Transcriptome of control and FTO-knockdown HOC313 cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE133857 | GEO | 2020/03/23
REPOSITORIES: GEO
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Project description:Genomic aberrations of Cyclin D1 (CCND1) and CDK4 in neuroblastoma indicate that dysregulation of the G1 entry checkpoint is an important cell cycle aberration in this pediatric tumor. Here we report that analysis of Affymetrix expression data of primary neuroblastic tumors shows an extensive over-expression of Cyclin D1 and CDK4 which correlates with histological subgroups and prognosis respectively. Immunohistochemical analysis demonstrated an over-expression of Cyclin D1 in neuroblasts and a low Cyclin D1 expression in all cell types in ganglioneuroma. This suggests an involvement of G1 regulating genes in neuronal differentiation processes which we further evaluated using RNA interference against Cyclin D1 and its kinase partner CDK4 in several neuroblastoma cell lines. This resulted in pRb pathway inhibition as shown by an almost complete disappearance of CDK4 specific pRb phosphorylation; reduction of E2F transcriptional activity and a decrease of Cyclin A protein levels. The Cyclin D1 and CDK4 knock-down resulted in a significant reduction in cell proliferation, a G1 specific cell cycle arrest and moreover an extensive neuronal differentiation. Affymetrix microarray profiling of siRNA treated cells revealed a shift in expression profile towards a neuronal phenotype. Several new potential downstream players are identified. We conclude that neuroblastoma functionally depend on over-expression of G1 regulating genes to maintain their undifferentiated phenotype. Keywords: Neuroblastoma, CCND1, Cyclin D1, CDK4
2008-08-20 | GSE8866 | GEO
Project description:Genomic aberrations of Cyclin D1 (CCND1) and CDK4 in neuroblastoma indicate that dysregulation of the G1 entry checkpoint is an important cell cycle aberration in this pediatric tumor. Here we report that analysis of Affymetrix expression data of primary neuroblastic tumors shows an extensive over-expression of Cyclin D1 and CDK4 which correlates with histological subgroups and prognosis respectively. Immunohistochemical analysis demonstrated an over-expression of Cyclin D1 in neuroblasts and a low Cyclin D1 expression in all cell types in ganglioneuroma. This suggests an involvement of G1 regulating genes in neuronal differentiation processes which we further evaluated using RNA interference against Cyclin D1 and its kinase partner CDK4 in several neuroblastoma cell lines. This resulted in pRb pathway inhibition as shown by an almost complete disappearance of CDK4 specific pRb phosphorylation; reduction of E2F transcriptional activity and a decrease of Cyclin A protein levels. The Cyclin D1 and CDK4 knock-down resulted in a significant reduction in cell proliferation, a G1 specific cell cycle arrest and moreover an extensive neuronal differentiation. Affymetrix microarray profiling of siRNA treated cells revealed a shift in expression profile towards a neuronal phenotype. Several new potential downstream players are identified. We conclude that neuroblastoma functionally depend on over-expression of G1 regulating genes to maintain their undifferentiated phenotype. Experiment Overall Design: The Cell line IMR-32 at time point 0 and transiently transfected with siRNA against GFP, Cyclin D1 and CDK4 at time point 48 hours. All experiments are biological triplicates.
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Project description:Interferons have been ascribed to mediate antitumor effects. IRF-1 is a major target gene of interferons. It inhibits cell proliferation and oncogenic transformation. Here we show that 60% of all mRNAs deregulated by oncogenic transformation mediated by c-myc and H-ras are reverted to the expression levels of non-transformed cells by IRF-1. These include cell cycle regulating genes. Activation of IRF-1 decreases cyclin D1 expression and CDK4 kinase activity concomitant with dephosphorylation of pRb. These effects of IRF-1 are mediated by inhibition of the MEK-ERK pathway and a transcriptional repression of cyclin D1. IRF-1 mediated effects on cell cycle progression were found to be overridden by ectopic expression of cyclin D1. Ablation of cyclin D1 by RNA interference experiments prevents transformation and tumor growth in nude mice. The data demonstrate that cyclin D1 is a key target for IRF-1 mediated tumor suppressive effects. Keywords: NIH3T3 cells, myc, ras, IRF-1, proliferation, transformation, G1/S cell cycle ceckpoint
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Project description:During the life cycle of Enterovirus 71 (EV71), N6-methyladenosine (m6A) modification has an important impact on viral replication and the cellular response to viral infection, but the effect of m6A modification on cellular RNAs and pathways during infection is still unknown. The purpose of this research was to investigate the role of m6A in regulating host cell inflammatory response and disease progression during EV71 infection. Methylation RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) results show the m6A methylation modification map of control and EV71-infected groups of RD cells. And multilevel validation showed that decreased expression of demethylase FTO (fat mass and obesity-associated protein) was responsible for the elevated total m6A levels in EV71-infected RD cells and that TXNIP may be a target gene for demethylase FTO action. Further functional experiments showed that demethylase FTO silencing promoted TXNIP expression, activation of NLRP3 inflammasome and promoted the release of pro-inflammatory factors in vitro, and demethylase FTO overexpression showed the opposite result. Further tested in an animal model of severe EV71 infection, with results consistent with in vitro. In conclusion, our findings suggested that depletion of demethylase FTO in the presence of EV71 infection increased the m6A level of TXNIP mRNA 3ʹ UTR, increased mRNA stability, and promoted TXNIP expression, which activated the NLRP3 inflammasome and led to the release of pro-inflammatory factors, ultimately promoted HFMD progression. This could help with the development and prevention of m6A modification inhibitor-based drugs for viral-related inflammation and disease.
