Persistent mRNA and miRNA expression changes in irradiated baboons.
ABSTRACT: We examined the transcriptome/post-transcriptome for persistent gene expression changes after radiation exposure in a baboon model. Eighteen baboons were irradiated with a whole body equivalent dose of 2.5 or 5?Gy. Blood samples were taken before, 7, 28 and 75-106 days after radiation exposure. Stage I was a whole genome screening for mRNA combined with a qRT-PCR platform for detection of 667 miRNAs. Candidate mRNAs and miRNAs differentially up- or down-regulated in stage I were chosen for validation in stage II using the remaining samples. Only 12 of 32 candidate genes provided analyzable results with two mRNAs showing significant 3-5-fold differences in gene expression over the reference (p?
Project description:Based on gene expression changes measured in the peripheral blood within the first 2 days after irradiation, we predicted a pancytopenia in a baboon model. Eighteen baboons were irradiated with 2.5 or 5 Gy. According to changes in blood cell counts, the surviving baboons (n = 17) exhibited a hematological acute radiation syndrome (HARS) either with or without a pancytopenia. We used a two stage study design where stage I was a whole genome screen (microarrays) for mRNA combined with a qRT-PCR platform for simultaneous detection of 667 miRNAs using a part of the samples. Candidate mRNAs and miRNAs differentially upregulated or downregulated (>2-fold, p < 0.05) during the first 2 days after irradiation were chosen for validation in stage II using the remaining samples and using throughout more sensitive qRT-PCR. We detected about twice as many upregulated (mean 2128) than downregulated genes (mean 789) in baboons developing an HARS either with or without a pancytopenia. From 51 candidate mRNAs altogether, 11 mRNAs were validated using qRT-PCR. These mRNAs showed only significant differences between HARS groups and H0, but not between HARS groups with and without pancytopenia. Six miRNA species (e.g., miR-574-3p, p = 0.009, ROC = 0.94) revealed significant gene expression differences between HARS groups with and without pancytopenia and are known to sensitize irradiated cells. Hence, in particular, the newly identified miRNA species for prediction of pancytopenia will support the medical management decision making.
Project description:Radiosensitivity differs in humans and likely among primates. The reasons are not well known. We examined pre-exposure gene expression in baboons (n = 17) who developed haematologic acute radiation syndrome (HARS) without pancytopenia or a more aggravated HARS with pancytopenia after irradiation. We evaluated gene expression in a two stage study design where stage I comprised a whole genome screen for messenger RNAs (mRNA) (microarray) and detection of 667 microRNAs (miRNA) (real-time quantitative polymerase chain reaction (qRT-PCR) platform). Twenty candidate mRNAs and nine miRNAs were selected for validation in stage II (qRT-PCR). None of the mRNA species could be confirmed during the validation step, but six of the nine selected candidate miRNA remained significantly different during validation. In particular, miR-425-5p (receiver operating characteristic = 0.98; p = 0.0003) showed nearly complete discrimination between HARS groups with and without pancytopenia. Target gene searches of miR-425-5p identified new potential mRNAs and associated biological processes linked with radiosensitivity. We found that one miRNA species examined in pre-exposure blood samples was associated with HARS characterized by pancytopenia and identified new target mRNAs that might reflect differences in radiosensitivity of irradiated normal tissue.
Project description:For effective medical management of radiation-exposed persons after a radiological/nuclear event, blood-based screening measures in the first few days that could predict hematologic acute radiation syndrome (HARS) are needed. For HARS severity prediction, we used microRNA (miRNA) expression changes measured on days one and two after irradiation in a baboon model. Eighteen baboons underwent different patterns of partial or total body irradiation, corresponding to an equivalent dose of 2.5 or 5 Gy. According to changes in blood cell counts (BCC) the surviving baboons (n = 17) exhibited mild (H1-2, n = 4) or more severe (H2-3, n = 13) HARS. In a two Stage study design we screened 667 miRNAs using a quantitative real-time polymerase chain reaction (qRT-PCR) platform. In Stage II we validated candidates where miRNAs had to show a similar regulation (up- or down-regulated) and a significant 2-fold miRNA expression difference over H0. Seventy-two candidate miRNAs (42 for H1-2 and 30 for H2-3) were forwarded for validation. Forty-two of the H1-2 miRNA candidates from the screening phase entered the validation step and 20 of them showed a statistically significant 2-4 fold up-regulation relative to the unexposed reference (H0). Fifteen of the 30 H2-3 miRNAs were validated in Stage II. All miRNAs appeared 2-3 fold down-regulated over H0 and allowed an almost complete separation of HARS categories; the strongest candidate, miR-342-3p, showed a sustained and 10-fold down-regulation on both days 1 and 2. In summary, our data support the medical decision making of the HARS even within the first two days after exposure where diagnostic tools for early medical decision are required but so far missing. The miRNA species identified and in particular miR-342-3p add to the previously identified mRNAs and complete the portfolio of identified mRNA and miRNA transcripts for HARS prediction and medical management.
