Up-regulation of CatSper genes family by selenium

<p>Abstract</p> <p>Background</p> <p>CatSper1-4 are a unique family of sperm cation channels, which are exclusively expressed in the testis and play an important role in sperm motility and male fertility. Despite their vital role in male fertility, almost nothing is known about the factors regulating their expression. Here, we investigated the effects of selenium (Se) on the expression of CatSper genes and sperm parameters in aging versus young male mice.</p> <p>Methods</p> <p>Forty 11-13 months aging male mice and forty 2-3 months young adult male mice were used. The animals were divided in two experimental groups: first group including aging males and second group comprising of young adult males, both treated with Se. The experimental groups were injected intra-peritoneally with Se (0.2 mg/kg) daily, for up to 5 weeks. Two other groups, aging and young adult mice without Se treatment were used as controls. All the animals were rapidly sacrificed by cervical dislocation on the days 21, 28, 35 and 42 after Se treatment. Subsequently, the morphology of the collected sperms was analyzed, and one of the testes from each mouse used for semi-quantitative RT-PCR. The significancy of the data was analyzed using ANOVA.</p> <p>Results and Discussion</p> <p>Our data revealed that there was a significant up-regulation of CatSper genes in the experimental groups compared to the control ones. Furthermore, the results of sperm analysis showed that the sperm parameters were improved in the aging as well as young adult male mice following Se treatment.</p> <p>Conclusion</p> <p>Se treatment in the aging subjects could up-regulate the expression of CatSper genes, and therefore results in elevation of sperm motility. Furthermore, Se treatment improved sperm parameters, especially morphology and viability rates.</p

MiR-218 as a multifunctional regulator of oncogenic processes in different solid tumors

MicroRNAs are highly conserved small non-coding regulatory RNAs that involve in post transcriptional regulating of gene expression during different cellular mechanisms. Aberration of miR-218 expression during tumorigenesis of different solid tumors has been reported by numerous studies. In current systematic review article, by using the terms “miR-218” and “cancer” we first searched for English language articles in the PubMed database, published from 1993 to April 2014. Then by a comprehensive review of related articles, we provided some new insights that highlight novel features and functions of miR-218 in initiation and progression of solid tumors. The majority of these studies propose a tumor suppressing role for miR-218 considering the fact that it is significantly down-regulated in tumor tissues compared with normal specimens. Despite accumulating body of evidence regarding tumor suppressor functions of miR-218 in solid tumors; more intensive reviewing about available miR-218 recent original studies and interpretation of existing data, revealed the multifunctional role of miR-218 in these kinds of malignancies by targeting different corresponding target genes. Take all together, MiR-218 targets different cellular processes in cancer cells and its expression pattern is in an important association with various states and features of tumors. It seems that miR-218 can increase the speed of cell cycle and cell division in lower sample grades and along with progression of cancer cells it's function changes to stabilization the cancer cells and not allowing them to invade. thats why it often shows up-regulation in lower grades and down-regulation in metastatic phase. Therefore, it seems of great importance to check samples stage, grade, lymph node metastasis status and other tumor features before evaluation of miR-218 as a prognostic or diagnostic biomarker. © 2016, Iranian Neurogenetics Society. All rights reserved

Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastoris

Background: Human alpha 1-antitrypsin (AAT) is a potent inhibitor of multiple serine proteases, and protects tissues against their harmful effects. Individuals with reduced or abnormal production of this inhibitor need intravenous administration of exogenous protein. In this study, we employed the methylotrophic (methanol utilizing) yeast Pichia pastoris (P. pastoris) as a preferential host for efficient production and secretion of recombinant AAT. Furthermore, we examined different strategies to maximize the yield of the secreted protein. Results: Our findings revealed that optimizing the codon usage of AAT gene for P. pastoris had positive effects on the level of secreted AAT under the control of inducible alcohol oxidase 1 (AOX1) and constitutive glycerol aldehyde phosphate dehydrogenase (GAP) promoters. Compared to AOX1, the GAP promoter increased the yield of AAT by more than two fold. It was also demonstrated that the human AAT native signal sequence was more effective than the well-known yeast signal sequence, alpha mating factor (\u3b1-MF). Doubling gene dosage nearly doubled the production of AAT, though dosages exceeding this limit had negative effects on the yield. Conclusion: P. pastoris is shown to be an efficient expression system for production of recombinant and biologically active AAT. Also different strategies could be used to elevate the amount of this secretable protein

