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Background: Coronary heart disease (CHD), a major cause of death worldwide, is defined as a narrowing or blockage of the coronary arteries that supply oxygen and blood to the heart. We aimed to find potential biomarkers for coronary artery disease, by comparing the expression profile of blood exosomes of both normal and CHD samples., Methods: Datasets of 6 CHD and 6 normal samples of blood exosomes were downloaded, and differentially expressed RNAs, with adjusted P <0.01 and log2FoldChange≥1 were achieved. Moreover, gene ontology (GO) and pathway analysis were accomplished by PANTHER database for datasets., Results: Our data analysis found 119 differentially expressed genes between two datasets. By comparing transcriptome profiles, we candidate the highest downregulated gene, ACSBG1, and the highest upregulated one , DEFA4, as specific biomarkers for CHD . Furthermore, GO and pathway analysis depicted that aforementioned differentially expressed genes are mostly involved in different molecular metabolic process, inflammation, immune system process and response to stimulus pathways which all cause cardiovascular diseases., Conclusion: We have provided new potential biomarkers for CHD, though experimental validation is still needed to confirm the suitability of the candidate genes for early detection of CHD., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2022 Gholipour et al. Published by Tehran University of Medical Sciences.)

Purpose: LncRNAs play essential roles in the cellular and molecular biology of glioma. Some LncRNAs exert their role through sponging miRNAs and regulating multiple signaling pathways. LINC02381 is involved in several cancer types as either oncogene or tumor suppressor. Here, we intended to find the molecular mechanisms of the LINC02381 effect during the glioma progression in related cell lines., Methods and Results: RNA-seq data analysis indicated the oncogenic characteristics of LINC02381, and RT-qPCR results confirmed its upregulation compared to normal tissues. Besides its expression was relatively stronger in invasive glioma cell lines. Furthermore, in silico analysis revealed LINC02381 is concentrated in the cytoplasm and predicted its sponging effect against miR-128 and miR-150, which was verified through dual luciferase assay. When LINC02381 was overexpressed in 1321N1, U87, and A172 cell lines, IGF1R and TrkC receptors as well as their downstream pathways (PI3K and RAS/MAPK), were upregulated, detected by RT-qPCR, and verified by western analysis. Consistently, LINC02381 overexpression was followed by an increased proliferation rate of transfected glioma cell lines, detected by flow cytometry and MTT assay, and RT-qPCR. It also resulted in elevated EMT and stemness markers expression level, increased migration rate, and reduced apoptosis rate, detected by RT-qPCR, western analysis, scratch test, and Annexin/PI flow cytometry analysis, respectively., Conclusion: The overall results indicated that LINC02381 exerts its oncogenic effect in glioma cells through sponging miR-128 and miR-150 to upregulate the IGF1R signaling pathway. Our results introduce LINC02381 and miR-128, and miR-150 as potential prognosis and therapy targets for the treatment of glioma., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Evidence indicates that the dysregulation of extracellular matrix (ECM) components can lead to cardiovascular diseases. The Talin-1 (TLN1) gene is a major component of the ECM, and it mediates integrin adhesion to the ECM. In this study, we aimed to determine microRNAs (miRs) that regulate the expression of TLN1 and determine expression alterations in TLN1 and its targeting miRs in coronary artery disease (CAD)., Methods: Data sets of CAD and normal samples of blood exosomes were downloaded, and TLN1 was chosen as one of the genes with differential expressions in an in silico analysis. Next, miR-182-5p and miR-9-5p, which have a binding site on 3´-UTR of TLN1, were selected using bioinformatics tools. Then, the miR target site was cloned in the psiCHECK-2 vector, and direct interaction between the miR target site and the TLN1 3'-UTR putative target site was investigated by luciferase assay. The expression of miR-182-5p, miR-9-5p, and TLN1 in the serum samples of CAD and non-CAD individuals was assessed via a real-time quantitative polymerase chain reaction., Results: Our data revealed that miR-182-5p directly regulated the expression of TLN1. Moreover, miR-182-5p and miR-9-5p were significantly upregulated in the CAD group. Hence, both bioinformatics and experimental analyses determined the downregulated expression of TLN1 in the CAD samples., Conclusions: Our findings demonstrated that miR-182-5p and miR-9-5p could play significant roles in TLN1 regulation and participate in CAD development by targeting TLN1. These findings introduce novel biomarkers with a potential role in CAD pathogenesis., (© 2022 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Background: ErbB/PI3K signaling is widely recognized as a critical modulator of malignancy and miRNAs have been found to play a crucial role in the regulation of this pathway., Objective: This study aimed to identify novel miRNAs related to the ErbBs loci and investigate the functional effects of these miRNAs on ErbB/PI3K signaling in cancer progression., Materials and Methods: Bioinformatics tools and RNA-seq data were used to discover novel miRNAs in breast and colon cancer cells. Gene expression levels were determined using RT-qPCR. Western blotting and dual-luciferase assays were used to identify the regulatory mechanism between ErbB4-miR1/2 and related genes. The effects of ErbB4-miR1/2 on cell proliferation, viability, ROS production, and migration were assessed by PI-flow cytometry, colony formation, MTT, ROS, scratch, and transwell assays in SKBR3 and SW480 cells., Results: MicroRNA prediction tools, RNA-seq data, RT-qPCR, and sequencing results identified ErbB4-miR1 and ErbB4-miR2 (ErbB4-miR1/2) as novel miRNAs encoded by ErbB4 gene. ErbB4-miR1/2 were downregulated in breast and colon tumor tissues and also in different cancerous cells. RT-qPCR and dual-luciferase assays revealed that ErbB2 and ErbB3 genes are regulated by ErbB4-miR1/2. Consistently, a decrease in the p-AKT/AKT protein ratio verified the suppressive effect of ErbB4-miR1/2 on ErbB/PI3K activity. Furthermore, ErbB4-miR1/2 overexpression suppressed cell proliferation, viability, and migration, and increased ROS production., Conclusions: ErbB4-miR1/2 are novel tumor suppressor miRNAs which attenuate ErbB/PI3K signaling in breast and colon cancer cells.

Background: Long non-coding RNAs (LncRNAs) are eminent genes in the human genome that interfere with the regulation of many complexities of organisms and control many of the various biological processes. As a result, it is considered that they may play an important role in different cancers. With regard to the high prevalence of breast cancer and the role of lncRNA, the present study aimed at investigating the expression of various lncRNAs., Method: Fresh tissues were obtained from operating rooms of Shariati, Khatamolanbia, and Milad Hospitals (Tehran, Iran) by a surgeon. A total of 45 tumor samples and 45 non-tumor samples (from the margin of tumor) were obtained from the same patients. Relative expression evaluation method was used in Real time PCR. Estrogenn receptor (ER), progesterone receptor (PR), and HER2 expression were analyzed using IHC analyses of each cell block., Results: Participants included 44 female and 1 male with the mean age ± SD of 50 ± 12.0 years (range: 23-74). A majority of participants (41/45) were Ductal carcinoma type. Our results showed significant expressions for CBR3-AS1 (P-value=0.0139), RAB6C-AS1 (P-value=0.0023), and ZEB2-AS1 (P-value=0.0289) in comparison with the healthy cells. ROC curve analysis for CBR3-AS1 LncRNA revaled sensitivity more than 70%., Conclusion: Although CBR3-AS1, RAB6C-AS1, and ZEB2-AS1 lncRNAs were found to have high expressions in the breast cancer cells, only CBR3-AS1 lncRNA has a high chance to be a breast cancer biomarker., .