Project description:It is still difficult to predict the probability of tumor recurrence after resection of hepatocellular carcinoma (HCC). In this study, we set out to identify specific microRNA (miRNA) in microdissected hepatitis B virus (HBV)-related HCC tissue from formalin-fixed paraffin-embedded (FFPE) samples which might be used in predicting early recurrence after HCC resection. Taqman low density arrays were used to detect the 667 miRNA profiles in both the microdissected tumorous and adjacent non-tumorous liver tissues from 20 HCC patients (discovery set) including 10 patients with early tumor recurrence and 10 without early tumor recurrence and to identify the differentially expressed miRNAs related to HCC recurrence. Then quantitative real-time PCR (qRT-PCR) was used to verify the findings in 106 patients (training set), and to develop a predictive assay. The identified miRNAs were further validated in an independent cohort of 112 patients (validation set). Thirty seven miRNAs were identified from 20 HCC patients and validated in 106 HCC patients using qRT-PCR. A significant association was found between miR-29a-5p level in HCC tissues and early tumor recurrence (P = 0.0002). This association was further confirmed in the independent validation set of 112 patients (P = 0.0154). MiR-29a-5p level was significantly associated with both time to tumor recurrence (TTR) (P = 0.0015) and overall survival (OS) (P = 0.0079) in validation set. In the multivariate analyses, miR-29a-5p was identified as an independent factor for TTR, particularly for those patients with early stage of HCC. The sensitivity and specificity of miR-29a-5p for the prediction of early HCC recurrence of BCLC 0/A stage HCC were 74.2% and 68.2%, respectively. These suggest that miR-29a-5p might be a useful marker for the prediction of early tumor recurrence after HCC resection, especially in BCLC 0/A stage HCCs.
Project description:Background:Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is a rapid and sensitive approach to identify miRNA and protein-coding gene expression in plants. However, because of the specially designated reverse transcription and shorter PCR products, very few reference genes have been identified for the quantitative analysis of miRNA expression in plants, and different internal reference genes are needed to normalize the expression of miRNAs and mRNA genes respectively. Therefore, it is particularly important to select the suitable common reference genes for normalization of quantitative PCR of miRNA and mRNA. Results:In this study, a modified reverse transcription PCR protocol was adopted for selecting and validating universal internal reference genes of mRNAs and miRNAs. Eight commonly used reference genes, four stably expressed novel genes in Populus tremula, three small noncoding RNAs and three conserved miRNAs were selected as candidate genes, and the stability of their expression was examined across a set of 38 tissue samples from four developmental stages of poplar clone 84K (Populus alba?×?Populus glandulosa). The expression stability of these candidate genes was evaluated systematically by four algorithms: geNorm, NormFinder, Bestkeeper and DeltaCt. The results showed that Eukaryotic initiation factor 4A III (EIF4A) and U6-2 were suitable for samples of the callus stage; U6-1 and U6-2 were best for the seedling stage; Protein phosphatase 2A-2 (PP2A-2) and U6-1 were best for the plant stage; and Protein phosphatase 2A-2 (PP2A-2) and Oligouridylate binding protein 1B (UBP) were the best reference genes in the adventitious root (AR) regeneration stage. Conclusions:The purpose of this study was to identify the most appropriate reference genes for qRT-PCR of miRNAs and mRNAs in different tissues at several developmental stages in poplar. U6-1, EIF4A and PP2A-2 were the three most appropriate reference genes for qRT-PCR normalization of miRNAs and mRNAs during the plant regeneration process, and PP2A-2 and UBP represent the best reference genes in the AR regeneration stage of poplar. This work will benefit future studies of expression and function analysis of miRNAs and their target genes in poplar.
