There is increasing evidence that the expression of non-coding RNA and mRNA (messenger RNA) is significantly altered following high-dose ionizing radiation (IR), and their expression may play a critical role in cellular responses to IR. However, the role of non-coding RNA and mRNA in radiation protection, especially in the nervous system, remains unknown. In this study, microarray profiles were used to determine microRNA (miRNA), long non-coding RNA (lncRNA), and mRNA expression in the hypothalamus of mice that were pretreated with amifostine and subsequently exposed to high-dose IR. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. We found that fewer miRNAs, lncRNAs, and mRNAs were induced by amifostine pre-treatment in exposed mice, which exhibited antagonistic effects compared to IR, indicating that amifostine attenuated the IR-induced effects on RNA profiles. GO and KEGG pathway analyses showed changes in a variety of signaling pathways involved in inflammatory responses during radioprotection following amifostine pre-treatment in exposed mice. Taken together, our study revealed that amifostine treatment altered or attenuated miRNA, lncRNA, and mRNA expression in the hypothalamus of exposed mice. These data provide a resource to further elucidate the mechanisms underlying amifostine-mediated radioprotection in the hypothalamus.

High-dose ionizing radiation (IR) alters the expression levels of non-coding RNAs (ncRNAs). However, the roles of ncRNAs and mRNAs in mediating radiation protection by radioprotectants remain unknown. Microarrays were used to determine microRNA (miRNA), long ncRNA (lncRNA), and mRNA expression profiles in the bone marrow of irradiated mice pretreated with amifostine, CBLB502, and nilestriol. Differentially expressed mRNAs were functionally annotated by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Some histone cluster genes were validated by real-time PCR, and the effects of radioprotectant combinations were monitored by survival analysis. We found that these radioprotectants increased the induction of lncRNAs and mRNAs. miRNA, lncRNA, and mRNA expression patterns were similar with amifostine and CBLB502, but not nilestriol. The radioprotectants exhibited mostly opposite effects against IR-induced miRNAs, lncRNAs, and mRNAs while inducing a common histone gene downregulation following IR, mainly via nucleosome assembly and related signaling pathways. Notably, the effects of nilestriol significantly complemented those of amisfostine or CBLB502; low-dose drug combinations resulted in better radioprotective effects in pretreated mice. Thus, we present histone gene downregulation by radioprotectants, together with the biological functions of miRNA, lncRNA, and mRNA, to explain the mechanism underlying radioprotection.

目的 通过小单链RNA高通量测序方法分析小单链RNA分子在血清中的稳定性,为获得研发小RNA药物的序列特点提供基础.方法 人工合成21nt随机序列小单链RNA分子库,用血清孵育后回收小单链RNA分子,进行高通量RNA测序,对测序结果进行比较分析.结果与结论 人工合成的不同序列小单链RNA分子在血清中存在稳定性差异,稳定性小单链RNA分子呈现出碱基偏性和基序特征. 上述结果将为小RNA分子药物设计和筛选提供参考.

The protein p53 plays a crucial role in the regulation of cellular responses to diverse stresses. Thus, a major priority in cell biology is to define the mechanisms that regulate p53 activity in response to stresses or maintain it at basal levels under normal conditions. Moreover, further investigation is required to establish whether RNA participates in regulating p53's interaction with other proteins. Here, by conducting systematic experiments, we discovered a p53 interactor-hnRNPC-that directly binds to p53, destabilizes it, and prevents its activation under normal conditions. Upon doxorubicin treatment, the lncRNA SNHG1 is retained in the nucleus through its binding with nucleolin and it competes with p53 for hnRNPC binding, which upregulates p53 levels and promotes p53-dependent apoptosis by impairing hnRNPC regulation of p53 activity. Our results indicate that a balance between lncRNA SNHG1 and hnRNPC regulates p53 activity and p53-dependent apoptosis upon doxorubicin treatment, and further indicate that a change in lncRNA subcellular localization under specific circumstances is biologically significant.