Obesity is one of the prevalent health-threatening conditions; however, it is preventable by lifestyle interventions such as exercise. The molecular mechanisms underlying physiological adaptation to physical activity are not fully understood. It has been documented that both intracellular and extracellular (circulating) microRNAs (miRNAs) are involved in both obesogenic and exercise adaptation mechanisms. We aimed to conduct a systematic review of publications that examined the effect of exercise on the expression of miRNAs in individuals with obesity. In addition, bioinformatics analysis was performed on most repetitive miRNAs. PubMed, Scopus, and Google Scholar were searched with relevant keywords. We only included studies that utilized exercise as a modality for the health management of human subjects with obesity to evaluate the changes in expression of obesity-related miRNAs. Through checking of 211 retrieved articles, we reached 12 eligible studies. Some studies reported a statistically significant correlation between the change of miRNAs and clinical parameters such as body mass index and fasting glucose. In silico analysis of most repetitive miRNAs i.e. miR-126, miR-21, miR-146a, miR-221, and miR-223 resulted in the molecular signaling pathways that potentially involve in cellular adaption to exercise in people with obesity. miRNAs partake in health-related benefits of physical activity on obesity-associated cellular and molecular phenomena. However, our understanding of the exact mechanism is still in its infancy. Consistently, the clinicians waiting for the result of more integrated experiments to develop a miRNAs panel as a predictive biomarker of exercise in patients with obesity.

The oncogenic role of long intergenic non-coding RNA for kinase activation (LINK-A) has been appraised in triple-negative breast cancer. However, the molecular function of LINK-A is still unclear in most cancers including lung cancer. The present study aimed to evaluate the impact of down-regulation of LINK-A in A549 and Calu-3 cell lines as cellular models of non-small cell lung carcinoma (NSCLC). We used the RNA interference system to knock down LINK-A. LINK-A expression was significantly reduced by siRNA transfection in A549 and Calu-3 cell lines. LINK-A down-regulation significantly reduced cell viability, colony-forming ability and cell migration, as measured by MTT, colony formation and invasion assays. Finally, cell cycle analysis and Annexin-V/7AAD staining indicated that apoptosis was influenced by LINK-A silencing. Taken together, LINK-A can be proposed as an oncogene in NSCLC.

Familial hypercholesterolemia (FH) is a common, yet underdiagnosed, genetic disorder characterized by lifelong elevated low-density lipoprotein cholesterol levels, which can increase the risk of early-onset coronary artery disease (CAD). In the present study, we screened the nucleotide variations of the LDLR and PCSK9 genes, as well as a part of the APOB gene, in Iranian patients with FH and premature CAD to find the genetic cause of the disorder. Fifteen unrelated individuals with a clinical diagnosis of FH and premature CAD were recruited. Direct DNA sequencing was applied to screen the whole coding exons and exon-intron boundaries of the LDLR and PCSK9 genes and the main parts of their introns, together with exon 26 of the APOB gene. The pathogenicity of the identified mutations was investigated via either segregation analyses in the family or in silico predictive software. Six different point mutations (p.Cys148Tyr, p.Cys216Tyr, p.Cys302Trp, p.Cys338Trp, p.Leu479Gln, and p.G593Afs ∗ 72) in LDLR and a double mutation (p.Asp172His and p.Ala53Val) in both LDLR and PCSK9 genes were identified in seven families with clinically diagnosed FH (43%), whereas no pathogenic mutations were found in eight families with clinically diagnosed FH. This study is the first to identify 1 pathogenic mutation in the LDLR gene (c.1014C > G [p.Cys338Trp]) and to cosegregate it from the affected individual in the family. No mutations were found in the APOB gene, whereas several silent mutations/polymorphisms were identified in the LDLR and PCSK9 genes. Genetic testing and reports on nucleotide alterations in the Iranian population are still limited. Our findings not only further confirm the significant role of FH in the incidence of premature CAD but also enlarge the spectrum of LDLR and PCSK9 variations and exhibit the heterogeneity of FH in Iranians. In patients with no mutation in the examined genes, the disease could be begotten either by a polygenic cause or by gene defects occurring in other related genes and regions not targeted in this study., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Moradi, Maleki, Ghaemmaghami, Khajali, Noohi, Moghadam, Kalyinia, Mowla, Seidah and Malakootian.)

Some miRNAs, including the miR-302 cluster, are critical regulators of the stemness state of embryonic stem cells and cell fate patterning. In this study, we evaluated the activity of the miR-302 core promotor in mice and human pluripotent stem cells, somatic tissue derivatives, and generated transgenic mice expressing EGFP under a miR-302 promoter. The expression of EGFP under the control of the miR-302 promotor was examined in the cell lines and somatic tissues of transgenic mice, transgenic blastocysts, and embryonic stem cells derived from transgenic blastocysts. Our results showed that the miR-302 promoter is highly expressed in the mouse and human pluripotent cells, weakly expressed in the somatic tissue derivatives, is highly expressed in both blastocysts and the first passages of transgenic embryonic stem cells, and lowly expressed in the somatic tissues of transgenic mice. It can be concluded that different temporal and spatial gene expression patterns occur during the embryonic and adult stages of cells in mice.

Oxidative stress is one of the crucial mediators of varicocele-related male infertility. Recently, roles of long noncoding RNAs (lncRNAs) in oxidative stress have begun to emerge, however, little is known about their role in male infertility. The aim of this study was to determine the role of lncRNA SLC7A11-AS1 in varicocele-related male infertility. Through a high-throughput bioinformatics investigation, we predicted that lncRNA SLC7A11-AS1 might be involved in this type of infertility. The reactive oxygen species (ROS) levels and expression levels of SLC7A11-AS1 isoforms were evaluated in ejaculated spermatozoa of 25 infertile men with varicocele and 17 fertile individuals as control. Isoform 6 of SLC7A11-AS1 that showed a significant elevation in infertile men with varicocele relative to the fertile group was overexpressed in testicular-derived carcinoma cell lines (NT2 and NCCIT) followed by assessment of ROS, glutathione (GSH), lipid peroxidation, and cell viability. Overexpression of SLC7A11-AS1 isoform 6 in NT2 and NCCIT cell lines resulted in a significant downregulation of SLC7A11 gene expression, which consequently decreased GSH levels and concomitantly increased ROS levels and enhanced lipid peroxidation, which jeopardized cell survival and promoted cell death. Our finding revealed a potential role of oxidative-related lncRNAs in the pathophysiology of male infertility associated with varicocele., (Copyright © 2020 Sanei-Ataabadi, Mowla and Nasr-Esfahani.)

