miR-34 Cooperates with p53 in Suppression of Prostate Cancer by Joint Regulation of Stem Cell Compartment

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Interdependent regulation of p53 and miR-34a in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) has an incidence 4/100 000 people in the western world and is one of the first cancers reported to be associated with deregulated miRNA expression. microRNAs are small non coding RNAs that are important regulators of protein expression through binding to their untranslated 3'-UTR region. The miR-34 family was demonstrated to be induced by the tumor suppressor p53 and to elicit p53-like responses like senescence, cell cycle arrest and apoptosis depending on the cell type. We have shown in a recent paper that miR-34a is severely increased in the TCL1-mouse model of CLL. This finding was reflected in human CLL. Moreover, it is demonstrated that its expression is dependent on the presence of the SNP309 in the intronic promoter of the MDM2 gene. In addition, low miR-34a expression was associated with shorter time to treatment (log-rank P = 0.003) in CLL. When reintroduced into CLL cells, miR-34a was able to induce apoptosis. Interestingly, this was dependent on an intact p53 pathway. Here, we present data showing that knockdown of p53 in HCT-116 cells severely reduces miR-34a induced apoptosis. In conclusion, miR-34a is proposed as a marker for the activity of the p53 pathway in CLL.     

Triptolide Induces Growth Inhibition and Apoptosis of Human Laryngocarcinoma Cells by Enhancing p53 Activities and Suppressing E6-Mediated p53 Degradation

Triptolide, an active compound extracted from Chinese herb Leigongteng (Tripterygium wilfordii Hook F.), shows a broad-spectrum of anticancer activity through its cytotoxicity. However, the efficacy of triptolide on laryngocarcinoma rarely been evaluated, and the mechanism by which triptolide-induced cellular apoptosis is still not well understood. In this study, we found that triptolide significantly inhibited the laryngocarcinoma HEp-2 cells proliferation, migration and survivability. Triptolide induces HEp-2 cell cycle arrest at the G1 phase and apoptosis through intrinsic and extrinsic pathways since both caspase-8 and -9 are activated. Moreover, triptolide enhances p53 expression by increasing its stability via down-regulation of E6 and E6AP. Increased p53 transactivates down-stream target genes to initiate apoptosis. In addition, we found that short time treatment with triptolide induced DNA damage, which was consistent with the increase in p53. Furthermore, the cytotoxicity of triptolide is decreased by p53 knockdown or use of caspases inhibitor. In conclusion, our results demonstrated that triptolide inhibits cell proliferation and induces apoptosis in laryngocarcinoma cells by enhancing p53 expression and activating p53 functions through induction of DNA damage and suppression of E6 mediated p53 degradation. These studies indicate that triptolide is a potential anti-laryngocarcinoma drug.     

miR-186-5p在酒精诱导的心肌细胞中高表达并通过靶基因XIAP调控细胞凋亡水平

目的:证实酒精可诱导AC16心肌细胞凋亡及其与酒精浓度和作用时间的关系,研究不同浓度酒精干预下AC16心肌细胞中miR-186-5p与X连锁凋亡抑制蛋白(XIAP)表达水平以及心肌细胞凋亡水平的改变,探究miR-186-5p以XIAP为靶基因调控酒精诱导的心肌细胞凋亡。方法:流式细胞术检测细胞凋亡水平,Western blot、实时定量PCR技术分别在蛋白质及基因水平检测细胞miR-186-5p与XIAP表达水平的变化,双萤光素酶报告基因靶基因荧光检测miR-186-5p与XIAP的靶际关系。结果:酒精诱导AC16心肌细胞发生凋亡,且与酒精浓度及作用时间呈正相关;酒精摄入上调AC16心肌细胞中miR-186-5p表达,下调XIAP表达; miR-186-5p参与酒精诱导的AC16心肌细胞凋亡过程,XIAP抑制酒精诱导的AC16心肌细胞凋亡; miR-186-5p以XIAP为靶基因调控酒精诱导的心肌细胞凋亡。结论:AC16心肌细胞经过酒精处理后,细胞的凋亡水平升高,并且随着酒精作用浓度和作用时间的延长,凋亡水平进一步升高;酒精处理后心肌细胞中miR-186-5p表达量上调,XIAP表达量下调,miR-186-5p以XIAP为靶基因,调控酒精处理后心肌细胞的凋亡。     

