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Multiple sclerosis (MS) is a chronic myelin destructive disease which affects central nerves system. CD4+ T cells are a group of adaptive immune system cells that have Pivotal role in immune response against the foreign agents. Th17 (T helper 17) cells are one of the subsets of CD4+ T cells which increased in multiple sclerosis patients. MicroRNAs are single stranded non-coding RNAs that regulate protein expression by targeting their mRNA. The aim of this work was to determine miRNAs which probably have effect on theTh17 differentiation pathway by means of bioinformatics methods to suppress this pathway and decrease MS symptoms. by using miRWalk and miRTarBase databases, the probable and validated interactions between some miRNAs and Th17 differentiation pathway proteins were investigated .Disregulated expression of this miRNAs clinically have been shown previously in MS patients. Results showed that miR-9 probably could induce Th17 differentiation from naïve T cells by suppressing negative regulator of Th17 differentiation pathway. In contrast, miR-17and miR-106a/b probably could inhibit Th17 differentiation pathway by suppressing positive regulator of this pathway. Thus, this miRNAs can be considered as potential therapeutic targets for suppression or symptom reduction and also diagnostic markers in MS patients.

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Aims: The induction of artificial over-expression of miRNAs is an appropriate approach to more effective cell differentiation. The significant role of microRNA-1(miR-1) has been reported in the development and differentiation of cardiac cells. Lentivirus is an effective vector for stable cell line production. The aim of this study was the production of recombinant HEK293T with miR-1 overexpression as a biological model for cardiac studies.
Materials & Methods: In this experimental study, HEK 293T cells were cultured in DMEM medium with 10% Fetal Bovine Serum (FBS) and L-glutamine 2mM and Penicillin-Streptomycin 1X in incubator medium. After cloning of miR-1 gene, recombinant clones were selected and the recombination was confirmed by sequencing. The miR-1 carrying vector and auxiliary vectors were packaged in the HEK293T to produce the recombinant virus. The infection of HEK293T by recombinant virus was performed in order to achieve stable cell line. Then, GFP fluorescent marker evaluated the efficiency of transfection and effective virus dilution. Finally, the alteration in expression level of miR-1 was assessed by qPCR. Data analysis was performed by comparing the threshold cycle and Pfaffl method.
Findings: The most GFP expression was detected in transfected cells by 150 micromole dilution. GFP fluorescent marker facilitated optimization and purification of recombinant cells. qPCR investigation demonstrated the significant increase in expression of miR-1 in transfected cells in comparison to controls.
Conclusion: The stable recombinant HEK293T miR-1 over-expressing cell line in lentivirus can be utilized as a suitable biological model for investigation of cardiac evolution and development processes.

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 ABSTRACT
Aims: Epithelial to mesenchymal transition (EMT) is an essential step in the developmental process, wound healing and cancer progression. In many cancers, EMT can increase aggressive properties including invasion, metastasis and Tumor resistance to apoptosis. Recently, miRNAs as a new class of non-coding RNAs that post-transcriptionally regulate gene expression have been demonstrated to have a crucial role in the regulation of EMT. However, the detailed mechanisms of miRNAs involvement in EMT in human cancer cells are still unclear. This study aimed to clarify this issue by using bioinformatics tools for predicting competent miRNAs target the main gens in EMT.
Materials and Methods:  To ascertain an effective miRNA for the EMT, we assessed five genes from EMT/MET as key genes. Then, to predict the most suitable miRNA: target interactions, different online databases including DIANA, TargetScan, and miRSystem were applied.
Results: Possible targeting effects of different miRNAs on candidate genes were analyzed. Merging data from databases has shown that 11 miRNAs with strong possibility communally can be involved in EMT/MET.
Conclusion: To conclude, it can be predicted that according to high interaction scores of these elected miRNAs with candidate genes in the above-mentioned databases, these miRNAs probably can have critical roles in EMT/MET. Hence, these miRNAs can be introduced as appropriate candidates for future investigations.