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect. Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model. The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology."

Alzheimer disease (AD) is a complex neurodegenerative disorder with no definite treatment. The expression of miR-29 family is significantly reduced in AD, suggesting a part for the family members in pathogenesis of the disease. The recent emergence of microRNA (miRNA)-based therapeutic approaches is emphasized on the efficiency of miRNA transfer to target cells. The endogenously made secretory vesicles could provide a biological vehicle for drug delivery. Characteristics such as small sizes, the ability to cross the blood-brain barrier, the specificity in binding to the right target cells, and most importantly the capacity to be engineered as drug carriers have made exosomes desirable vehicles to deliver genetic materials to the central nervous system. Here, we transfected rat bone marrow mesenchymal stem cells and HEK-293T cells (human embryonic kidney 293 cells) with recombinant expression vectors, carrying either mir-29a or mir-29b precursor sequences. A significant overexpression of miR-29 and downregulation of their targets genes, BACE1 (β-site amyloid precursor protein cleaving enzyme 1) and BIM [Bcl-2 interacting mediator of cell death (BCL2-like 11)], were confirmed in the transfected cells. Then, we confirmed the packaging of miR-29 in exosomes secreted from the transfected cells. Finally, we investigated a possible therapeutic effect of the engineered exosomes to reduce the pathological effects of amyloid-β (Aβ) peptide in a rat model of AD. Aβ-treated model rats showed some deficits in spatial learning and memory. However, in animals injected with miR-29-containing exosomes at CA1 (cornu ammonis area), the aforementioned impairments were prevented. In conclusion, our findings provide a new approach for the packaging of miR-29 in exosomes and that the engineered exosomes might have a therapeutic potential in AD., (Copyright © 2020 Jahangard, Monfared, Moradi, Zare, Mirnajafi-Zadeh and Mowla.)

One of the main molecular causes that contributes to varicocele-related male infertility is excess production of reactive oxygen species (ROS). It is believed that hypoxia is an important stimulator of ROS in this condition. Recently, the significant roles of long non-coding RNAs (lncRNAs) in hypoxia response have emerged. Despite the investigation of hypoxia, there is scant information about the role of hypoxia-responding lncRNAs in varicocele-related male infertility. In the present study, we deduced eight hypoxia-responding lncRNAs based on high-throughput RNA sequencing data from two Gene Expression Omnibus (GEO) datasets. We used qRT-PCR to assess the expression levels of some of these lncRNAs in 42 ejaculated spermatozoa samples from 25 infertile men with varicocele and 17 fertile men as controls. We identified significant increases in expression levels of hypoxia-related lncRNAs, MIR210HG and MLLT4-AS1 in ejaculated spermatozoa of infertile men with varicocele. These lncRNAs also showed significant positive correlations with ROS levels and meaningful negative correlations with sperm parameters (count and motility). Besides, in silico studies identified several hypoxia response elements (HREs) within selected lncRNAs promoters. Delineation of hypoxia-related lncRNAs in varicocele-related infertility provides a valuable insight into male infertility., Competing Interests: MHNE is a shareholder and chief embryologist at private infertility center “the Isfahan Fertility and Infertility Center”. MHNE and other authors have no conflict of interest to declare. The Isfahan Fertility and Infertility Center does not alter our adherence to PLOS ONE policies on sharing data and materials.

Frequent abnormalities in 7p12 locus in different tumors like lung cancer candidate this region for novel regulatory elements. MiRNAs as novel regulatory elements encoded within the human genome are potentially oncomiRs or miR suppressors. Here, we have used bioinformatics tools to search for the novel miRNAs embedded within human chromosome 7p12. A bona fide stem loop (named mirZa precursor) had the features of producing a real miRNA (named miRZa) which was detected through RT-qPCR following the overexpression of its precursor. Then, endogenous miRZa was detected in human cell lines and tissues and sequenced. Consistent to the bioinformatics prediction, RT-qPCR as well as dual luciferase assay indicated that SMAD3 and IGF1R genes were targeted by miRZa. MiRZa-3p and miRZa-5p were downregulated in lung tumor tissue samples detected by RT-qPCR, and mirZa precursor overexpression in SW480 cells resulted in increased sub-G1 cell population. Overall, here we introduced a novel miRNA which is capable of targeting SMAD3 and IGF1R regulatory genes and increases the cell population in sub-G1 stage.

Long non-coding RNAs (lncRNAs) are important modulators of various cellular and molecular events, including cancer-associated pathways. The Anti-differentiation ncRNA (ANCR) is a key regulator of keratinocyte differentiation, where its expression is necessary to maintain epidermal progenitor's cells. Herein, we investigated the expression pattern of ANCR in the course of neural differentiation. Moreover, we used published RNAseq data and clinical samples to evaluate the alteration of ANCR expression in different cell types and brain tumors. Furthermore, we manipulated ANCR expression in glioma cell lines to clarify a potential functional role for ANCR in tumorigenesis. Our qRT-PCR results revealed a significant upregulation of ANCR in more malignant and less differentiated types of brain tumors (P = 0.03). This data was in accordance with down regulation of ANCR during neural differentiation. ANCR suppression caused an elevation in apoptosis rate, as well as a G1 cell cycle arrest in glioblastoma cell line. Altogether, our data demonstrated that ANCR may play a role in glioma genesis and that it could be considered as a potential diagnostic and therapeutic target to combat brain cancers.

Background: Human mesenchymal stem cells (MSCs) have been shown to be involved in the formation and modulation of tumor stroma and in interacting with tumor cells, partly through their secretome. Exosomes are nano-sized intraluminal multi-vesicular bodies secreted by most types of cells and have been found to mediate intercellular communication through the transfer of genetic information via coding and non-coding RNAs to recipient cells. Since exosomes are considered as protective and enriched sources of shuttle microRNAs (miRNAs), we hypothesized that exosomal transfer of miRNAs from MSCs may affect tumor cell behavior, particularly angiogenesis., Methods: Exosomes derived from MSCs were isolated and characterized by scanning electron microscopy analyses, dynamic light scattering measurements, and Western blotting. Fold changes in miR-100 expression levels were calculated in exosomes and their corresponding donor cells by qRT-PCR. The effects of exosomal transfer of miR-100 from MSCs were assessed by qRT-PCR and Western blotting of the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells. The quantification of secreted VEGF protein was determined by enzyme-linked immunosorbent assay. The putative paracrine effects of MSC-derived exosomes on tumor angiogenesis were explored by in vitro angiogenesis assays including endothelial cell proliferation, migration and tube formation assays., Results: We found that MSC-derived exosomes induce a significant and dose-dependent decrease in the expression and secretion of vascular endothelial growth factor (VEGF) through modulating the mTOR/HIF-1α signaling axis in breast cancer-derived cells. We also found that miR-100 is enriched in MSC-derived exosomes and that its transfer to breast cancer-derived cells is associated with the down-regulation of VEGF in a time-dependent manner. The putative role of exosomal miR-100 transfer in regulating VEGF expression was substantiated by the ability of anti-miR-100 to rescue the inhibitory effects of MSC-derived exosomes on the expression of VEGF in breast cancer-derived cells. In addition, we found that down-regulation of VEGF mediated by MSC-derived exosomes can affect the vascular behavior of endothelial cells in vitro., Conclusions: Overall, our findings suggest that exosomal transfer of miR-100 may be a novel mechanism underlying the paracrine effects of MSC-derived exosomes and may provide a means by which these vesicles can modulate vascular responses within the microenvironment of breast cancer cells.

