Down-regulatory effects of miR-211 on long non-coding RNA SOX2OT and SOX2 genes in esophageal squamous cell carcinoma

Objective: MicroRNAs (miRNAs) are a class of non-coding RNAs (ncRNAs) that transcriptionally or post-Transcriptionally regulate gene expression through degradation of their mRNA targets and/or translational suppression. However, there are a few reports on miRNA-mediated expression regulation of long ncRNAs (lncRNAs). We have previously reported a significant upregulation of the lncRNA SOX2OT and its intronic coding gene, SOX2, in esophageal squamous cell carcinoma (ESCC) tissue samples. In this study, we aimed to evaluate the effect of induced overexpression of miR-211 on SOX2OT and SOX2 expression in vitro. Materials and Methods: In this experimental study, we performed both bioinformatic and experimental analyses to examine whether these transcripts are regulated by miRNAs. From the list of potential candidate miRNAs, miR-211 was found to have complementary sequences to SOX2OT and SOX2 transcripts. To validate our finding experimentally, we transfected the NT-2 pluripotent cell line (an embryonal carcinoma stem cell) with an expression vector overexpressing miR-211. The expression changes of miR-211, SOX2OT, and SOX2 were then quantified by a real-Time polymerase chain reaction (RT-PCR) approach. Results: Compared with mock-Transfected cells, overexpression of miR-211 caused a significant down-regulation of both genes (P<0.05). Furthermore, flow-cytometry analysis revealed a significant elevation in sub-G1 cell population following ectopic expression of miR-211 in NT-2 cells. Conclusion: We report here, for the first time, the down-regulation of SOX2OT and SOX2 genes by an miRNA. Considering the vital role of SOX2OT and SOX2 genes in pluripotency and tumorigenesis, our data suggest an important and inhibitory role for miR-211 in the aforementioned processes

The effect of microRNA-375 overexpression, an inhibitor of Helicobacter pylori-induced carcinogenesis, on lncRNA SOX2OT

Background: Helicobacter pylori is a major human pathogenic bacterium in gastric mucosa. Although the association between gastric cancer and H. pylori has been well-established, the molecular mechanisms underlying H. pylori-induced carcinogenesis are still under investigation. MicroRNAs (miRNAs) are small noncoding RNAs that modulate gene expression at the posttranscriptional level. Recently, studies have revealed that miRNAs are involved in immune response and host cell response to bacteria. Also, microRNA-375 (miR-375) is a key regulator of epithelial properties that are necessary for securing epithelium-immune system crosstalk. It has been recently reported that miR-375 acts as an inhibitor of H. pylori-induced gastric carcinogenesis. There are few reports on miRNA-mediated targeting long noncoding RNAs (lncRNAs). Objectives: This study aimed to examine the possible effect of miR-375 as an inhibitor of H. pylori-induced carcinogenesis on the expression of lncRNA SOX2 overlapping transcript (SOX2OT) and SOX2, a master regulator of pluripotency of cancer stem cells. Materials and Methods: In a model cell line, NT-2 was transfected with the constructed expression vector pEGFP-C1 contained miR- 375. The RNA isolations and cDNA synthesis were performed after 48 hours of transformation. Expression of miR-375 and SOX2OT and SOX2 were quantified using real-time polymerase chain reaction and compared with control cells transfected with pEGFP-C1-Mock clone. Cell cycle modification was also compared after transfections using the flow cytometry analysis. Results: Following ectopic expression of miR-375, SOX2OT and SOX2 expression analysis revealed a significant decrease in their expression level (P < 0.05) in NT-2 cells compared to the control. Cell cycle analysis following ectopic expression of miR-375 in the NT-2 cells using propidium iodine staining revealed significant extension in sub-G1 cell cycle. Conclusions: This is the first report to show down-regulation of SOX2OT and SOX2 following induced expression of miR-375. This findingmaysuggest expression regulation potential between different classes of ncRNAs, for example between miR-375andSOX2OT. This data not only extends our understanding of possible ncRNA interactions in cancers but also may open novel investigation lines towards elucidation of molecular mechanisms controlling H. pylori inflammation and carcinogenesis. © 2016, Ahvaz Jundishapur University of Medical Sciences

Evaluating the miR-302b and miR-145 expression in formalin-fixed paraffin-embedded samples of esophageal squamous cell carcinoma