Project description:PURPOSE: The aim of the present study was to examine the biological differences between seminomas with occult and clinically apparent metastases at the time of diagnosis of the primary tumor to gain insight into the biology of these tumors and facilitate the identification of novel predictors of seminoma metastasis. MATERIALS AND METHODS: Total RNA including small RNAs was isolated from testicular tumors of patients with pure seminoma presenting with lymphogenic metastasis (n?=?5, clinical stage IIb/c) and occult metastasis (n?=?5, clinical stage I). The regulation of biological processes was examined (1) throughout the mRNA transcriptome (whole genome microarrays, 8×60 K Array, Agilent with 4 samples/group) and (2) the miRNA transcriptome employing small RNA next generation sequencing (SOLID, Life Technologies with 5 samples/group). Protein coding genes (mRNAs) and small RNAs showing a significant (?2-fold) difference between the groups were identified. Finally (3), we examined 95 candidate miRNAs in 36 apparent metastasized and another 5 occult metastasized seminoma using logistic regression analysis. RESULTS: Among 19,596 genes, on average 12,894 mRNAs appeared expressed (65.8%, SD+/-2.4; range, 62.0-69.3%) and 16.99×106/13.94×106 small RNA reads were identified for apparent/occult metastasized seminoma. These reads on average convert into 9,901/9,675 small RNAs including 422/404 mature microRNAs. None of these mRNAs/small RNAs met our selection criteria for candidate genes. From 95 candidate miRNAs 44 appeared expressed, with 3 of them showing weak but significant (p?=?0.05) differences among both groups. CONCLUSIONS: Occult and apparent metastasized seminomas are biologically almost indistinguishable and probably represent no separate tumor entities. These findings may simplify future research on seminoma metastasis.
Project description:This research was performed to estimate the potential effects of Di (2-ethylhexyl) phthalate (DEHP) on changes of ovarian miRNA expression profile during mouse primordial follicle assembly using miRNAs-seq analysis. The ovaries of newborn mice were collected and in vitro cultured with different concentration of DEHP for 72 h. Then they were prepared for miRNAs-seq analysis. The results indicated that DEHP exposure altered ovarian miRNA expression profile of newborn mice. Eighteen differentially expressed miRNAs were screened after 100 ?M DEHP exposure. The target mRNAs of differentially expressed miRNAs were predicted and further analyzed through gene ontology (GO) enrichment analysis and pathway enrichment analysis. Our results showed that the differentially expressed miRNAs from DEHP exposure can regulate ovarian development by targeting mRNAs involved in MAPK, mTOR, FoxO signaling pathways. Three miRNAs of miR-32-5p, miR-19a-3p, and miR-141-3p were randomly selected from the differentially expressed miRNAs to quantify their expression level by miRNA qRT-PCR. The results of qRT-PCR and miRNA-seq were consistent. Considering one of its target gene PTEN of miR-19a-3p and the decreased level of pAKT and increased Bax/Bcl-2 under DEHP exposure, we speculated that the altered expression of miR-19a-3p by DEHP exposure affected mouse primordial follicle assembly via PI3K/AKT1/mTOR signaling pathway. Epigenetic changes are one of the most important targets of toxicant exposure. The effects of DEHP exposure on microRNA (one of the epigenetic regulators) expression profile were uncovered to enrich the research on relationship of epigenetics and toxicant exposure.
Project description:BACKGROUND:MicroRNAs (miRNAs) play vital roles in acute inflammatory and antiviral responses during influenza A virus (IAV) infection. The Xijiao Dihuang decoction combined with Yinqiao powder (XDY) is applied to remedy viral pneumonia in China and its therapeutic efficacy in pneumonic mice challenged with IAV was demonstrated; however, the underlying mechanisms remain elusive. Thus, this study aimed to explore the miRNA-mRNA profiles in the lungs of IAV-infected mice and investigate the therapeutic mechanisms of XDY involving miRNAs and associated pathways. METHODS:We detected the cellular miRNA contents in the lungs of mice treated with XDY (23?g/kg/d) for A/FM/1/47 (H1N1) (FM1) infection at 4?days postinoculation (dpi) and 7 dpi. MiRNA and mRNA high-throughput sequencing analyses, and miRNA and mRNA qRT-PCR analyses were used to detect and verify the relevant miRNAs and mRNAs. Conjoint analysis, GO enrichment analysis, and KEGG database analysis were applied to identify the miRNA-mRNA regulatory relationships. RESULTS:The quantities of differentially expressed miRNAs and mRNAs were upregulated over time. The data showed that 104 miRNAs and 3485 mRNAs were differentially expressed after challenge with FM1 on day 4, while 191 miRNAs and 6126 mRNAs were differentially expressed on day 7. The GO enrichment analysis and KEGG database data showed that the differentially expressed miRNAs and mRNAs were mainly enriched in JNK activity, MAPK phosphatase activity, and the TLR, Jak-STAT and TNF signalling pathways after treatment of FM1 infection with XDY. Generally, the expression trends of differentially expressed miRNAs and mRNAs based on the qRT-PCR results exhibited good consistency with the results of the high-throughput sequencing analysis. CONCLUSIONS:MiRNAs and mRNAs were differentially expressed during FM1 infection. The therapeutic mechanisms of XDY in FM1-infected mice, might be related to regulating antiviral immunity and ameliorating excessive inflammatory responses by modulating the expression of dysregulated miRNAs and mRNAs involved in the ERK/JNK-AP-1, and IFN-?/STAT signalling pathways.