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MicroRNAs are a group of small non-coding RNAs that regulate gene expression in eukaryotes at the post-transcriptional level. MicroRNAs, through regulating the expression of large numbers of mRNAs, act as major regulators of various biological processes such as embryonic development, cell proliferation, differentiation, and apoptosis. Therefore, the identification of microRNAs and their target genes is very effective in finding the mechanisms of embryonic development, growth, and also the processes underlying the induction and progression of various diseases. Because of the high costs of molecular experiments, the identification of effective microRNAs through bioinformatics tools and computational biology is faster and cheaper than the experimental methods. Several online bioinformatics tools and databases have been developed and are freely available for predicting microRNAs target genes. The available online tools use a broad range of information, including sequencing data, gene expression data, and computational algorithms for predicting microRNAs target genes. Some of the most important of these online tolls are miRWalk, TargetScan, RNAhybrid, Diana-microT, miRanda, and MirTarget. The four main features of the interaction between a microRNA and an mRNA, including seed pairing, sequence conservation, free energy, and access to the binding site in a target are used in the algorithm of all of these prediction tools. This stud aimed to review the latest findings on the characteristics and capabilities of microRNA target prediction tools, comparing the performance of these tools, and finally introducing the most efficient tool in the field of target gene prediction for bioinformatics, biomedicine, and molecular medicine studies.

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Ras signaling is an important intracellular signaling pathway that is key regulator of several aspects of normal cell growth and malignant transformation. The RAS gene family consists of three small G proteins; H-Ras, N-Ras, and K-Ras that play a central role in cell signaling for growth, proliferation, and migration. Mutation of the Ras oncogenes creates the malignant properties that are needed for cancer to grow and spread. MicroRNAs (miRNAs) that are encoded within the Ras genes might also have roles in cancer development. Here, novel microRNAs located in the human N-Ras gene were bioinformatically predicted. SSC profiler program was utilized to predict the stem-loop structures within the genomic area of interest. UCSC genome browser database was useed to analyze the conservation status of the putative miRNA and its precursor sequence. Furthermore, the N-Ras-miRs prediction was also performed by using MatureBayes online tool. In addition, RNAFOLD online software which applies the minimum-free energy (MFE) RNA structure prediction algorithm, was used for approximate prediction of the stem-loop structure. Our results demonstrate that N-Ras with about 5Kb length has some predicted miRNA stem-loop-like structures that have relatively conserved sequences. Overall, accumulative pieces of evidence indicated the presence of novel miRNAs encoded within the N-Ras oncogene.

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Objective: The current methods for bladder cancer diagnosis suffer from low sensitivity and specificity. Therefore, finding a novel tumor markers with high specificity and sensitivity is of great interest. MicroRNAs (miRNA, miR) are small endogenously-produced, non-coding RNAs with an important role in regulating gene expression. Recent studies show that miRNAs expression profiles represent significant tumor-specific changes that are unique for most cancers. The aim of this study was to optimize miRNA containing total RNA extraction from urine and use it as a reliable and repeatable technique for miRNA detection in urine of patients with bladder cancer. Materials and Methods: Total RNA was extracted from the urine of patients with bladder cancer and normal individuals using RNX and Trizol solutions with and without modifications of original protocols. Real-time quantitative RT-PCR was then used to detect miRNAs with a potential link to bladder tumorigenesis. Results: RNX and the modified Trizol are practical methods for RNA extraction from urine samples. The mir-21 amplification of the extracted RNAs using modified Trizol method was more efficient than that of RNX method. It is noteworthy that, the levels of miRNAs expression were much higher in the frozen urines compared to the fresh ones. Conclusion: We have succeeded to set-up a protocol to easily amplify miRNAs in urine samples. Based on the data, microRNAs seem to be good biomarkers for early detection and screening of bladder cancer.

