A novel signaling role for miR-451 in esophageal tumor microenvironment and its contribution to tumor progression.

OBJECTIVE: We evaluated miR-451 expression in serum and tissue samples of esophageal squamous cell carcinoma (ESCC) patients. Then, we examined a secretory role of miR-451 in esophageal tumor microenvironment. METHODS: miR-451 expression was evaluated in 39 serum samples from esophageal SCC patients compared to 39 normal individuals as well as 26 pairs of fresh-frozen tumor and adjacent normal tissues from patients with ESCC, using qRT-PCR. In a co-culture system of human normal fibroblasts (HFSF-PI3) and esophageal cancer cell line (KYSE-30), we evaluated exosomal miR-451 secretion into the conditioned medium (CM) of both cell lines. Then, we analyzed the effect of primiR-451-transfected fibroblasts on the migration potency of their neighboring KYSE-30 cells. RESULTS: We detected miR-451 over-expression in serum samples of esophageal cancer patients compared to the normal group (P = 0.005). Interestingly, fresh-frozen tumor tissues from the same patients showed miR-451 down-regulation compared to their adjacent normal counterparts (P = 0.043). Co-culturing the KYSE-30 cell line with normal fibroblasts significantly induced miR-451 exosomal secretion into the CM. Moreover, co-culture of KYSE-30 cell line with miR-451-over-expressing fibroblasts significantly induced migration tendency in KYSE-30 cell line compared to the mock-transfected fibroblasts (P < 0.0001). In this system, MIF expression (a validated target of miR-451) in the KYSE-30 cell line was increased although this alteration was not statistically significant (fold change = 4.44). CONCLUSIONS: Our data suggest that cancer-associated fibroblasts use exosomal miR-451 as a signaling molecule to provide a favorable niche for tumor cell migration and cancer progression. Our findings provide new insights into the stromal role of miR-451 in the esophageal tumor microenvironment as a communicatory molecule and suggest a signaling role for miR-451 in extracellular matrix cross-talks.

Two lung development-related microRNAs, miR-134 and miR-187, are differentially expressed in lung tumors.

INTRODUCTION: MicroRNAs (miRNAs) are involved in various cellular events needed for embryonic development and tumorigenesis. As some of the development-specific gene expression patterns could be observed in cancers, we speculated that the expression pattern of lung development-specific miRNAs miR-134 and miR-187 might be altered in lung tumor samples. Lung cancer is the first cause of cancer related deaths worldwide, mostly due to its late diagnosis. Therefore, finding a reliable diagnostic tumor marker, based on molecular profile of tumorigenesis, would be critical in lowering lung cancer mortality. METHODS: We employed a real-time RT-PCR approach to evaluate the expression alteration of two lung development-related miRNAs in lung tumor tissues. The suitability of miRs expression alterations as lung tumor biomarkers was tested by receiver operating characteristic (ROC) curve analysis. The effect of miR-187 overexpression on a lung carcinoma cell cycle was assessed using flow cytometry analysis. RESULTS: Our data revealed a significant upregulation (7.8 times, p<0.02) of miR-134 in lung tumors. However, its expression level failed to discriminate different tumor types and grades of malignancies from each other. Moreover, the ROC curves analysis did not give it a good score as a reliable biomarker (AUC=0.522, P=0.729). In contrast, miR-187 showed a significant down-regulation (P=0.008) in lung tumors. Similarly, its expression level failed to differentiate different tumor types or grades of malignancies. Nevertheless, ROC curve analysis gave it an AUC score of 0.669 (P=0.012), which suggests its suitability as a potential biomarker for lung cancer. Furthermore, ectopic expression of miR-187 in A549 cells caused a cell cycle arrest in G1 phase (P=0.013). CONCLUSION: Altogether, our data demonstrated an altered expression of two development-related miRNAs namely miR-134 and miR-187 in lung tumors for the first time. Moreover we have shown that miR-134 and miR-187 expression alternation were in accordance with their approved regulatory roles, therefore these miRNAs could serve as new biomarkers with potential usefulness in lung cancer diagnosis and treatments. In addition, miR-187 expression in tumor cells could perturb cell cycle which supported its possible role as tumor suppressor.

The stem cell self-renewal gene, Musashi 1, is highly expressed in tumor and non-tumor samples of human bladder.

