Bioinformatics study of the MIR-200 family and the target genes in Prostate Cancer

Introduction: Considering the limitations of the common diagnostic test for prostate cancer prostate cancer, the introduction of higher-specific biomarkers for a more accurate and timely diagnosis of prostate cancer is desired. In this study, we aimed to investigate the miR-200 family [miR-200a, miR-200b, miR-200c, miR-141, and miR-429] and their target genes using bioinformatics prediction tools in order to propose potential diagnostic biomarkers for prostate cancer

Method: In this theoretical study, based on bioinformatics study of micro RNA, the DIANA TOLLS-mirPath v.3 database was used to search the target genes of the miR-200 family in the signaling pathways known in prostate cancer pathogenesis. Then, we confirmed the suggested genes by the online predictive tools such as MiRWalk, Targetscan and RNAhybrid. Finally, the functional role of target genes was investigated on the DAIVID bioinformatics database

Results: According to the results of this study, the E2F3 gene is the target of all family members of miR-200, BCL2 is the common target of miR-200b / miR-200c / miR-429 and CCNE2 is the common target of miR-200a / miR-141 subsets. Also, miR-200c and miR-429 can modulate the pathogenesis of prostate cancer by targeting CDKN1B and KRAS genes, respectively

Conclusion: The results of this study showed that members of the miR-200 family targeting the genes involved in important biological processes and molecular pathways of prostate cancer pathogenesis are likely to be effective diagnostic biomarkers in future experimental studies

Hippocampal expression of connexin36 and connexin43 during epileptogenesis in pilocarpine model of epilepsy

Background: Gap junctions [GJs] provide direct intercellular communications that are formed by hexameric protein subunits, called connexin [Cx]. The role of Cxs in epileptogenesis has not received sufficient attention. Hippocampus with critical function in epileptogenesis has a wide network of GJs. We examined the protein expression levels of hippocampal Cx36 [the prominent Cx present between GABAergic interneurons] and Cx43 [the main Cx expressed by astrocytes] during epileptogenesis in the pilocarpine model of epilepsy

Methods: Male Wistar rats received scopolamine [1 mg/kg, s.c.]. Pilocarpine [380 mg/kg, i.p.] was administered 30 min thereafter to induce status epilepticus [SE]. SE was stopped 2 h later by diazepam [10 mg/kg, i.p.]. Cx36 and Cx43 protein expression was assessed by Western blot analysis in the hippocampus of SE-experienced rats, after injection of diazepam [F0 subgroup], after acquisition of focal seizures [F3 subgroup], and after development of generalized seizures [F5 subgroup]. The control subgroups, C0, C3, and C5, were aged-matched rats, which received saline [1 ml/kg, i.p.] instead of pilocarpine. Injection of scopolamine and diazepam, and dissection of hippocampi were carried out at the same time interval as the test subgroups

Results: SE emerged in 67.1% of pilocarpine-treated animals. Focal and generalized seizures developed 3.8 +/- 0.4 and 7.0 +/- 0.5 days after SE, respectively. Cx36 protein abundance was not significantly different between test and control groups in the three time points. However, Cx43 protein level showed 40% increase in F3 subgroup [P<0.05 compared to C3, P<0.01 compared to F0 and F5]

Conclusion: Hippocampal Cx43 is overexpressed in pilocarpine model of epileptogenesis after acquisition of focal seizures

[Bioinformatic evaluations for locating the microRNA suppressing PI3K/AKT pathway and analysis in prostate cancer cell lines]

Objective: Prostate cancer is the fifth most common cancer. In 2012, it was the second leading cause of cancer death for men worldwide. The PI3K/AKT pathway plays an essential role in pathogenesis of prostate cancer; the key role of this pathway in cancer progression makes it an attractive target for prostate cancer therapy. MicroRNAs [miRNAs] that regulate gene expression have a special ability to simultaneously control multiple genes and pathways which make them candidates for therapeutics. This study aims to determine miRNAs which target the PI3K/AKT pathway and evaluate them in prostate cancer cell lines

Methods: In order to determine an effective miRNA for the PI3K/AKT pathway, we assessed six genes from this pathway which have been proposed as drug targets in ten different prediction algorithms. Next, the candidate miRNAs were analyzed in expression profile and pathway analysis databases. Expression of candidate miRNAs in control and prostate cancer cell lines were subsequently evaluated

Results: According to bioinformatics, the miR-29 family could target the most genes from this list. Other bioinformatic estimates confirmed these results. The miR-29 family showed significant downregulation in prostate cancer cell lines LNCAP, PC3 and DU-145 compared to control samples