Background: Bacterial small regulatory RNAs (sRNAs) play important roles in sensing environment changes through sRNA-target mRNA interactions. However, the current strategy for detecting sRNA-mRNA interactions usually combines bioinformatics prediction and experimental verification, which is hampered by low prediction accuracy and low-throughput. Additionally, among the 4736 sequenced bacterial genomes, only about 2164 sRNAs from 319 strains have been described. Furthermore, target mRNAs of only 157 sRNAs have been uncovered. Obviously, highly efficient methods were required to detect sRNA-mRNA interactions in the sequenced genomes. This study aimed to apply a modified CLASH (cross-linking, ligation and sequencing hybrids) method to detect RNA-RNA interactions in E. coli, a model bacterial organism.Results: Statistically significant interactions were detected in 29 transcript pairs. To the best of our knowledge, 24 pairs were reported for the first time and were novel RNA interactions, including tRNA-tRNA, tRNA-ncRNA (non-coding RNA), tRNA-rRNA, rRNA-mRNA, rRNA-ncRNA, rRNA-rRNA, rRNA-IGT (intergenic transcript), and tRNA-IGT interactions.Conclusions: Discovery of novel RNA-RNA interactions in the present study demonstrates that RNA-RNA interactions might be far more complicated than ever expected. New methods may be required to help discover more novel RNA-RNA interactions. The present work describes a high-throughput protocol not only for discovering new RNA interactions, but also directly obtaining base-pairing sequences, which should be useful in assessing RNA structure and interactions.

Objective To study the effect of Ro60 on migration and invasion of lung adenocarcinoma A 549 cells. Methods We knocked down Ro60 and analyzed the ability of migration and invasion of A 549 cells.Quantitative real time ( RT)-PCR and Western blotting were performed to detect mRNA and protein levels of selected molecules associated with migration and invasion of neoplasm .Results When Ro60 was downregulated , the migration and invasion of A 549 cells decreased markedly.Meanwhile,the mRNA expression of matrix metalloproteinase (MMP)9,c-Src etc was observably reduced.Furthermore, downregulation of Ro60 diminished the expression of Src protein and the activation of MMP-9 protein.Conclusion Downregulation of Ro60 inhibits migration and invasion of A549 cells by regulating Src protein and activating MMP-9 protein.

Objective To conduct a pilot study on genome-wide in vivo protein-RNA interactions in E.coli.Methods Bacterial lysate was treated with RNase before the RNA fragments protected by proteins were extracted from treated lysate and used to construct cDNA library that was applied to high-throughput sequencing .Finally, the transcripts bound by proteins were obtained by bioinformatics analysis .Results A total of 3193 transcripts were obtained , including 2234 mRNAs, 47 sRNAs, 39 tRNAs, 11 rRNAs, and 862 intergenic regions .Conclusion Some information of transcripts interacting with proteins in E.coli is acquired , which will facilitate further studies of protein-RNA interactions .

DNA damage responses(DDR) cellular defense mechanism acts as a potent barrier for tumor initiation and progression,containing transcriptional regulation,cell cycle checkpoint activation,DNA damage repair,apoptotic and non-apoptotic cell death,which is the cornerstone of genome stability.Dysfunction of DDR signaling is associated with genomic instability diseases,such as tumor,senescence and complex diseases.The promyelocytic leukemia protein(PML) is the indispensable scaffold of PML nuclear bodies(PML-NBs),which are matrix associated multi-protein subnuclear structures.Previous studies have showed that PML-NBs are dynamic sensor of DNA damage,and regulate a wide variety of biological processes,including chromatin remodeling,epigenetic modification,gene transcription and post-transcriptional RNA stability and translation,post-translational protein modification and location.PML-NBs function as pivotal hub of signal transduction and network coordination in p53-dependent and p53-independent checkpoint activation,damage repair and apoptosis induction in DNA damage responses(DDR).

TO THE EDITOR—Human brucellosis is caused by transmission of Brucella from animal reservoirs to humans by direct contact with infected animals or consumption of raw animal products such as unpasteurized milk or cheese. In many countries, brucellosis is underreported, and thus, official statistics reflect only a fraction of its true incidence [1]. Although its true incidence in China remains largely unknown, the incidence of human brucellosis is estimated to vary from <0.03 to >160 individuals per 100 000 population [2, 3]. Various sanitary, socioeconomic, and political factors have led the epidemiology of human brucellosis to change drastically over the past 2 decades [4, 5]. One important factor has been increased domestic and international travel [6]. This, together with the lack of awareness of brucellosis in lowincidence areas, impeded its recognition in these areas. Such initial lack of recognition typically results in delayed diagnosis and treatment, which may lead to chronic infection, and thus great economic and health problems for sufferers [2]. Increasing awareness of brucellosis and its distribution is important for both clinicians and travelers for achieving prevention, early diagnosis, and treatment. To assist in these endeavors, this study analyzed the spatial and time distribution of human brucellosis cases in China to identify high-risk regions. Information of the cases reported from January 2004 to December 2010 from the national surveillance system was collected. The geographic data for each case were extracted and a spatial incidence database was constructed. The incidence for each county was calculated, and space and time distribution and cluster identification were analyzed by using SaTScan and ArcGIS software as described previously [7]. As shown in Figure 1, 2 clusters were identified: a primary cluster (cluster I) and a secondary cluster (cluster II). Cluster I includes a total of 117 counties distributed at the junctions of Inner Mongolia, Hebei, Liaoning, Jilin, and Heilongjiang provinces, which were inhabited by 37 416 402 residents and reported 61 067 cases during the study period. Analysis of the data revealed the relative risk to be 40.093 and the log-likelihood ratio to be 151 573 (P < .001). Cluster II includes 331 counties at the junctions of Hebei, Shanxi, Inner Mongolia, Henan, Shanxi, and Ningxia provinces, which were inhabited by 104 103 364 residents and reported 29 595 cases during the study period. The relative risk and log-likelihood ratio were 5.236 and 23 130 (P < .001), respectively. The relative risk of transmission in cluster I is approximately 7.6 times that of cluster II. Investigation of high-risk factors for human brucellosis in 5 counties in cluster I identified animal contact and consumption of products from infected animals as the most significant factors. The diagnosis of human brucellosis in cluster I is relatively timely compared with that in other regions (P < .001). Because treatment within 3 months is generally efficacious [8], we recommend that clinicians and travelers remain aware