Regulation of Neuronal Cell Cycle and Apoptosis by MicroRNA 34a

The cell cycle of neurons remains suppressed to maintain the state of differentiation and aberrant cell cycle reentry results in loss of neurons, which is a feature in neurodegenerative disorders like Alzheimer''s disease (AD). Present studies revealed that the expression of microRNA 34a (miR-34a) needs to be optimal in neurons, as an aberrant increase or decrease in its expression causes apoptosis. miR-34a keeps the neuronal cell cycle under check by preventing the expression of cyclin D1 and promotes cell cycle arrest. Neurotoxic amyloid β_1–42 peptide (Aβ₄₂) treatment of cortical neurons suppressed miR-34a, resulting in unscheduled cell cycle reentry, which resulted in apoptosis. The repression of miR-34a was a result of degradation of TAp73, which was mediated by aberrant activation of the MEK extracellular signal-regulated kinase (ERK) pathway by Aβ₄₂. A significant decrease in miR-34a and TAp73 was observed in the cortex of a transgenic (Tg) mouse model of AD, which correlated well with cell cycle reentry observed in the neurons of these animals. Importantly, the overexpression of TAp73α and miR-34a reversed cell cycle-related neuronal apoptosis (CRNA). These studies provide novel insights into how modulation of neuronal cell cycle machinery may lead to neurodegeneration and may contribute to the understanding of disorders like AD.     

Regulation of p53 by Mdm2: fate is in the numbers

The p53 tumor suppressor protein is normally restrained by the Mdm2 oncoprotein, which promotes p53 ubiquitination. In a recent issue of Science, report that p53 may face two alternative fates, depending on Mdm2 levels: high Mdm2 drives p53 polyubiquitination and degradation within the cell nucleus, whereas low Mdm2 promotes p53 monoubiquitination and nuclear exclusion.     

Hypoxia induces accumulation of p53 protein, but activation of a G1-phase checkpoint by low-oxygen conditions is independent of p53 status.

It has been convincingly demonstrated that genotoxic stresses cause the accumulation of the tumor suppressor gene p53. One important consequence of increased p53 protein levels in response to DNA damage is the activation of a G1-phase cell cycle checkpoint. It has also been shown that G1-phase cell cycle checkpoints are activated in response to other stresses, such as lack of oxygen. Here we show that hypoxia and heat, agents that induce cellular stress primarily by inhibiting oxygen-dependent metabolism and denaturing proteins, respectively, also cause an increase in p53 protein levels. The p53 protein induced by heat is localized in the cytoplasm and forms a complex with the heat shock protein hsc70. The increase in nuclear p53 protein levels and DNA-binding activity and the induction of reporter gene constructs containing p53 binding sites following hypoxia occur in cells that are wild type for p53 but not in cells that possess mutant p53. However, unlike ionizing radiation, the accumulation of cells in G1 phase by hypoxia is not strictly dependent on wild-type p53 function. In addition, cells expressing the human papillomavirus E6 gene, which show increased degradation of p53 by ubiquitination and fail to accumulate p53 in response to DNA-damaging agents, do increase their p53 levels following heat and hypoxia. These results suggest that hypoxia is an example of a "nongenotoxic" stress which induces p53 activity by a different pathway than DNA-damaging agents.     

Down-Regulation of miR-129-5p Inhibits Growth and Induces Apoptosis in Laryngeal Squamous Cell Carcinoma by Targeting APC

miRNAs regulate gene expression and are key mediators of tumourigenesis. miR-129 has diverse effects in tumours, but its role in laryngeal squamous cell carcinoma (LSCC) remains unknown. This article focuses on the role of miR-129-5p in LSCC. We show miR-129-5p is upregulated in primary LSCC tumours and correlated with advanced disease. Down-regulating miR-129-5p suppressed cell proliferation and migration, and caused cell cycle arrest in Hep-2 cell lines. Downregulation of miR-129-5p alone is sufficient to induce apoptosis both in vivo and in vitro. Moreover, the growth of LSCC xenograft exposed to miR-129-5p antisense oligonucleotides (ASO) in BALB/c mice was markedly inhibited. In addition, we found that miR-129-5p targeted adenomatous polyposis coli (APC) to release inhibition of Wnt signalling causing cell growth and tumourigenesis. Our results suggest miR-129-5p functions as an oncogene in LSCC by repressing APC and is a potential therapeutic target for LSCC.     

Regulation of p53 stability and apoptosis by a ROR agonist

Activation of p53 function leading to cell-cycle arrest and/or apoptosis is a promising strategy for development of anti-cancer therapeutic agents. Here, we describe a novel mechanism for stabilization of p53 protein expression via activation of the orphan nuclear receptor, RORα. We demonstrate that treatment of cancer cells with a newly described synthetic ROR agonist, SR1078, leads to p53 stabilization and induction of apoptosis. These data suggest that synthetic ROR agonists may hold utility in the treatment of cancer.     

