Depletion of intermediate filament protein Nestin,a target of microRNA-940, suppresses tumorigenesis by inducing spontaneous DNA damage accumulation in human nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a major malignant tumor of the head and neck region in southern China. The understanding of its underlying etiology is essential for the development of novel effective therapies. We report for the first time that microRNA-940 (miR-940) significantly suppresses the proliferation of a variety of cancer cell lines, arrests cells cycle, induces caspase-3/7-dependent apoptosis and inhibits the formation of NPC xenograft tumors in mice. We further show that miR-940 directly binds to the 3′-untranslated regions of Nestin mRNA and promotes its degradation. Likewise, depletion of Nestin inhibits tumor cell proliferation, arrest cells at G2/M, induces apoptosis and suppresses xenograft tumor formation in vivo. These functions of miR-940 can be reversed by ectopic expression of Nestin, suggesting that miR-940 regulates cell proliferation and survival through Nestin. Notably, we observed reduced miR-940 and increased Nestin levels in NPC patient samples. Protein microarray revealed that knockdown of Nestin in 5-8F NPC cells alters the phosphorylation of proteins involved in the DNA damage response, suggesting a mechanism for the miR-940/Nestin axis. Consistently, depletion of Nestin induced spontaneous DNA damage accumulation, delayed the DNA damage repair process and increased the sensitivity to irradiation and the chemotherapeutic agent doxorubicin. Collectively, our findings indicate that Nestin, which is downregulated by miR-940, can promote tumorigenesis in NPC cells through involvement in the DNA damage response. The levels of microRNA-940 and Nestin may serve as indicators of cancer status and prognosis.Nasopharyngeal carcinoma (NPC), a major malignant tumor of the head and neck region, is endemic to Southeast Asia, southern China, the Arctic, the Middle East and North Africa.¹ Low differentiation and high metastatic potential and recurrence rates are major pathologic features of NPC. The incidence of NPC in southern China has remained very high, with a 5-year overall survival rate of approximately 70%.² Within 4 years after radiation therapy, about 30–40% of NPC patients develop distant metastasis, which is associated with poor prognosis.³ Therefore, an understanding of the underlying etiology is essential for the development of novel effective therapies for NPC.MicroRNAs (miRNAs), a class of small (∼22 nucleotides) noncoding RNAs, reduce mRNA stability and/or suppress translation by binding to the 3′-untranslated regions (3′-UTRs) or coding sequences of target mRNAs.⁴ As such, miRNAs are involved in the majority of basic biologic processes, including cell proliferation, apoptosis, differentiation and development.⁵ Cumulative evidence also suggests that miRNAs can function as potential oncogenes or tumor suppressor genes.^{6, 7} Abnormal expression of miRNAs and mutations of their genes have been documented in various types of tumors.⁸ Recently, a growing number of miRNAs have been implicated in the development of NPC. For instance, the decreased expression of miR-100 has been reported to cause Plk1 overexpression, which in turn contributes to NPC progression.⁹ MiR-200a upregulation in the undifferentiated cell line C666-1 inhibits cell growth, migration and invasion by targeting ZEB2 and CTNNB1.¹⁰ Inhibition of miR-141, which is upregulated in NPC specimens, may affect cell cycle, apoptosis, cell growth, migration and invasion through targeting of BRD3, UBAP1 and PTEN.¹¹ In addition, reduced levels of let-7 in NPC might have a role in the proliferation through DNA methylation.¹² In view of the roles of miRNAs in tumorigenesis, identification of key miRNAs and their targets that contribute to NPC progression may provide novel targets for NPC diagnosis and treatment.Nestin, a member of the type VI intermediate filament protein family, is widely expressed in mammalian nervous tissue, some immortalized mammalian stem cell lines¹³ and precursor cells of some tissues, for which expression is decreased with differentiation.^{14, 15, 16} As a stem cell/progenitor cell marker,¹⁷ Nestin is essential for mitogen-stimulated proliferation of neural progenitor cells,¹⁸ and loss of Nestin leads to apoptosis of neural progenitor cells in zebrafish.¹⁹ Recently, Nestin has been detected in various cell lines established from human solid tumors²⁰ and has been associated with aggressive nervous system tumors.²¹ All of these findings suggest that Nestin is associated with tumorigenesis; however, the precise role of Nestin and the relationship between Nestin and NPC progression are still unknown.In this study, we screen 350 different miRNAs and determined that miR-940 inhibits the proliferation of the NPC cell lines 5-8F and CNE2. Furthermore, miR-940 expression induces G2/M arrest, promotes apoptosis and suppresses xenograft tumor growth. Bioinformatic and luciferase reporter assays revealed that miR-940 targets two putative binding sites in the Nestin 3′-UTR region. A physiologic role for miR-940 was suggested by its common downregulation in NPC tissues, whereas Nestin showed a converse pattern of upregulation. Knockdown of Nestin in 5-8F and CNE2 cells induces G2/M arrest and apoptosis and inhibits cell proliferation and xenograft tumor growth; conversely, ectopic expression of Nestin partially reverses the effects of miR-940 on cell proliferation, cell cycle and apoptosis. Interestingly, knockdown of Nestin induces spontaneous DNA damage accumulation, delays DNA damage repair and enhances sensitivity to ionizing radiation (IR) of 5-8F cells both in vitro and in vivo. These results elucidate a pathway by which miR-940 regulates tumor progression in NPC by targeting Nestin.     