Background: MicroRNAs are involved in key cellular processes regulating, and their misregulation is linked to cancer. The miR-302-367 cluster is exclusively expressed in embryonic stem and carcinoma cells. This cluster also promotes cell reprogramming and stemness process. In contrast, miR-145 is mostly regarded as a tumor suppressor, where it regulates cellular functions such as cell division, differentiation, and apoptosis. By suppressing the main pluripotency factors (OCT4, SOX2, MYC and KLF4), miR-145 silences the self-renewal program in ESCs. Therefore, the main aim of this study is to find a potential link between the expression level of hsa-miR-302b and hsa-miR-145 with tumor vs. non-tumor as well as high-grade vs. low-grade states of the esophageal tissue samples. Methods: A total number of 40 formalin-fixed, paraffin-embedded (FFPE) samples of esophageal squamous-cell carcinoma (ESCC) were obtained, and the tumor and marginal non-tumor areas delineated and punched off by an expert pathologist. Total RNA was extracted with Trizol, and cDNA synthesized using the miRCURY LNA™ Universal RT microRNA PCR Kit. Real-time reverse transcription polymerase chain reaction (RT-PCR) assays were performed using specific LNA-primers and SYBR Green master mix. Results: The expression level of miR-302b failed to show any significant difference, neither between tumor and their non-tumor counterparts, nor among tumors with different grades of malignancies (P > 0.05). In contrast, miR-145 was significantly down regulated in all grades of tumor samples (P 0.05). CONCLUSION: Our data revealed a significant down-regulation of miR-145 in ESCC tissue samples. Based on our ROC curve analysis data (AUC = 0.74, P < 0.001) miR-145 could be regarded as a potential tumor marker for diagnosis of esophageal cancer. © 2015, Academy of Medical Sciences of I.R. Iran. All rights reserved

Analysis of Opportunities and Challenges for R&D Management and the Role of the R&D Society for its Improvement – A Case Study in Iran

Research and Development (R&D) management in Iran is faced to many barriers and obstacles, in which R&D units are considered as the basic core of the product development and innovation. Due to structural shortcomings, a great number of organizations and industries have not been able to find their actual status. There are about different 1141 R&D units with a dispersion pattern in Iran. This paper considers and analyzes the R&D case study in one of the provinces located in the north part of Iran in order to enhance the potential R&D activities in respect with the industrialized areas and zones. In this province, there are about 2504 industrial units of which there are only 44 R&D units certified by the state government. However, there are limit numbers of these R&D units that are extensively active. This paper also addresses the current status in respect with the R&D activities to find out why there is a lack and depression of these activities in the industrial units. By considering the opportunity and challenges of these R&D units, there is a need to change these units to be active in order to quickly respond the market and demand requirements. Finally, a few alternative solutions and improvement plans are proposed, in which the Iranian R&D Society is responsible for supporting and succeeding these action plans towards the organization goals.R&D Management

Expression, Tissue Distribution and Function of miR-21 in Esophageal Squamous Cell Carcinoma

Objective:MiR-21 is an oncomir expressed by malignant cells and/or tumor microenvironment components. In this study we focused on understanding the effects of stromal miR-21 on esophageal malignant cells.Design:MiR-21 expression was evaluated in formalin-fixed paraffin-embedded samples from patients with esophageal squamous-cell carcinoma (SCC) by quantitative RT-PCR. MiR-21 tissue distribution was visualized with in situ hybridization. A co-culture system of normal fibroblasts and esophageal cancer cells was used to determine the effects of fibroblasts on miR-21 expression levels, and on SCC cell migration and invasion.Results:MiR-21 was overexpressed in SCCs, when compared to the adjacent non-tumor tissues (P = 0.0007), and was mainly localized in the cytoplasm of stromal cells adjacent to malignant cells. Accordingly, miR-21 expression was increased in tumors with high versus low stromal content (P = 0.04). When co-cultured with normal fibroblasts, miR-21 expression was elevated in SCC cells (KYSE-30), while its expression was restricted to fibroblasts when co-cultured with adenocarcinoma cells (OE-33 and FLO-1). MiR-21 was detected in conditioned media of cancer cell lines, illustrating the release of this miRNA into the environment. Co-culturing with normal fibroblasts or addition of fibroblast conditioned media caused a significant increase in cell migration and invasion potency of KYSE-30 cells (P<0.0001). In addition, co-culturing cancer cells with fibroblasts and expression of miR-21 induced the expression of the cancer associated fibroblast (CAF) marker S100A4.Conclusions:MiR-21 expression is mostly confined to the SCC stroma and its release from fibroblasts influences the migration and invasion capacity of SCC cells. Moreover, miR-21 may be an important factor in "activating" fibroblasts to CAFs. These findings provide new insights into the role of CAFs and the extracellular matrix in tumor microenvironment formation and in tumor cell maintenance, and suggest miR-21 may contribute to cellular crosstalk in the tumor microenvironment. © 2013 Nouraee et al