Conclusion: These results propose the possibility of using the miR-29 family to inhibit the PI3K/AKT pathway in prostate cancer

effect of MDMA and pentoxifylline drug on bad/bcl-xl gene dosage expression changes on rat liver

MDMA generally known as ecstasy, have deleterious effects on the serotonergic neurotransmitter system. Recent findings suggest that the liver and brain are major target organs of MDMA-related toxicities. Although most research is being dynamically performed on brain, however, the molecular mechanisms by which MDMA elicits adverse effects in both organs are poorly undrestood.The present study was performed to obtain evidence for molecular mechanism of apoptosis involved in MDMA-induced hepatotoxicity in rat liver after MDMAadministration. Moreover, the antagonistic effect of pentoxifylline was assessed on hepatotoxicity after MDMA administration. In this experimental study, sample size and power in each group were calculated as 10 rats with 95% confidence level and 5% confidence interval. In the study, four experimental groups were selected including Control Normal, MDMA, MDMA+PTX and PTX+MDMA. MDMA was dissolved in PBS and intraperitoneally injected three doses of 7.5mg/kg with two hours gap between doses. Pentoxyfilline also was injected as 100mg/kg, simultaneously with third dose of MDMA. After treatment, total RNA was isolated from liver tissue [5mg]. Absorbance at 260nm, 280nm and 230nm were measured and immediately reverse transcription was performed. Included target genes were BAD and BCL-XL as pro-apoptotic and anti-apoptotic gene, respectively. After set up and validation, Real-Time PCR were performed and obtaining data were statistically analyzed to determine significantly differences between groups. Using Real-Time quantitative PCR results, BCL-XL gene expression ratio significantly increased in MDMA+PTX group. Moreover, BAD gene expression ratio increased and up-regulated in PTX+MDMA group [P-value <0.001].Our study focused on molecular mechanism of MDMA in programmed cell death using gene expression quantification of a pro-apoptotic and anti-apoptoic gene in MDMA-induced hepatotoxocity. The results shown MDMA prompted apoptosis in liver and pentoxifylline protects hepatotoxicity after and befor taking MDMA

SULF 1 gene polymorphism, rs6990375 is in significant association with fetus failure in IVF technique

Sulfatase 1 [SULF1] function is to remove the 6-O-sulphate group from heparan sulfate. This action changes the binding sites of extracellular growth factors. SULF1 expression has been reported to be changed in angiogenesis. We hypothesized that single nucleotide polymorphisms [SNPs] of SULF1 would impact clinicopathologic characteristics. Study of SULF1 gene polymorphism with fetus failure in in vitro fertilization [IVF] technique. We studied one common [minor allele frequency >0.05] regulatory SNP, rs6990375, with polymerase chain reaction and restriction fragment length polymorphism method, in 53 infertile women with fetus failure in IVF technique and 53 women with at least one healthy child as controls. We found that rs6990375 is significantly associated with an early failure in IVF and frequency of G allele is high in women with fetus failure in IVF technique [p<0.001]. These findings suggest that SULF1genetic variations may play a role in IVF technique fetus failure. Further studies with large sample sizes on SULF1 SNPs may be useful in support of this claim

Nasal intermittent positive pressure ventilation vs. nasal continuous positive airway pressure in preterm infants with respiratory distress

The trend today is to minimize the use of mechanical ventilation. Nasal continuous positive airway pressure [NCPAP] and nasal intermittent positive pressure ventilation [NIPPV] are 2 non-invasive treatments for respiratory distress syndrome [RDS]. There is little study in literature comparing early use of NIPPV with NCPAP as primary modes of respiratory support. The aim of this study is to determine whether NIPPV and NCPAP would have different survival rates and possible complications. In this prospective clinical trial study, 120 preterm neonates [gestational age 28-36 weeks] who were admitted due to respiratory distress between January and May 2012 in the neonatal intensive care unit of Afzalipour hospital. Sixty infants were randomized to NCPAP and comparable infants to NIPPV [birth weight 1807.05 +/- 52 vs. 1882.50 +/- 56 g, gestational age 32.05 +/- 2.94 vs. 32.16 +/- 2.08 weeks, respectively]. Patients were randomly allocated into 2 treatment groups using minimization technique. One group was treated by NCPAP and the second one treated by NIPPV. Survival analysis was applied to estimate and compare survival rates. Infants treated initially with NIPPV needed less endotracheal ventilation than infants treated with NCPAP [13.3% vs. 41.7%, p=0.001]. Estimated survival rates at 24 h in NIPPV were 97% versus 82% for NCPAP group. We have seen that the risk of failure for those received NCPAP was nearly 4 times higher than NIPPV group. According to our results, among preterm infants with suspected [RDS], the use of NIPPV reduces the need for intubation and mechanical ventilation in comparison to NCPAP