MicroRNAs are small non-coding RNAs that posttranscriptionally regulate mRNA expression. hsa-miR-6165 which was previously discovered in our group is located in the forth intron of p75NTR gene and its function is still under investigation. As P75NTR has diverse cellular functions, some of the complexity of its function could be attributed to the internally located microRNA. Our analysis revealed that treatment of HCT116 cells with 5-azacytidine promoted differential expression of hsa-miR-6165 from its host gene which is consistent with the bioinformatic prediction of an independent promoter for hsa-miR-6165. In addition, hsa-miR-6165 promoter is capable of driving GFP reporter gene in HeLa cells. The putative target gene expression level which was detected using RT-qPCR is inversely proportional to the expression level of hsa-miR-6165 during NT2 cell neural differentiation. Furthermore, hsa-miR-6165 overexpression resulted in significant downregulation of ABLIM-1, PVRL1, and PDK1 target genes, while it attenuates NT2 neural differentiation. Hsa-miR-6165 overexpression in SW480 cells also resulted in significant downregulation of PKD1, DAGLA, and PLXNA2 putative target genes, while it increases the sub-G1 cell population of SW480 and HEK293T cells as detected by flow cytometry. Overall, in this study, we report an independent promoter for hsa-miR-6165 which is active in HeLa cells. Additionally, hsa-miR-6165 targets ABLIM-1, PVRL1, PKD1, PLXNA2, and PDK1 genes, and unlike in HEK293T and SW480 cells, hsa-miR-6165 overexpression does not affect HeLa cells while its downregulation reduces sub-G1 cell population. Our results validate that hsa-miR-6165 affects the cell cycle progression and could increase apoptosis in human cell lines.

Bone marrow stromal cells (BMSCs) are attractive cellular sources for cell therapy of many diseases, specifically neurodegenerative ones. The potential capability of BMSCs could be further augmented by enhancing their neuroprotective property, differentiation potential, and survival rate subsequent to transplantation. Therefore, a concurrent upregulation of neurotrophin-3 (NT-3) and its high affinity receptor, tyrosin kinase C (TrkC), was utilized in our study. BMSCs were cotransfected with pDsRed1-N1-NT-3 and pCMX-TrkC plasmids before induction of neural differentiation. pEGFP-N1-transfected BMSCs were also employed as a control. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed for gene expression analysis. Cell viability was evaluated by MTT assay, while apoptosis rate was assessed by flow cytometry after PI and Annexin V staining. NT-3 and TrkC mRNA levels were greatly elevated following cotransfection of cells with pDsRed1-N1-NT-3 and pCMX-TrkC vectors. The expression of neural markers (i.e., NFM, and NeuroD1) was augmented in cotransfected BMSCs, compared to the control ones, after neural induction. At each time point, the viability and apoptosis rates of the cells over-expressing NT-3 and TrkC showed increased and reduced patterns, respectively. Our data demonstrated that NT-3/TrkC-co-transfected BMSCs, compared to those of intact cells, could be more beneficial graft candidates for the upcoming treatment strategies of neurogenic disorders due to their increased viability and expression of neural markers. This may be due to their increased level of neural differentiation potential and/or their enhanced rate of survival and/or their useful capacity to secrete NT-3.

Gold nanoparticles (GNPs) have been applied as radiosensitizer in radiotherapy. Limited reports have shown that GNPs may be effective as a dose enhancer agent for electron radiation therapy. Some Monte Carlo Simulation studies have shown that selecting suitable size of GNPs and electron energies are critical for effective dose enhancement. The aim of this study was to assess possible radiosensitization effect of GNPs on cancer cell treated with 4MeV electron beams. Approximately 10nm GNPs were synthesized and characterized by electron microscope and dynamic light scattering. MCF-7 and MDA-MB-231 breast cancer cells were used and their viability was measured by MTT assay. Radiosensitization effect of GNPs under 4MeV electron beams was measured by clonogenic assay. The result showed a concentration dependent uptake of GNPs without reducing cell viability at concentrations ≤50mg/L. Incubation of cancer cells with GNPs caused a significant decrease in their viability following exposure to electron beams as well as a decrease in their survival fraction when compared to control. The sensitizer enhancement ratio (SER) by electron beams in MCF-7 cells was 1.43 and 1.40 in presence of 25 and 50mg/L GNPs, respectively. For MDA-MB-231 cells, it was 1.62 in presence of 25mg/L GNPs. Our data demonstrated the significant dose enhancement of the GNPs in combination with 4MeV electron beams that could be applicable for the treatment of superficial tumors and intra operative radiation therapy., (Copyright © 2017. Published by Elsevier Ltd.)

Neurotrophin receptors play a crucial role in neuronal survival, differentiation and regeneration. Nerve growth factor receptor (NGFR) or P75 NTR is a neurotrophin receptor that is involved in many pathological conditions including cancers. Genetic factors that are involved in regulation of neurotrophin receptors are under intense investigation. MiRNAs are novel regulators of signalling pathways that are candidates for regulation of neurotrophin receptors. Computational programs predicted that NGFR gene is a bona fide target for hsa-miR- 939. RT-qPCR, Western analysis and dual luciferase assay evidences indicated that NGFR transcript is targeted by hsa-miR-939. Also, hsa-miR-939 overexpression brought about down-regulation of NGFR expression in U87 cell line, followed by cell death rate reduction, detected by flow cytometry. Taken together, here for the first time, hsa-miR-939 is introduced as a novel key regulator of NGFR expression and its involvement in cell death/survival processes is suggested.

