Bioinformatics analysis of the biological changes involved in the osteogenic differentiation of human mesenchymal stem cells

The mechanisms underlying the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) remain unclear. In the present study, we aimed to identify the key biological processes during osteogenic differentiation. To this end, we downloaded three microarray data sets from the Gene Expression Omnibus (GEO) database: GSE12266, GSE18043 and GSE37558. Differentially expressed genes (DEGs) were screened using the limma package, and enrichment analysis was performed. Protein‐protein interaction network (PPI) analysis and visualization analysis were performed with STRING and Cytoscape. A total of 240 DEGs were identified, including 147 up‐regulated genes and 93 down‐regulated genes. Functional enrichment and pathways of the present DEGs include extracellular matrix organization, ossification, cell division, spindle and microtubule. Functional enrichment analysis of 10 hub genes showed that these genes are mainly enriched in microtubule‐related biological changes, that is sister chromatid segregation, microtubule cytoskeleton organization involved in mitosis, and spindle microtubule. Moreover, immunofluorescence and Western blotting revealed dramatic quantitative and morphological changes in the microtubules during the osteogenic differentiation of human adipose‐derived stem cells. In summary, the present results provide novel insights into the microtubule‐ and cytoskeleton‐related biological process changes, identifying candidates for the further study of osteogenic differentiation of the mesenchymal stem cells.     

Human umbilical cord Wharton's jelly mesenchymal stem cells do not transform to tumor-associated fibroblasts in the presence of breast and ovarian cancer cells unlike bone marrow mesenchymal stem cells

Human bone marrow mesenchymal stem cells (hBMMSCs) were shown to transform into tumor-associated fibroblasts (TAFs) when in the vicinity of breast cancer tumors and played an important role in tumor enhancement and metastasis. In early human development MSCs migrating from the yolk sac and aorta-gonad-mesonephros (AGM) via the umbilical cord to the placenta and back to the fetal bone marrow were shown to get trapped in the gelatinous Wharton's jelly of the umbilical cord. The common origin of the Wharton's jelly MSCs and the finally homed hBMMSCs prompted us to evaluate whether hWJSCs are also involved in TAF transformation. hWJSCs and hBMMSCs were grown in the presence of breast and ovarian cancer cell conditioned medium (MDA-TCM, TOV-TCM) for 30 days. No changes were observed in the hWJSCs but the hBMMSCs transformed from short to thin long fibroblasts, their proliferation rates increased and CD marker expression decreased. The transformed hBMMSCs showed positive staining for the tumor-associated markers FSP, VEGF, EGF, and Tn-C. Real-time PCR and multiplex luminex bead analysis showed upregulation of TAF-related genes (FSP, FAP, Tn-C, Tsp-1, EGF, bFGF, IL-6, α-SMA, VEGF, and TGF-β) for hBMMSCs with low expression for hWJSCs. The luciferase assay showed that hWJSCs previously exposed to MDA-TCM or TOV-TCM had no stimulatory growth effect on luciferase-tagged MDA or TOV cells unlike hBMMSCs. The results confirmed that hWJSCs do not transform to the TAF phenotype and may therefore not be associated with enhanced growth of solid tumors making them a safe MSC for cell based therapies.     

