The oncogene Musashi1 encodes novel miRNAs in breast cancer.

RNA-binding protein Musashi1 (MSI1) shows an increased expression level in several cancers and has been introduced as a prognostic marker in some malignancies. It is expected that if any miRNA is encoded by this gene, it might have a role in cancer development or could be considered as a prognostic biomarker. Accordingly, in this study, we aimed to find novel miRNA(s) inside the intronic regions of the MSI1 gene. Here, we report two novel miRNAs within intron 4 of MSI1 gene, named MSM2 and MSM3, which were selected among several miRNA precursors predicted by bioinformatic studies. For experimental analysis, corresponding precursor miRNAs were transfected into HEK293T cells and exogenous expression of the mature miRNAs were detected. Two mature miRNAs, MSM3-3p and MSM3-5p were generated by MSM3 precursor and one, MSM2-5p was derived from MSM2. Besides, endogenous expression of MSM2-5p and MSM3-3p was detected in MCF-7 and SH-SY5Y cell lines. Expression of both mature miRNAs was also detected in clinical samples of breast cancer. Additionally, the interaction between the MSM3-3p and 3'UTR region of PDE11A was confirmed by dual luciferase assay. Overall, our data demonstrated that MSI1 gene encodes two novel miRNAs in breast cancer cells.

Melatonin accelerates the developmental competence and telomere elongation in ovine SCNT embryos.

SCNT embryos suffer from poor developmental competence (both in vitro and in vivo) due to various defects such as oxidative stress, incomplete epigenetic reprogramming, and flaws in telomere rejuvenation. It is very promising to ameliorate all these defects in SCNT embryos by supplementing the culture medium with a single compound. It has been demonstrated that melatonin, as a multitasking molecule, can improve the development of SCNT embryos, but its function during ovine SCNT embryos is unclear. We observed that supplementation of embryonic culture medium with 10 nM melatonin for 7 days accelerated the rate of blastocyst formation in ovine SCNT embryos. In addition, the quality of blastocysts increased in the melatonin-treated group compared with the SCNT control groups in terms of ICM, TE, total cell number, and mRNA expression of NANOG. Mechanistic studies in this study revealed that the melatonin-treated group had significantly lower ROS level, apoptotic cell ratio, and mRNA expression of CASPASE-3 and BAX/BCL2 ratio. In addition, melatonin promotes mitochondrial membrane potential and autophagy status (higher number of LC3B dots). Our results indicate that melatonin decreased the global level of 5mC and increased the level of H3K9ac in the treated blastocyst group compared with the blastocysts in the control group. More importantly, we demonstrated for the first time that melatonin treatment promoted telomere elongation in ovine SCNT embryos. This result offers the possibility of better development of ovine SCNT embryos after implantation. We concluded that melatonin can accelerate the reprogramming of telomere length in sheep SCNT embryos, in addition to its various beneficial effects such as increasing antioxidant capacity, reducing DNA damage, and improving the quality of derived blastocysts, all of which led to a higher in vitro development rate.

Assessment of MUSASHI 1 and MUSASHI 2 expression in spermatozoa and testicular tissue.

MUSASHI (MSI) family plays the main role in the spermatogenesis process. The purpose of this study was the assessment of sperm MSI1 and MSI2, and sperm functional tests in infertile men (n = 30) with varicocele and fertile men (n = 30). Furthermore, MSI1 and MSI2 proteins were assessed in testicular tissue of azoospermic men (n = 9) as well as epididymal spermatozoa and testis of mice. Expression of MSI1 and MSI2 was assessed at RNA and protein levels in human spermatozoa. Sperm concentration and motility were significantly lower, while abnormal sperm morphology, lipid peroxidation, DNA fragmentation and protamine deficiency were significantly higher in men with varicocele compared to fertile individuals. Any significant difference was not observed in the expression of MSI1 and MSI2 mRNA between the two groups. Unlike MSI1 protein that was not detectable in humans, the relative expression of MSI2 protein was similar in varicocele and fertile individuals. The expression level of both Msi1 and Msi2 proteins was also observable in mouse spermatozoa. No significant relationship was observed between sperm functional parameters with expression of these genes. The data of this study demonstrated that although MSI1 and MSI2 play important roles during spermatogenesis, their relative expression in spermatozoa was not affected by varicocele.

