. Several microRNAs have been linked to the development and proliferation of glioblastomas, associated with different molecular mechanisms, among them programmed cell death. Studies have shown that these microRNAs in brain tumors exhibit altered expression levels, one of the essential mechanisms for the regulation of tumor stem cells (CSCs).
Objectives
To evaluate the effect of ionizing radiation and temozolomide, alone or associated, on the expression of miRNAs that act as tumor suppressors (miR‐15 and miR‐16) and as oncogene (miR‐21), and adhered cells in neurospheres in culture of glioblastoma cell line (U343‐MG).
Materials and Methods
The trypan blue was used to examine cell viability before and after treatment, and method of real time PCR to check the expression of microRNAs in time 0h, considered to be immediately after the treatments, and 48h after exposure to treatments.
Results and Conclusions
The presented microRNAs are differentially expressed when comparing the different treatment modalities and time. We also observed differences between adhered cells and neurospheres. The oncogene miR‐21 remained with higher expression on treatment with ionizing radiation in neurospheres compared with the group of adherent cells.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Introduction
Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults, characterized by aggressive growth, limited response to therapy and inexorable recurrence. Due ...to the extremely unfavorable prognosis of GBM, it is important to develop more effective diagnostic and therapeutic strategies. The discovery of a subpopulation of cells in the tumor called cancer stem cells (CSCs) opens a wide field in understanding the aggressiveness of GBM. In addition to self‐renewal and tumorigenesis, these cells are known for their resistance to radiotherapy and chemotherapy compared to other cancer cells. In this context, many studies have evaluated the role of microRNAs associated with various molecular mechanisms and the development and proliferation of various types of diseases, including cancer. Studies have shown that in brain tumors these miRNAs have altered levels of expression. In addition, they are essential in the regulation of tumor stem cells.
Objectives
To evaluate the expression of microRNAs in neurospheres and attached cells established after cell culture of samples from ten patients diagnosed with GBM submitted to treatments with temozolomide and ionizing radiation, isolated or associated.
Materials and Methods
Initially, cell cultures were established from the GBMs samples and then these were treated with temozolomide and ionizing radiation. The cell viability evaluation was made with trypan blue 48 hours after treatments and the quantification of microRNA expression was made by Taqman Low Density Array (TLDA®). A description of clinical and epidemiological data was also carried out on 10 patients, who provided tumor samples for this study, followed by immunohistochemical evaluation of the expression of IDH1‐mutated in these samples.
Results
After treatment with temozolomide and ionizing radiation, we found some statistically significant differences between the cell viability of neurospheres and attached cells. Regarding the expression of the microRNAs, five were differentially expressed between neurospheres and attached cells. miRNAs‐101, ‐124a and ‐1275 showed increased expression in neurospheres treated with ionizing radiation. miR‐155 showed increased expression in attached cells treated with ionizing radiation. miR‐138 showed increased expression in attached cells treated with temozolomide. The median overall survival of the series of cases was 56.5 years. There was expression of IDH1‐mutated in 90% of cases, and death in 40%.
Conclusion
After treatments with temozolomide and ionizing radiation, no statistically significant differences were observed between the cell viability of neurospheres and attached cells. Regarding the overall expression of miRNAs, these presented a differential expression pattern in the cell groups, and may be modulated by the proposed treatments.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
Abstract
Background
Cancer is a group of heterogeneous diseases characterized by several disruptions of the genetic and epigenetic components of cell biology. Some types of cancer have been shown to ...be constituted by a mosaic of cells with variable differentiation states, with more aggressive tumors being more undifferentiated. In most cases, undifferentiated tumor cells express associated embryonic markers such as the OCT4, NANOG, SOX2, and CARM1 genes. The ectopic or reminiscent expression of some master regulator genes of pluripotency has been indicated as the cause of the poorly differentiated state of tumors, and based on the evidence of some reports, can be used as a possible therapeutic target. Considering this information, a more detailed investigation of the expression of pluripotency-associated genes is necessary to evaluate the roles of these genes in the etiology of some tumors and their use targets of therapy.
Methods
The expression of four pluripotency-related genes was investigated (OCT4, NANOG, SOX2, and CARM1) in the most malignant primary human brain tumor, glioblastoma (GBM).
Results and Conclusion
The results demonstrated a signature of OCT4/SOX2/CARM1 genes and a significant increase of CARM1 expression in GBM cases.