Lower expression of miR-218 in human breast cancer is associated with lymph node metastases, higher grades, and poorer prognosis.

Breast cancer is considered as the most prevalent malignancy in women worldwide. Despite emergence of several prognosticators for better management of patients, there are still limitations for their clinical application due to the complexity of breast tumors, and therefore, new biomarkers for better prognosis of clinical outcomes would be of the great essence. MicroRNAs are highly conserved small non-coding regulatory RNAs involved in post-transcriptional regulating of gene expression during different cellular mechanisms. Accumulating studies suggest that miR-218 plays a multifunctional role in various cancer types and different stages. Here, to address prognostic significance of miR-218 in breast cancer, we investigate the expression profile of miR-218 and B-cell-specific Moloney murine leukemia virus integration site 1 ( BMI1) gene, as one of the putative targets of miR-218, in 33 paired breast tumors and their adjacent normal tissues with respect to the clinicopathological features of patients using quantitative real-time polymerase chain reaction. The correlation of both miR-218 and BMI1 gene expression with overall survival of breast cancer patients was also examined recruiting OncoLNC data portal. Finally, to better understand biological function of miR-218 in breast cancer, we performed in silico Gene Ontology and signaling pathway enrichment analysis on miR-218 targetome. According to our data, significant elevation of the expression of miR-218 and downregulation of BMI1 were observed in clinical breast cancer specimens compared with normal tissues ( p < 0.0001). The lower expression of miR-218 was associated with lymph node metastases, higher grades, and poorer prognosis (logrank p = 0.00988), whereas no significant difference in overall survival was observed between patients with higher and lower expression of BMI1 (logrank p = 0.254). These findings suggest that miR-218 expression profiling might be clinically applicable as a prognostic biomarker in breast cancer. In addition, our in silico enrichment analyses revealed that the association of miR-218 expression with breast cancer prognosis might be through its involvement in endocytosis and gap junction biological pathways

Comparing The Effects of Small Molecules BIX-01294, Bay K8644, RG-108 and Valproic Acid, and Their Different Combinations on Induction of Pluripotency Marker-Genes by Oct4 in The Mouse Brain

Objective: Every cell type is characterized by a specific transcriptional profile together with a unique epigenetic landscape. Reprogramming factors such as Oct4, Klf4, Sox2 and c-Myc enable somatic cells to change their transcriptional profile and convert them to pluripotent cells. Small molecules such as BIX-01294, Bay K8644, RG-108 and valproic acid (VPA) are reported as effective molecules for enhancing induction of pluripotency in vitro, however, their effects during in vivo reprogramming are addressed in this experimental study. Materials and Methods: In this experimental study, Oct4 expressing lentiviral particles and small molecules BIX-01294, Bay K8644 and RG-108 were injected into the right ventricle of mice brain and VPA was systematically administered as oral gavages. Animals treated with different combinations of small molecules for 7 or 14 days in concomitant with Oct4 exogenous expression were compared for expression of pluripotency markers. Total RNA was isolated from the rims of the injected ventricle and quantitative polymerase chain reaction (PCR) was performed to evaluate the expression of endogenous Oct4, Nanog, c-Myc, klf4 and Sox2 as pluripotency markers, and Pax6 and Sox1 as neural stem cell (NSC) markers. Results: Results showed that Oct4 exogenous expression for 7 days induced pluripotency slightly as it was detected by significant enhancement in expression of Nanog (p<0.05). Combinatorial administration of Oct4 expressing vector and BIX-01294, Bay K8644 and RG-108 did not affect the expression of pluripotency and NSC markers, but VPA treatment along with Oct4 exogenous expression induced Nanog, Klf4 and c-Myc (p<0.001). VPA treatment before the induction of exogenous Oct4 was more effective and significantly increased the expression of endogenous Oct4, Nanog, Klf4, c-Myc (p<0.01), Pax6 and Sox1 (p<0.001). Conclusion: These results suggest VPA as the best enhancer of pluripotency among the chemicals tested, especially when applied prior to pluripotency induction by Oct4