Increased expression of MUSASHI1 in epithelial breast cancer cells is due to down regulation of miR-125b.

BACKGROUND: Musashi1 (MSI1) is an oncogenic protein with a crucial role in the proliferation and characteristics of the epithelial cells in breast cancer. The change in expression of MSI1 has a role in solid tumor progression. There are different factors that regulate MSI1 expression in various cancer tissues including microRNAs which are considered as one of the most important of these factors. The aim of our study is identification of the molecular cause of maximal expression of MSI1 in epithelial breast cancer cell lines. RESULTS: Among predicted microRNAs, miR-125b, miR-637 and miR-802 were able to significantly reduce the luciferase activity. In addition, the relative expression of these three miRNAs were measured in the cancerous cell lines that results showed a significant reduction in expression of all microRNAs. On the other hand, only the overexpression of miR-125b caused a change in the expression pattern of MSI1 in breast epithelial cancer cell lines. Accordingly, our results demonstrated that the exogenous expression of miR-125b decreased not only the MSI1 protein but also expression of epithelial markers in breast cancer cells. CONCLUSIONS: The results of luciferase reporter assay showed that MSI1 is a direct target for miR-125b in epithelial breast cancer cells. Moreover, higher amount of MSI1 in those cell lines seems due to the reduced amount of miR-125b, which is responsible for epithelial features of those kinds of cancer cells. Therefore, the modulation of miR-125b may be a potential approach to help to combat against epithelial breast tumors.

Intracellular functions of RNA-binding protein, Musashi1, in stem and cancer cells.

RNA-binding protein, musashi1 (MSI1), is a main protein in asymmetric cell division of the sensory organ precursor cells, whereas its expression is reported to be upregulated in cancers. This protein is a critical element in proliferation of stem and cancer stem cells, which acts through Wnt and Notch signaling pathways. Moreover, MSI1 modulates malignancy and chemoresistance of lung cancer cells via activating the Akt signaling. Due to the main role of MSI1 in metastasis and cancer development, MSI1 would be an appropriate candidate for cancer therapy. Downregulation of MSI1 inhibits proliferation of cancer stem cells and reduces the growth of solid tumors in several cancers. On the other hand, MSI1 expression is regulated by microRNAs in such a way that several different tumor suppressor miRNAs negatively regulate oncogenic MSI1 and inhibit migration and tumor metastasis. The aim of this review is summarizing the role of MSI1 in stem cell proliferation and cancer promotion.

New insights into the cellular activities of Fndc5/Irisin and its signaling pathways.

Fndc5, a well-defined myokine and also identified as an adipokine, has a critical role in modulation of metabolism and protection against obesity. These important functions are mediated by irisin, a secretory peptide produced from proteolytic processing of Fndc5. The other beneficial physiological effects of irisin are alleviation of oxidative stress, neuroprotective effects, and anti-inflammatory properties and associated anti-metastatic effects. Fndc5/irisin exerts its biological effects through several intracellular signaling pathways. The major signaling pathway is thought to be MAPK signaling pathways which are involved in neural differentiation, browning of white adipocytes, as well as osteoblast proliferation and differentiation. Other essential functions of Fndc5/irisin are mediated through additional pathways including AMPK pathway, PI3K/AKT, and STAT3/Snail. Thorough understanding of the mechanisms of irisin actions are essential in order to develop Fndc5/irisin for therapeutic purposes. In the present review, we focus on the current knowledge of the signaling pathways that elicit irisin actions.

An efficient method for bacterial production and activity assessment of recombinant human insulin like growth factor 1.

