Lin-28 homologue A (LIN28A) promotes cell cycle progression via regulation of cyclin-dependent kinase 2 (CDK2), cyclin D1 (CCND1), and cell division cycle 25 homolog A (CDC25A) expression in cancer

The RNA-binding protein LIN28A regulates the translation and stability of a large number of mRNAs as well as the biogenesis of certain miRNAs in embryonic stem cells and developing tissues. Increasing evidence indicates that LIN28A functions as an oncogene promoting cancer cell growth. However, little is known about its molecular mechanism of cell cycle regulation in cancer. Using tissue microarrays, we found that strong LIN28A expression was reactivated in about 10% (7.1-17.1%) of epithelial tumors (six tumor types, n = 369). Both in vitro and in vivo experiments demonstrate that LIN28A promotes cell cycle progression in cancer cells. Genome-wide RNA-IP-chip experiments indicate that LIN28A binds to thousands of mRNAs, including a large group of cell cycle regulatory mRNAs in cancer and embryonic stem cells. Furthermore, the ability of LIN28A to stimulate translation of LIN28A-binding mRNAs, such as CDK2, was validated in vitro and in vivo. Finally, using a combined gene expression microarray and bioinformatics approach, we found that LIN28A also regulates CCND1 and CDC25A expression and that this is mediated by inhibiting the biogenesis of let-7 miRNA. Taken together, these results demonstrate that LIN28A is reactivated in about 10% of epithelial tumors and promotes cell cycle progression by regulation of both mRNA translation (let-7-independent) and miRNA biogenesis (let-7-dependent).     

The Intermediate Filament Vimentin Mediates MicroRNA miR-378 Function in Cellular Self-renewal by Regulating the Expression of the Sox2 Transcription Factor

MicroRNAs are short noncoding RNAs that are implicated in cell self- renewal and cancer development. We show that miR-378 is up-regulated in human cancers and found that tumor cells transfected with miR-378 acquired properties of tumor stem cells, including cell self-renewal. Overexpression of miR-378 enhanced cell survival and colony formation. Isolated from a single-cell colony, the miR-378-expressing cells formed tumors in nude mice at low cell densities. These cells expressed higher levels of miR-378 and formed more and larger spheres and colonies. We found that the miR-378-expressing cells contained a large number of side population cells and could undergo differentiation. Cells transfected with miR-378 expressed increased levels of Sox2. Expression of miR-378 and Sox2 was found correlated significantly in cancer cell lines and in cancer patient specimens. We also observed opposite levels of vimentin in the cancer cell lines and human breast carcinoma specimens. We further demonstrated that vimentin is a target of miR-378, and ectopic transfection of vimentin inhibited Sox2 expression, resulting in decreased cell survival. Silencing vimentin promoted Sox2 expression and cell survival. Our study demonstrates that miR-378 is a regulator of stem cell marker Sox2 by targeting vimentin, which may serve as a new tool in studying the role of stem cells in tumorigenesis.     

Gene and microRNA expression signatures of human mesenchymal stromal cells in comparison to fibroblasts

Human mesenchymal stromal cells (MSCs) offer great hope for the treatment of tissue degenerative and immune diseases, but their phenotypic similarity to dermal fibroblasts may hinder robust cell identification and isolation from diverse tissue harvests. To identify genetic elements that can reliably discriminate MSCs from fibroblasts, we performed comparative gene and microRNA expression profiling analyses with genome-wide oligonucleotide microarrays. When taken globally, both gene and microRNA expression profiles of MSCs were highly similar to those of fibroblasts, accounting well for their extensive phenotypic and functional overlaps. Scattered expression differences were pooled to yield an MSC-specific molecular signature, consisting of 64 genes and 21 microRNAs whose expressions were at least 10-fold and two-fold higher, respectively, in MSCs compared with fibroblasts. Genes either encoding transmembrane proteins or associated with tumors were relatively abundant in this signature. These data should provide the molecular basis not only for the discovery of novel diagnostic markers discriminating MSCs from fibroblasts, but also for further studies on MSC-specific signaling mechanisms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sohyun Bae and Jung Hoon Ahn contributed equally to this work. This research was supported in part by a grant (SC-2140) from Stem Cell Research Center of the 21st Century Frontier Research Program, and in part by Korea Science and Engineering Foundation grant (M10641000037) funded by the Ministry of Education, Science and Technology, Republic of Korea.     