EAF2 Suppresses Hypoxia-Induced Factor 1α Transcriptional Activity by Disrupting Its Interaction with Coactivator CBP/p300

Previous studies revealed that the potential tumor suppressor EAF2 binds to and stabilizes pVHL, suggesting that EAF2 may function by disturbing the hypoxia signaling pathway. However, the extent to which EAF2 affects hypoxia and the mechanisms underlying this activity remain largely unknown. In this study, we found that EAF2 is a hypoxia response gene harboring the hypoxia response element (HRE) in its promoter. By taking advantage of the pVHL-null cell lines RCC4 and 786-O, we demonstrated that hypoxia-induced factor 1α (HIF-1α), but not HIF-2α, induced EAF2 under hypoxia. Subsequent experiments showed that EAF2 bound to and suppressed HIF-1α but not HIF-2α transactivity. In addition, we observed that EAF2 inhibition of HIF-1α activity resulted from the disruption of p300 recruitment and that this occurred independently of FIH-1 (factor inhibiting HIF-1) and Sirt1. Furthermore, we found that EAF2 protected cells against hypoxia-induced cell death and inhibited cellular uptake of glucose under hypoxic conditions, suggesting that EAF2 indeed may act by modulating the hypoxia-signaling pathway. Our findings not only uncover a unique feedback regulation loop between EAF2 and HIF-1α but also provide a novel insight into the mechanism of EAF2 tumor suppression.     

The influences of PRG-1 on the expression of small RNAs and mRNAs

Background

In metazoans, Piwi-related Argonaute proteins play important roles in maintaining germline integrity and fertility and have been linked to a class of germline-enriched small RNAs termed piRNAs. Caenorhabditis elegans encodes two Piwi family proteins called PRG-1 and PRG-2, and PRG-1 interacts with the C. elegans piRNAs (21U-RNAs). Previous studies found that mutation of prg-1 causes a marked reduction in the expression of 21U-RNAs, temperature-sensitive defects in fertility and other phenotypic defects.

Results

In this study, we wanted to systematically demonstrate the function of PRG-1 in the regulation of small RNAs and their targets. By analyzing small RNAs and mRNAs with and without a mutation in prg-1 during C. elegans development, we demonstrated that (1) mutation of prg-1 leads to a decrease in the expression of 21U-RNAs, and causes 35 ~ 40% of miRNAs to be down-regulated; (2) in C. elegans, approximately 3% (6% in L4) of protein-coding genes are differentially expressed after mutating prg-1, and 60 ~ 70% of these substantially altered protein-coding genes are up-regulated; (3) the target genes of the down-regulated miRNAs and the candidate target genes of the down-regulated 21U-RNAs are enriched in the up-regulated protein-coding genes; and (4) PRG-1 regulates protein-coding genes by down-regulating small RNAs (miRNAs and 21U-RNAs) that target genes that participate in the development of C. elegans.

Conclusions

In prg-1-mutated C. elegans, the expression of miRNAs and 21U-RNAs was reduced, and the protein-coding targets, which were associated with the development of C. elegans, were up-regulated. This may be the mechanism underlying PRG-1 function.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-321) contains supplementary material, which is available to authorized users.     

A Genetically Modified Protein-Based Hydrogel for 3D Culture of AD293 Cells

Hydrogels have strong application prospects for drug delivery, tissue engineering and cell therapy because of their excellent biocompatibility and abundant availability as scaffolds for drugs and cells. In this study, we created hybrid hydrogels based on a genetically modified tax interactive protein-1 (TIP1) by introducing two or four cysteine residues in the primary structure of TIP1. The introduced cysteine residues were crosslinked with a four-armed poly (ethylene glycol) having their arm ends capped with maleimide residues (4-armed-PEG-Mal) to form hydrogels. In one form of the genetically modification, we incorporated a peptide sequence ‘GRGDSP’ to introduce bioactivity to the protein, and the resultant hydrogel could provide an excellent environment for a three dimensional cell culture of AD293 cells. The AD293 cells continued to divide and displayed a polyhedron or spindle-shape during the 3-day culture period. Besides, AD293 cells could be easily separated from the cell-gel constructs for future large-scale culture after being cultured for 3 days and treating hydrogel with trypsinase. This work significantly expands the toolbox of recombinant proteins for hydrogel formation, and we believe that our hydrogel will be of considerable interest to those working in cell therapy and controlled drug delivery.     