Favorable results after conservative management of 316 valproate intoxicated patients

Valproic acid (VPA) is an effective antiepileptic drug widely used worldwide. Despite several studies indicating the usefulness of intravenous L-carnitine in the treatment of VPA poisoning, this drug is not readily available in Iran. The aim of this study was to determine whether supportive care without antidote would result in acceptable outcomes in VPA poisoned patients. Materials and Methods: In an observational, retrospective, single-center case series, all patients &gt;12-year-old with VPA overdose who had referred to a tertiary center between 2009 and 2013 were consecutively enrolled. Patients� demographic and presenting features, physical examinations, clinical management, laboratory data, and outcomes were recorded. Results: A total of 316 patients were enrolled with pure VPA toxicity. The most common presenting signs/symptoms were drowsiness, nausea and vomiting, vertigo, and headache. In the course of the disease, 14 patients (4.4) were intubated and three (0.9) required hemodialysis with mean dialysis sessions of two. Fourteen patients were admitted to Intensive Care Unit, and seizures occurred in five. The initial level of consciousness was lower in patients with poor outcome. The median ingested dose of VPA in patients who required dialysis was significantly higher (20 vs. 6 g; P = 0.006). Multivariate analyses revealed that coma on presentation was associated with a worse outcome (P = 0.001; odds ratio = 61.5, 95 CI = 5.8-646.7). Conclusion: Prognosis of VPA poisoned patients appears to be good even with supportive care. According to our study, older age, ingestion of higher amounts of VPA and lower PCO2, HCO3, base excess, and CPK levels prone the patients to more severe toxicities in univariate analysis, but the main poor prognostic factor is coma on presentation in multivariate analysis. © 2015 Journal of Research in Medical Sciences

Two novel splice variants of SOX2OT, SOX2OT-S1, and SOX2OT-S2 are coupregulated with SOX2 and OCT4 in esophageal squamous cell carcinoma

Long noncoding RNAs (lncRNAs) have emerged as new regulators of stem cell pluripotency and tumorigenesis. The SOX2 gene, a master regulator of pluripotency, is embedded within the third intron of a lncRNA known as SOX2 overlapping transcript (SOX2OT). SOX2OT has been suspected to participate in regulation of SOX2 expression and/or other related processes; nevertheless, its potential involvement in tumor initiation and/or progression is unclear. Here, we have evaluated a possible correlation between expression patterns of SOX2OT and those of master regulators of pluripotency, SOX2 and OCT4, in esophageal squamous cell carcinoma (ESCC) tissue samples. We have also examined its potential function in the human embryonic carcinoma stem cell line, NTERA2 (NT2), which highly expresses SOX2OT, SOX2, and OCT4. Our data revealed a significant coupregulation of SOX2OT along with SOX2 and OCT4 in tumor samples, compared to the non-tumor tissues obtained from the margin of same tumors. We also identified two novel splice variants of SOX2OT (SOX2OT-S1 and SOX2OT-S2) which coupregulated with SOX2 and OCT4 in ESCCs. Suppressing SOX2OT variants caused a profound alteration in cell cycle distribution, including a 5.9 and 6.9 time increase in sub-G1 phase of cell cycle for SOX2OT-S1 and SOX2OT-S2, respectively. The expression of all variants was significantly diminished, upon the induction of neural differentiation in NT2 cells, suggesting their potential functional links to the undifferentiated state of the cells. Our data suggest a part for SOX2OT spliced variants in tumor initiation and/or progression as well as regulating pluripotent state of stem cells. © AlphaMed Press 2013

Hubble imaging of the ionizing radiation from a star-forming galaxy at z=3.2 with fesc>50%