Effects of simulated microgravity on metabolic activities related to DNA damage and repair in lymphoblastoid cells

We adopted a simple experimental framework to follow the dependence of structural aberrations and the modifications in selected metabolic processes correlated with the exposure of cells to microgravity. Alterations to the cellular metabolism induced by exposure to microgravity are evidentiated in the modification of PARP activity (strongly dependent to the presence of DNA damages and to the altered gene expression), in the modification of the repair ability and in the cell's energy homeostasis (NAD and ATP). Cells are exposed continuously to microgravity in a Random Positioning Machine (RPM) in complete medium for 48 hours. At the end of this period a part of these cells are immediately analysed for the parameters reported above and the remaining were furtherly incubated in standard laboratory conditions to document eventual defects during the phases of the recovery process. A part of cells, just after exposure to microgravity, were also subjected to treatment with a strong damaging agent, KBrO3, and these cells were subsequently analyzed. This final treatment was meant to amplify the eventual deficiencies experienced by microgravity-exposed cells in the DNA repair process also in dependence with the alterated metabolic conditions resulting after the exposure to microgravity.     

Effects of simulated microgravity on DU 145 human prostate carcinoma cells

The high aspect rotating-wall vessel (HARV) was recently designed by NASA to cultivate animal cells in an environment that simulates microgravity. This work examines the effects of HARV cultivation on DU 145 human prostate carcinoma cells. In the HARV, these prostate cells grew in suspension on Cytodex-3 microcarrier beads to form bead aggregates with extensive three-dimensional growth between beads and on the aggregate surface. HARV and spinner-flask control cultures of DU 145 cells had similar doubling times, but the former was characterized by a higher percentage of G(1)-phase cells, larger bead aggregates, enhanced development of filopodia and microvilli-like structures on the aggregate surface, and stronger staining for select cytoskeletal proteins (cytokeratins 8 and 18, actin, and vimentin). When compared with static controls grown in a T-flask and Transwell insert, HARV cultures grew more slowly and differences in the cell cycle and immunostaining became more pronounced. These results suggest that HARV cultivation produced a culture that was less aggressive from the perspective of proliferation, more differentiated and less pliant than any of the three control cultures examined in this work. Possible factors effecting this change are discussed including turbulence and three-dimensional growth. (c) 1996 John Wiley & Sons, Inc.     

Regulation of Expression of Deoxyhypusine Hydroxylase (DOHH), the Enzyme That Catalyzes the Activation of eIF5A,by miR-331-3p and miR-642-5p in Prostate Cancer Cells

The enzyme deoxyhypusine hydroxylase (DOHH) catalyzes the activation of eukaryotic translation initiation factor (eIF5A), a protein essential for cell growth. Using bioinformatic predictions and reporter gene assays, we have identified a 182-nt element within the DOHH 3′-untranslated region (3′-UTR) that contains a number of target sites for miR-331-3p and miR-642-5p. Quantitative RT-PCR studies demonstrated overexpression of DOHH mRNA and underexpression of miR-331-3p and miR-642-5p in several prostate cancer cell lines compared with normal prostate epithelial cells. Transient overexpression of miR-331-3p and/or miR-642-5p in DU145 prostate cancer cells reduced DOHH mRNA and protein expression and inhibited cell proliferation. We observed synergistic growth inhibition with the combination of miR-331-3p and miR-642-5p and mimosine, a pharmacological DOHH inhibitor. Finally, we identified a significant inverse relationship between the expression of miR-331-3p or miR-642-5p and DOHH in a cohort of human prostate cancer tissues. Our results suggest a novel role for miR-331-3p and miR-642-5p in the control of prostate cancer cell growth via the regulation of DOHH expression and eIF5A activity.     

Cell shape and gene expression in human intervertebral disc cells: in vitro tissue engineering studies

The objective of the present study was to examine the relation between gene expression and the shape of human intervertebral disc cells cultured in vitro in three-dimensional (3D) scaffolds. Disc cells from 19 subjects were seeded into either a collagen sponge or collagen gel and cultured for 10 days. In situ hybridization was performed on serial sections of paraffin embedded specimens and assessed for expression of selected genes important for extracellular matrix formation: Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase. Rounded cells grown in collagen gel showed expression of Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase; expression of these genes was absent in spindle shaped cells. Cells in the collagen sponge that lay on the sponge margin were frequently spindle shaped; these cells expressed type I collagen, but not type II collagen, aggrecan or chondroitin-6 sulfotransferase. Results presented here provide novel data concerning disc cell gene expression with collagen 3D constructs. This information is useful for future tissue engineering studies that have the challenging goal of selectively modulating gene expression.     