Genome-wide identification and expression profiling of ankyrin-repeat gene family in maize

Members of the ankyrin repeats (ANK) gene family encode ANK domain that are common in diverse organisms and play important roles in cell growth and development, such as cell-cell signal transduction and cell cycle regulation. Recently, genome-wide identification and evolutionary analyses of the ANK gene family have been carried out in Arabidopsis and rice. However, little is known regarding the ANK genes in the entire maize genome. In this study, we described the identification and structural characterization of 71 ANK genes in maize (ZmANK). Then, comprehensive bioinformatics analyses of ZmANK genes family were performed including phylogenetic, domain and motif analysis, chromosomal localization, intron/exon structural patterns, gene duplications and expression profiling. Domain composition analyses showed that ZmANK genes formed ten subfamilies. Five tandem duplications and 14 segmental duplications were identified in ZmANK genes. Furthermore, we took comparative analysis of the total ANK gene family in Arabidopsis, rice and maize, ZmANKs were more closely paired with OsANKs than with AtANKs. At last, expression profile analyses were performed. Forty-one members of ZmANK genes held EST sequences records. Semi-quantitative expression and microarray data analysis of these 41 ZmANK genes demonstrated that ZmANK genes exhibit a various expression pattern, suggesting that functional diversification of ZmANK genes family. The results will present significant insights to explore ANK genes expression and function in future studies in maize.     

BmCaspase-8-like regulates autophagy by suppressing BmDREDD-mediated cleavage of BmATG6

Autophagy plays an important role in tissue remodeling during insect development. The interplay between autophagy-related (ATG) proteins and caspases regulates the autophagic activity of ATGs, thereby modulating the process of autophagy. Our previous study characterized BmCaspase-8-like (BmCasp8L) as a caspase suppressor that inhibits apoptosis and immune signaling by suppressing the activation of death-related ced-3/Nedd2-like caspase (DREDD), a caspase-8 homolog in silkworm. In this study, we explored the regulatory role of BmCasp8L in autophagy. We found that the expression of Bmcasp8l increased from the late spinning stage to the pupa stage in the posterior silk gland (PSG), correlating with the expression patterns of Bmatg8 and Bmatg6. RNA interference-mediated downregulation of BmCasp8L expression significantly decreased starvation-induced autophagic influx as determined by the levels of BmATG8–phosphatidylethanolamine and the percentage of cells displaying punctate enhanced green fluorescent protein-BmATG8. Conversely, the overexpression of BmCasp8L significantly increased autophagic influx. We also found that BmCasp8L underwent autophagic degradation induced by starvation and that it was colocalized with BmATG8. Lastly, we demonstrated that BmDREDD attenuated autophagy and BmCasp8L suppressed BmDREDD-mediated cleavage of BmATG6. Taken together, our results demonstrated that BmCasp8L is a novel proautophagic molecule which suppresses BmDREDD-mediated cleavage of BmATG6 and is a target for autophagy.     

Food constraints explain the restricted distribution of wintering Lesser White‐fronted Geese Anser erythropus in China

More than 90% of the Lesser White‐fronted Geese Anser erythropus in the Eastern Palearctic flyway population winter at East Dongting Lake, China. To explain this restricted distribution and to understand better the winter feeding ecology and habitat requirements of this poorly known species, we assessed their food availability, diet and energy budgets at this site through two winters. Lesser White‐fronted Geese maintained a positive energy budget when feeding on above‐ground green production of Eleocharis and Alopecurus in recessional grasslands in autumn and spring to accumulate fat stores. Such food was severely depleted by late November and showed no growth in mid‐winter. Geese fed on more extensive old‐growth Carex sedge meadows in mid‐winter where they were in energy deficit and depleted endogenous fat stores. Geese failed to accumulate autumn fat stores in one year when high water levels prevented the Geese from using recessional grassland feeding areas. Fat stores remained lower throughout that winter and Geese left for breeding areas later in spring than in the previous year, perhaps reflecting the need to gain threshold fat stores for migration. Sedge meadows are widespread at other Yangtze River floodplain wetlands, but recessional grasslands are rare and perhaps restricted to parts of East Dongting Lake, which would explain the highly localized distribution of Lesser White‐fronted Geese in China and their heavy use of these habitats at this site. Sympathetic management of water tables is essential to maintain the recessional grasslands in the best condition for Geese. Regular depletion of fat stores whilst grazing sedge meadows in mid‐winter also underlines the need to protect the species from unnecessary anthropogenic disturbances that enhance energy expenditure. The specialized diet of the Lesser White‐fronted Goose may explain its highly restricted winter distribution and global rarity.     

城乡景观中土壤生态系统微塑料的来源、迁移特征及其风险

土壤微塑料污染的持久性、复杂性及其对土壤生态系统的影响受到越来越广泛的关注,但对其来源、迁移过程等仍有许多问题尚未理清。从复合生态系统的角度,通过对城乡景观中土壤微塑料的来源、迁移过程及其风险等相关研究进展进行了系统梳理,分析了城乡不同景观中土壤微塑料的主要来源和特点,讨论了土壤微塑料在城乡景观中的迁移特征及其驱动力,探讨了土壤微塑料在城乡景观中的生态和健康风险。今后需进一步明确城乡景观中土壤微塑料污染的物质流过程与环境归趋特征,加强微塑料对土壤生态系统的作用过程及生态系统服务影响的研究,揭示土壤微塑料对人类健康的直接或间接影响,建立城乡复合生态系统土壤微塑料污染预测模型,以维护土壤生态安全与人居环境健康。