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.

Site-specific integration as an efficient method for production of recombinant human hyaluronidase PH20 in semi-adherent cells.

PH20 is a hyaluronidase enzyme that can hydrolyze the glycosidic bond in hyaluronic acid as the major proteoglycan found in extracellular matrices. In the present study, we constructed and characterized two donor plasmids, one of them with one and the second with two PH20 expression cassettes. The expression vectors were site specifically integrated into the genome of HEK293T cells using PhiC31 integrase system to develop HEK293T stable cell lines secreting His-tagged recombinant human PH20 (rhPH20) in the culture supernatant. The produced rhPH20 was quantified using ELISA and turbidimetric assay tests, and its catalytic activity was also assessed by treating the mouse cumulus-oocyte complexes. Our results showed that the secreted rhPH20 in the culture supernatant had the specific activity of 16,660 IU/mg and the recombinant enzyme was able to remove the cumulus cells from oocytes. The results also indicated that phiC31 enzyme inserted the PH20-expressing donor vectors into the specific pseudo attP sites including 10q21.2 and 20q11.22 in the genome of the target cells with different copy numbers. Taken together, our findings demonstrate that PhiC31 integrase system is able to be applied as a robust tool for efficient production and secretion of soluble and active rhPH20 by HEK293T cells as a semi-adherent human cell line. KEY POINTS: • Efficient production of human recombinant PH20 in a semi-adherent human cell line • Successful application of PhiC31 integrase system for generation of stable recombinant clones • Use of a human cell line for expression of a recombinant human protein due to complex and efficient post-translational modifications and protein folding.

Smart co-delivery of miR-34a and cytotoxic peptides (LTX-315 and melittin) by chitosan based polyelectrolyte nanocarriers for specific cancer cell death induction.

A novel polyelectrolyte nanocarrier was synthesized via layer-by-layer self-assembly of polycationic and polyanionic chains. The nanocarrier is composed of polyglutamate grafted chitosan core, dextran sulfate as a complexing agent, and polyethyleneimine shell decorated with folic acid. This polyelectrolyte complex has unique physicochemical properties so that the core is considered as an efficient carrier for LTX-315 and melittin peptides, and the shell is suitable for delivery of miR-34a. The spherical nanocarriers with an average size of 123 ± 5 nm and a zeta potential of -36 ± 1 mV demonstrated controlled-release of gene and peptides ensured a synergistic effect in establishing multiple cell death pathways on chemoresistance human breast adenocarcinoma cell line, MDA-MB-231. In vitro cell viability assays also revealed no cytotoxicity for the nanocarriers, and an IC50 of 15 µg/mL and 150 µg/mL for melittin and LTX-315, respectively, after 48 h, whereas co-delivery of melittin with miR-34a increased smart death induction by 54%.

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.

Improving germline transmission efficiency in chimeric chickens using a multi-stage injection approach.

Although different strategies have been developed to generate transgenic poultry, low efficiency of germline transgene transmission has remained a challenge in poultry transgenesis. Herein, we developed an efficient germline transgenesis method using a lentiviral vector system in chickens through multiple injections of transgenes into embryos at different stages of development. The embryo chorioallantoic membrane (CAM) vasculature was successfully used as a novel route of gene transfer into germline tissues. Compared to the other routes of viral vector administration, the embryo's bloodstream at Hamburger-Hamilton (HH) stages 14-15 achieved the highest rate of germline transmission (GT), 7.7%. Single injection of viral vectors into the CAM vasculature resulted in a GT efficiency of 2.7%, which was significantly higher than the 0.4% obtained by injection into embryos at the blastoderm stage. Double injection of viral vectors into the bloodstream at HH stages 14-15 and through CAM was the most efficient method for producing germline chimeras, giving a GT rate of 13.6%. The authors suggest that the new method described in this study could be efficiently used to produce transgenic poultry in virus-mediated gene transfer systems.

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.

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.

Rational Development of A Polycistronic Plasmid with A CpG-Free Bacterial Backbone as A Potential Tool for Direct Reprogramming.