CONTEXT: The stem cell model for cancer assumes that a key event in tumorigenesis is the deregulation of genes involved in the regulation of stem cell self-renewal. The Musashi family is an evolutionarily conserved group of neural RNA-binding proteins. In mammals, the family consists of two individual genes, Musashi 1 (MSI1) and MSI2, encoding the Musashi 1 and Musashi 2 proteins. Musashi 1 is involved in the regulation of self-renewal of stem cells. Recently, its over-expression has also been reported in a variety of human tumors. AIMS: To investigate a potential expression of the stem cell self-renewal gene, Musashi 1, in human bladder cancer, we examined its gene expression in a series of tumor and non-tumor tissue samples of bladder. MATERIALS AND METHODS: Relative expression of MSI1 was determined by the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 70 surgical samples of bladder. RESULTS: Using specific primers for MSI1 and TBP (as an internal control) for qRT-PCR technique, we found a relatively high expression level of MSI1 in all examined tumor and non-tumor bladder tissue specimens. However, our data did not show any correlation between the level of gene expression and tumor/non-tumor states of the samples (P>0.05). CONCLUSIONS: All together, our data demonstrated that Musashi 1 is highly and un-differentially expressed in both examined tumoral and apparently normal bladder tissues.

Germ cell apoptosis induced by experimental cryptorchidism is mediated by molecular pathways in mouse testis.

The aim of the study was to characterise the alterations in expression of some apoptosis regulators in unilaterally and bilaterally heat-treated mouse testes at different time intervals to 42 days after surgery. Cryptorchidism was induced in immature mice by returning the testis to the abdominal cavity via a surgical procedure. Transcript levels of Bax, Bcl-2 proper, p53 and survivin mRNA and protein were determined in normal and cryptorchid testes using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. RT-PCR data verified the elevation of p53 expression and decrease of Bax and Bcl-2 proper mRNA in the cryptorchid testis in a time-dependent manner. The expression of survivin 140 and 40 variants strongly decreased in the bilateral groups compared with unilateral and control groups. These changes were significantly different in the bilateral groups in comparison with the unilateral groups. Immunohistochemistry data showed that the intensity of p53 and Bax expression mainly increased in the remainder cells in the cryptorchid testis and the rates of Bcl-2 proper and survivin expression decreased mainly in the bilateral groups. These observations suggest that multiple molecular pathways participate in the germ cell apoptosis induced by cryptorchidism.

Differential effects of Nucleostemin suppression on cell cycle arrest and apoptosis in the bladder cancer cell lines 5637 and SW1710.

OBJECTIVES: The Nucleostemin (NS) gene encodes a nucleolar protein enriched in adult and embryonic stem cells. NS is thought to regulate cancer cell proliferation, but the mechanisms involved are poorly understood. In this study, we have investigated the role of NS in bladder cancer. MATERIALS AND METHODS: Expression of NS was determined by quantitative reverse transcription-polymerase chain reaction in bladder carcinoma cell lines and in normal uro-epithelial cell cultures. We used an RNAi strategy to investigate the function of NS in two selected carcinoma cell lines. RESULTS: High NS expression was found in most bladder carcinoma cell lines and normal uro-epithelial cells. Knockdown of NS expression induced a severe decline in cell proliferation in 5637 and SW1710 cell lines, both with mutant p53. Apoptosis was more strongly enhanced in 5637 cells lacking RB1 than in SW1710 cells lacking p16(INK4A). Moreover, NS-siRNA-treated 5637 cells accumulated mainly in G(2)/M, whereas SW1710 cells arrested in G(0)/G(1). CONCLUSION: Our data indicate that NS expression is necessary for cell proliferation and evasion of apoptosis in bladder cancer cells, independent of its effect on p53. Also, we speculate that the precise effect of NS on cell cycle regulation may relate to functional status of RB1 and CDKN2A/p16(INK4A).

Differential gene expression and alternative splicing of survivin following mouse sciatic nerve injury.