Objective To investigate the expression and subcellular localization of RNA-binding protein Ro60 in neoplasms before and after γ-ray irradiation,and the function of Ro60 in tumor cell proliferation and radio-sensitivity.Methods The eukaryotic expression plasmid of mGFP-Ro60 was constructed and transfected into HCT116 and MCF7 tumor cells.The cellular localization of Ro60 was examined before and after irradiation.Cell proliferation and radio-sensitivity were detected by CCK8 and trypan blue assay.Results The result of immunoblotting showed that tumor cells expressed Ro60 protein.The irradiation increased Ro60 expression and induced significantl nuclear aggregation of Ro60.The cell proliferation before and after irradiation was drastically reduced while cell death increased in Ro60 overexpressed tumor cells.Conclusion γ-ray irradiation alters Ro60 expression and localization,and Ro60 plays an important role in tumor cell proliferation and radio-sensitivity.

The cyclin-dependent kinase inhibitor p21WAF1/Cip1 is a critical cell cycle regulator which translocates into the nucleus to participate in DNA repair during DNA damage responses. In the present study, we showed that the tumor suppressor, promyelocytic leukemia protein (PML) contributes to the up-regulation of p21 in a p53-independent pathway. Knock-down of PML in p53-null H1299 and HCT 116 (p53−/−) tumor cells by specific siRNA resulted in down-regulation of p21 protein expression, inhibition of γ-irradiation-induced p21 up-regulation, and a decrease in p21 protein half-life. In PML knockdown H1299 cells, the down-regulation of p21 protein expression was reversed by MG132 treatment indicating that the proteasomal degradation of p21 protein was increased. Thus, PML positively regulates p21 expression by inhibiting proteasome-mediated proteolysis. Knockdown of PML decreased the repair of γ-irradiation-induced double strand breaks (DSBs) as indicated by the delayed disappearance of γ-H2AX foci and a decreased association between p21 and proliferating cell nuclear antigen (PCNA). Over-expression of p21 significantly restored the delayed DSB repair function. Taken together, these data provide evidence for a p53-independent functional relationship between PML and p21 in γ-irradiation-induced DNA damage responses, and identify PML as a positive post-translational regulator of p21 in p53-deficient tumor cells.

Lung resistance-related protein (LRP) has roles in multi-drug resistance of tumor cells. Understanding the mechanisms that regulate LRP expression in tumor cells is an important research area. A putative p53 response element in the LRP promoter has been found. Thus, p53-related regulation of LRP expression was explored in this study. We first demonstrated that p53 overexpression inhibited LRP expression both at the protein and mRNA levels. Then, using a dual-luciferase reporter assay, we located the p53 response element to the Y-box (-263 similar to-407) of the LRP promoter, the YB-1 binding site, but not the putative p53 response element. Furthermore, coimmunoprecipitation and chromatin immunoprecipitation showed p53 could bind to the Y-box of the LRP promoter through interaction of p53 with YB-1. YB-1 coexpression with p53 facilitated p53-induced suppression of endogenous LRP expression in MCF-7 cells. HDAC2, a corepressor of p53, was found to also interact with YB-1, and this interaction was mediated by p53. These results showed that the p53-HDAC2 transcriptional repressor complex can bind to the Y-box of the LRP promoter and repress LRP expression through interaction with YB-1. p53-related suppression of LRP expression was completely reversed by doxorubicin treatment and Adr, whereas CP and VP-16 treatment induced LRP expression increased significantly. Inhibition of LRP expression by siRNA facilitated Adr induced apoptosis of MCF-7 cells. All these findings indicated that loss of p53-related suppression of LRP may be the reason for LRP expression increase, and, therefore, chemotherapy resistance in tumor cells. J. Cell. Physiol. 226: 3433-3441, 2011. (C) 2011 Wiley Periodicals, Inc.