Apoptosis of fetal testicular cells is regulated by both p53-dependent and independent mechanisms

A portion of fetal germ cells undergoes apoptosis in the physiological context, but the molecular mechanisms of their apoptosis are largely unknown. Because p53 tumor suppressor gene product promotes apoptosis in various types of cells, we have investigated the expression of p53 in fetal gonads and examined the influence of loss of p53 function in fetal gonad cells using mice deficient in the p53 gene. We found that the expression of p53 protein in fetal testis was induced after 15.5 dpc (days post coitum), while the expression was not detected in fetal ovary. The number of apoptotic cells found in the seminiferous tubules of fetal testes was not significantly different between p53-deficient and wild-type mice until 16.5 dpc. At 17.5 dpc, however, more apoptotic cells were observed in wild-type testes than in the p53-deficient mice. In contrast, a similar number of apoptotic cells was found in fetal ovaries throughout these developmental stages. These observations indicate that p53 promotes apoptosis of fetal testicular cells after 16.5 dpc.     

Inhibition of p53 by Lentiviral Mediated sh RNA Abrogates G1 Arrest and Apoptosis in Retinal Pigmented Epithelial Cell Line

We silenced p53 gene expression in ARPE-19, a human retinal pigmented epithelial cell line using RNA interference. The effect of silencing the p53 gene in proliferating ARPE-19 cells was studied. Four short hairpin RNAs (shRNAs) targeting different regions of human p53 mRNA were delivered individually into ARPE-19 cells using lentiviral vector to produce stable cell lines. p53 mRNA and protein levels were reduced to varying extents in the four shRNA-transduced ARPE-19 cell lines. The cell line that showed greatest reduction (85-90%) of p53 expression showed decreased p21 promoter activation after DNA damage with camptothecin, etoposide and MMS. Whereas treatment of wild type ARPE-19 cells with camptothecin resulted in apoptosis, silencing p53 expression increased their survival. Cell cycle analyses indicated that irradiation resulted in a G1 arrest in ARPE-19 cells, and that the arrest was significantly reduced in p53-silenced cells. Thus, p53 plays a central role in the response of ARPE-19 cells to DNA damaging agents that act via different mechanisms. Additionally, ARPE-19 cells with reduced p53 expression behave similar to tumor cell lines with mutated or non-functional p53. The present data demonstrate the utility of lentiviral vectors to create stable isogenic cell lines with reduced expression of a specific gene, thereby permitting the study of the function of a gene, the pathways controlled by it, and the effect of therapeutics on a cell with altered genetic makeup in a pair-wise fashion.     

The over-expression of miR-34a fails to block DoHH2 lymphoma cell proliferation by reducing p53 via c-MYC down-regulation

MicroRNAs (miRNAs) might behave as tumor suppressors and for that they are under consideration as novel therapeutic drugs. We tested the tumor suppressor activity of miRNA-34a (miR-34a) by measuring cell proliferation of the follicular lymphoma cell line DoHH2 transfected with this miRNA. We report that miR-34a did not inhibit cell proliferation notwithstanding a marked down-regulation of c-MYC. Interestingly, DoHH2 transfected cells showed a significant p53 down-regulation, suggesting that c-MYC positively controls p53 and the failed inhibition of cell proliferation is probably due to the down-regulation of the c-MYC/p53 axis. In keeping with this, c-MYC silencing also down-regulated p53 and had no effect on cell proliferation. In accordance with this hypothesis, etoposide or nutlin-3 treatment or a small interfering RNA (siRNA) against BCL6 (B-cell lymphoma 6) inhibited the proliferation of DoHH2 cells by up-regulating p53 without affecting either miR-34a or c-MYC levels. These results indicate that the proliferation is controlled by the regulatory axis c-MYC/p53 and suggest that paradoxically miR-34a behaves as a pro-proliferative rather than an anti-proliferative miRNA in DoHH2 cells.     

过表达UHRF2通过上调p53及p21表达引起HEK293细胞G1期阻滞

UHRF2（ubiquitin like with PHD and ring finger domains 2）是新近发现的具有多个结构域的核蛋白,在细胞周期调控和表观遗传学中发挥重要作用．近期研究提示,UHRF2是肿瘤抑制蛋白p53的1个E3连接酶,在体内外能与p53相互结合并促进其泛素化,过表达UHRF2能使细胞周期停滞于G1期．然而,UHRF2介导的G1期阻滞及其与p53联系尚不清楚．通过共转染UHRF2质粒及p53特异性小干扰RNA(siRNAs)到HEK293细胞构建细胞模型,探索UHRF2引起细胞周期停滞与p53之间的关系．结果显示,UHRF2能促进HEK293细胞中p53的稳定,从而引起p21 (CIP1/WAF1)基因表达,并使细胞周期停滞于G1期;但在siRNA抑制p53的表达后p21(CIP1/WAF1)表达降低,UHRF2引起的细胞周期阻滞消除．研究结果提示,UHRF2可通过稳定p53,上调p21的表达,从而介导细胞周期阻滞于G1期;同时UHRF2可能参与细胞周期调控及DNA损伤反应（DNA damage response, DDR）．UHRF2稳定p53的具体分子机制及其在DDR中的作用有待进一步研究证明．