STUDY DESIGN: In vivo studies using an axotomy model in adult male Naval Medical Research Institute mice. OBJECTIVE: Survivin, a unique member of the inhibitor of the apoptosis (IAP) protein family, is expressed during embryonal development, but is undetectable in terminally differentiated cells and tissues. Owing to the vital role of survivin in cellular proliferation and apoptotic cell death, and also to the necessity of treatment of the nervous system injuries, we have monitored survivin gene expression as well as its alternative splicing changes at different time points within injured mouse sciatic nerves. SETTING: Department of Genetics, School of Basic Sciences, Tarbiat Modares University, Tehran, Iran. METHODS: The sciatic nerves of adult male Naval Medical Research Institute mice were transected and the expression of survivin was examined in the distal and proximal parts of the dissected nerves as well as in the corresponding segments within the spinal cord of the animals, using semi-quantitative RT-PCR and immunohistochemistry. RESULTS: Survivin is not expressed in undamaged sciatic nerves, but, after sciatic nerve injury, it is gradually upregulated in proximal and distal parts of the dissected nerve (P<0.05). Survivin140 is the main variant expressed after injury, accompanied by a low expression of survivin40 (P<0.05). There was no expression of the survivin121 variant after injury. CONCLUSIONS: Survivin is differentially expressed and spliced in damaged nerve and spinal cord. Future works on the manipulation of expression and/or the splicing of survivin could decipher the potential effects of survivin variants on the regeneration of nerve and/or spinal cord injuries.

Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions.

The aim of this study was to compare the in vitro effects of glial cell line-derived neurotrophic factor, stem cell factor, granulocyte macrophage-colony stimulating factor, and co-culture with Sertoli cells on the efficiency of adult mouse spermatogonial stem cells colony formation. For these purpose, both Sertoli and spermatogonial cells were isolated from adult mouse testes. The identity of the cells was confirmed through analysis of alkaline phosphatase activity, immunocytochemistry against OCT-4, c-kit, and vimentin, and also by transplantation of these cells in the recipient testes. The isolated spermatogonial cells were treated either with various concentrations of the above mentioned factors or co-cultured with Sertoli cells for 3 wk. The spermatogonial cells of the resulting colonies were transplanted via rete testis into the mouse testes, which were irradiated with 14 Gy. The results indicated that glial cell line-derived neurotrophic factor is the most appropriate factor for in vitro colonization of adult mice spermatogonial cells compared with other cytokines and growth factors. A short-term co-culture with Sertoli cells showed a significant increase in the number and diameter of the colonies compared with the treated growth factors and the control group. We have also demonstrated that mouse spermatogonial stem cells in the colonies after co-culturing with Sertoli cells could induce spermatogenesis in the recipient testes after transplantation.

CatSper genes expression, semen characteristics and histology of testes in the contusive spinal cord-injured mice model.

STUDY DESIGN: Experimental study. OBJECTIVE: A recently characterized CatSper genes, encodes for unique Ca(2+) channels in the testes, where they play essential roles in sperm motility. The aim of this research is to evaluate potential changes in the expression of CatSper genes, sperm parameters and testis histology following spinal cord injury (SCI). SETTING: Department of Anatomical Sciences, Tarbiat Modares University, Tehran, Iran. METHODS: A total of 75 adult NMRI mice were divided into three groups (25 in each group) of SCI, sham and control. Following laminectomy, SCI group was subjected to injury at the ninth thoracic vertebra. The epididymal sperm parameters were studied at day 1 and at weeks 1, 2, 4 and 6 after injury. One testis was removed for morphological study and the other testis were used for molecular study. Reverse transcription-PCR was performed at different time in all the groups. RESULTS: Our results revealed that SCI affects spermatogenesis as well as sperm quality and quantity. In gene expression analysis, there was a significant downregulation of CatSpers 1 and 2 at 4 and 6 weeks following contusion. There were no differences between the semen parameters and CatSper genes expression at the different time points in the sham and control groups. CONCLUSIONS: Our data indicated that the SCI mouse model causes a significant reduction in all sperm parameters, along with deleterious effects on spermatogenesis as well as the expression of CatSpers 1 and 2.

Knocking-down the expression of nucleostemin significantly decreases rate of proliferation of rat bone marrow stromal stem cells in an apparently p53-independent manner.

OBJECTIVES: Nucleostemin (NS) is a recently identified GTP-binding protein, predominantly expressed in embryonic and adult stem cells but not in terminally differentiated cells. NS is expressed in bone marrow-derived mesenchymal stem cells, and its expression ceases upon induction of neural differentiation. The major aim of this study was to determine whether down-regulation of NS expression acts as a promoter, or otherwise as a by-product of differentiation and senescence processes. MATERIALS AND METHODS: We used RNA interference protocols to specifically knock down NS in rat bone marrow-derived stromal stem cells. Changes in rate of proliferation and cell cycle profile after knocking-down of NS were measured. In addition, changes in expression of associated genes were studied by semiquantitative RT-PCR, Western blotting and immunocytochemistery. RESULTS: Knocked-down expression of NS caused a significant decrease in the rate of cell proliferation with concomitant shutting off of expression of cyclin D1 and survivin, two other well-known regulators of cell proliferation. Interestingly, we noticed no obvious changes in expression level of p21, the main effector of p53 for its cell cycle repressing function. CONCLUSION: Our findings revealed a master role for NS in promoting proliferation of rat bone marrow-derived stromal stem cells. Moreover, we suggest that despite previous proposals, the cell cycle arrest/inhibitory role of NS is unlikely to be related to its proposed property of interaction with p53.

