Real-time quantitative PCR analysis of human malignant pleural mesothelioma tissue specimens
ABSTRACT: BACKGROUND: Malignant pleural mesothelioma (MPM) is difficult to distinguish from reactive mesothelial proliferations (RMPs). MicroRNAs (miRNAs) are small non-coding RNA-strands (~22 nucleotides) that post-transcriptionally regulate gene-expression. Studies have shown that miRNAs are potential diagnostic markers in other cancers; however, it is uncertain whether miRNAs are useful biomarkers for differentiating MPM from RMP. OBJECTIVE: To identify differentially expressed microRNAs which can aid in the diagnostics of MPM. METHODS: We screened with a quantitative RT-PCR (RT-qPCR)-based platform the expression of 742 miRNAs in formalin-fixed paraffin-embedded (FFPE) preoperative diagnostic biopsies, surgically resected MPM-specimens previously treated with chemotherapy, and corresponding non-neoplastic pleura (NNP) from 5 patients. RESULTS: By comparing the change in microRNA expression in patient-matched tissue samples, we identify 14 miRNAs which exhibit statistically significant deregulation between sample groups. CONCLUSION: Based on this initial screening of miRNA expression, we have identified 14 miRNAs which are potential diagnostic biomarkers and may aid in diffferentiating RMP from MPM. qPCR microRNA expression profiling. MicroRNA expression in surgical tissue samples and presurgery diagnostic biopsies of malignant pleural mesotheliomas was compared to corresponding patient-matched non-neoplastic pleura. Patient-matched diagnostic biopsies from still chemotherapy-naïve patients were included to test for chemotherapy-induced changes in microRNA expression.
Project description:BACKGROUND: Malignant pleural mesothelioma (MPM) is difficult to distinguish from reactive mesothelial proliferations (RMPs). MicroRNAs (miRNAs) are small non-coding RNA-strands (~22 nucleotides) that post-transcriptionally regulate gene-expression. Studies have shown that miRNAs are potential diagnostic markers in other cancers; however, it is uncertain whether miRNAs are useful biomarkers for differentiating MPM from RMP. OBJECTIVE: To identify differentially expressed microRNAs which can aid in the diagnostics of MPM. METHODS: We screened with a quantitative RT-PCR (RT-qPCR)-based platform the expression of 742 miRNAs in formalin-fixed paraffin-embedded (FFPE) preoperative diagnostic biopsies, surgically resected MPM-specimens previously treated with chemotherapy, and corresponding non-neoplastic pleura (NNP) from 5 patients. RESULTS: By comparing the change in microRNA expression in patient-matched tissue samples, we identify 14 miRNAs which exhibit statistically significant deregulation between sample groups. CONCLUSION: Based on this initial screening of miRNA expression, we have identified 14 miRNAs which are potential diagnostic biomarkers and may aid in diffferentiating RMP from MPM. Overall design: qPCR microRNA expression profiling. MicroRNA expression in surgical tissue samples and presurgery diagnostic biopsies of malignant pleural mesotheliomas was compared to corresponding patient-matched non-neoplastic pleura. Patient-matched diagnostic biopsies from still chemotherapy-naïve patients were included to test for chemotherapy-induced changes in microRNA expression.