Enrichment of A Rare Subpopulation of miR-302-Expressing Glioma Cells by Serum Deprivation

Objective: MiR-302-367 is a cluster of polycistronic microRNAs that are exclusively expressed in embryonic stem (ES) cells. The miR-302-367 promoter is functional during embryonic development but is turned off in later stages. Motivated by the cancer stem cell hypothesis, we explored the potential expression of miR-302 in brain tumor cell lines. Materials and Methods: In the present experimental study, we have tried to expand our knowledge on the expression pattern and functionality of miR302 cluster by quantifying its expression in a series of glioma (A-172, 1321N1, U87MG) and medulloblastoma (DAOY) cell lines. To further assess the functionality of miR-302 in these cell lines, we cloned its promoter core region upstream of the enhanced green fluorescent protein (EGFP) or luciferase encoding genes. Results: Our data demonstrated a very low expression of miR-302 in glioma cell lines, compared with that of embryonal carcinoma cell line NT2 being used as a positive control. The expression of miR-302 promoter-EGFP construct in the aforementioned cell lines demonstrated GFP expression in a rare subpopulation of the cells. Serum deprivation led to the generation of tumorospheres, enrichment of miR-302 positive cells and upregulation of a number of pluripotency genes. Conclusion: Taken together, our data suggest that miR-302 could potentially be used as a novel putative cancer stem cell marker to identify and target cancer stem cells within tumor tissues

BCL11B Is Involved in Stress-Induced Differentiation of Keratinocytes and Has A Potential Role in Psoriasis Pathogenesis

Objective: Psoriasis is a common, auto-immune skin disease characterized by abnormal proliferation and differentiationof keratinocytes. Studies revealed the role of stress stimulators in the pathogenesis of psoriasis. Oxidative stressand heat shock are two important stress factors tuning differentiation and proliferation of keratinocytes, regardingto psoriasis disease. BCL11B is a transcription factor with critical role in embryonic keratinocyte differentiation andproliferation. Given this, in keratinocytes we have investigated potential role of BCL11B in stress-induced differentiation.Furthermore, we searched for a potential intercommunication between BCL11B expression and psoriasis-relatedkeratinocyte stress factors.Materials and Methods: In this experimental study, data sets of psoriatic and healthy skin samples were downloadedin silico and BCL11B was chosen as a potential transcription factor to analyze. Next, a synchronized in vitro model wasdesigned for keratinocyte proliferation and differentiation. Oxidative stress and heat shock treatments were employed onHaCaT keratinocytes in culture, and BCL11B expression level was measured. Cell proliferation rate and differentiationwere analyzed by synchronized procedure test. Flow cytometry was done to analyze cell cycle alterations due to theoxidative stress.Results: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) data revealed a significantupregulation of BCL11B expression in keratinocytes, by 24 hours after initiating differentiation. However, it was followedby a significant down-regulation in almost all the experiments, including the synchronized model. Flow cytometer datademonstrated a G1 cell cycle arrest in the treated cells.Conclusion: Results indicated a remarkable role of BCL11B in differentiation and proliferation of HaCaT keratinocytes.This data along with the results of flow cytometer suggested a probable role for BCL11B in stress-induced differentiation,which is similar to what is happening during initiation and progression of normal differentiation