Effect of bone morphogenetic protein-4 (BMP-4) on adipocyte differentiation from mouse embryonic stem cells.

Embryonic stem (ES) cells can differentiate spontaneously into various lineages in vitro. However, spontaneous commitment of ES cells to the adipocyte lineage is rare. In the present study, bone morphogenic protein-4 (BMP-4) is described as a factor inducing adipocyte differentiation from ES cells at a high rate. For this reason, ES-cell-derived embryoid bodies (EBs) in suspension cultures were exposed to different doses of BMP-4 for 5 days before they were plated onto gelatin-coated tissue culture plates. Moreover, the effect of serum-containing and serum-free media in three different combinations was assessed. Plated EBs, stained with Sudan Black and processed for transmission and scanning electron microscopy, were observed daily for adipocyte formation. Treatment with BMP-4 resulted in the appearance of adipocyte clusters in EBs' outgrowth, depending on the doses applied. Early in differentiation, many small fat droplets were observed in adipocytes, while later on they coalesced and formed a few large fat droplets. Adipocyte clusters had a fibrillar and vascular stroma, and each adipocyte was surrounded with a reticular external lamina. Furthermore, the appearance and development of adipocytes and their changes following 2-3 weeks of starvation mimicked live adipose tissue. In fact, understanding the biological activity of growth and differentiation factors is needed to regulate and direct stem cell differentiation to specific cell types in vitro.

Biosynthesis and post-translational processing of the precursor to brain-derived neurotrophic factor.

We examined the biosynthesis and post-translational processing of the brain-derived neurotrophic factor precursor (pro-BDNF) in cells infected with a pro-BDNF-encoding vaccinia virus. Metabolic labeling, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis reveal that pro-BDNF is generated as a 32-kDa precursor that is N-glycosylated and glycosulfated on a site, within the pro-domain. Some pro-BDNF is released extracellularly and is biologically active as demonstrated by its ability to mediate TrkB phosphorylation. The precursor undergoes N-terminal cleavage within the trans-Golgi network and/or immature secretory vesicles to generate mature BDNF (14 kDa). Small amounts of a 28-kDa protein that is immunoprecipitated with BDNF antibodies is also evident. This protein is generated in the endoplasmic reticulum through N-terminal cleavage of pro-BDNF at the Arg-Gly-Leu-Thr(57)- downward arrow-Ser-Leu site. Cleavage is abolished when Arg(54) is changed to Ala (R54A) by in vitro mutagenesis. Blocking generation of 28-kDa BDNF has no effect on the level of mature BDNF and blocking generation of mature BDNF with alpha(1)-PDX, an inhibitor of furin-like enzymes, does not lead to accumulation of the 28-kDa form. These data suggest that 28-kDa pro-BDNF is not an obligatory intermediate in the formation of the 14-kDa form in the constitutive secretory pathway.

Neurotrophin-3 sorts to the constitutive secretory pathway of hippocampal neurons and is diverted to the regulated secretory pathway by coexpression with brain-derived neurotrophic factor.

Hippocampal neurons release nerve growth factor (NGF) through the constitutive secretory pathway, thus allowing the protein to be continuously available for promoting nerve cell survival. In contrast, hippocampal neurons use the regulated secretory pathway to process brain-derived neurotrophic factor (BDNF), which alters synaptic activity when released acutely from dense-core vesicles. Thus, understanding how neurons sort and deliver neurotrophins may provide clues to their functions in brain. In this study, we monitored the processing and delivery of neurotrophin-3 (NT-3). Pulse-chase studies, immunocytochemistry, and secretagogue-induced release experiments were performed on cultured hippocampal neurons and AtT-20 cells infected with vaccinia viruses encoding the NT-3 precursor (pro-NT-3). Results show that most newly synthesized NT-3 is released through the constitutive secretory pathway as a result of furin-mediated endoproteolytic cleavage of pro-NT-3 in the trans-Golgi network. Pro-NT-3 can also be diverted into the regulated secretory pathway when cells are treated with alpha1-PDX, a selective inhibitor of furin-like enzymes, or when pro-NT-3 expression is increased by transient transfection methods. In cells coinfected with viruses coding for pro-NT-3 and pro-BDNF, NT-3 is sorted into the regulated pathway, stored in secretory granules, and released in response to extracellular cues together with BDNF, apparently as a result of heterodimerization, as suggested by coimmunoprecipitation data. Taken together, these data show that sorting of the NT-3 precursor can occur in both the constitutive and regulated secretory pathways, which is consistent with NT-3 having both survival-promoting and synapse-altering functions.