Project description:Malignant pleural mesothelioma (MPM) is an aggressive tumor with a dismal overall survival (OS) and to date few molecular markers are available to guide patient management. This study aimed to identify a prognostic miRNA signature in MPM patients who did not undergo tumor resection. Whole miRNA profiling using a microarray platform was performed on 26 unresected MPM with distinct clinical outcome: 15 patients had aggressive disease (OS<12 months) and 11 patients indolent disease (OS>36 months). Three prognostic miRNAs (mir-99a, let-7c and miR-125b) encoded at the same cluster (21q21) were selected for further validation and tested on publicly available miRNA sequencing data (TCGA) from 72 MPM patients with survival data. A risk model was built based on these 3 miRNAs that was validated by quantitative PCR in an independent set of 30 MPM patients. High risk patients had shorter median OS (7.6 months) as compared with low-risk patients (median not reached). In the multivariate Cox model, a high risk score was an independently associated with shorter OS (HR=3.14; 95% CI, 1.18–8.34; P=0.022). Our study identified that the downregulation of the miR-99a/let-7/miR-125b miRNA cluster predicts poor outcome in unresected MPM. Overall design: In the present study the whole miRNA profile was evaluated in a cohort of 26 malignant pleural mesothelioma (MPM) sample derived from patients who did not undergo surgery and in 3 nonmalingnat pleura (NMP) as normal control (NC). The comparison between MPM and NC samples allowed to identify miRNA linked to tumorigenesis. The second aim was the identification of miRNA correlated with overall survival (OS); for this purpose the MPM patients were divided based on the survival rate in: i) long survivors (LS; patients whose OS was greater than 36 months); and ii) short survivors (SS; patients died within 12 months from the diagnosis).
Project description:The objectives of the study: 1. Does the phase of the menstrual cycle alter microRNA (miRNA) plasma profiles in healthy women of reproductive age and in women with endometriosis? 2. Does this alter prospects for development of a miRNA-based diagnostic test for endometriosis? Prospectively recruited asymptomatic control women and women with surgically diagnosed endometriosis (n = 8 in each group) were included. Each patient provided blood samples in the early proliferative, late proliferative and mid luteal phases of the menstrual cycle (n = 47 total plasma samples). The cycle phase was verified by hormonal profile. RNA was extracted from each sample and expression of microRNAs was assessed using TaqMan Low Density Human miRNA arrays.
Project description:Osteosarcoma (OS) is the primary bone tumor in children and young adults. Currently, there are no reliable, non-invasive biological markers to detect the presence or progression of disease, assess therapy response, or provide upfront prognostic insights. Using a qPCR-based platform that analyzes more than 750 miRNAs, we analyzed control and diseased-associated plasma from a genetically engineered mouse model of OS to identify a profile of four plasma miRNAs. Plasma from mice with OS were profiled for miRNAs and compared with the profile of plasma from disease-free mice
Project description:Osteosarcoma (OS) is the primary bone tumor in children and young adults. Currently, there are no reliable, non-invasive biological markers to detect the presence or progression of disease, assess therapy response, or provide upfront prognostic insights. Using a qPCR-based platform that analyzes more than 750 miRNAs, we analyzed control and diseased-associated plasma from human cases of OS to identify a profile of differentially expressed miRNAs. Comprehensive miRNA expression profiling was completed using the Exiqon miRNome platform (human panels I+II, V3) on 20 disease samples (10 localized, 10 metastatic) and 15 healthy controls.
Project description:Purpose: There is a quest for novel non-invasive diagnostic markers for the detection of breast cancer. The goal of this study is to identify circulating microRNA signatures using a cohort of Asian Chinese breast cancer patients, and to compare microRNA profiles between tumour and serum samples. Experimental design: MicroRNAs from paired breast cancer tumours, normal tissue and serum samples derived from 32 patients were comprehensively profiled using microarrays (1300 microRNAs against tumour and normal tissues) or LNA RT-PCR panels (742 microRNAs against serum samples). Serum samples from healthy individuals (n=22) were also employed as normal controls. Significant serum microRNAs, identified by logistic regression, were validated in an independent set of serum samples from patients (n=82) and healthy controls (n=53). Results: The 20 most significant microRNAs differentially expressed in breast cancer tumours included miR-21, miR-10b, and miR-145, previously shown to be dysregulated in breast cancer. Interestingly, 16 of the 20 most significant microRNAs differentially expressed in serum samples were novel. MiR-1, miR-92a, miR-133a and miR-133b were identified as the most important diagnostic markers, and were successfully validated; receiver operating characteristic curves derived from combinations of these microRNAs exhibited areas under the curves of 0.944-0.946. Only seven microRNAs were overexpressed in both tumours and serum, suggesting that microRNAs may be released into the serum selectively. Conclusion: The clinical employment of microRNA signatures as a non-invasive diagnostic strategy is promising, but should be further validated for different subtypes of breast cancers. Blood samples were collected in Becton Dickinson (Franklin Lakes, NJ) Vacutainer SST tubes. Serum was harvested by centrifugation at 2200g after allowing blood to clot for 30mins. 32 patient samples and 22 samples from healthy controls were obtained for profiling. Sera samples were stored at -80oC.