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Project description:Zinc-finger homeobox 3 (ZFHX3, also known as ATBF1) suppresses prostatic tumorigenesis. ZFHX3 is frequently found to have numerous deletions in human prostate cancer (PCa). However, the underlying molecular function of ZFHX3 during prostatic tumorigenesis is not well understood. N6-methyladenosine (m6A) modification in RNA plays a critical role in the development of cancers; however, the relationship between ZFHX3 and m6A modification is largely unknown in PCa. In this study, we found that ZFHX3 knockdown decreased total m6A levels through enhancing the transcriptional activity of FTO in PCa cells. Importantly, FTO inhibition suppressed cell proliferation and rescued the promoting function of ZFHX3 knockdown on cell proliferation. In vivo, we verified that FTO was upregulated and ZFHX3 was decreased in PCa patients and that a high level of ZFHX3 is indispensable for low FTO expression and is correlated with better patient survival. Through transcriptome sequencing and Me-RIP sequencing, we revealed that E2F2 and CDKN2C were the direct targets of FTO-mediated m6A modification and ZFXH3 was required for the regulation of FTO on E2F2 and CDKN2C expression. Unexpectedly, we uncovered that ZFHX3 expression was in return regulated by FTO in a m6A-dependent way. These findings establish a novel crosstalk mechanism between ZFHX3 and FTO in prostatic tumorigenesis.
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Project description:The cyclin D1 oncogene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the Rb protein and promotes progression through G1 to S phase of the cell cycle. Several prostate cancer cell lines and a subset of primary prostate cancer samples have increased cyclin D1 protein expression. However, the relationship between cyclin D1 expression and prostate tumor progression has yet to be clearly characterized. This study examined the effects of manipulating cyclin D1 expression in either human prostatic epithelial or stromal cells using a tissue recombination model. The data showed that overexpression of cyclin D1 in the initiated BPH-1 cell line increased cell proliferation rate, but did not elicit tumorigenicity in vivo. However, overexpression of cyclin D1 in Normal Prostate Fibroblasts (NPF) that were subsequently recombined with BPH-1 did induce malignant transformation of the epithelial cells. The present study also showed that recombination of BPH-1 + cyclin D1 overexpressing fibroblasts (NPF cyclin D1) resulted in permanent malignant transformation of epithelial cells (BPH-1 NPF-cyclin D1 cells) similar to that seen with Carcinoma Associated Fibroblasts (CAFs). Microarray analysis showed that the expression profiles between CAFs and NPF cyclin D1 cells were highly concordant including cyclin D1 upregulation. These data indicated that the tumor-promoting activity of cyclin D1 may be tissue-specific. Keywords: cyclin D1; stromal-epithelial interactions; prostate cancer; cDNA microarray
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Project description:Diabetic retinopathy (DR), a leading cause of irreversible vision loss in the working-age population, is an inflammatory, neuro-vascular complication of diabetes with poorly understood mechanism. N6-methyladenosine (m6A) modification plays crucial roles in biological and pathological events, while its role in DR remain elusive. Herein, we identified the pathological involvement of m6A demethylase FTO in DR. FTO expression was elevated in proliferative membranes of DR patients, endothelial cells (EC) under diabetic stress, and retinal vessels of diabetic murine models. FTO overexpression in EC promoted EC cycle progression and tip cell formation to facilitate angiogenesis in vitro, in mice and in zebrafish. FTO also regulated EC-pericyte crosstalk to trigger diabetes-induced microvascular leakage, and mediated EC-microglia crosstalk to induce retinal inflammation and subsequent neurodegeneration in vivo and in vitro. Mechanistically, FTO regulated EC features depending on its demethylation activity via modulating CDK2 mRNA stability with YTHDF2 as the reader. Moreover, FTO up-regulation in EC under diabetic conditions was driven by lactic acid mediated histone lactylation. FB23-2, which inhibits FTO’s m6A demethylase activity, suppressed diabetes associated endothelial phenotypes in vitro and in vivo. Noteworthy, we developed a novel macrophage membrane coated and PLGA-Dil based nanoplatform encapsulating FB23-2 for systemic administration, and confirmed its targeting and therapeutic efficacy in mice. Collectively, our study demonstrated an FTO-mediated regulatory network that coordinates EC biology and retinal homeostasis in DR, providing a promising nanotherapeutic approach for DR treatment. Diabetic retinopathy (DR), a leading cause of irreversible vision loss in the working-age population, is an inflammatory, neuro-vascular complication of diabetes with poorly understood mechanism. N6-methyladenosine (m6A) modification plays crucial roles in biological and pathological events, while its role in DR remain elusive. Herein, we identified the pathological involvement of m6A demethylase FTO in DR. FTO expression was elevated in proliferative membranes of DR patients, endothelial cells (EC) under diabetic stress, and retinal vessels of diabetic murine models. FTO overexpression in EC promoted EC cycle progression and tip cell formation to facilitate angiogenesis in vitro, in mice and in zebrafish. FTO also regulated EC-pericyte crosstalk to trigger diabetes-induced microvascular leakage, and mediated EC-microglia crosstalk to induce retinal inflammation and subsequent neurodegeneration in vivo and in vitro. Mechanistically, FTO regulated EC features depending on its demethylation activity via modulating CDK2 mRNA stability with YTHDF2 as the reader. Moreover, FTO up-regulation in EC under diabetic conditions was driven by lactic acid mediated histone lactylation. FB23-2, which inhibits FTO’s m6A demethylase activity, suppressed diabetes associated endothelial phenotypes in vitro and in vivo. Noteworthy, we developed a novel macrophage membrane coated and PLGA-Dil based nanoplatform encapsulating FB23-2 for systemic administration, and confirmed its targeting and therapeutic efficacy in mice. Collectively, our study demonstrated an FTO-mediated regulatory network that coordinates EC biology and retinal homeostasis in DR, providing a promising nanotherapeutic approach for DR treatment.
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