We show here that γ-irradiation leads to the translocation of endogenous Werner syndrome helicase (WRN) from nucleoli to nucleoplasmic DNA double strand breaks (DSBs), and WRN plays a role in damage repair. The relocation of WRN after irradiation was perturbed by promyelocytic leukemia protein (PML) knockdown and enhanced by PML IV over-expression. PML IV physically interacted with WRN after irradiation. Amino acids (a.a.) 394 to 433 of PML were necessary for this interaction and the nucleoplasmic translocation of WRN and were involved in DSB repair and cellular sensitivity to γ-irradiation. Taken together, our results provide molecular support for a model in which PML IV physically interacts with and regulates the translocation of WRN for DNA damage repair through its 394–433 a.a. domain.

It is well established that promyelocytic leukaemia nuclear bodies (PML NBs) play important roles in DNA damage responses (DDR). After irradiation, PML NBs dynamically recruit or release important proteins involved in cell-cycle regulation, DNA repair and apoptosis. As PML protein is the key molecule of PML NBs' dynamic assembling, we aimed to characterize the PML-interacting proteins in (60)Co-irradiated MCF-7 cells. A proteomic approach using CoIP, mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 124 proteins that may associate with PML after irradiation. Bioinformatic analysis of the identified proteins showed that most of them were related to characterized PML functions, such as transcriptional regulation, cell-cycle regulation, cell-death regulation and response to stress. Four proteins, B23, MVP, G3BP1 and DHX9, were verified to co-localize with PML differentially before and after ionizing radiation (IR) treatment. The proteins identified in this study will significantly improve our understanding of the dynamic organization and multiple functions of PML NBs in DDR.

AIM:Investigate the molecular mechanism of regulating survivin expression and related signal transduction pathway, molecular cascade reaction and biological effects in activated PBMC.METHODS:The expression of survivin and related proteins were detected by Western blot in PBMC stimulated by PHA and rhIL-2 with or without JAK inhibitor-AG490 treatment, and FCM was performed to analyze cell cycle and cell division.RESULTS:Our results indicated that molecular and cellular reactions in PBMC activated by PHA and rhIL-2 were dependent on time series. At first, the phosphorylation of Stat3 and Stat5 were observed, then, protein levels of CyclinD3 and CyclinE increased, and the stimulated PBMC began to enter to S phage with survivin protein expression was initiated, which at last resulted in cell division with dramatically increasing expression of survivin protein. AG490 could significantly inhibit all these reactions but had no effect on the expressions of the cell cycle inhibitor-P21 and anti-apoptosis protein-Bcl-2.CONCLUSION:The expression of survivin in stimulated PBMC was dependent on the primarily activated JAK-STAT pathway, which upregulated CyclinD3 and CyclinE protein levels, initiated the cell cycle progression, and induced cell cycle-dependent survivin expression, and so survivin was involved in cell division and cell proliferation.

Genomic stability is frequently associated with tumor genesis and progression. Maintaining genomic stability is very important for cells exerting their physiological functions. By it's RBCC domain, promyelocytic leukemia (PML)protein can interact with nearly 50 proteins to form PML nuclear bodies (PML-NBs) which are dynamic, sub-nuclear and multi-protein complex combined with nuclear matrix. As a function unit of interchromatin compartment belonging to compartmentalized nuclear architecture, PML can meet the time-space demands at high level of regulation of eukaryotic gene expression. Recent studies have shown that PML is a new partner of p53, as gatekeeper of genomic stability. It can cooperate with p53 to induce cell apoptosis in DNA damage response. And it can recruit several repair molecules to take part in DNA damage repair. Moreover, PML participates in centrosome duplication checkpoint by regulating aurora A activity and spindle-assembly checkpoint by regulating survivin expression. There fore, PML plays an important role in maintaining genomic stability by regulating cell apoptosis, DNA repair, chromosome duplication and mitosis. And loss or insufficiency of PML expression has been found in several tumors and carcinoma cells. All of those showed that PML may be a key molecule in maintaining genomic stability. Here, we review and discuss the recent development in research of the relationship between PML and genomic stability.