Project description:Malignant Pleural Mesothelioma (MPM) is an aggressive cancer that is often diagnosed at an advanced stage and is characterized by a long latency period (20-40 years between initial exposure and diagnosis) and prior exposure to asbestos. Currently accurate diagnosis of MPM is difficult due to the lack of sensitive biomarkers, and despite minor improvements in treatment, median survival rates do not exceed 12 months. Accumulating evidence suggests that aberrant expression of long non-coding RNAs (lncRNAs) play an important functional role in cancer biology. LncRNAs are a class of recently discovered non-protein coding RNAs >200 nucleotides in length with a role in regulating transcription. Here we used NCode long noncoding microarrays to identify differentially expressed lncRNAs potentially involved in MPM pathogenesis. High priority candidate lncRNAs were selected on the basis of statistical (P<0.05) and biological significance (>3-fold difference). Expression levels of 9 candidate lncRNAs were technically validated using RT-qPCR, and biologically validated in three independent test sets: (1) 57 archived MPM tissues obtained from extrapleural pneumonectomy patients, (2) 15 cryopreserved MPM and 3 benign pleura, and (3) an extended panel of 10 MPM cell lines. RT-qPCR analysis demonstrated consistent up-regulation of these lncRNAs in independent datasets. ROC curve analysis showed that two candidates were able to separate benign pleura and MPM with high sensitivity and specificity, and were associated with nodal metastases and survival following induction chemotherapy. These results suggest that lncRNAs have potential to serve as biomarkers in MPM. To identify mRNA and lncRNA biomarkers associated with malignant pleural mesothelioma (MPM), we performed gene expression array analysis on 4 MPM cell lines (H28, MM05, MSTO-211H, H226) compared to the immortalised mesothelial line (MeT-5A). All cell lines were profiled in duplicate. Synthesis of the labelled first strand cDNA was conducted using the Superscript Plus Direct cDNA labeling system with starting material of 10ug total RNA. The labeled dNTP mix was added to the reaction to generate labeled second strand cDNA. Following the hydrolysis reaction, single-stranded cDNA was purified using low elution volume spin cartridges included in the purification module. Samples were labelled using Alexa Fluor 555 dyes. Samples were then hybridised to NCode Long Noncoding RNA Microarrays. Slides were scanned using an Agilent Scanner. Genes differentially expressed between Met-5A and the MPM cell lines were identified on the basis of P-value and fold change.
