Pre-Exposure Gene Expression in Baboons with and without Pancytopenia after Radiation Exposure.
ABSTRACT: 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: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: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:Differently expressed microRNAs (miRNAs) in the plasma of lung adenocarcinoma (LA) patients might serve as biomarkers for LA detection. MiRNA expression profiling was performed using Exiqon panels followed by the verification (30 LA VS. 10 healthy controls (HCs)) with quantitative reverse transcription polymerase chain reaction (qRT-PCR) in the screening phase. Identified miRNAs were confirmed through training (42 LA VS. 32 HCs) and testing stages (66 LA VS. 62 HCs) by using qRT-PCR based absolute quantification methods. A total of six up-regulated plasma miRNAs (miR-19b-3p, miR-21-5p, miR-221-3p, miR-409-3p, miR-425-5p and miR-584-5p) were identified. The six-miRNA panel could discriminate LA patients from HCs with areas under the receiver operating characteristic curve of 0.72, 0.74 and 0.84 for the training, testing and the external validation stage (33 LA VS. 30 HCs), respectively. All the miRNAs identified except miR-584-5p were significantly up-regulated in LA tissues. MiR-19-3p, miR-21-5p, miR-409-3p and miR-425-5p showed high expression in arterial plasma with borderline significance. Additionally, miR-19-3p, miR-21-5p and miR-221-3p were significantly up-regulated in exosomes extracted from LA peripheral plasma samples. In conclusion, we identified a six-miRNA panel in peripheral plasma which might give assistance to the detection of LA at least for Asian population to a certain extent.
Project description:INTRODUCTION: Circulating microRNAs (miRNAs) are easily accessible and have already proven to be useful as prognostic markers in cancer patients. However, their origin and function in the circulation is still under discussion. In the present study we analyzed changes in the miRNAs in blood plasma of head and neck squamous cell carcinoma (HNSCC) patients in response to radiochemotherapy and compared them to the changes in a cell culture model of primary HNSCC cells undergoing simulated anti-cancer therapy. MATERIALS AND METHODS: MiRNA-profiles were analyzed by qRT-PCR arrays in paired blood plasma samples of HNSCC patients before therapy and after two days of treatment. Candidate miRNAs were validated by single qRT-PCR assays. An in vitro radiochemotherapy model using primary HNSCC cell cultures was established to test the possible tumor origin of the circulating miRNAs. Microarray analysis was performed on primary HNSCC cell cultures followed by validation of deregulated miRNAs via qRT-PCR. RESULTS: Unsupervised clustering of the expression profiles using the six most regulated miRNAs (miR-425-5p, miR-21-5p, miR-106b-5p, miR-590-5p, miR-574-3p, miR-885-3p) significantly (p?=?0.012) separated plasma samples collected prior to treatment from plasma samples collected after two days of radiochemotherapy. MiRNA profiling of primary HNSCC cell cultures treated in vitro with radiochemotherapy revealed differentially expressed miRNAs that were also observed to be therapy-responsive in blood plasma of the patients (miR-425-5p, miR-21-5p, miR-106b-5p, miR-93-5p) and are therefore likely to stem from the tumor. Of these candidate marker miRNAs we were able to validate by qRT-PCR a deregulation of eight plasma miRNAs as well as miR-425-5p and miR-93-5p in primary HNSCC cultures after radiochemotherapy. CONCLUSION: Changes in the abundance of circulating miRNAs during radiochemotherapy reflect the therapy response of primary HNSCC cells after an in vitro treatment. Therefore, the responsive miRNAs (miR-425-5p, miR-93-5p) may represent novel biomarkers for therapy monitoring. The prognostic value of this exciting observation requires confirmation using an independent patient cohort that includes clinical follow-up data.
Project description: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?<?0.0001). From 667 candidate miRNAs, 290 miRNA were eligible for analysis with 21 miRNAs independently validated using qRT-PCR. These miRNAs showed persistent expression changes on each day and over days 7-106 days after exposure (n?=?7). In particular miR-212 involved in radiosensitivity and immune modulation appeared persistently and 48-77-fold up-regulated over the entire time period. We are finally trying to put our results into a context of clinical implications and provide possible hints on underlying molecular mechanisms to be examined in future studies.
Project description:Dysregulated expression of specific microRNAs (miRNAs) in serum has been recognised as promising diagnostic biomarkers for colorectal cancer (CRC). In the initial screening phase, a total of 32 differentially expressed miRNAs were selected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) based Exiqon panel with 3 CRC pool samples and 1 normal control (NC) pool. Using qRT-PCR, selected serum miRNAs were further confirmed in training (30 CRC VS. 30 NCs) and testing stages (136 CRC VS. 90 NCs). We identified that serum levels of miR-19a-3p, miR-21-5p and miR-425-5p were significantly higher in patients with CRC than in NCs. The areas under the receiver operating characteristic (ROC) curve of the three-miRNA panel were 0.86, 0.74 and 0.87 for the training, testing and the external validation stages (30 CRC VS. 18 NCs), respectively. Significantly, elevated expression of the three miRNAs was also observed in CRC tissues (n = 24). Furthermore, the expression levels of the three miRNAs were significantly elevated in exosomes from CRC serum samples (n = 10). In conclusion, we identified a serum three-miRNA panel for the diagnosis of CRC.