The Wnt signaling pathway is hyperactivated in most colorectal cancers (CRC). Finding new regulators of this pathway represents the potential for cancer diagnosis or treatment. OCC-1 was initially reported as an up-regulated gene in colon carcinoma, without knowing its mechanism of action. Here, two novel transcript variants and an exonic microRNA that originated from the OCC-1 gene are reported, showing positive effects on Wnt activity. Up-regulation of the known OCC-1 variant (assigned as OCC-1A/B) was limited to CRC, and its overexpression increased survival of CRC-originated SW480 cells (Wnt + ), while resulting in apoptosis of Wnt-suppressed SW480 cells or HeLa cells (Wnt - ) detected by PI staining. Immunocytochemistry showed that the OCC-1A/B-encoded peptide was localized to the nucleus, where its overexpression resulted in Wnt signaling up-regulation, detected by TOP/FOPflash assay. The noncoding portion of the OCC-1A/B transcript had a suppressive effect on Wnt activity and had a negative correlation with APPL2 neighboring gene expression. Unlike OCC-1A/B, the novel OCC-1C splice variant had no expression alteration in CRC, and it seemed to encode a smaller peptide with cytoplasmic localization. A 60-nucleotide (nt) fragment containing an AUG start codon is spliced out to produce an OCC-1D noncoding RNA variant. The 60-nt RNA was validated as the precursor of a novel microRNA, which we named miR-ex1 Both OCC-1D and miR-ex1 were coordinately up-regulated in CRC. MiR-ex1 functional analysis revealed that it is targeting the APC2 tumor suppressor gene and is an activator of the Wnt signaling pathway. Overall, the OCC-1 gene is now introduced as a novel Wnt signaling regulator and as a potential therapeutic target., (© 2016 Najafi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The new claim about the origin of cancer known as Cancer Stem Cell theory states that a somatic differentiated cell can dedifferentiated or reprogrammed for regaining the cancer cell features. It has been recently shown that expression of stemness factors such as Oct4, Sox2, Nanog and Klf4, in a variety of somatic cancers can leads to development of tumorogenesis. Here, the expression of Oct4 variants were evaluated in brain tumor tissues by quantitative RT-PCR and immunohistochemical (IHC) analysis. In next phase of our study, the expression of Oct4B1 was knock-down in brain cancer cell lines and its effect on cell cycle was assessed. Finally, in order to get insights into sequence-structure-function relationships of Oct4 isofroms, their sequences were analysed using bioinformatic tools. Our data revealed that all three variants of Oct4 are expressed in different types of brain cancer. The expression level of Oct4B1, in contast to Oct4B, was much higher in high-grade brain tumors compared with low-grade ones. In line with qPCR, the expression of Oct4A and B isofroms was confirmed with IHC in different types of brain tumors. Moreover, as a result of the suppression of Oct4B1 expression, the brain cancer cells were arrested in G2/M phase of cell cycle. Bioinfromatics data indicated that the predicted Oct4B1 protein have DNA binding properties. All together, our findings suggest that Oct4B1 has a potential role in tumorigenesis of brain cancer and can be considered as a new tumor marker with potential value in diagnosis and treatment of brain cancer.

Long non-coding RNAs are manifested as a new paradigm of molecular effectors in a wide range of human diseases. Human SOX2 overlapping transcript (SOX2OT) gene can generate six lncRNA transcript variants which are functionally assumed to be correlated with cellular differentiation and carcinogenesis. However, the circumstances determining expressional and functional differences between SOX2OT transcript variants remain to be explored. Here, we studied the expression of all SOX2OT transcript variants specifically in five human cancer cell lines by real-time RT-PCR. Changes of the new SOX2OT transcript variants expression were measured during the NT2 teratocarcinoma cell line neuronal-like differentiation and were compared to pluripotency regulators, SOX2 and OCT4A gene expressions. Surprisingly, we identified two new SOX2OT transcripts, named SOX2OT-7, SOX2OT-8 which lack exon 8. We discovered that beside active proximal and distal SOX2OT promoters, different cancer cell lines express high levels of some SOX2OT transcript variants differentially by alternative splicing. Significantly, both SOX2OT-7 and SOX2OT-8 are highly expressed in human cancer cell lines coinciding with SOX2, one of the pluripotency regulators. Our results revealed that SOX2OT-7 is almost the most abundant form of SOX2OT transcript variants in the examined cancer cell lines particularly in NT2 teratocarcinoma cell line where its expression falls upon neuronal-like differentiation similar to SOX2 and OCT4A. We suggest that at least some of SOX2OT transcripts are significantly associated with cancer and stem cell related pathways.

Purpose: Many cell signaling pathways essential for normal stem cell development are involved in cancer initiation and progression. In the present study, motivated by a possible contribution of reprogramming process in induction of cancer, we compared the expression level of main genes involved in iPS generation, i.e., miR-302, miR-145, SOX2, c-MYC, and P21, in a series of tumor and non-tumor tissues of stomach., Methods: A total number of 34 tumors and their matched non-tumor (as control) gastric surgical specimens were obtained. The expression of the candidate genes was evaluated by using real-time PCR and immunohistochemistry (IHC) techniques., Results: Our data revealed a significant downregulation of miR-302b, P21, and miR-145 genes in intestinal and SOX2 gene in diffuse type of tumor samples. SOX2, but not the other genes, showed a significant downregulation in both proximal (cardia and fundus) and distal (body and antrum) sites of stomach. Based on receiver-operating characteristic (ROC) analyses, the highest total area under the curve (AUC) was found for SOX2 (AUC = 82 %, P < 0.001). Interestingly, all tumor samples revealed a negative signal for c-MYC expression, while non-tumor samples represented an intense cytoplasmic staining., Conclusions: Despite the fact that some hESC-specific genes are upregulated in tumors, our data revealed a significant downregulation of all candidate genes, except for c-MYC, in tumor samples of stomach. Moreover, ROC data demonstrated that SOX2 gene expression index is a better potential biomarker of gastric cancer, compared to other tested genes. SOX2 expression has a good sensitivity and specificity to discriminate correctly between tumor/non-tumor and also high/low grades of tumor malignancy. It seems downregulation of miR-302b, miR-145, and P21 could contribute to gastric tumor initiation and progression.

Objective: In spite of accumulating information about pathological aspects of sulfur mustard (SM), the precise mechanism responsible for its effects is not well understood. Circulating microRNAs (miRNAs) are promising biomarkers for disease diagnosis and prognosis. Accurate normalization using appropriate reference genes, is a critical step in miRNA expression studies. In this study, we aimed to identify appropriate reference gene for microRNA quantification in serum samples of SM victims., Materials and Methods: In this case and control experimental study, using quantitative real-time polymerase chain reaction (qRT-PCR), we evaluated the suitability of a panel of small RNAs including SNORD38B, SNORD49A, U6, 5S rRNA, miR-423-3p, miR-191, miR-16 and miR-103 in sera of 28 SM-exposed veterans of Iran-Iraq war (1980-1988) and 15 matched control volunteers. Different statistical algorithms including geNorm, Normfinder, best-keeper and comparative delta-quantification cycle (Cq) method were employed to find the least variable reference gene., Results: miR-423-3p was identified as the most stably expressed reference gene, and miR- 103 and miR-16 ranked after that., Conclusion: We demonstrate that non-miRNA reference genes have the least stabil- ity in serum samples and that some house-keeping miRNAs may be used as more reliable reference genes for miRNAs in serum. In addition, using the geometric mean of two reference genes could increase the reliability of the normalizers.