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ntroduction: Colorectal cancer (CRC) is one of the most important cancers and the second leading cause of cancer mortality in Iran. CRC is spread through genetic and epigenetic changes. Understanding the molecular pathways in these genetic and epigenetic changes can provide a clear perspective on cancer treatment.
Materials and Methods: This study was performed on 40 samples of intestinal tumor growths (polyps), 30 tumor samples and 40 normal tissues adjacent to the tumor. RNA and tissue DNA extraction, study of gene expression, miR-22, miR-194 LncRNA MINCR using Real time PCR was performed.
Results: In this study, the expression of MINCR LncRNA in tumor and polyp samples increased compared to the adjacent normal tissue and the expression of miR-194 miR-22 was decreased. Negative correlation coefficient was observed between miR-22, miR-194 and MINCR expression levels and also a significant relationship was observed between the expression of these lncRNAs and microRNAs.

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Leguminosae is one of the most important plant families. In this study, we searched Arachis hypogaea, Glycine max, G. soja, Lotus japonicas, Medicago truncatula, Phaseolus vulgaris, and Vigna unguiculata conserved miRNAs through Expressed Sequence Tag (EST) based-homology method. All candidate sequences with appropriate fold-back structures were screened and characterized according to several filtering criteria. Chromosomal MIR locus and their distributions determined. The Dollo maximum parsimony was employed to construct the species relationship based on the MIR birth and death. Also, the number of MIRs gains and losses in the evolutionary process. In addition, we estimated the numbers of MIRs in their ancestral species using Dollo maximum parsimony in their phylogenetic tree. We found 414 novel miRNAs from 130 MIR families meeting the restricted filtering criteria. Either evolutionary time or the number of miRNAs gains and losses are estimated and characterized in the ancestral species based on the taxon-based phylogenetic tree. It speculates that gains of miRNA gene families within Leguminosae have accelerated during its evolutionary time. In addition, several taxon-specific MIR families find to assign diverse taxon and their species. Our thorough analyses resulted in the definition of some miRNA families as being lineage-specific. Therefore, they can use as markers in future systematic studies.

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Objective: Infection with herpes simplex virus type 1 induces viral latency in neuron trigeminal ganglions. The late associated transcript (LAT) is uniquely expressed in infected neural cells, however no coding protein associated with these transcripts has been identified in infected cells. It has been shown that six microRNAs transcribed from LAT have the capabilities to affect the cell signaling pathways, thus interfering in pathways such as those of cell differentiation and proliferation. Transforming growth factor beta (TGF-β) pathway is a critical pathway among cell signaling circuits. The Smad4 protein, as an important member of the TGF-β signaling pathway, mediates the connection between membrane receptors, cytoplasmic kinases, and nuclear transcription factors. Methods: This study bioinformatically and experimentally evaluated LAT microRNA expression and assessed microRNA targeting of Smad4 transcripts in human neuroblastoma cells by using real-time PCR. Results: Analysis of two different softwares results showed that the Smad4 gene was targeted by LAT-derived microRNAs at multiple sites. Over-expression of LAT microRNAs in BE2(c) cells caused reduction in Smad4 transcripts. Conclusion: The results of bioinformatical analysis with relative quantification of Smad4 transcripts and its downstream-related genes such as cyclinD, CDK2, and Myc showed that the LAT transcript could control Smad4 expression.

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Objective: The aim of the present study was the production of recombinant lentviruses that express miR-16. After transduction, altered expression levels of miRNA and its target protein were analyzed. Methods: A DNA fragment that contained the miR-16 precursor was cloned in a lentiviral plasmid. Lentiviral vector particles were produced by transient calcium phosphate co-transfection of 293T cells with the combined lenti-miR, structural and packaging plasmids. Viral supernatants were harvested and concentrated by ultracentrifuge. Virus titration was determined by fluorescent microscopy and flow cytometry. Altered expression levels of miR-16 were evaluated by real-time PCR; its protein target was evaluated by Western blot. Results: The identity of DNA was established by colony-PCR, enzymatic digestion of positive clones, and DNA sequencing. After co-transfection of 293T cells with the combined lenti-miR, structural and packaging plasmids, viral particles were concentrated and the virus titer determined. Maximum expression of the GFP reporter gene was obtained in more than 80% of the cells transduced with lentivirus at MOI=1. Real-time PCR assay showed that miR-16 expression levels significantly increased in transduced cells compared with the control group. As shown by Western blot analysis, miR-16 overexpression downregulated Bcl-2 expression at the protein level. Conclusion: This lentivirus expression system could be considered as a tool for efficient delivery of produced miRNAs to cells.