To evaluate the effect of radiotherapy and temozolomide on the expression of miRNAs apoptotic (miRNAs-21, -221, -222 (anti-apoptotic) and miRNAs-15a, -16 (pro-apoptotic)) and the gene MGMT in ...glioblastoma cell lines.
The limited knowledge of the molecular biology of malignant gliomas may hinder the development of therapeutic modalities. In this scenario, one of the greatest advances of recent years was the identification of microRNAs. These molecules have an important role in biological processes involving cancer, including glioblastoma.
Trypan blue was used to verify the cell viability, and real time PCR to quantify the expression of microRNAs and gene 24, 48 and 120 h after exposure to treatments.
There was a statistically significant decrease of expression of miR-15a between 48 and 120 h in line T98 G treated with radiation, increased expression of miR-15a between 24 and 120 h in line U251 treated with radiation and temozolomide, and increased expression of miR-16 between 24 and 120 h in line U251 treated with radiation alone and when combined with temozolomide. There was a decrease in MGMT gene expression, between 24 and 48 h in U343 cells treated with temozolomide.
Ionizing radiation and temozolomide modified the expression of miRNAs studied and MGMT.
Cerebral ischemia has not only a high mortality rate, which is the second leading cause of death worldwide, but is also responsible for severe disabilities in working age individuals, generating ...enormous public expending for treatment and rehabilitation of the affected individuals. The role of microRNAs in the pathophysiology of cerebral ischemia has been highlighted in current investigations. In addition, recent studies have also highlighted physical exercise as a possible protective factor both in the prevention and in the effects of cerebral ischemia, placing it as an important study resource. Thus, we investigated the role of physical exercise in experimental cerebral ischemia associated with the expression of microRNA-27b. 16 animals were used, divided into four experimental groups: Control, Physical Exercise, Cerebral Ischemia and Cerebral Ischemia associated with Physical Exercise. The real-time PCR methodology was used to analyze the expression of microRNA-27b. Although there were no statistically significant differences in the expression of microRNA-27b between the groups studied, the increased expression of microRNA-27b in the Physical Exercise group indicates its neuroprotective role in the pathophysiology of cerebral ischemia.
MicroRNAs (miRNAs) are short noncoding RNA molecules that regulate gene expression and are related to endothelial dysfunction (EnD). Recently, miRNAs have also been explored as potential biomarkers ...and target molecular therapy of erectile dysfunction (ED). Could the miRNAs be the tip of the iceberg of chronic arterial disease foreshadowed by the ED?
To investigate the expression of miR-15b, miR-16, miR-138, miR-221, and miR-222 in corpus cavernosum (CC) and peripheral blood in a rat model of endothelium dysfunction secondary to diabetes (DM) and alcohol consumption to assess potential endothelial lesion biomarkers.
Twenty males Wistar rats were divided into 4 groups: control group (C), alcohol consumption group (A), diabetic group (D), diabetic-alcohol consumption group (D + A). DM was alloxan-induced and alcohol consumption was through progressive increase of ethanol concentration in drinkable water. After 7 weeks, miRNAs expressions from CC and blood sample were evaluated by real-time PCR. Functional assessment of CC was performed in an acetylcholine endothelium-dependent relaxation pharmacological study.
miRNA expression in CC and blood were evaluated; pharmacological study in CC strips was conducted to validate EnD.
We found that 3 miRNAs (miR-16, miR-221, and miR-222) were downregulated in the CC in the D+A group, while all 5 miRNAs were downregulated in the blood of D and D + A groups. The endothelium-dependent relaxation induced by acetylcholine was significantly decreased in groups A, D, and D + A. Diagnostic accuracy estimated by AUC, to discriminating groups A, D, and D + A from controls, was superior to >0.9 in all plasmatic miRNAs.
miRNAs downregulation was identified in both CC and blood notably in DM associated with alcohol consumption animals (D + A), the greatest endothelial injury potential group. Serum miRNAs have also demonstrated high diagnostic accuracy properties in predicting CC relaxation dysfunction labeling EnD. RB Tiraboschi, FSL Neto, DP da Cunha Tirapelli, et al. Expression of MicroRNAs (miR-15b, miR-16, miR-138, miR-221, and miR-222) as Biomarkers of Endothelial Corpus Cavernosum Dysfunction in a Diabetic Alcoholic Murine Model. Sex Med 2021;9:100326.
