ABSTRACT: To identify factors involved in OPC senescence, we compared gene expressions between OPC-CG4, OPC-FCS and OPC-Rev. Overall design: We established OPC senescence model system, in which OPC become senescent in the presence of high concentration of FCS. This phenotypes were kept even when the medium was switched to their optimal serum-free medium.
Project description:To identify factors involved in OPC senescence, we compared gene expressions between OPC-CG4, OPC-FCS and OPC-Rev. We established OPC senescence model system, in which OPC become senescent in the presence of high concentration of FCS. This phenotypes were kept even when the medium was switched to their optimal serum-free medium.
Project description:Noncoding RNAs include small transcripts, such as microRNAs and piwi-interacting RNAs, and a wide range of long noncoding RNAs (lncRNAs). Although many lncRNAs have been identified, only a small number of lncRNAs have been characterized functionally. Here, we sought to identify lncRNAs differentially expressed during replicative senescence. We compared lncRNAs expressed in proliferating, early-passage, 'young' human diploid WI-38 fibroblasts [population doubling (PDL) 20] with those expressed in senescent, late-passage, 'old' fibroblasts (PDL 52) by RNA sequencing (RNA-Seq). Numerous transcripts in all lncRNA groups (antisense lncRNAs, pseudogene-encoded lncRNAs, previously described lncRNAs and novel lncRNAs) were validated using reverse transcription (RT) and real-time, quantitative (q)PCR. Among the novel senescence-associated lncRNAs (SAL-RNAs) showing lower abundance in senescent cells, SAL-RNA1 (XLOC_023166) was found to delay senescence, because reducing SAL-RNA1 levels enhanced the appearance of phenotypic traits of senescence, including an enlarged morphology, positive β-galactosidase activity, and heightened p53 levels. Our results reveal that the expression of known and novel lncRNAs changes with senescence and suggests that SAL-RNAs play direct regulatory roles in this important cellular process. RNA was extracted from both young and senescent WI-38 cells and used for total RNA-Seq.
Project description:Cellular senescence disables the proliferation of damaged cells and it is relevant for cancer and aging. Here, we show that cellular senescence occurs during mammalian embryonic development. Specifically, we have focused on the mouse regressing mesonephros and the endolymphatic sac of the inner ear. Senescence is characterized by SAβG activity, heterochromatinization, and proliferative arrest. Mechanistically, developmentally-programmed senescence at the mesonephros and endolymphatic sac is strictly dependent on p21, but independent of DNA damage, p53 or other cell cycle inhibitors, and it is regulated by the TGFβ/SMAD and PI3K/FOXO pathways. Developmentally-programmed senescence is followed by macrophage infiltration and clearance of senescent cells. Abrogation of senescence by p21 deletion is only partially compensated by apoptosis and originates detectable developmental abnormalities. Importantly, high levels of p21 are also associated to the regressing mesonephros and endolymphatic sac in human embryos. These findings place cellular senescence as a relevant morphogenic process during embryonic development. We microdissected mesonephric tubules from senescent (WT) and non-senescent (p21-null) embryos to get information about this new senescence that occurs during embryogenesis.
Project description:In this study we analyzed the myeloma cell contact-mediated changes on the transcriptome of skeletal precursor cells. Therefore, human mesenchymal stem cells (MSC) and osteogenic precursor cells (OPC) were co-cultured with the representative myeloma cell line INA-6 for 24 h. Afterwards, MSC and OPC were separated from INA-6 cells by fluorescence activated cell sorting. Total RNA of MSC and OPC fractions was used for whole genome array analysis. Overall design: Primary MSC from the cancellous bone from the acetabulum received from donors after total hip arthroplasty were isolated as described by Noth et al., 2002 (PMID:12382974) and expanded in DMEM/Ham´s F12 medium including 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin and 50 µg/ml L-Ascorbic acid 2-phosphate. MSC were passaged at least once before they were used for experiments. Osteogenic precursor cells (OPC) were generated by incubating MSC with DMEM High Glucose medium for two weeks supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mmol/l beta-glycerophosphate, 100 nmol/l dexamethasone, and 50 µg/ml L-Ascorbic acid-2-phosphate. The myeloma cell medium consisted of RPMI 1640 medium, 20% FCS, 100 µg/ml gentamicin, 2 mmol/l L-glutamine, and 1 mmol/l sodium pyruvate. One day before co-culturing, confluent MSC and OPC monolayers were adapted to cell culturing conditions by incubating them with a 1:1 (v/v) mixture of MSC/myeloma cell medium and OPC/myeloma cell medium and INA-6 cells were stained with 5 µmol/l Cell Tracker® Green 5-chloromethylfluorescein diactetate. Afterwards, INA-6 cells were washed with PBS and used for co-culture. After 24 h, cells were trypsinized and separated by fluorescence activated cell sorting (BD FACS AriaTM III cell sorter). The control cells were treated accordingly. Cell pellets of MSC and OPC fractions were lysed in RA1 buffer containing 1% 2-mercaptoethanol and stored at -80°C until total RNA extraction. Total RNA was isolated with the NucleoSpin® RNA II kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) according to the manufacturer´s instructions. The whole genome array analysis was performed using the Affymetrix HG-U133 Plus 2.0 GeneChips (Affymetrix, High Wycombe, United Kingdom). Each 5 control specimens and 5 co-cultured samples of both MSC and OPC were used for hybridization.