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Objective: Different signaling pathways have been identified that are involved in the cellular response to opiates. The mitogen-activated protein kinase pathway is one of the most important signaling pathways underlying the neuronal response to opiates. MicroRNAs (miRNAs) are considered to be post-transcriptional regulators of gene expression with paramount significance, which plays key roles in modulating cellular processes such as neuronal plasticity and synaptic consolidation. The purpose of this study is to identify miRNAs that are differentially expressed in response to chronic morphine treatment, and predict those genes that have a possible role in this process. Because the MAPK pathway in involved in morphine dependence and participates in hypersensitivity to pain, determining miRNAs that modulate this pathway could be insightful in morphine dependence treatment and pain control. Methods: In this study, the BE(2)-C neuroblastoma cell line was chronically treated with morphine sulphate and the changes in expression of 750 miRNAs were analyzed by real time PCR. Results: Two up- and down- regulated groups of  miRNAs were determined to be differentially expressed in response to morphine: i) has-mir-193a-3p, -212, -181c, -362-3p, -639, -646  and ii) has-mir-412, -937, -558, -552, -943, -628-5p, -593, -555, -636, -643, 566, -571, -642, -653, -611, -31, let7-g. Conclusion: The analysis of differentially expressed miRNAs showed that the MAPK signaling pathway could be regarded as a signaling pathway with utmost significance in chronic morphine response. Due to the role played by MAPK pathway in cellular response to morphine exposure, we can propose that protein phosphorylation has a presumable part in this response.

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Objective: microRNAs (miRNAs) are noncoding RNAs that function as key regulators of diverse biological activities such as cellular metabolism, cell proliferation and cell cycle regulation. Recent studies have indicated the high potential of these small molecules to control stem cell differentiation into desired cells. The aim of present study is to investigate the possible effect of let-7f on expression of hepatic nuclear factor 4 alpha (HNF4a) and some hepatic specific factors such as albumin (ALB), alpha fetoprotein (AFP), cytokeratin18 (CK18) and cytokeratin19 (CK19) in human adipose tissue derived stem cells (hADSCs). Methods: ADSCs were isolated from human adipose tissue using collagenase type I and were transduced by recombinant lentiviruses that contained human inhibitor let-7f and Scramble (negative control). Afterward, the expressions of HNF4a, ALB, AFP, CK18 and CK19 were evaluated by Real-time PCR at different time points. Results: Transduction efficiency of lentiviral vectors into ADSCs was more than 80% as judged by the expression of the GFP reporter gene. Real-time PCR analysis revealed that inhibition of let-7f in hADSCs resulted in significant up regulation of hepatic specific genes compared with the negative control. The expression level of HNF4a also increased in experimental cells at day 14, which supported the suppression of HNF4a expression by let-7f. Conclusion: The results of this study identified let-7f as a negative regulator of HNF4a expression in hADSCs and increased the expression of hepatocyte specific factors through silencing of let-7f. Therefore, suppression of let-7f could be a considerable tool for hepatic differentiation of hADSCs.