OBJECTIVE: Induced pluripotent stem cells are generated from somatic cells by direct reprogramming. These reprogrammed pluripotent cells have different applications in biomedical fields such as regenerative medicine. Although viral vectors are widely used for efficient reprogramming, they have limited applications in the clinic due to the risk for immunogenicity and insertional mutagenesis. Accordingly, we designed and developed a small, non-integrating plasmid named pLENSO/Zeo as a 2A-mediated polycistronic expression vector. MATERIALS AND METHODS: In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of human LIN28, NANOG, SOX2 and OCT4 along with an EGFP reporter gene. Each reprogramming factor is separated by an intervening sequence that encodes a 2A self-processing peptide. The reprogramming cassette is located downstream of a CMV promoter. The vector is easily propagated in the E. coli GT115 strain through a CpG-depleted vector backbone. We evaluated the stability of the constructed vector bioinformatically, and its ability to stoichiometric expression of the reprogramming factors using quantitative molecular methods analysis after transient transfection into HEK293 cells. RESULTS: In the present study, we developed a nonviral episomal vector named pLENSO/ Zeo. Our results demonstrated the general structural stability of the plasmid DNA. This relatively small vector showed concomitant, high-level expression of the four reprogramming factors with similar titers, which are considered as the critical parameters for efficient and consistent reprogramming. CONCLUSION: According to our experimental results, this stable extrachromosomal plasmid expresses reliable amounts of four reprogramming factors simultaneously. Consequently, these promising results encouraged us to evaluate the capability of pLENSO/Zeo as a simple and feasible tool for generation of induced pluripotent stem cells from primary cells in the future.

Nurr1 and PPARγ protect PC12 cells against MPP(+) toxicity: involvement of selective genes, anti-inflammatory, ROS generation, and antimitochondrial impairment.

Parkinson's disease (PD) can degenerate dopaminergic (DA) neurons in midbrain, substantia-nigra pars compacta. Alleviation of its symptoms and protection of normal neurons against degeneration are the main aspects of researches to establish novel therapeutic strategies. PPARγ as a member of PPARs have shown neuroprotection in a number of neurodegenerative disorders such as Alzheimer's disease and PD. Nuclear receptor related 1 protein (Nurr1) is, respectively, member of NR4A family and has received great attentions as potential target for development, maintenance, and survival of DA neurons. Based on neuroprotective effects of PPARγ and dual role of Nurr1 in anti-inflammatory pathways and development of DA neurons, we hypothesize that PPARγ and Nurr1 agonists alone and in combined form can be targets for neuroprotective therapeutic development for PD in vitro model. 1-Methyl-4-phenylpyridinium (MPP(+)) induced neurotoxicity in PC12 cells as an in vitro model for PD studies. Treatment/cotreatment with PPARγ and Nurr1 agonists 24 h prior to MPP(+) induction enhanced the viability of PC12 cell. The viability of PC12 cells was determined by MTS test. Mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) were detected by flow cytometry. In addition, the relative expression of four genes including TH (the marker of DA neurons), Ephrin A1, Nurr1, and Ferritin light chain were assessed by RT-qPCR. In the MPP(+)-pretreated PC12 cells, PPARγ and Nurr1 agonists and their combined form resulted in a decrease in the cell death rate. Moreover, production of intracellular ROS and MMP modulated by MPP(+) was decreased by PPARγ and Nurr1 agonists' treatment alone and in the combined form.

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.

Fndc5 overexpression facilitated neural differentiation of mouse embryonic stem cells.

Fndc5 has been recently recognized as a myokine which could be cleaved and secreted into blood stream. It is termed as irisin with an important role in thermogenesis and energy homeostasis. Increased expression of Fndc5 has been reported upon retinoic acid treatment during neural differentiation and its knockdown decreased neural differentiation and neurite outgrowth. This study tries to evaluate the effect of Fndc5 overexpression on rate of neural differentiation in mouse. (Thus, transduced cell line of mouse embryonic stem cell with ability to express Fndc5 under Doxycycline treatment was established. Subsequently, the effect of overexpression of Fndc5 on different stages of neural differentiation was studied). Our study showed an increase enhancement in neuronal precursor markers and mature neuron markers upon overexpression of Fndc5, concluding that Fndc5 facilitates neural differentiation. This effect might be related to increased expression of BDNF following overexpression of Fndc5. Our findings are consistent with recent studies reporting a similar role for Fndc5 in proliferation of neural cells and increase in the expression of neurotrophins like BDNF.

Induced expression of Fndc5 significantly increased cardiomyocyte differentiation rate of mouse embryonic stem cells.

Fibronectin type III domain-containing 5 protein (Fndc5) is an exercise hormone and its transcript profile in mouse showed high degree of expression in heart, skeletal muscle and brain. Our previous studies indicated a significant increase (approximately 10 fold) in mRNA level of Fndc5 when embryonic stem cells were differentiated into beating bodies. As a step closer to identify the involvement of Fndc5 in the process of cardiomyocyte differentiation, we generated a stably inducible transduced mouse embryonic stem cell (mESC) line that overexpressed Fndc5 following Doxycycline induction. Our results indicated that the overexpression of Fndc5 during spontaneous cardiac differentiation significantly increased not only at RNA levels for mesodermal markers but also at the transcriptional levels for cardiac progenitor and cardiac genes. These data suggest that Fndc5 may be involved in cardiomyocyte differentiation. Therefore, a new hope will be arisen for potential application of this myokine for regeneration of damaged cardiac tissues especially in cardiac failure.