靶向抑制CPEB4对鼻咽癌细胞的影响及放射增敏的机制研究

目的 本研究于2016年3~6月期间探讨靶向抑制CPEB4对鼻咽癌细胞生物学行为以及放射敏感度的影响。方法 利用RNAi技术构建慢病毒载体靶向抑制CPEB4基因的干扰序列;采用CCK-8法绘制CNE-2-shCPEB4、5-8F-shCPEB4、CNE-2及5-8F的生长曲线（增殖能力）;采用Transwell法检测CNE-2-shCPEB4、5-8F-shCPEB4迁移能力的改变;采用流式细胞术法检测CNE-2-shCPEB4、5-8F-shCPEB4凋亡率的改变;采用克隆形成实验检测CNE-2-shCPEB4、5-8F-shCPEB4、CNE-2及5-8F细胞的放射敏感度差异。采用Western blot法检测CNE-2-shCPEB4,5-8F-shCPEB4、CNE-2及5-8F细胞EMT相关蛋白表达改变。结果 CNE-2-shCPEB4、5-8F-shCPEB4分别与CNE-2及5-8F对照,增殖速度以及迁移能力减弱;凋亡率无明显改变,放射敏感度增加。Western blot法检测了CPEB4靶向抑制后EMT表型相关蛋白表达改变,结果提示E-cadherin表达上调,slug、snail以及vimentin的表达下调相关,推测靶向抑制CPEB4后鼻咽癌放射敏感度增强可能与EMT相关蛋白及因子的表达改变相关。结论 靶向抑制CPEB4蛋白表达后,鼻咽癌细胞系的增殖能力、体外迁移能力显著减弱,放射敏感度明显增强,其机制可能与CPEB4抑制EMT表型相关,E-cadherin表达上调,vimentin表达下调,E-cadherin表达上调可能与抑制E-cadherin表达的转录因子slug、snail表达下调相关。     

CPEB regulation of human cellular senescence, energy metabolism, and p53 mRNA translation

Cytoplasmic polyadenylation element-binding protein (CPEB) stimulates polyadenylation and translation in germ cells and neurons. Here, we show that CPEB-regulated translation is essential for the senescence of human diploid fibroblasts. Knockdown of CPEB causes skin and lung cells to bypass the M1 crisis stage of senescence; reintroduction of CPEB into the knockdown cells restores a senescence-like phenotype. Knockdown cells that have bypassed senescence undergo little telomere erosion. Surprisingly, knockdown of exogenous CPEB that induced a senescence-like phenotype results in the resumption of cell growth. CPEB knockdown cells have fewer mitochondria than wild-type cells and resemble transformed cells by having reduced respiration and reactive oxygen species (ROS), normal ATP levels, and enhanced rates of glycolysis. p53 mRNA contains cytoplasmic polyadenylation elements in its 3′ untranslated region (UTR), which promote polyadenylation. In CPEB knockdown cells, p53 mRNA has an abnormally short poly(A) tail and a reduced translational efficiency, resulting in an ~50% decrease in p53 protein levels. An shRNA-directed reduction in p53 protein by about 50% also results in extended cellular life span, reduced respiration and ROS, and increased glycolysis. Together, these results suggest that CPEB controls senescence and bioenergetics in human cells at least in part by modulating p53 mRNA polyadenylation-induced translation.     

MicroRNA-107: a novel promoter of tumor progression that targets the CPEB3/EGFR axis in human hepatocellular carcinoma

MicroRNAs (miRNAs) are dysregulated in many types of malignancies, including human hepatocellular carcinoma (HCC). MiR-107 has been implicated in several types of cancer regulation; however, relatively little is known about miR-107 in human HCC. In the present study, we showed that the overexpression of miR-107 accelerates the tumor progression of HCC in vitro and in vivo through its new target gene, CPEB3. Furthermore, our results demonstrated that CPEB3 is a newly discovered tumor suppressor that acts via the EGFR pathway. Therefore, our study demonstrates that the newly discovered miR-107/CPEB3/EGFR axis plays an important role in HCC progression and might represent a new potential therapeutic target for HCC treatment.     