OCT4B1 is a newly discovered spliced variant of OCT4 which is primarily expressed in pluripotent and tumor cells. Based on our previous studies, OCT4B1 is significantly overexpressed in tumors, where it endows an anti-apoptotic property to tumor cells. However, the mechanism by which OCT4B1 regulates the apoptotic pathway is not yet elucidated. Here, we investigated the effects of OCT4B1 suppression on the expression alteration of 84 genes involved in apoptotic pathway. The AGS (gastric adenocarcinoma), 5637 (bladder tumor), and U-87MG (brain tumor) cell lines were transfected with OCT4B1 or irrelevant siRNAs. The expression level of apoptotic genes was then quantified using a human apoptosis panel-PCR kit. Our data revealed an almost similar pattern of alteration in the expression profile of apoptotic genes in all three studied cell lines, following OCT4B1 suppression. In general, the expression of more than 54 apoptotic genes (64 % of arrayed genes) showed significant changes. Among these, some up-regulated (CIDEA, CIDEB, TNFRSF1A, TNFRSF21, TNFRSF11B, TNFRSF10B, and CASP7) and down-regulated (BCL2, BCL2L11, TP73, TP53, BAD, TRAF3, TRAF2, BRAF, BNIP3L, BFAR, and BAX) genes had on average more than tenfold gene expression alteration in all three examined cell lines. With some minor exceptions, suppression of OCT4B1 caused upregulation of pro-apoptotic and down-regulation of anti-apoptotic genes in transfected tumor cells. Uncovering OCT4B1 down-stream targets could further elucidate its part in tumorigenesis, and could lead to finding a new approach to combat cancer, based on targeting OCT4B1.

Objectives: OCT4 is a transcription factor required for pluripotency during early embryogenesis and the maintenance of identity of embryonic stem cells and pluripotent cells. Therefore, the effective expression regulation of this gene is highly critical. UTR regions are of great significance to gene regulation. In this study, we aimed to investigate the potential regulatory role played by 5´UTR and 3´UTR of the Oct4 gene in mouse BMSC and P19 cells., Materials and Methods: The Oct4 5´UTR and 3´UTR sequences were cloned into pGL3 luciferase plasmid which led to the generation of pGL3 5´-UTR, pGL3 5´&3´-UTRs and pGL3 3´-UTR vectors. The vectors were transfected into BMSC and P19 cells followed by luciferase assay., Results: The assay of luciferase expression exhibited a direct link between the presence of Oct4 3´- UTR and the decrease of luciferase count in both cell lines; whereas 5´UTR indicated diverse behaviors in two cells. This discrepancy could be explained in view of the difference of cellular contexts in which the Oct4 UTRs act., Conclusion: This study sheds some light on the role of UTR regions of mouse Oct4 in regulating post-transcriptional gene expression in pluripotent cells. These data represent potential to be used for the development of novel therapeutic approaches for a variety of malignancies.

Nucleostemin (NS) is a nucleolar protein involved in stem cell (SC) self-renewal by controlling cell cycle progression. In addition to SCs, NS is also expressed in some highly proliferating cells including several adult stem cells and cancer cell lines. NS knock-down in different cell lines demonstrated its cell type-dependent function in arresting cell cycle in either G1 or G2/M phases. Here, we have evaluated the expression of NS and iPS genes in 36 gastric cancer and their matched marginal nontumor tissues by means of real-time polymerase chain reaction (RT-PCR). We have also examined a potential causative role of NS in gastric tumorigenesis by suppressing its expression in a gastric cancer cell line, AGS. Our data revealed that NS expression level is much higher in tumor tissues (p = 0.046), especially in high-grade ones (p < 0.001), whereas the expression of Klf4 and Sox2 is downregulated in tumor tissues compared to marginal nontumor samples (p < 0.001). Furthermore, NS suppression in the AGS cell line caused some morphological alterations, a cell cycle arrest at G1 phase, and an upregulation of iPS genes: Nanog, Sox2, and Klf4. Based on our results, NS overexpression seems to have a causative role in gastric tumorigenesis and/or progression, and it could be considered as a potential tumor marker for diagnosis, molecular classification, and molecular therapy of gastric adenocarcinoma.

Background: Catsper proteins are responsible for entering Ca(2+) to the cell and play an important role in sperm motility and male fertility. Antioxidants are vital for sperm motility too. Escanbil (Calligonum) extract possess some of the important antioxidant like Catechin and Quercetin., Objective: Here we investigated the effects of Escanbil (Calligonum) extract on the sperm parameters and the expressing of Catsper gene in aging male mice., Materials and Methods: In this animal study, firstly, dose response was performed by using these three doses of Escanbil (Calligonum) (10, 30 and 50 mg/kg). 5 mice in each group were considered and Intra Peritoneal injection was done for 5 weeks. the sperm parameters analyzed and dUTP nick end labeling (TUNEL )staining was done. 30 mg/kg dose was considered as optimum dose. Secondly: fifteen aging male mice (11-13 months) were divided into three groups: control, sham and experiment. The experiments were injected Intra peritonealy with Escanbil (Calligonum) extract (30mg/kg) weekly for up to 5 weeks. The sham group was injected Intra Peritoneal (DMSO). Sperm parameters were analyzed. Expression of Catsper genes was analyzed by Real time PCR., Results: Our results showed that after Escanbil (Calligonum) treatment (30 mg/kg), the sperm parameters were improved in experimental group (p<0.05). Our data showed that there was a statistical significance difference between the expressions of Catsper 2, 4 in aging experiment group comparison with aging control group (p<0.05)., Conclusion: We investigated that the Escanbil (Calligonum) extract (30 mg/kg) can improve sperm parameters and change the expression of Catsper genes in aging male mice. This herbal extract can be used as an antioxidant component for clinical usages.