Project description:Malignant pleural mesothelioma (MPM) is a highly lethal, poorly understood neoplasm that is typically associated with asbestos exposure. We performed transcriptional profiling using high-density oligonucleotide microarrays containing approximately 22,000 genes to elucidate potential molecular and pathobiological pathways in MPM using discarded human MPM tumor specimens (n=40), normal lung specimens (n=4), normal pleura specimens (n=5), and MPM and SV40-immortalized mesothelial cell lines (n=5). In global expression analysis using unsupervised clustering techniques, we found two potential subclasses of mesothelioma which correlate loosely with tumor histology. We also identified sets of genes with expression levels that distinguish between multiple tumor subclasses, normal and tumor tissues, and tumors with different morphologies. Microarray gene expression data were confirmed using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and protein analysis for three novel candidate oncogenes (NME2, CRI1, and PDGFC) and one candidate tumor suppressor (GSN). Finally, we used bioinformatics tools (i.e. software) to create and explore complex physiological pathways that may be relevant in mesothelioma tumorigenesis, pathobiology, or both. Tissues and cell lines profiled using microarrays. Discarded MPM surgical specimens (n=40), normal pleura specimens (n=5), and normal lung specimens (n=4) were freshly collected (and snap frozen) from patients who underwent surgery at Boston’s Brigham and Women’s Hospital (BWH) between October 1998 and August 2000. All of these patients underwent extrapleural pneumonectomy with heated intra-pleural cisplatin chemotherapy delivered after the specimens were removed. All normal specimens were obtained from patients who were never diagnosed with MPM. Two human MPM cell lines (MS589 and MS428) were kindly provided by Jonathan A. Fletcher, M.D., Department of Pathology, BWH. The JMN1B MPM cell line19,20 has been described previously. The SV40-immortalized, non-tumorigenic mesothelial cell line (Met-5A)21 and the MPM cell line MSTO-211H22 were purchased from the American Type Culture Collection. Normal tissues were obtained from additional consented patients undergoing treatment for diseases other than MPM. All MPM samples used in these studies contained relatively pure tumor (greater than 50% tumor cells per high power field examined in a section adjacent to the tissue used). The microscopic slides from the patients’ resection specimens were reviewed by one of the authors (J.G.), and the diagnosis and histologic subclassification of MPM confirmed in all cases. Linked clinical and pathological data were obtained for all patients who contributed tumor specimens. Specimens and data were rendered anonymous to protect patient confidentiality. Studies utilizing human tissues were approved by and conducted in accordance with the policies of the Institutional Review Board at BWH. SUBMITTER_CITATION: Gordon, G. J., Rockwell, G. N., Jensen, R. V., Rheinwald, J. G., Glickman, J.N., Aronson, J. P., Pottorf, B. J., Nitz, M. D., Richards, W. G., Sugarbaker, D. J., and Bueno, R. Identification of novel candidate oncogenes and tumor suppressors in malignant pleural mesothelioma using large-scale transcriptional profiling. American Journal of Pathology, 166: 1827-1840, 2005.
Project description:MicroRNA expression profiles of malignant pleural mesothelioma (MPM) specimens were analyzed to identify novel microRNA that are potentially involved in the oncogenic transformation of human pleural cells. In addition to several novel MPM-associated microRNAs, we observed that the expression level of microRNA-1 was significantly lower in tumors as compared to normal pleural specimens. Subsequently, pre-mir of microRNA-1 was introduced into MPM cell lines to overexpress this microRNA. Phenotypic changes of these altered cells were assayed. The cellular proliferation rate was significantly inhibited after overexpression of microRNA-1. Early and late apoptosis were measured by Annexin V and TUNEL assays, respectively. Taken together, these data suggested that overexpression of microRNA-1 induced apoptosis in these MPM cell lines, acting as a tumor suppressor. We confirmed our observations by assessing in the transduced MPM cells cell cycle-related genes, pro-apoptotic and anti-apoptotic genes, which all showed coordinated, significant changes characteristic of the apoptotic phenotype. Thus, further investigation and validation of our microRNA database of MPM may elucidate previously unrecognized molecular pathways and/ or mechanisms by identifying novel microRNAs that are involved in malignant transformation. Our study has now found microRNA-1 to be one of these MPM-associated microRNAs, with potential pathogenic and therapeutic significance. MicroRNA microarray transcriptional profiling studies of 25 MPM primary tumors as well 6 normal pleural samples from an unmatched patient cohort as normal comparators were performed.
Project description:Mature microRNA transcripts were analyzed in 9 CHO cell lines growing at different proliferation rates. The goal was to identify microRNAs that correlate positively or negatively with CHO proliferation rates. A common reference design was chosen, where all samples were hybridized against a pool of all RNA samples.