Ageing genetic signature of hypersomatotropism

Acromegaly is a pathological condition that is caused by over-secretion of growth hormone (GH) and develops primarily from a pituitary adenoma. Excess GH exposure over a prolonged period of time leads to a wide range of systemic manifestations and comorbidities. Studying the effect of excess GH on the cellular level could help to understand the underlying causes of acromegaly health complications and comorbidities. In our previous publications, we have shown that excess GH reduces body side population (SP) stem cells and induces signs of premature ageing in an acromegaly zebrafish model. Here, we study acromegaly ageing in greater depth at the level of gene expression. We investigated whether acromegaly induces an ageing genetic signature in different organs. Using the GenAge database, our acromegaly model showed a significant enrichment of ageing genetic datasets in the muscle but not in other organs. Likewise, the hierarchical clustering of wild type (WT), acromegaly and aged RNA data from various organs revealed the similarity of gene expression profiles between the acromegaly and the aged muscles. We therefore identified overlapping differentially expressed genes (DEGs) in different organs between acromegaly and aged zebrafish. Importantly, about half of the muscle, liver and brain acromegaly DEGs overlapped with aged zebrafish DEGs. Interestingly, overlapping was observed in the same way; acromegaly-up DEGs overlapped with aged zebrafish up DEGs, not down DEGs, and vice versa. We then identified the biological functions of overlapping DEGs. Enrichment database analysis and gene ontology showed that most overlapping muscle genes were involved in ageing metabolism, while overlapping liver DEGs were involved in metabolic pathways, response to hypoxia and endoplasmic reticulum stress. Thus, this study provides a full ageing genetic signature of acromegaly at the gene expression level.     

A stress-induced cellular aging model with postnatal neural stem cells

Aging refers to the physical and functional decline of the tissues over time that often leads to age-related degenerative diseases. Accumulating evidence implicates that the senescence of neural stem cells (NSCs) is of paramount importance to the aging of central neural system (CNS). However, exploration of the underlying molecular mechanisms has been hindered by the lack of proper aging models to allow the mechanistic examination within a reasonable time window. In the present study, we have utilized a hydroxyurea (HU) treatment protocol and effectively induced postnatal subventricle NSCs to undergo cellular senescence as determined by augmented senescence-associated-β-galactosidase (SA-β-gal) staining, decreased proliferation and differentiation capacity, increased G0/G1 cell cycle arrest, elevated reactive oxygen species (ROS) level and diminished apoptosis. These phenotypic changes were accompanied by a significant increase in p16, p21 and p53 expression, as well as a decreased expression of key proteins in various DNA repair pathways such as xrcc2, xrcc3 and ku70. Further proteomic analysis suggests that multiple pathways are involved in the HU-induced NSC senescence, including genes related to DNA damage and repair, mitochondrial dysfunction and the increase of ROS level. Intriguingly, compensatory mechanisms may have also been initiated to interfere with apoptotic signaling pathways and to minimize the cell death by downregulating Bcl2-associated X protein (BAX) expression. Taken together, we have successfully established a cellular model that will be of broad utilities to the molecular exploration of NSC senescence and aging.     

Senescence induced by RECQL4 dysfunction contributes to Rothmund–Thomson syndrome features in mice

Cellular senescence refers to irreversible growth arrest of primary eukaryotic cells, a process thought to contribute to aging-related degeneration and disease. Deficiency of RecQ helicase RECQL4 leads to Rothmund–Thomson syndrome (RTS), and we have investigated whether senescence is involved using cellular approaches and a mouse model. We first systematically investigated whether depletion of RECQL4 and the other four human RecQ helicases, BLM, WRN, RECQL1 and RECQL5, impacts the proliferative potential of human primary fibroblasts. BLM-, WRN- and RECQL4-depleted cells display increased staining of senescence-associated β-galactosidase (SA-β-gal), higher expression of p16^INK4a or/and p21^WAF1 and accumulated persistent DNA damage foci. These features were less frequent in RECQL1- and RECQL5-depleted cells. We have mapped the region in RECQL4 that prevents cellular senescence to its N-terminal region and helicase domain. We further investigated senescence features in an RTS mouse model, Recql4-deficient mice (Recql4^HD). Tail fibroblasts from Recql4^HD showed increased SA-β-gal staining and increased DNA damage foci. We also identified sparser tail hair and fewer blood cells in Recql4^HD mice accompanied with increased senescence in tail hair follicles and in bone marrow cells. In conclusion, dysfunction of RECQL4 increases DNA damage and triggers premature senescence in both human and mouse cells, which may contribute to symptoms in RTS patients.     