Comparison of nasal intermittent positive pressure ventilation and nasal continuous positive airway pressure treatments using parametric survival models

Objective: The Cox model is the dominant tool in clinical trials to compare treatment options. This model does not specify any specific form to the hazard function. On the other hand, parametric models allow the researcher to consider an appropriate shape of hazard function for the event of interest. The aim of this article is to compare performance of Cox and parametric models

Methods: We used data collected in a prospective clinical trial that aimed to compare performance of nasal intermittent positive pressure ventilation [NIPPV] and nasal continuous positive airway pressure [NCPAP] treatments in terms of survival of newborn infants who had respiratory distress syndrome [RDS]. Performance of Cox, exponential, Weibull, and log-logistic models were compared in terms of goodness of fit

Findings: Fitting the Cox model, we have seen that infants who received NCPAP were 4.23 [Hazard Ratio= 4.23, 95% Confidence Interval: 1.87-9.59] times more likely to fail than those received NIPPV [P=0.001]. Adequacy of the exponential model was rejected. We have seen a decreasing hazard rate over time, in both treatment groups. This decrease was sharper in NCPAP group. Akiake information criterion corresponded to the log-logistic model and was lower than all other models followed by Weibull model

Conclusion: Our results demonstrate the benefit of parametric survival models over traditional Cox regression model in terms of modeling of shape of hazard function. We saw a decreasing hazard that confirms the flexibility of parametric models in terms of the modeling of hazard rate

Evaluation of cisplatin and cisplatin-loaded magnetic iron oxide nanoparticles on BCL2 and BAX genes in the breast cancer T47D cell line

Breast cancer is the second leading cause of cancer death in women. Cisplatin is a traditional cancer drug commonly used in chemotherapy for killing cancer cells. Modulation at the mRNA levels of apoptotic related genes often correlate with the sensitivity of various types of cancer cells to chemotherapeutic agents. Nanoparticulate drug delivery systems are being developed to effectively deliver smaller doses of chemotherapeutic agents and control drug distribution in the body. In this study, we evaluate the expressions of BCL2 and BAX genes in T47D treated with cisplatin and cisplatin nanoparticles, which can result in a new approach to breast cancer therapy. In this study, we treated T47D cells with different concentrations of cisplatin and cisplatin nanoparticles at 48 h. The IC50 was determined. We extracted RNA by using RNX solution, after which cDNA was synthesized. The precise primers for the BCL2, BAX and TBP genes were designed by specific software. The quantity of BCL2 and BAX gene expression compared to TBP gene [reference gene] was analyzed using real-time PCR. BCL2 and BAX gene expression levels in T47D cells treated by cisplatin were 0.7 [BCL2] and 1.48 [BAX], in T47D cells treated with cisplatin-loaded nanoparticles, the gene expressions were 0.03 [BCL2] and 2.41 [BAX]. In this study, the results have shown that cisplatin-loaded nanoparticles are effective anticancer agents. Cisplatin nanoparticles induce apoptosis in human breast cancer cell lines. We have shown that cisplatin nanoparticles strongly increased cytotoxicity in comparison to the free drug in the T47D cell line

Effect of pentoxifylline on bcl-2 gene expression changes in hippocampus after ischemia-reperfusion in wistar rats by a quantitative RT-PCR method

Ischemia-reperfusion injury is the tissue damage caused when blood supply returns to the tissue after a period of ischemia or lack of oxygen. Ischemia-reperfusion brain injury initiates an inflammatory response involving the expression of adhesion molecules and cytokines. Twenty-four male Wistar rats [250-300 g body wt] were used in this study. The animals were divided into four groups of 6 rats each: I: Control group that was subjected to ischemia-reperfusion, II: Ischemia-reperfusion group that was subjected to all surgical procedures, III: Drug group that received pentoxifylline [200, 400 and 600 mg/kg] 60 min before and after ischemia and IV: Vehicle group that received saline. Seventy two h after ischemia-reperfusion, the hippocampus was taken for studying the changes in bcl-2 gene expression. We used quantitative real-time PCR for the detection of bcl-2 gene expression in ischemia and drug groups and then compared them to normal samples. The results showed the gene dosage ratio of 0.66 and 1.5 for ischemia group and the drug groups, respectively. The results also showed the bcl-2 gene expression declined in ischemia group as compared to the drug group. Furthermore, we observed a significant difference in the bcl-2 gene expression between ischemia and drug groups. These findings are consistent with anti-apoptotic properties of bcl-2 gene. Furthermore this method provides a powerful tool for the investigators to study brain ischemia and respond to the treatment drugs with anti-apoptotic agents