miR-891a-5p通过CPEB1调控结肠癌细胞生物过程的机制研究

目的 探讨miR-891a-5p通过细胞质多聚腺苷酸化元件结合蛋白1(CPEB1)调控结肠癌细胞增殖、凋亡、迁移、侵袭的机制。方法 运用实时荧光定量逆转录聚合酶链反应（qPCR）检测结肠癌组织、癌旁组织、人正常结肠上皮细胞和人结肠癌细胞中miR-891a-5p的表达;将LOVO细胞分为A组（不做任何处理）、B组（转染anti-miRcon）、C组（转染anti-miR-891a-5p）、D组（共转染anti-miR-891a-5p和si-con）、E组（共转染anti-miR-891a-5p和siCPEB1）、F组（转染miR-con）、G组（转染miR-891a-5p）;CCK-8法检测细胞增殖率、Annexin V-FITC/PI法检测细胞凋亡率;蛋白免疫印迹实验检测细胞周期蛋白D1(Cyclin D1)、胱天蛋白酶-3酶原（Pro-caspase-3）、裂解的胱天蛋白酶-3(C-caspase-3)、N-钙黏蛋白（N-cadherin）、上皮细胞钙黏蛋白（E-cadherin）和波形蛋白（Vimentin）的蛋白表达;Transwell实验检测细胞迁移、侵袭;双荧光素酶报告基因检...     

“The seven sins” of the Hebbian synapse: Can the hypothesis of synaptic plasticity explain long-term memory consolidation?

Memorizing new facts and events means that entering information produces specific physical changes within the brain. According to the commonly accepted view, traces of memory are stored through the structural modifications of synaptic connections, which result in changes of synaptic efficiency and, therefore, in formations of new patterns of neural activity (the hypothesis of synaptic plasticity). Most of the current knowledge on learning and initial stages of memory consolidation ("synaptic consolidation") is based on this hypothesis. However, the hypothesis of synaptic plasticity faces a number of conceptual and experimental difficulties when it deals with potentially permanent consolidation of declarative memory ("system consolidation"). These difficulties are rooted in the major intrinsic self-contradiction of the hypothesis: stable declarative memory is unlikely to be based on such a non-stable foundation as synaptic plasticity. Memory that can last throughout an entire lifespan should be "etched in stone." The only "stone-like" molecules within living cells are DNA molecules. Therefore, I advocate an alternative, genomic hypothesis of memory, which suggests that acquired information is persistently stored within individual neurons through modifications of DNA, and that these modifications serve as the carriers of elementary memory traces.     

CPEB1, a novel risk gene in recent-onset schizophrenia,contributes to mitochondrial complex I defect caused by a defective provirus ERVWE1

BACKGROUNDSchizophrenia afflicts 1% of the world population. Clinical studies suggest that schizophrenia patients may have an imbalance of mitochondrial energy metabolism via inhibition of mitochondrial complex I activity. Moreover, recent studies have shown that ERVWE1 is also a risk factor for schizophrenia. Nevertheless, there is no available literature concerning the relationship between complex I deficits and ERVWE1 in schizophrenia. Identifying risk factors and blood-based biomarkers for schizophrenia may provide new guidelines for early interventions and prevention programs. AIMTo address novel potential risk factors and the underlying mechanisms of mitochondrial complex I deficiency caused by ERVWE1 in schizophrenia. METHODSQuantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay were used to detect differentially expressed risk factors in blood samples. Clinical statistical analyses were performed by median analyses and Mann-Whitney U analyses. Spearman’s rank correlation was applied to examine the correlation between different risk factors in blood samples. qPCR, western blot analysis, and luciferase assay were performed to confirm the relationship among ERVWE1, cytoplasmic polyadenylation element-binding protein 1 (CPEB1), NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2), and NDUFV2 pseudogene (NDUFV2P1). The complex I enzyme activity microplate assay was carried out to evaluate the complex I activity induced by ERVWE1. RESULTSHerein, we reported decreasing levels of CPEB1 and NDUFV2 in schizophrenia patients. Further studies showed that ERVWE1 was negatively correlated with CPEB1 and NDUFV2 in schizophrenia. Moreover, NDUFV2P1 was increased and demonstrated a significant positive correlation with ERVWE1 and a negative correlation with NDUFV2 in schizophrenia. In vitro experiments disclosed that ERVWE1 suppressed NDUFV2 expression and promoter activity by increasing NDUFV2P1 level. The luciferase assay revealed that ERVWE1 could enhance the promoter activity of NDUFV2P1. Additionally, ERVWE1 downregulated the expression of CPEB1 by suppressing the promoter activity, and the 400 base pair sequence at the 3′ terminus of the promoter was the minimum sequence required. Advanced studies showed that CPEB1 participated in regulating the NDUFV2P1/NDUFV2 axis mediated by ERVWE1. Finally, we found that ERVWE1 inhibited complex I activity in SH-SY5Y cells via the CPEB1/NDUFV2P1/NDUFV2 signaling pathway. CONCLUSIONIn conclusion, CPEB1 and NDUFV2 might be novel potential blood-based biomarkers and pathogenic factors in schizophrenia. Our findings also reveal a novel mechanism of ERVWE1 in the etiology of schizophrenia.