Project description:Blood-stasis syndrome (BSS) is one of the Traditional Chinese medicine (TCM) syndrome differentiations that are commonly seen in stroke and ischemic heart diseases; however, the BSS differentiation criterion is not standardized. More objective biomarkers for BSS diagnosis are needed.Acute ischemic stroke (AIS) or unstable angina (UA) patients with BSS and healthy controls were enrolled. The miRNA and mRNA expression profiles of UA patients and AIS patients were compared to those of healthy controls to identify the differentially expressed miRNA and mRNA of BSS. Bioinformatics analysis was used to identify significantly deregulated miRNAs and mRNAs correlated to BSS. QRT-PCR was performed to validate the bioinformatics analysis results.Approximately 401 mRNAs and 11 miRNAs were differentially expressed in both UA and AIS patients compared to healthy controls. Gene ontology (GO) functional analysis was performed, and multiple GO terms were enriched. Among the overlapping DE miRNAs and mRNAs, miR-146b-5p, -199a-5p and 23 targeted mRNAs were pivotal genes in the BSS genomic characteristics. These 2 miRNAs and 23 mRNAs formed network-type biomarkers for BSS.The genomic characteristics of BSS were shown in this study. miR-146b-5p, -199a-5p and the 23 targeted mRNAs formed a diagnostic network for BSS. Further improvement and validation of this diagnostic network might lead to more objective diagnostic criteria for BSS.
Project description:Breast cancer (BC) is the most common invasive cancer in women, and it imposes a heavy burden on patients. microRNAs (miRNAs/miRs) have been found to play an important role in the development of tumors, but their role in the malignant progression of BC is unclear. In the present study, the expression level of miR?425?5p was examined in patients with BC, and its association with prognosis was investigated. In vitro experiments were performed to examine role of miR?425?5p in the development of BC cells. A downstream target gene of miR?425?5p was predicted using a miRNA target prediction tool and validated with a luciferase reporter assay. It was found that miR?425?5p expression was increased in BC tissues and cell lines, and was associated with tumor size, clinical stage, lymph node metastasis, distant metastasis and poor overall survival in patients with BC. Knockdown of miR?425?5p in BC cell lines inhibited proliferation and migration. PTEN was identified as a downstream target gene of miR?425?5p. Overexpression of PTEN was demonstrated to partially inhibit the promotional effect of miR?425?5p on cell proliferation and migration. Taken together, miR?425?5p is associated with poor prognosis, and promotes cell proliferation and migration via PTEN. Thus, miR?425?5p may serve as a therapeutic and prognostic marker for BC.
Project description:Purpose:Cervical squamous cell carcinoma (CSCC) seriously affects women's health worldwide, and it is of great significance to illuminate the specific role of circRNAs in CSCC. Materials and Methods:Three mRNA datasets, two miRNA datasets and one circRNA dataset of CSCC, downloaded from GEO, were utilized in this study. Differentially expressed mRNAs (DEmRNAs), miRNAs (DEmiRNAs) and circRNAs (DEcircRNAs) were identified, and a ceRNA (DEcircRNA-DEmiRNA-DEmRNA) regulatory network was constructed. GO and pathway analyses of DEcircRNAs and DEmRNAs in the ceRNA regulatory network were performed. Quantitative real-time polymerase chain reaction (qRT-PCR) validation of the expression of the selected DEmRNAs, DEmiRNAs and DEcircRNAs was performed. Results:A total of 1356 DEmRNAs, 13 DEmiRNAs and 77 DEcircRNAs were obtained. The ceRNA network contained 3 circRNA-miRNA pairs and 158 miRNA-mRNA pairs, including 3 circRNAs, 3 miRNAs, and 138 mRNAs. Functional annotation of DEmRNAs in the ceRNA regulatory network revealed that these DEmRNAs were significantly enriched in cell cycle, p53 signalling pathway and DNA replication. The qRT-PCR results were generally consistent with those of our integrated analysis. Conclusion:In conclusion, we speculate that the regulation of the hsa_circ_0000069/hsa-miR-125b-5p/CDKN2A and hsa_circ_0020594/hsa-let-7c-5p/CCNB2 axes may be involved in CSCC.
Project description:Blood stasis syndrome (BSS) has been considered to be the major type of syndromes in unstable angina (UA) patients. The aim of this study was to find the systems biology-based microRNA (miRNA) and mRNA expression biomarkers for BSS of UA. We identified 1081 mRNAs and 25 miRNAs differentially expressed between BSS of UA patients and healthy controls by microarrays. We used DAVID, miRTrail, and the protein-protein interactions method to explore the related pathways and networks of differentially expressed miRNAs and mRNAs. By combining the results of pathways and networks, we found that the upregulation of miR-146b-5p may induce the downregulation of CALR to attenuate inflammation and the upregulation of miR-199a-5p may induce the downregulation of TP53 to inhibit apoptosis in BSS of UA patients. The expression patterns of miR-146b-5p, miR-199a-5p, CALR, and TP53 were confirmed by qRT-PCR in an independent validation cohort including BBS of UA patients, non-BBS of UA patients, and healthy controls. miR-146b-5p, miR-199a-5p, CALR, and TP53 could be significant biomarkers of BSS of UA patients. The systems biology-based miRNA and mRNA expression biomarkers for the BSS of UA may be helpful for the further stratification of UA patients when deciding on interventions or clinical trials.