Acute dyskerin depletion triggers cellular senescence and renders osteosarcoma cells resistant to genotoxic stress-induced apoptosis

Dyskerin is a conserved, nucleolar RNA-binding protein implicated in an increasing array of fundamental cellular processes. Germline mutation in the dyskerin gene (DKC1) is the cause of X-linked dyskeratosis congenita (DC). Conversely, wild-type dyskerin is overexpressed in sporadic cancers, and high-levels may be associated with poor prognosis. It was previously reported that acute loss of dyskerin function via siRNA-mediated depletion slowed the proliferation of transformed cell lines. However, the mechanisms remained unclear. Using human U2OS osteosarcoma cells, we show that siRNA-mediated dyskerin depletion induced cellular senescence as evidenced by proliferative arrest, senescence-associated heterochromatinization and a senescence-associated molecular profile. Senescence can render cells resistant to apoptosis. Conversely, chromatin relaxation can reverse the repressive effects of senescence-associated heterochromatinization on apoptosis. To this end, genotoxic stress-induced apoptosis was suppressed in dyskerin-depleted cells. In contrast, agents that induce chromatin relaxation, including histone deacetylase inhibitors and the DNA intercalator chloroquine, sensitized dyskerin-depleted cells to apoptosis. Dyskerin is a core component of the telomerase complex and plays an important role in telomere homeostasis. Defective telomere maintenance resulting in premature senescence is thought to primarily underlie the pathogenesis of X-linked DC. Since U2OS cells are telomerase-negative, this leads us to conclude that loss of dyskerin function can also induce cellular senescence via mechanisms independent of telomere shortening.     

RNA-Seq Analysis Reveals MAPKKK Family Members Related to Drought Tolerance in Maize

The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved signal transduction pathway that is involved in plant development and stress responses. As the first component of this phosphorelay cascade, mitogen-activated protein kinase kinase kinases (MAPKKKs) act as adaptors linking upstream signaling steps to the core MAPK cascade to promote the appropriate cellular responses; however, the functions of MAPKKKs in maize are unclear. Here, we identified 71 MAPKKK genes, of which 14 were novel, based on a computational analysis of the maize (Zea mays L.) genome. Using an RNA-seq analysis in the leaf, stem and root of maize under well-watered and drought-stress conditions, we identified 5,866 differentially expressed genes (DEGs), including 8 MAPKKK genes responsive to drought stress. Many of the DEGs were enriched in processes such as drought stress, abiotic stimulus, oxidation-reduction, and metabolic processes. The other way round, DEGs involved in processes such as oxidation, photosynthesis, and starch, proline, ethylene, and salicylic acid metabolism were clearly co-expressed with the MAPKKK genes. Furthermore, a quantitative real-time PCR (qRT-PCR) analysis was performed to assess the relative expression levels of MAPKKKs. Correlation analysis revealed that there was a significant correlation between expression levels of two MAPKKKs and relative biomass responsive to drought in 8 inbred lines. Our results indicate that MAPKKKs may have important regulatory functions in drought tolerance in maize.     

Retracted: Intra‐arterial transplantation of human bone marrow mesenchymal stem cells (hBMMSCs) improves behavioral deficits and alters gene expression in rodent stroke model