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Objective: MicroRNAs (miRNAs) are single-stranded small RNAs 18-25 nucleotides in length that regulate gene expression through translational inhibition and mRNA cleavage. Aberrant expression of miRNAs contribute to several diseases. This has increased interest in profiling the expressions of these molecules. Real-time quantitative PCR (RQ-PCR) is a sensitive, quantitative technique for gene expression assessment. To correct for systematic variables such as the amount of starting template, RNA quality and enzymatic efficiencies, RQ-PCR data is commonly normalized to an endogenous control gene which is stably-expressed across the test sample set. To avoid occurring further error in the quantification of gene expression data, it is necessary that candidate endogenous controls be validated in the samples of interest. In this study the expression of miRNA-16 and small nuclear RNA (snRNA)-U6 in hepatocellular carcinoma (HCC) cell lines under dendrosomal curcumin treatment were evaluated to identify appropriate endogenous controls for dendrosomal curcumin-related miRNA expression assays. Methods:  HCC cell lines were treated with dendrosomal curcumin. Dendrosomal curcumin entry into HepG2 and HuH-7 cells was assessed by fluorescent microscopy images. RNA was extracted and cDNA, after polyA polymerization, was synthesised. Then, we performed gene expression assays using RQ-PCR. Results: In this treatment condition, miRNA-16 for HepG2, snRNA-U6 and the combined miRNA-16 and snRNA-U6 for HuH-7 were suitable endogenous controls. Conclusion: These genes are appropriate endogenous controls for miRNA expression assays in HCC cell lines under treatment with dendrosomal curcumin. There are stable, non-significant expression changes of these genes.  

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Aims: A decrease or an increase in the expression of non-coding RNAs (ncRNAs) in the nervous system, via affecting the expression of certain molecules, induces dysfunctions in signaling pathways leading to neuronal death and neurodegenerative movement disorders. The aim of this study was to evaluate the role and underlying molecular mechanisms of ncRNAs in movement disorders.
Materials & Methods: Literature in PubMed between 2000-2020 with keywords of ncRNA, miRNA, lncRNA, and neurodegenerative movement disorder were searched. Then, the related data on the role and molecular mechanisms of the involvement of ncRNAs in Parkinson’s disease, Huntington’s disease, and Amyotrophic Lateral Sclerosis (ALS) were reviewed.
Findings: The findings indicate that the alterations in the expression of microRNAs and lncRNAs through affecting the expression of essential molecules for neuronal survival such as neurotrophic factors and anti-oxidants cause neuronal cell death and induce neurodegenerative movement disorders, including Parkinson’s disease, Huntington’s disease, and ALS. Besides, pre-clinical and clinical studies have shown that ncRNAs levels in the body fluids may serve as biomarkers in early diagnostic and monitoring the progression of movement disorders. New therapeutics based on targeting ncRNAs, especially miRNAs, have been developed and examined in animal models, which makes a hope to be appropriate candidates in controlling the progression of movement disorders in human patients in the near future.
Conclusion: It can be concluded that knowing effective ncRNAs and the related molecular mechanisms involved in movement disorders will result in the development of novel diagnostic and therapeutic approaches for the movement disorders.  

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MicroRNAs are endogenous noncoding RNA that play vital roles in all plant cellular metabolic processes by mediating target gene expression. To date, miRNAs in Taraxacum spp., which is an important industrial plant, have remained largely unknown. In the present study, 970 miRNAs from 399 families were identified in Taraxacum spp by conducting computational approaches. The most frequent miRNAs in Taraxacum spp was miR5021. According to the KEGG results, miR5021, miR838, and miR1533 are related to the terpene biosynthesis pathway, while miR5015b, and miR1436 are involved in the starch and sucrose biosynthesis pathways. Quantitative real-time PCR assay was performed to validate the expression levels of five predicted miRNAs and 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGCR) and invertase as the target genes. Results indicated that the highest relative expression of miR1533 and miR1436 occurred in the flower, while the highest transcripts levels of miR5015b were observed in the stem. In addition, the higher relative expression level of the miR5021 and miR838 was consistent with the lower expression level of the HMGCR gene in all tissue, suggesting that miR5021 and miR838 are involved in regulating HMGCR gene expression. Since mevalonate pathway is the main source of isopentenyl pyrophosphate, which is used in the synthesis of rubber, miR5021 and miR838 play an important role in the production of rubber by regulating the expression of HMGCR enzyme. These findings will accelerate future perspective studies on the regulatory mechanisms of miRNAs in Taraxacum kok-saghyz.