Previous studies from our group described the consequences of using ethanol on penile erection. Nevertheless, the molecular mechanisms surrounding microRNAs, apoptosis process and their relationship ...with erectile dysfunction associated with alcohol consumption are still poorly understood. The objective of this analysis was to evaluate the mechanism of apoptosis by the expression of AIF and PARP, as well as their regulatory microRNAs: miR-145, miR-210 and miR-486, in the corpus cavernosum of rats submitted to a semivoluntary alcoholism model. For this study 24 Wistar rats were divided into two groups: control (C) and treated with 20 % ethanol (A) for seven weeks. The corpus cavernosum samples were prepared for immunohistochemical analysis of AIF and PARP protein expression, and microRNAs miR-145, miR-210, miR-486 gene expression in cavernous tissue was performed by real time PCR. The immunohistochemical analysis showed little nuclear positive labeling for the protein PARP and AIF in the corpus cavernosum of control and ethanol treated animals. After analysis of miR-145, -210 and -486 microRNA expression in the 12 animals studied, no results were found with significant statistical difference between the control and alcoholized groups. The expression of AIF and PARP and their regulatory microRNAs involved in apoptotic process (miR-145, miR-210 and miR-486) were not altered in the corpus cavernosum of rats submitted to semivoluntary alcoholism.
The differential diagnosis, treatment and prognosis of pancreatic ductal adenocarcinoma (PA) are still a challenge in clinical practice. The aim of the current study was to assess the role of select ...plasma and tissue microRNAs (miRs‐21, ‐23a, ‐100, ‐107, ‐181c e ‐210) as biomarkers for the diagnosis of PDCA. Samples of plasma (PAp Group, n=13), pancreatic tumors (PAt Group, n=18) and peritumoral regions (PPT Group, n=9) were collected from patients during the surgical procedure. The control group consisted of samples collected from patients submitted to pancreatic surgery for trauma or from cadaveric organ donors (PC Group, n=7). Healthy volunteers donated blood for plasma collection (PCp Group, n=6). The relative abundance of six miRNAs was measured in all groups using real‐time PCR, and serum CA 19‐9 levels were determined in Groups PA and PC, with the difference being considered significant when p <0.05. In tissue samples, there was a difference in the expression of miRNA‐21 (p=0.005) and miRNA‐210 (p=0.008) across the PAt, PC and PPT groups; miRNA‐21 distinguished group PAt from PC and PPT and miRNA‐210 distinguished group PAt from PC. Serum CA 19‐9 showed 88% accuracy (ROC‐AUC 0.95; 0.84 – 1), 83% sensitivity, and 100% specificity at a cut‐off of 37 ng/dl. The PAp group showed overexpression of miR‐181c (95% CI1.67 – 17.68; p<0.00001) and miR‐210 (95% CI0.16 – 5,20; p=0.03) compared to the PCp group. The combination of tissue expression of miR‐21, ‐210 and serum CA 19‐9 showed 100% accuracy for the diagnosis of PA, and the accuracy of plasma miR‐181c (PAp × PCp) was also 100%. These findings suggest that the combination of tissue miRNAs and serum CA 19‐9 can improve accuracy, but plasma miRNA expression can be used as a noninvasive diagnostic test for PA, although further studies are necessary to validate these results as well as to evaluate the utility of miRNA expression as a biomarker related to survival and to the response to therapy in PA.
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
This study aimed to investigate the morphometric and the pattern of protein and gene expression related to the extrinsic apoptotic pathway in experimental focal cerebral ischemia and the hole of ...neuroprotection with hypothermia and ketoprofen. For this analysis, 120 rats were randomly divided into 3 groups (20 animals each): control - no surgery (20 animals); sham - simulation of surgery (20 animals); ischemic - focal ischemia for 1 hour, without reperfusion (80 animals) and divided into four subgroups with 20 animals each: ischemic + intraischemic hypothermia; ischemic + previous intravenous ketoprofen, and ischemic + hypothermia and ketoprofen. The infarct volume was measured using morphometric analysis of infarct areas defined by triphenyl tetrazolium chloride and the patterns of expression of the apoptosis genes (Fas, c-Flip, caspase-8 and caspase-3) and the apoptosis protein caspase-3 were evaluated by quantitative real-time PCR and immunohistochemistry, respectively. Hypo expression of genes of extrinsic pathway of apoptosis was observed: Fas receptor, c-Flip and caspase-8 in the ischemics areas. Increases in the gene and protein caspase-3 in the ischemic areas were also observed, and these increases were reduced by hypothermia and ketoprofen, also noted in the morphometric study. The caspases-3 increase suggests that this gene plays an important role in apoptosis, probably culminating in cell death and that the neuroprotective effect of hypothermia and ketoprofen is involved.
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