effect of pentoxifylline on bcl-2 gene expression changes in hippocampus after long-term use of ecstasy in Wistar rats

3,4- Methylenedioxymethamphetamine [MDMA or "Ecstasy"] is a psychoactive and hallucinogenic drug of abuse. MDMA has been shown to produce neurotoxicity both in animals and humans. Recently, the vasodilator drugs such as pentoxifylline is one of the new strategies which have been considered as neuroprotector. In this study effect of pentoxifylline on bcl-2 gene expression changes in hippocampus of rat following long- term use of ecstasy was investigated. 30 male Wistar rats weighing 250-300g were randomly divided into 5 groups: control [normal], sham [MDMA injection], experimental 1[MDMA and then PTX injections], experimental 2[PTX injection and after 1 week, MDMA injection] and vehicle [saline injection] groups. All drugs were injected intraperitoneally. Two weeks later, the hippocampi were removed for studying the changes in bcl-2 gene expression. We used quantitative real time PCR for detection of bcl-2 gene expression in treated groups and then compared them to control samples. The results showed the gene dosage ratio of 0.49, 0.78 and 1.17 for sham, experimental 1 and experimental 2 groups, respectively. The results also showed the bcl-2gene expression declined in sham group as compared to the experimental groups. Furthermore, we observed a significant difference in the bcl-2 gene expression between sham and experimental 2 groups. We conclude that quantitative real time PCR could be used as a direct method for the detection of bcl-2 gene expression in tested and normal samples

Effect of chronic intracerebroventricluar administration of lipopolysaccharide on connexin43 protein expressionin rat hippocampus

Hippocampal damages, which are accompanied by inflammation, are among the main causes of epilepsy acquisition. We previously reported that chronic intracerebroventricular [i.c.v.] injection of lipopolysaccharide [LPS] modulates epileptogenesis in rats. There is a network of gap junction channels in the hippocampus that contribute to epileptogenesis. Gap junction channels are formed by oligomeric protein subunits called connexins [Cx]. Astrocytic Cx43 and neuronal Cx36 are expressed in the hippocampus. In order to find out the possible role of gap junctions in seizure-modulating effect of LPS and neuroinflammation, we studied the effect of central administration of LPS on expression of Cx36 and Cx43 in rat hippocampus. LPS, 2.5 micro g/rat/day, was injected i.c.v. to male Wistar rats for 14 days. mRNA and protein abundance of Cx36, Cx43 and IL1-beta were measured in rat hippocampus by real time-PCR, Western blot and ELISA techniques, at the beginning, in the middle, and at the end of the treatment period. IL1-beta protein level was significantly increased 6 h after first injection of LPS. Cx36 and Cx43 mRNA expression did not alter during chronic administration of LPS. A selective decrease in Cx43 protein expression was observed after 7 injections of LPS. It is suggested that Cx43 containing gap junctions in the hippocampus is down-regulated in response to chronic injection of LPS. This event can inhibit propagation of toxic and noxious molecules to neighboring cells and modulate hippocampal excitability and epileptogenesis

Upregulation of connexins 30 and 32 gap junctions in rat hippocampus at transcription level by chronic central injection of lipopolysaccharide

Gap junctions composed of connexins [Cx] are functional in cell defense by propagation of toxic/death molecules to neighboring cells. Hippocampus, one of the brain regions with particular vulnerability to damage, has a wide network of gap junctions. Functional response of astrocytic Cx30 and neuronal Cx32 to hippocampal damage is unknown. We infused lipopolysaccharide [LPS] intracerebroventricularly [2.5 microg/rat] once daily for two weeks to create neuroinflammation. The mRNA and protein levels of the Cx were measured in the hippocampus after 1[st], 7[th] and 14[th] injection by real-time PCR and Western-blot techniques. A significant increase in Cx32 and Cx30 gene expression was observed after 7[th] and 14[th] injection of LPS with no significant change in their protein abundance. Transcriptional overexpression of hippocampal Cx30 and Cx32 could be an adaptive response to production of intracellular toxic molecules but it is not accompanied with post- transcriptional overexpression and might have no functional impact

