RNA Profiling Reveals a Common Mechanism of Histone Gene Downregulation and Complementary Effects for Radioprotectants in Response to Ionizing Radiation.

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.

Effects of Amifostine Pre-treatment on MIRNA, LNCRNA, and MRNA Profiles in the Hypothalamus of Mice Exposed to 60Co Gamma Radiation.

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.

Detecting RNA-RNA interactions in E. coli using a modified CLASH method.

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.

Nuclear retention of the lncRNA SNHG1 by doxorubicin attenuates hnRNPC-p53 protein interactions.

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.

Promyelocytic leukemia protein interacts with werner syndrome helicase and regulates double-strand break repair in γ-irradiation-induced DNA damage responses.

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 overexpression. 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.

p53 Suppresses lung resistance-related protein expression through Y-box binding protein 1 in the MCF-7 breast tumor cell line.

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~-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.

Functional proteomic analysis of promyelocytic leukaemia nuclear bodies in irradiation-induced MCF-7 cells.

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.

[The molecular mechanism of survivin expression in activated human peripheral lymphocytes].

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.

[The mechanisms of p21WAF1/Cip-1 expression in MOLT-4 cell line induced by TSA].

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.