Glioblastoma is an invasive tumor of the central nervous system. Tumor recurrence resulting from ineffective current treatments, mainly due to the blood-brain barrier, highlights the need for innovative therapeutic alternatives. The recent availability of nanotechnology represents a novel targeted strategy in cancer therapy. Natural products have received considerable attention for cancer therapy because of general lower side effects. Curcumin is a new candidate for anticancer treatment, but its low bioavailability and water solubility represent the main disadvantages of its use. Here, curcumin was efficiently encapsulated in a nontoxic nanocarrier, termed dendrosome, to overcome these problems. Dendrosomal curcumin was prepared as 142 nm spherical structures with constant physical and chemical stability. The inhibitory role of dendrosomal curcumin on the proliferation of U87MG cells, a cellular model of glioblastoma, was evaluated by considering master genes of pluripotency and regulatory miRNA (microribonucleic acid). Methylthiazol tetrazolium assay and flow cytometry were used to detect the antiproliferative effects of dendrosomal curcumin. Annexin-V-FLUOS and caspase assay were used to quantify apoptosis. Real-time polymerase chain reaction was used to analyze the expression of OCT4 (octamer binding protein 4) gene variants (OCT4A, OCT4B, and OCT4B1), SOX-2 (SRY [sex determining region Y]-box 2), Nanog, and miR-145. Dendrosomal curcumin efficiently suppresses U87MG cells growth with no cytotoxicity related to dendrosome. Additionally, the accumulation of cells in the SubG1 phase was observed in a time- and dose-dependent manner as well as higher rates of apoptosis after dendrosomal curcumin treatment. Conversely, nonneoplastic cells were not affected by this formulation. Dendrosomal curcumin significantly decreased the relative expression of OCT4A, OCT4B1, SOX-2, and Nanog along with noticeable overexpression of miR-145 as the upstream regulator. This suggests that dendrosomal curcumin reduces the proliferation of U87MG cells through the downregulation of OCT4 (octamer binding protein 4) variants and SOX-2 (SRY [sex determining region Y]-box 2) in an miR-145-dependent manner.

Objective: Endothelial progenitor cells (EPCs) have a potential application for cell therapy, however, their biological nature is not well-understood. EPCs also possess some stemness features, such as their clonogenicity and differentiation capacity. The main aim of this study was to evaluate the expression of certain transcription factors regulating selfrenewal property of stem cells., Materials and Methods: In this experimental study, peripheral blood mononuclear cells were isolated from fresh human blood of several volunteers and were cultured in fibronectin- coated plates. EPCs were identified based on their morphology and growth characteristic. Then, the expression of some markers implicated in self-renewal capacity was assessed in the isolated cells using reverse transcription-polymerase chain reaction (RTPCR) and immunocytochemistry., Results: Expression of the cell surface markers, CD31 and CD34, was determined by RT-PCR and immunocytochemistry. Furthermore, these cells had the ability for Di-ACLDL incorporation as well as attachment to lectin I. EPCs did not express the main stem cell markers, like OCT4-A, Nanog, and Sox2; nevertheless, they expressed the weaker pluripotent markers, including OCT4B and OCT4-B1 spliced variants, such as Nucleostemin and ZFX. Furthermore, the expression of Nucleostemin and ZFX genes revealed a decreasing pattern from days 4(th) to 11(th)., Conclusion: The main regulators of stem cell self-renewal genes, including OCT4-A, Nanog, and Sox2 are not expressed in EPCs. Forced expression of these genes can elevate the stemness property and clinical application of EPCs.

Low-density quantitative real-time PCR (qPCR) arrays are often used to profile expression patterns of microRNAs in various biological milieus. To achieve accurate analysis of expression of miRNAs, non-biological sources of variation in data should be removed through precise normalization of data. We have systematically compared the performance of 19 normalization methods on different subsets of a real miRNA qPCR dataset that covers 40 human tissues. After robustly modeling the mean squared error (MSE) in normalized data, we demonstrate lower variability between replicates is achieved using various methods not applied to high-throughput miRNA qPCR data yet. Normalization methods that use splines or wavelets smoothing to estimate and remove Cq dependent non-linearity between pairs of samples best reduced the MSE of differences in Cq values of replicate samples. These methods also retained between-group variability in different subsets of the dataset.

Objective: microRNAs (miRNAs) are a new class of non-coding RNAs involved in regulating various biological processes including proliferation, differentiation, and apoptosis, among others. Alterations in miRNA expression are reported in several human cancers, which suggests their potential roles in tumor initiation and progression. Members of the miR-302 cluster are highly expressed in embryonic stem cells (ESC), where they regulate cell self-renewal and pluripotency. Based on the cancer stem cell (CSC) hypothesis, mis-expression of such genes might contribute to tumorigenicity. This study aims to find a potential link between the expression level of human/homo sapiens miR-302b (has-miR- 302b) and tumor/grade state of gastric tissues., Materials and Methods: A matched based case-control study was conducted that included tumor and matched marginal non-tumor surgical specimens from 34 patients diagnosed with gastric adenocarcinoma. Randomly selected samples were obtained from the Iran National Tumor Bank. cDNA synthesis was carried out on total RNA, by using the miRCURY LNA(TM) Universal RT microRNA PCR Kit. Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) assays were performed with specific LNA(TM) primers and SYBR Green master mix. The human embryonic carcinoma cell line, NTERA2 (NT2) and a human gastric adenocarcinoma cell line, AGS, were used to optimize the PCR reactions. A comparative evaluation of miR-302b expression in tumor and non-tumor gastric samples was performed by either paired t test or Wilcoxon non-parametric test. The ability of miR-302b to discriminate tumor from non-tumor gastric samples was evaluated using the area under the receiver operating characteristic (ROC) curve., Results: According to our data, miR-302b expression (normalized to that of the U6 snRNA housekeeping gene) in the pluripotent cell line NT2 was more than 500 times greater than that of the AGS cell line. The level of expression was even lower in tumor and non-tumor gastric tissue samples. The data further revealed a down-regulation of miR-302b in gastric tumor samples (p=0.001), particularly in high-grade adenocarcinoma (p=0.009). However, ROC analysis data demonstrated a low sensitivity and specificity of miR-302b expression to discriminate between the tumor and non-tumor state of the samples (AUC=0.63)., Conclusion: Despite the upregulation of some hESC-specific genes in tumors, our data revealed a down-regulation of miR-302b in high-grade tumors. This data suggested a potential tumor-suppressor role for miR-302b in tumorigenesis of gastric tissue.

Objectives: The key transcriptional regulator Oct4 is one of the self-renewal and differentiation-related factors in cancer stem cells, where it maintains "stemness" state. Cancer stem cells have been identified in a variety of solid malignancies. They are a small population of tumor cells with stem cell characteristics, which are a likely cause of relapse in cancer patients. Due to high incidence, mortality, and recurrence rates of bladder cancer and the necessity of accurate prediction of malignant behavior of the tumors, we evaluated the prognostic value of Oct4 expression in formalin-fixed paraffin-embedded (FFPE) tissues of bladder cancer., Materials and Methods: In this study, Oct4 expression was evaluated in 52 (FFPE) tissues of bladder cancer. RNA extraction from samples of 30 patients from the archive of Labbafi-Nejad Medical Centre in Tehran was performed and Oct4 expression levels were examined by semi-quantitative RT-PCR. The intracellular distribution of Oct4 protein was also determined by immunohistochemistry (IHC)., Results: The results revealed a significant correlation between the expression level of Oct4 and the tumors' grade and stage. A mostly cytoplasmic distribution of Oct4 protein was also confirmed by IHC., Conclusion: All together, our data indicate that the expression level of Oct4 gene is correlated with the clinical and histopathological prognostic indexes of tumors and thus can be considered as a potential prognostic tumor marker.