Project description:With advancing age, senescent cells accumulate in tissues and critically influence aging-associated diseases. We report the first catalog of a novel class of regulatory RNAs, circular RNAs, differentially expressed in senescent (non-dividing) compared with dividing human fibroblasts. Among them, we focused on the circular RNA CircPVT1, as it promoted proliferation by binding and neutralizing let-7, a microRNA capable of triggering senescence. Interestingly, by suppressing let-7, CircPVT1 selectively elevated the expression of several proliferative proteins which were otherwise repressed by let-7. In summary, we have uncovered a novel mechanism whereby circular RNAs control gene expression programs in senescent cells. Overall design: Identification and validation of Senescence-Associated Circular RNAs (SAC-RNAs) in human diploid WI-38 fibroblasts
Project description:During the progress of senescence, cells sequentially acquire diverse senescent phenotypes together with several gene reprogramming steps. It is still unclear what will be the key regulator in charge of collective gene expression changes at the initial senescent reprogramming. In this study, we show that suppression of DNA methyltransferase 1 (DNMT1)-mediated maintenance DNA methylation activity was an initial event developed prior to gain of senescent phenotypes by employing time-series gene expression profiles of two different senescence models of human diploid fibroblast (HDF), replicative senescence (RS; GSE41714) and H2O2-induced senescence (HS). Overall design: The analysis of gene expression profiling among cellular senescence induced by hydrogen peroxide. two different data sets; a sample set of senescence by H2O2 (n =7 including control sample) and a set of siRNA-mediated knockdown experiment (n=5 including control sample).
Project description:Cellular senescence is a tumor suppressor mechanism, and immortalization facilitates neoplastic transformation. Both mechanisms may be highly relevant to hepatocellular carcinoma (HCC) development and its molecular heterogeneity. Cellular senescence appears to play a major role in liver diseases. Chronic liver diseases are associated with progressive telomere shortening leading senescence that is observed highly in cirrhosis, but also in some HCC. We previously described the generation of immortal and senescence-programmed clones from HCC-derived Huh7 cell line. We used microarrays to detail the global programme of gene expression profiles of immortal and senescent-programmed clones. Overall design: Immortal C11I and G11I clones were tested after PD 150. Senescence-programmed clones were tested at proliferating presenescent (C3P, G12P) and senescent (C3S, G12S) stages. Proliferating presenescent cells were tested at PD 20-30 with >85% BRDU positivity, and senescent cells were <5% BRDU positive and >50% SABG positive. The clones were plated in triplicate and subjected to RNA extraction.
Project description:We used IMR90 ER:RAS cells infected with an empty vector or an shRNA for ARID1B and induced senescence by addition of 4OHT. 6 days later RNA was collected for gene expression analysis. With a functional screen we previously identified ARID1B as a new regulator of cellular senescence. By performing gene expression analysis we confirmed this finding and showed that knockdown of ARID1B prevents the expression of genes induced during senescence. Overall design: 3 samples examined: Non-senescent cells (vector - 4OHT), Senescent cells (vector + 4OHT), Senescent cells expressing shRNA for ARID1B (shARID1B + 4OHT)
Project description:Cellular senescence is classified into two types; replicative and premature senescence. Gene expression and epigenetic changes are different in types of senescence, replicative and premature senescence, and cell types. Normal human diploid fibroblast TIG-3 cells were often used in cellular senescence research, however, their epigenetic profiles were not fully understood. To elucidate how cellular senescence is epigenetically regulated in TIG-3 cells, we analyzed gene expression and DNA methylation profiles among three types of senescent cells, namely, replicative senescent, RAS-induced senescent (RIS) and non-permissive temperature-induced senescent SVts8 cells, using gene expression and methylation microarrays. The expression of genes involved in cell cycle and immune response were commonly either down- or up-regulated among three types of senescent cells, respectively. The sequential alteration of DNA methylation level was observed only in replicative senescent cells in a time-dependent manner, but not in premature senescent cells. The integrated analysis of gene expression and methylation in replicative senescent cells demonstrated that the expression of 759 genes involved in cell cycle and immune response was associated with methylation. Furthermore, hypomethylation occurred at non-CpG island regions (open sea) on the genes with increased expression as well as non-CpG promoter of the genes related to immune response. Several miRNAs regulated by DNA methylation were found to affect the expression of their target genes. Taken together, these results indicate that DNA methylation contributes to introduction and establishment of replicative senescence partly by regulating gene expression. Overall design: We examined the gene expression and epigenetic difference following senescence among three different types of senescent cells, replicative senescent, RAS-induced senescent (RIS) and senescent SVts8 cells, derived from Normal human diploid fibroblast TIG-3 cells. Gene expression and genome-wide DNA methylation profiles were obtained using SurePrint G3 Human GE microarray 8×60K Ver. 2.0 (Agilent) and Infinium HumanMethylation27/450 BeadChip (Illumina), respectively.