[Development and application of real-time quantitative polymerase chain reaction technique using SYBR green I in the diagnosis of Down syndrome]

Rapid diagnosis of Trisomy 21 Syndrome [Down Syndrome] patients using Real-Time quantitative Polymerase Chain Reaction [Real-Time qPCR] in order to establish a novel method for prenatal diagnosis in the future. A total of 5 ml of peripheral blood was obtained from each patient and normal controls [NR]. Then, genomic DNA from lymphocytes was extracted using the salting out procedure. Gene dosage levels of DSCAM and [PMP22, DSCAM] in Down Syndrome and NR were analyzed using real-time quantitative PCR. The DSCAM/ PMP22 ratio was calculated according to the 2-delta delta Ct formula for all samples. Real-time PCR showed a DSCAM/PMP22 ratio of 1.48 +/- 0.18 and 1.01 +/- 0.10 [p<0.001] in Down Syndrome and normal samples, respectively, demonstrating three copies of the target [DSCAM] gene in Trisomy 21 Syndrome. DSCAM/PMP22 ratio is increased significantly in Down Syndrome patients than NR [1.5 times]. Therefore, the real-time quantitative PCR technique can be used as a sensitive, accurate and reliable technique for rapid and prenatal diagnosis of Trisomy 21 Syndrome

Use of Real-Time PCR method on the amniocyte samples for prenatal diagnosis of Down syndrome

In this study, the possibility of prenatal diagnosis of Down syndrome with Real-Time PCR method was evaluated. In this context, optimization of a suitable method for purification of high quality DNA from amniotic fluid samples was also considered. Pregnant women who had the high risk of having babies with Down syndrome were selected according to the biochemical and sonographic data and referred to the amniocentesis center. The DNA of total 59 amniotic fluid samples were extracted with different methods including boiling method, salting out method, Procedures of DNA extraction from Blood and Cell Culture by DNP_ Kit [CitmaGen], Procedure of DNA extraction from cells by DNA Isolation Kit for cells and tissues [Roche], Procedure of DNA extraction from Tissue by MagNa Pure DNA Isolation kit [Roche], and QIAamp DNA Micro Kit [Qiagen]. Then, the quality and quantity of the extracted DNA were evaluated by the NanoDrop ND- 1000 spectrophotometer device. Real-Time PCR reaction using fluorescent dye SYBR Green I [Applied Biosystems, UK] was performed to specifically amplify DSCAM and DYRK1A2 genes and the reference gene [PMP22]. Data analysis was performed using comparative cycle threshold method for the determination of the gene dosage and determining the number of copies of chromosome 21. This study showed that DNA extracted from amniotic fluid samples using QIAamp DNA Micro Kit [Qiagen] has the desirable quantity and quality for Real-Time PCR. Specific proliferation of targets and reference genes was achieved and difference between normal and affected groups based on differences between their gene dosages was determined. Prenatal diagnosis of Down syndrome is feasible by the Real-Time PCR method using DNA samples from amniotic fluid cells extracted by QIAamp DNA Micro Kit [Qiagen]. The results are comparable to the corresponding results from conventional cytogenetic methods

[Detection of unknown deletions in alpha globin genes in alpha thalassemia carriers using real-time PCR]

Alpha-thalassemia is one of the most prevalent hemoglobin disorders in the world and it is a common hereditary condition caused by deletion of one or more alpha-globin genes. Common alpha-thalassemia deletions like 3.7 kb, 4.2 kb, 20.5 kb and Med can be detected by Multiplex PCR. There are, however, some unknown deletions that can not be detected by the mentioned method or even by direct DNA sequencing. In the present study, Real-time PCR was used to determine the presence or absence of unknown deletions. Real-time PCR was performed using intercalating dye SYBR Green I and alpha1, alpha2 and CLCN7 genes were amplified. Data analysis was conducted using comparative threshold method [delta delta CT] for determination of Gene dosage of alpha1-globin and alpha2-globin genes. The results showed the ratio of 0.90 +/- 0.16 for normal individuals and the ratio of 0.32 +/- 0.15 for carrier samples with deletions. In addition, Melting curve analysis confirmed the specific amplification of target genes. The Real-time PCR assay is simple, rapid, and reliable. It can be applied for direct determination of unknown deletions in Alpha-thalassemia carriers