The prosegments of furin and PC7 as potent inhibitors of proprotein convertases. In vitro and ex vivo assessment of their efficacy and selectivity.

All proprotein convertases (PCs) of the subtilisin/kexin family contain an N-terminal prosegment that is presumed to act both as an intramolecular chaperone and an inhibitor of its parent enzyme. In this work, we examined inhibition by purified, recombinant bacterial prosegments of furin and PC7 on the in vitro processing of either the fluorogenic peptide pERTKR-MCA or the human immunodeficiency virus envelope glycoprotein gp160. These propeptides are potent inhibitors that display measurable selectivity toward specific proprotein convertases. Small, synthetic decapeptides derived from the C termini of the prosegments are also potent inhibitors, albeit less so than the full-length proteins, and the C-terminal P1 arginine is essential for inhibition. The bacterial, recombinant prosegments were also used to generate specific antisera, allowing us to study the intracellular metabolic fate of the prosegments of furin and PC7 expressed via vaccinia virus constructs. These vaccinia virus recombinants, along with transient transfectants of the preprosegments of furin and PC7, efficiently inhibited the ex vivo processing of the neurotrophins nerve growth factor and brain-derived neurotrophic factor. Thus, we have demonstrated for the first time that PC prosegments, expressed ex vivo as independent domains, can act in trans to inhibit precursor maturation by intracellular PCs.

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

Nerve growth factor (NGF) is released through the constitutive secretory pathway from cells in peripheral tissues and nerves where it can act as a target-derived survival factor. In contrast, brain-derived neurotrophic factor (BDNF) appears to be processed in the regulated secretory pathway of brain neurons and secreted in an activity-dependent manner to play a role in synaptic plasticity. To determine whether sorting differences are intrinsic to the neurotrophins or reflect differences between cell types, we compared NGF and BDNF processing in cultured hippocampal neurons using a Vaccinia virus expression system. Three independent criteria (retention or release from cells after pulse-chase labeling, depolarization-dependent release, and immunocytochemical localization) suggest that the bulk of newly synthesized NGF is sorted into the constitutive pathway, whereas BDNF is primarily sorted into the regulated secretory pathway. Similar results occurred with AtT 20 cells, including those transfected with cDNAs encoding neurotrophin precursor-green fluorescent protein fusions. The NGF precursor, but not the BDNF precursor, is efficiently cleaved by the endoprotease furin in the trans-Golgi network (TGN). Blocking furin activity in AtT 20 cells with alpha1-PDX as well as increasing the expression of NGF precursor partially directed NGF 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.

Mammalian subtilisin/kexin isozyme SKI-1: A widely expressed proprotein convertase with a unique cleavage specificity and cellular localization.

Using reverse transcriptase-PCR and degenerate oligonucleotides derived from the active-site residues of subtilisin/kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, rat, and mouse type I membrane-bound proteinase called subtilisin/kexin-isozyme-1 (SKI-1). Computer databank searches reveal that human SKI-1 was cloned previously but with no identified function. In situ hybridization demonstrates that SKI-1 mRNA is present in most tissues and cells. Cleavage specificity studies show that SKI-1 generates a 28-kDa product from the 32-kDa brain-derived neurotrophic factor precursor, cleaving at an RGLT downward arrowSL bond. In the endoplasmic reticulum of either LoVo or HK293 cells, proSKI-1 is processed into two membrane-bound forms of SKI-1 (120 and 106 kDa) differing by the nature of their N-glycosylation. Late along the secretory pathway some of the membrane-bound enzyme is shed into the medium as a 98-kDa form. Immunocytochemical analysis of stably transfected HK293 cells shows that SKI-1 is present in the Golgi apparatus and within small punctate structures reminiscent of endosomes. In vitro studies suggest that SKI-1 is a Ca2+-dependent serine proteinase exhibiting a wide pH optimum for cleavage of pro-brain-derived neurotrophic factor.