Human insulin like growth factor 1 directs physiological roles in cellular proliferation and differentiation process. The protein is considered as an important therapeutic target with clinical significance. In this study, to avoid production of human insulin like growth factor 1 as inclusion body, the thioredoxin was used as a solubilizing fusion tag. The expression of fusion human insulin like growth factor 1 was carried out in E. coli Rosetta-gami by transformation of pET-32b contained functional elements. The evaluation of different conditions involving protein expression including IPTG concentration, temperature and post induction time showed that 0.1 mM IPTG at 34 °C for 4 h was the optimum condition. The isolated fusion protein was purified using nickel affinity purification and digested by entrokinase to produce mature recombinant protein without any additional tag. The accuracy of produced recombinant protein was confirmed by western blot analysis. Biological activity of produced recombinant human insulin like growth factor 1 was determined by its proliferation effects on MCF-7 cells, expansion of bovine granulosa cells and activation of PI3K/Akt signaling pathway in these cells. The present study provides a simple and efficient method for high-level production of soluble, active recombinant human insulin like growth factor 1 in E. coli.

Super magnetic nanoparticles NiFe2O4, coated with aluminum-nickel oxide sol-gel lattices to safe, sensitive and selective purification of his-tagged proteins.

Super magnetic nanoparticle NiFe2O4 with high magnetization, physical and chemical stability was introduced as a core particle which exhibits high thermal stability (>97%) during the harsh coating process. Instead of multi-stage process for coating, the magnetic nanoparticles was mineralized via one step coating by a cheap, safe, stable and recyclable alumina sol-gel lattice (from bohemite source) saturated by nickel ions. The TEM, SEM, VSM and XRD imaging and BET analysis confirmed the structural potential of NiFe2O4@NiAl2O4 core-shell magnetic nanoparticles for selective and sensitive purification of His-tagged protein, in one step. The functionality and validity of the nickel magnetic nanoparticles were attested by purification of three different bioactive His-tagged recombinant fusion proteins including hIGF-1, GM-CSF and bFGF. The bonding capacity of the nickel magnetics nanoparticles was studied by Bradford assay and was equal to 250 ± 84 µg Protein/mg MNP base on protein size. Since the metal ion leakage is the most toxicity source for purification by nickel magnetic nanoparticles, therefor the nickel leakage in purified final protein was determined by atomic absorption spectroscopy and biological activity of final purified protein was confirmed in comparison with reference. Also, in vitro cytotoxicity of nickel magnetic nanoparticles and trace metal ions were investigated by MTS assay analysis. The results confirmed that the synthesized nickel magnetic nanoparticles did not show metal ion toxicity and not affected on protein folding.

Analysis of Promyelocytic Leukemia in Human Embryonic Carcinoma Stem Cells During Retinoic Acid-Induced Neural Differentiation.

BACKGROUND: Promyelocytic leukemia protein (PML) is a tumor suppressor protein that is involved in myeloid cell differentiation in response to retinoic acid (RA). In addition, RA acts as a natural morphogen in neural development. OBJECTIVES: This study aimed to examine PML gene expression in different stages of in vitro neural differentiation of NT2 cells, and to investigate the possible role of PML in pluripotency and/or neural development. MATERIALS AND METHODS: RA was used as a neural inducer for in vitro neural differentiation of NT2 cells. During this process PML mRNA and protein levels were assessed by quantitative real time RT-PCR (QRT-PCR) and Immunoblotting, respectively. Furthermore bisulfite sequencing PCR (BSP) was used to assess PML promoter methylation in NT2 cells and NT2 derived neuronal precursor cells (NT2.NPCs). RESULTS: QRT-PCR results showed that, PML had maximum expression with significant differences in NT2 derived neuronal precursor cells relative to NT2 cells and NT2 derived neural cells (NT2.NCs). Numerous isoforms of PML with different intensities appeared in immunoblots of pluripotent NT2 cells, NT2.NPCs, and NT2.NCs. Furthermore, the methylation of the PML promoter in NT2.NCs was 2.6 percent lower than NT2 cell. CONCLUSIONS: The observed differences in PML expression in different cellular stages possibly could be attributed to the fact that PML in each developmental state might be involved in different cell signaling machinery and different functions. The appearance of different PML isoforms with more intensity in neural progenitor cells; may suggest apossible role for this protein in neural development.