SNHG15 is a bifunctional MYC-regulated noncoding locus encoding a lncRNA that promotes cell proliferation, invasion and drug resistance in colorectal cancer by interacting with AIF

Background: Thousands of long noncoding RNAs (lncRNAs) are aberrantly expressed in various types of cancers, however our understanding of their role in the disease is still very limited. Methods: We applied RNAseq analysis from patient-derived data with validation in independent cohort of patients. We followed these studies with gene regulation analysis as well as experimental dissection of the role of the identified lncRNA by multiple in vitro and in vivo methods. Results: We analyzed RNA-seq data from tumors of 456 CRC patients compared to normal samples, and identified SNHG15 as a potentially oncogenic lncRNA that encodes a snoRNA in one of its introns. The processed SNHG15 is overexpressed in CRC tumors and its expression is highly correlated with poor survival of patients. Interestingly, SNHG15 is more highly expressed in tumors with high levels of MYC expression, while MYC protein binds to two E-box motifs on SNHG15 sequence, indicating that SNHG15 transcription is directly regulated by the oncogene MYC. The depletion of SNHG15 by siRNA or CRISPR-Cas9 inhibits cell proliferation and invasion, decreases colony formation as well as the tumorigenic capacity of CRC cells, whereas its overexpression leads to opposite effects. Gene expression analysis performed upon SNHG15 inhibition showed changes in multiple relevant genes implicated in cancer progression, including MYC, NRAS, BAG3 or ERBB3. Several of these genes are functionally related to AIF, a protein that we found to specifically interact with SNHG15, suggesting that the SNHG15 acts, at least in part, by regulating the activity of AIF. Interestingly, ROS levels, which are directly regulated by AIF, show a significant reduction in SNHG15-depleted cells. Moreover, knockdown of SNHG15 increases the sensitiveness of the cells to 5-FU, while its overexpression renders them more resistant to the chemotherapeutic drug. Conclusion: Altogether, these results describe an important role of SNHG15 in promoting colon cancer and mediating drug resistance, suggesting its potential as prognostic marker and target for RNA-based therapies

Differential processing and sorting of nerve growth factor and brain-derived neurotrophic factor in hippocampal neurons

We have examined the biosynthesis, post-translational processing and sorting of neurotrophins in both constitutive and regulated cells infected with a pro-NGF, pro-BDNF and/or pro-NT-3 encoding vaccinia virus. Our results show that: (1) pro-BDNF is generated as a 32 kDa precursor that is N-glycosylated and glyco-sulfated in its prodomain. The precursor undergoes N-terminal cleavage to generate mature BDNF (14 kDa) as well as a truncated form of the precursor (28 kDa). (2) Both 32 and 28 kDa BDNF are released into media and are able to stimulate TrkB auto-phosphorylation. (3) The production of 28 kDa BDNF, unlike that of mature BDNF, occurs in the ER and is generated by a novel enzyme called subtilisin/kexin isozyme-1 (SKI-1) at the RGLT 57&darr;SL site. (4) Results obtained from pulse-chase experiments, secretagogue-induced release, and immunocytochemistry suggest that, NGF and NT-3 are primarily secreted constitutively while BDNF is principally directed to the regulated secretory pathway. (5) In contrast to homodimeric NT-3, NT-3/BDNF heterodimer is primarily sorted to the regulated secretory pathway suggesting that for the NT-3/BDNF heterodimer, the presence of a single pro-BDNF chain is sufficient for packaging into large dense-core secretory vesicles and subsequent regulated release. (6) Blocking furin activity in AtT-20 cells with alpha1-PDX as well as increasing the level of expression of NGF and NT-3 precursors partially directed them into the regulated secretory pathway. Therefore, neurotrophins can be sorted into either the constitutive or regulated secretory pathways, and sorting may be regulated by the efficiency of furin cleavage in the TGN. This mechanism may explain how neuron-generated neurotrophins can act both as survival factors and as neuropeptides