Project description:Breast cancer is one of the most important causes of cancer-related deaths worldwide in women. In addition to gene expression studies, the progressing work in the miRNA area including miRNA microarray studies, brings new aspects to the research on the cancer development and progression. Microarray technology has been widely used to find new biomarkers in research and many transcriptomic microarray studies are available in public databases. In this study, the breast cancer miRNA and mRNA microarray studies were collected according to the availability of their data and clinical information, and combined by a newly developed ranking-based meta-analysis approach to find out candidate miRNA biomarkers (meta-miRNAs) that classify breast cancers according to their grades and explain the relation between miRNAs and mRNAs. This approach provided meta-miRNAs specific to breast cancer grades, pointing out let-7 family members as grade classifiers. The qRT-PCR studies performed with independent breast tumors confirmed the potential biomarker role of let-7 family members (meta-miRNAs). The concordance between the meta-mRNAs and miRNA target genes specific to tumor grade (common genes) supported the idea of mRNAs as miRNA targets. The pathway analysis results showed that most of the let-7 family miRNA targets, and also common genes, were significantly taking part in cancer-related pathways. The qRT-PCR studies, together with bioinformatic analyses, confirmed the results of meta-analysis approach, which is dynamic and allows combining datasets from different platforms.
Project description:BACKGROUND:Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease with various clinical manifestations. MicroRNAs (miRNAs) and immunometabolism are recognized as key elements in SLE pathogenesis; however, the relationship between miRNAs in peripheral blood mononuclear cells (PBMCs) and metabolism in SLE remains unclear. METHODS:We detected PBMC miRNA and mRNA profiles from 3 pooled SLE patients and 3 healthy controls (HCs) using next-generation sequencing, predicted miRNA targets in dysregulated mRNAs, predicted functions and interactions of differentially expressed genes using bioinformatics analysis, validated candidate miRNAs using qRT-PCR, and investigated the association between the expression of candidate miRNAs and SLE clinical characteristics. Moreover, we validated the direct and transcriptional regulatory effect of NovelmiRNA-25 on adenosine monophosphate deaminase 2 (AMPD2) using a dual-luciferase reporter assay and western blot and confirmed AMPD2 mRNA and protein expression in PBMCs using qRT-PCR and western blot, respectively. RESULTS:Multilayer integrative analysis of microRNA and mRNA regulation showed that 10 miRNAs were down-regulated and 19 miRNAs were up-regulated in SLE patient PBMCs compared with HCs. Bioinformatics analysis of regulatory networks between miRNAs and mRNAs showed that 19 miRNAs were related to metabolic processes. Two candidate miRNAs, NovelmiRNA-25 and miR-1273h-5p, which were significantly increased in the PBMCs of SLE patients (P?<?0.05), represented diagnostic biomarkers with sensitivities of 94.74% and 89.47%, respectively (area under the curve?=?0.574 and 0.788, respectively). NovelmiRNA-25 expression in PBMCs was associated with disease activity in SLE patients, in both active and stable groups (P?<?0.05). NovelmiRNA-25 overexpression downregulated AMPD2 expression in HEK293T cells through direct targeting of the AMPD2 3'UTR (P?<?0.01), while inhibition of NovelmiRNA-25 activity led to increased AMPD2 expression (P?<?0.01). NovelmiRNA-25 overexpression also downregulated AMPD2 protein expression in HEK293T cells; AMPD2 protein expression in SLE patient PBMCs was decreased. Our results show that differentially expressed miRNAs play an important role in SLE. CONCLUSIONS:Our data demonstrate a novel mechanism in SLE development that involves the targeting of AMPD2 expression by NovelmiRNA-25. miRNAs may serve as novel biomarkers for the diagnosis and evaluation of disease activity of SLE and represent potential therapeutic targets for this disease.