Stroke is a multi‐factorial polygenic disease and is a major cause of death and adult disability. Administration of bone marrow stem cells protects ischemic rat brain by facilitating recovery of neurological functions. But the molecular mechanism of stem cells action and their effect on gene expression is not well explored. In this study, we have transplanted 1 × 10⁶ human bone marrow mesenchymal stem cells (hBMMSCs) in middle cerebral artery occluded (MCAo) adult male Wistar rats through intracarotid artery route at 24 h after surgery. Motor behavioral tests (rotarod and open field) were performed to assess the changes in motor functions at day 0 and day1, 4, 8 and 14. The expression of studied genes at mRNA and protein level was quantified by using Q‐PCR and western blotting, respectively. Further, we have assessed the methylation pattern of promoter of these genes by using methylation‐specific PCR. Data were analyzed statistically and correlated. A significant improvement in behavioral deficits was observed in stem cells treated group after 14th day post stroke. Significantly (p < 0.05) increased mRNA and protein levels of brain derived neurotrophic factor and ANP genes in hBMMSCs treated group along with decrease in methylation level at their promoter was observed. On the other hand, significantly decreased mRNA and protein level of TSP1 and WNK1 in hBMMSCs treated group was observed. In conclusion, hBMMSCs administration significantly improves the behavioral deficits by improving motor and locomotor coordination. The promoter of TSP1 and WNK1 genes was found to be hyper‐methylated in hBMMSCs group resulting in their decreased expression while the promoter of ANP and brain derived neurotrophic factor was found to be hypo‐methylated. This study might shed a light on how hBMMSCs affect the gene expression by modulating methylation status.

     

Identification of hub genes and pathways in adrenocortical carcinoma by integrated bioinformatic analysis

Adrenocortical carcinoma (ACC), a rare malignant neoplasm originating from adrenal cortical cells, has high malignancy and few treatments. Therefore, it is necessary to explore the molecular mechanism of tumorigenesis, screen and verify potential biomarkers, which will provide new clues for the treatment and diagnosis of ACC. In this paper, three gene expression profiles (GSE10927, GSE12368 and GSE90713) were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were obtained using the Limma package. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched by DAVID. Protein‐protein interaction (PPI) network was evaluated by STRING database, and PPI network was constructed by Cytoscape. Finally, GEPIA was used to validate hub genes’ expression. Compared with normal adrenal tissues, 74 up‐regulated DEGs and 126 down‐regulated DEGs were found in ACC samples; GO analysis showed that up‐regulated DEGs were enriched in organelle fission, nuclear division, spindle, et al, while down‐regulated DEGs were enriched in angiogenesis, proteinaceous extracellular matrix and growth factor activity; KEGG pathway analysis showed that up‐regulated DEGs were significantly enriched in cell cycle, cellular senescence and progesterone‐mediated oocyte maturation; Nine hub genes (CCNB1, CDK1, TOP2A, CCNA2, CDKN3, MAD2L1, RACGAP1, BUB1 and CCNB2) were identified by PPI network; ACC patients with high expression of 9 hub genes were all associated with worse overall survival (OS). These hub genes and pathways might be involved in the tumorigenesis, which will offer the opportunities to develop the new therapeutic targets of ACC.     

Aging alters tissue resident mesenchymal stem cell properties

Tissue resident mesenchymal stem cells (MSCs) are known to participate in tissue regeneration that follows cell turnover, apoptosis, or necrosis. It has been long known that aging impedes an organism's repair/regeneration capabilities. In order to study the age associated changes, the molecular characteristics of adipose tissue derived MSCs (ASCs) from three age groups of healthy volunteers i.e., young, middle aged, and aged were investigated. The number and multilineage differentiation potential of ASCs declined with age. Aging reduces the proliferative capacity along with increases in cellular senescence. A significant increase in quiescence of G2 and S phase was observed in ASCs from aged donors. The expression of genes related to senescence such as CHEK1 and cyclin-dependent kinase inhibitor p16^ink4a was increased with age, however genes of apoptosis were downregulated. Further, an age-dependent abnormality in the expression of DNA break repair genes was observed. Global microRNA analysis revealed an abnormal expression of mir-27b, mir-106a, mir-199a, and let-7. In ubiquitously distributed adipose tissue (and ASCs), aging brings about important alterations, which might be critical for tissue regeneration and homeostasis. Our findings therefore provide a better understanding of the mechanism(s) involved in stem cell aging and regenerative potential, and this in turn may affect tissue repair that declines with aging.