Star-forming galaxies are considered to be the leading candidate sources that dominate the cosmic reionization at z>7, and the search for analogs at moderate redshift showing Lyman continuum (LyC) leakage is currently a active line of research. We have observed a star-forming galaxy at z=3.2 with Hubble/WFC3 in the F336W filter, corresponding to the 730-890A rest-frame, and detect LyC emission. This galaxy is very compact and also has large Oxygen ratio [OIII]5007/[OII]3727 (>=10). No nuclear activity is revealed from optical/near-infrared spectroscopy and deep multi-band photometry (including the 6Ms X-ray, Chandra). The measured escape fraction of ionizing radiation spans the range 50-100\%, depending on the IGM attenuation. The LyC emission is detected at S/N=10 with m(F336W)=27.57+/-0.11 and it is spatially unresolved, with effective radius R_e<200pc. Predictions from photoionization and radiative transfer models are in line with the properties reported here, indicating that stellar winds and supernova explosions in a nucleated star-forming region can blow cavities generating density-bounded conditions compatible with optically thin media. Irrespective to the nature of the ionizing radiation, spectral signatures of these sources over the entire electromagnetic spectrum are of central importance for their identification during the epoch of reionization, when the LyC is unobservable. Intriguingly, the Spitzer/IRAC photometric signature of intense rest-frame optical emissions ([OIII]+Hbeta) observed recently at z~7.5-8.5 is similar to what is observed in this galaxy. Only the James Webb Space Telescope will measure optical line ratios at z>7 allowing a direct comparison with lower redshift LyC emitters, as reported here.Comment: 6 pages, 5 figures, ApJ submitted (comments welcome

Aberrant expression of breast development-related microRNAs, miR-22, miR-132, and miR-212, in breast tumor tissues

Purpose: MicroRNAs (miRNAs) are a major class of small endogenous RNA molecules that posttranscriptionally regulate the expression of most genes in the human genome. miRNAs are often located in chromosomal fragile sites, which are susceptible to amplification or deletion. Chromosomal deletions are frequent events in breast cancer cells. Deletion and loss of heterozygosity at 17p13.3 have been reported in 49 of breast cancers. The aim of the current study was to evaluate potential expression alterations of miR-22, miR-132, and miR-212, which are located on the 17p13.3 locus and are required for mammary gland development. Methods: A matched case-control study was conducted, which included 36 pairs of tumor and matched nontumor surgical specimens from patients diagnosed with breast invasive ductal carcinoma. Formalin-fixed paraffin-embedded samples from archival collections at the pathology department of Shariati Hospital were prepared for RNA extraction using the xylene-ethanol method before total RNA was isolated with TRIzol Reagent. Specific primers were designed for cDNA synthesis and miRNA amplification. The expression of miRNAs was then evaluated by real-time polymerase chain reaction (RTPCR). Results: According to our RT-PCR data, the miR-212/ miR-132 family was downregulated in breast cancer (0.328-fold, p<0.001), and this reduced expression was the most prominent in high-grade tumors. In contrast, miR-22 exhibited a significant upregulation in breast tumor samples (2.183-fold, p=0.040). Conclusion: Consistent with the frequent deletion of the 17p13.3 locus in breast tumor cells, our gene expression data demonstrated a significant downregulation of miR-212 and miR-132 in breast cancer tissues. In contrast, we observed a significant upregulation of miR-22 in breast tumor samples. The latter conflicting result may have been due to the upregulation of miR-22 in stromal/cancer-associated fibroblasts, rather than in the tumor cells. © 2016 Korean Breast Cancer Society. All rights reserved

The Lyman Continuum Escape Fraction of The Cosmic Horseshoe: A Test of Indirect Estimates

High redshift star-forming galaxies are likely responsible for the reionization of the Universe, yet direct detection of their escaping ionizing (Lyman continuum) photons has proven to be extremely challenging. In this study, we search for escaping Lyman continuum of the Cosmic Horseshoe, a gravitationally lensed, star-forming galaxy at z=2.38 with a large magnification of $\sim24$. Transmission at wavelengths of low ionization interstellar absorption lines in the rest-frame ultraviolet suggest a patchy, partially transparent interstellar medium. This makes it an ideal candidate for direct detection of the Lyman continuum. We obtained a 10-orbit Hubble near-UV image using the WFC3/UVIS F275W filter that probes wavelengths just below the Lyman limit at the redshift of the Horseshoe in an attempt to detect escaping Lyman continuum radiation. After fully accounting for the uncertainties in the opacity of the intergalactic medium as well as accounting for the charge transfer inefficiency in the WFC3 CCDs, we find a $3 \sigma$ upper-limit for the relative escape fraction of $f_{esc,rel}<0.08$. This value is a factor of five lower than the value (0.4) predicted by the 40\% transmission in the low-ion absorption lines. We discuss the possible causes for this discrepancy and consider the implications for future attempts at both direct Lyman continuum detection as well as indirect estimates of the escape fraction.Comment: 10 pages, 8 Figures, submitted to the Astrophysical Journa