Cell shape and gene expression in human intervertebral disc cells: in vitro tissue engineering studies

The objective of the present study was to examine the relation between gene expression and the shape of human intervertebral disc cells cultured in vitro in three-dimensional (3D) scaffolds. Disc cells from 19 subjects were seeded into either a collagen sponge or collagen gel and cultured for 10 days. In situ hybridization was performed on serial sections of paraffin embedded specimens and assessed for expression of selected genes important for extracellular matrix formation: Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase. Rounded cells grown in collagen gel showed expression of Types I and II collagen, aggrecan and chondroitin-6 sulfotransferase; expression of these genes was absent in spindle shaped cells. Cells in the collagen sponge that lay on the sponge margin were frequently spindle shaped; these cells expressed type I collagen, but not type II collagen, aggrecan or chondroitin-6 sulfotransferase. Results presented here provide novel data concerning disc cell gene expression with collagen 3D constructs. This information is useful for future tissue engineering studies that have the challenging goal of selectively modulating gene expression.     

Effects of simulated microgravity on male gametophyte of Prunus, Pyrus, and Brassica species

Summary. In this study we evaluated the effect of simulated microgravity on pollen germination of both herbaceous and woody species in order to investigate the possibility of applying gametophytic selection for plant growth in the space environment. The behaviour of gametophytes exposed to the stress of clinostat rotation could be used to screen the degree of tolerance of the sporophyte to simulated microgravity. The use of male gametophyte selection overcomes the problems generally encountered by sporophytic selection in space especially for woody plants: the large size of plants and their long juvenile phase. In this experiment, pollen collected from just bloomed flowers of Prunus persica, P. avium, P. domestica, Pyrus communis, and Brassica rapa was subjected to tests assessing its viability by techniques such as fluorochromatic reaction. Once pollen viability was ascertained by fluorescence microscopy, pollen was placed on the growth medium in petri dishes both at 1 g and on the clinostat. After incubating for 1 day at room temperature, pollen was observed under a light microscope in order to detect parameters such as the percentage of germination and the growth direction. Then histochemical analyses were performed in order to verify the presence and distribution of nuclei, cytoplasm, and storage substances. Moreover, the presence, size, and morphology of callose plugs were observed. Results showed that the response of gametophytes to simulated microgravity is dependent on the species, some showing altered metabolism, others being unaffected. Correspondence and reprints: Laboratorio di Botanica ed Ecologia Riproduttiva, Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, Via Università 100, I 80055 Portici (Naples), Italy.     

Proliferation of human hematopoietic bone marrow cells in simulated microgravity

Expansion and/or maintenance of hematopoietic stem cell (HSC) potential following in vitro culture remains a major obstacle in stem cell biology and bone marrow (BM) transplantation. Several studies suggest that culture of mammalian cells in microgravity (micro-g) may reduce proliferation and differentiation of these cells. We investigated the application of these findings to the field of stem cell biology in the hopes of expanding HSC with minimal loss of hematopoietic function. To this end, BM CD34+ cells were cultured for 4-6 d in rotating wall vessels for simulation of micro-g, and assessed for expansion, cell cycle activation, apoptosis, and hematopoietic potential. While CD34+ cells cultured in normal gravity (1-g) proliferated up to threefold by day 4-6, cells cultured in micro-g did not increase in number. As a possible explanation for this, cells cultured in simulated micro-g were found to exit G0/G1 phase of cell cycle at a slower rate than 1-g controls. When assayed for primitive hematopoietic potential in secondary conventional 1-g long-term cultures, cells from initial micro-g cultures produced greater numbers of cells and progenitors, and for a longer period of time, than cultures initiated with 1-g control cells. Similar low levels of apoptosis and adhesion molecule phenotype in micro-g and 1-g-cultured cells suggested similar growth patterns in the two settings. These data begin to elucidate the effects of micro-g on proliferation of human hematopoietic cells and may be potentially beneficial to the fields of stem cell biology and somatic gene therapy.