Purpose: To investigate and compare the expression of OCT4B1 between tumor and non-tumor bladder tissues., Materials and Methods: We investigated the expression of OCT4B1 in 30 tumor and non-tumor surgical specimens of the bladder, using the TaqMan real-time polymerase chain reaction approach and by carefully designing primers and probes specific for the amplification of the variant., Results: Most tumor and non-tumor samples of the bladder showed OCT4B1 expression, but its expression level was significantly higher in the tumors (P < .002). Moreover, the up-regulation of OCT4B1 was more significant in high-grade tumors compared to the low-grade ones (P < .05). We have also employed the RNA interference strategy to evaluate the functional role of OCT4B1 in a bladder cancer cell line, 5637. Suppression of OCT4B1 caused some changes in cell cycle distribution, and significantly elevated the rate of apoptosis in the cells., Conclusion: Our findings suggest that OCT4B1 plays a potential role in tumor initiation and/or progression of the bladder cancer. Additionally, OCT4B1 can be regarded as a new tumor marker for detection, classification, and treatment of the bladder cancer. However, more experimental studies are needed to replicate our findings.

Purpose: The length of GT-repeats polymorphic region in the promoter of human Heme oxygenase-1 gene (HO-1) alters the level of its transcriptional activity in response to oxidative stresses. Decreased level of HO-1 protein in the seminal plasma has been reported to be associated with oligospermia and azoospermia in male infertility. This is the first study to investigate the association between GT-repeats expansion in the promoter of the HO-1 gene and male infertility., Methods: The frequencies of different GT-repeats alleles in the promoter of HO-1 gene were determined in 100 cases and 100 normal controls using PCR-PAGE, ABI fragment analysis genotyping and sequencing analysis., Results: All alleles were classified into S and L alleles. S alleles were specified as number 0 to 3 with <27 GT-repeats and L alleles were specified as number 4 to 6 with >27 repeats. The L allele frequency was significantly higher among case group (54.5%) than that was obtained in the normal control group (37.5%). Statistical analysis provided a significant relationship between L allele and male infertility (P < 0.001)., Conclusions: This study shows for the first time that GT-repeats expansion in promoter of the HO-1 gene is associated with oligospermia and azoospermia among Iranian infertile cases.

Embryoid bodies (EBs) are primitive embryonic structures derived from differentiating embryonic stem cells (ESCs). Many techniques have been used to obtain EBs. Improving the technique of EB formation can help in achieving better results in ESCs differentiation into neurons, myocardiocytes, haemopoeitic cells, and others. We evaluated the use of Sigmacote as a hydrophobic substrate to improve EB formation. CCE and P19 cell lines were used to obtain EBs and retinoic acid was used to induce neural differentiation. The results revealed that Sigmacote, as a hydrophobic substrate, can improve EB formation from ESCs. Our results demonstrate that the silicon-coating of glass petri dishes by Sigmacote is an easy and reproducible technique to enhance EB formation from murine ESCs and EC cells.

Introduction: A Polycomb group repressor protein named BMI1 represses the genes that induce cellular senescence and cell death, and it can contribute to cancer when improperly expressed. We aimed to evaluate expression of BMI1 gene in bladder tumors., Materials and Methods: Tissue specimens containing bladder tumor were evaluated and compared with intact tissues from tumor margins and normal bladders. There were 40 tumor specimens of patients with transitional cell carcinoma of the bladder, 20 tumor-free tissues taken from the margin of the tumors, and 8 specimens from patients without tumor. Specific primers for BMI1 and B2M (as an internal control) were used for reverse transcript polymerase chain reaction technique. The production and distribution of BMI1 protein was also examined by western blotting and immunohistochemistry techniques., Results: Polymerase chain reaction generated a 683-bp product, corresponding to the expected size of BMI1 amplified region. The identity of the amplified fragment was then confirmed by direct DNA sequencing. The mean of expression of BMI1 detected in tumor tissues was significantly higher than that in intact tissues, and there was also a significant association between the mean of gene expression and the stage of malignancy (P < .001). The expression of BMI1 at protein level was further confirmed by western blotting and immunohistochemistry., Conclusion: BMI1 is a potent repressor of retinoblastoma and p53 pathways, and hence, elucidating its role in tumorigenesis is very important. We reported for the first time the expression of BMI1 and its correlation with incidence and progress of bladder tumors.

Background: Cytotoxic therapy can lead to prolonged azoospermia or even sterility. In the present study, we investigated the morphological changes of mouse testes after gamma-Radiation., Methods: After anaesthetizing of NMRI mice, testes and their surrounding tissues were irradiated using a cobalt therapy machine. Four experimental groups were irradiated with fractionated doses of: 1.5 + 8, 1.5 + 12 and 1.5 + 16 Gy (with an interval of 24 h) and single dose of 14 Gy. Non-irradiated mice were considered as control group. Testes were removed 4, 6 and 8 weeks following irradiation, weighed and processed for light microscopic study. Diameters of seminiferous tubules and their lumens, epithelium thickness, percentage of different types of tubules and number of spermatogenic cell were measured. Moreover, sperm count motility and viability rates were evaluated in epididymis., Results: Number of normal tubules, epithelium thickness, tubules diameter and lumen diameter were significantly reduced with high dose irradiation in comparison with control testes. The recovery was observed after 8 weeks. Epididymal sperm count, motility and viability rates were significantly decreased in the irradiated mice comparing non-irradiated ones. These parameters were increased after 8 weeks., Conclusion: According to the results, irradiation can cause temporary azoospermia in mouse and this effect is reversible after 8 weeks.

Introduction: Survivin, an inhibitor of apoptosis (IAP), has been reported to be capable of regulating both cellular proliferation and apoptotic cell death. Survivin expression has been described during embryonic development and in adult cancerous tissues, with greatly reduced expression in adult normal differentiated tissues, particularly if their proliferation index is low. In the present study, the expression and potential diagnostic and prognostic value of survivin in bladder tumors was evaluated., Materials and Methods: Primary and recurrent bladder tumor specimens were obtained from patients referred to the Shaheed Labbafinejad Medical Center in Tehran, Iran. Total RNA was isolated from frozen tissues, reverse transcribed and amplified by means of a nested polymerase chain reaction technique., Results: Survivin was detected in 3 cases of primary tumors (42.8%) and 6 cases of recurrent tumors of bladder (60%). Survivin-DeltaEx3 expression was seen in 41.2% of the 17 cases with bladder cancer., Conclusion: Our findings suggest that the expression of survivin and survivin-DeltaEx3 is well associated with invasive and more-aggressive forms of bladder cancer. Our data also indicate that the presence of survivin-DeltaEx3 is better correlated with tumorigenesis of bladder cancer compared with survivin expression.