Construction of a new minicircle DNA carrying an enhanced green florescent protein reporter gene for efficient expression into mammalian cell lines.

The presence of a bacterial backbone in conventional eukaryotic expression plasmids may cause undesirable effects by triggering the immune responses in mammals and repression of episomal transgene expression. To avoid these problems, researchers have proposed the use of minicircle DNAs which are episomal vectors that have lost their bacterial backbone using a site-specific recombinase mediated recombination. In the present study, we have constructed a new minicircle DNA vector that carries an enhanced green florescent protein (EGFP) reporter gene using phage ΦC31 integrase-mediated recombination and homing endonuclease ISceI-mediated purification in E. coli. ΦC31 integrase expression was under the control of the araBAD promoter, whereas ISceI endonuclease was controlled by the tac promoter. This vector was transfected into CHO-K1 cells, which showed transient expression of EGFP up to 14 generations. Similar results were obtained upon transient transfection into HEK cells. In addition, PCR results on genomic DNA, demonstrated the EGFP-minicircle was episomal and did not integrate into the host genome. Our constructed parental plasmid expresses EGFP and could be used for the generation of episomal minicircle DNA with intent to carry out transient transfection of interested DNA fragments into the eukaryotic cells for various purposes.

Constructing a Mouse Oct4 Promoter/EGFP Vector, as a Whole-Cellular Reporter to Monitor the Pluripotent State of Cells.

BACKGROUND: The transcription factor Oct-4, is an important marker of undifferentiating level and a key regulating factor for maintenance of pluripotency in cells. Establishment of an Oct-4 promoter-based reporter system is an appropriate tool for monitoring the differentiation of embryonic stem cells both in vivo and in vitro. METHODS: In the present study, we report construction of a recombinant vector, pDB2 Oct4 promoter/EGFP, in which expression of Enhanced Green Fluorescent Protein (EGFP) was controlled by the mouse Oct-4 promoter. RESULTS: In transfected mouse embryonic stem cells with this vector, EGFP was predicted to be specifically expressed in pluripotency state. After transfection, high-level expression of EGFP under the control of Oct-4 promoter was observed in manipulated embryonic stem cells. CONCLUSION: Thus, our new cellular reporter showed that both the properties of embryonic cells and expression the EGFP could be of great help in studying the differentiating and reprogramming mechanisms of mESCs.

Characterization and Functional Assessment of Mouse PPARγ1 Promoter.

BACKGROUND: Peroxisome Proliferator Activated Receptor gamma (PPARγ), a member of nuclear receptor superfamily, comprises two isoforms in mouse. These two isoforms are encoded by different mRNAs, which are arisen by alternative promoter usage. There are two promoter regions upstream of PPARγ gene. A 3 kb fragment, containing several transcription factor binding sites, acts as PPARγ1 promoter region. Thus, expression pattern of PPARγ1 isoform is due to the potential transcription factors that could influence its promoter activity. PPARγ, Retinoid X Receptor (RXR) and Vitamin D Receptor (VDR), as nuclear receptors could influence PPARγ gene expression pattern during several differentiation processes. During neural differentiation, PPARγ1 isoform expression reaches to maximal level at neural precursor cell formation. METHODS: A vast computational analysis was carried out to reveal the PPARγ1 promoter region. The putative promoter region was then subcloned upstream of an EGFP reporter gene. Then the functionality of PPARγ1 promoter was assessed in different cell lines. RESULTS: Results indicated that Rosiglitazone increased PPARγ1 promoter regulated EGFP expression of neural precursor cells during Embryoid Body (EB) formation. Furthermore vitamin D reduced PPARγ1 promoter regulated EGFP expression of neural precursor cells during EB formation through binding to its receptor. CONCLUSION: This study suggests that there are potential response elements for PPAR/RXR and VDR/RXR heterodimers in PPARγ1 isoform promoter. Also VDR/RXR heterodimers may decrease PPARγ expression through binding to its promoter.