The promyelocytic leukemia (PML) can selectively and dynamically recruit a number of proteins including p53 to form a sub-nuclear multiprotein chamber named PML-NBs. In DNA damage response, p53 is recruited into PML-NBs and modified by phosphorylations and acetylations, which in turn potentiate its transcriptional and pro-apoptotic activities. In contrast, in carcinoma cells, the role of PML in the irradiation induced p53-mediated apoptosis is not precisely understood. In this study, we have used the breast carcinoma cell line, MCF-7, and stably suppressed the expression of PML. Inhibition of PML expression had no detectable effect on the expression of endogenous p53 at the mRNA level; however, a significant decrease of p53 protein was observed. There was also an increase in the p53-MDM2 complexes, which may facilitate p53 protein degradation by the ubiquitin-proteasome pathway, also in irradiation treated cells. The p53 transcriptional activity was attenuated both in unstressed and 10 Gy irradiation treated cells. Moreover, inhibition of PML expression in MCF-7 cells significantly reduced p53 downstream genes, cell cycle arrest gene p21(WAF/cip-1) and pro-apoptotic gene Bax expression, then irradiation-induced apoptosis. These results suggest that PML is a key regulator in the irradiation activated p53 apoptotic pathway in breast carcinoma cells.

To study the functions of survivin gene further,three siRNA segments (S1,S2,S3) specifically targeting survivin were selected.Accordingly RNAi plasmids pTet-U6-S1,pTet-U6-S2,pTet-U6-S3 were(constructed.) The changes of survivin expression were examined by semi-quantitative reverse transcription(polymerase) chain reaction(RT-PCR) and Western-blot analysis after the plasmids had been transfected into HeLaS3.The results show that survivin expression is inhibited at both mRNA and protein levels after the(plasmid) pTet-U6-S3 has been transfected,which targets on the 3' non-encoding region of survivin.The(inhibition) level is much higher than that of S1 sequence reported previously.Compared with that of the(control,the inhibition) ratios of S1,S2,S3 to mRNA of survivin are 20%,12.5% and 40% respectively,and 39.2%,17.0% and 58.6% to its protein.And the effect of the plasmid pTet-U6-S3 on the cell(apoptosis) was analyzed by flow cytometry.The apoptosis of HeLaS3 cells was greatly increased after(transfection) for 48 h.

To investigate the function and molecular mechanism of p21(WAF1/Cip-1) expression in MOLT-4 cells induced by HDAC inhibitor TSA, the expression pattern of p21(WAF1/Cip-1) and the distribution of cell cycle in TSA treated cells were analyzed. The results showed that TSA could effectively induce G(2)/M arrest and apoptosis of MOLT-4 cells. Kinetic experiments demonstrated that p21(WAF1/Cip-1) were upregulated quickly before cell arrested in G(2)/M and began decreasing at the early stage of apoptosis. Meanwhile, the proteasome inhibitor MG-132 could inhibit the decrease of p21(WAF1/Cip-1) at the early stage of apoptosis, which showed that proteasome pathway involved in p21(WAF1/Cip-1) degradation during the TSA induced G(2)/M arrest and apoptosis responses. This study also identified that the protein level of p21(WAF1/Cip-1) was highly associated with the cell cycle change induced by TSA. Compared to cells treated by TSA only, exposure MOLT-4 cells to TSA meanwhile treatment with MG-132 increased the protein level of p21(WAF1/Cip-1) and increased the numbers of cell in G(2)/M-phase, whereas the cell apoptosis were delayed. It is concluded that p21(WAF1/Cip-1) plays a significant role in G(2)/M arrest and apoptosis signaling induced by TSA in MOLT-4 cells.

目的:研究组蛋白去乙酰化酶抑制剂(HDIs)诱导肿瘤细胞产生周期阻滞以及诱导p21WAF1/CIP1表达的分子机制.方法:以人宫颈癌HeLa细胞为模型,用曲古抑菌素A(TSA)处理HeLa细胞,通过流式细胞术检测细胞周期及凋亡,通过Western印迹及RT-PCR检测周期相关分子的蛋白及mRNA的表达;并构建TSA靶分子启动子区的荧光素酶报告质粒,进一步检测TSA的主要作用位点,寻找相关作用途径.结果与结论:TSA可诱导HeLa细胞发生周期阻滞,并呈现明显的剂量和时间依赖关系,加大用药剂量及延长用药时间可诱导凋亡.TSA可显著诱导p21WAF1/CIP1的表达,表现为转录水平的调节,其变化可以指示周期阻滞的程度.p21WAF1/CIP1启动子区3′端是TSA作用的主要位点,同样被TSA诱导表达增加的p53很可能通过与其效应元件的结合而使TSA诱导p21WAF1/CIP1表达的总效果增强.