Association of MiR-126 with Soluble Mesothelin-Related Peptides, a Marker for Malignant Mesothelioma

BACKGROUND: Improved detection methods for diagnosis of malignant pleural mesothelioma (MPM) are essential for early and reliable detection as well as treatment. Since recent data point to abnormal levels of microRNAs (miRNAs) in tumors, we hypothesized that a profile of deregulated miRNAs may be a marker of MPM and that the levels of specific miRNAs may be used for monitoring its progress. METHODS AND RESULTS: miRNAs isolated from fresh-frozen biopsies of MPM patients were tested for the expression of 88 types of miRNA involved in cancerogenesis. Most of the tested miRNAs were downregulated in the malignant tissues compared with the normal tissues. Of eight significantly downregulated, three miRNAs were assayed in cancerous tissue and adjacent non-cancerous tissue sample pairs collected from 27 formalin-fixed, paraffin-embedded MPM tissues by quantitative RT-PCR. Among the miRNAs tested, only miR-126 significantly remained downregulated in the malignant tissues. Furthermore, the performance of the selected miR-126 as biomarker was evaluated in serum samples of asbestos-exposed subjects and MPM patients and compared with controls. MiR-126 was not affected by asbestos exposure, whereas it was found strongly associated with VEGF serum levels. Levels of miR-126 in serum, and its levels in patients' serum in association with a specific marker of MPM, SMRPs, correlate with subjects at high risk to develop MPM. CONCLUSIONS AND SIGNIFICANCE: We propose miR-126, in association with SMRPs, as a marker for early detection of MPM. The identification of tumor biomarkers used alone or, in particular, in combination could greatly facilitate the surveillance procedure for cohorts of subjects exposed to asbestos

MicroRNA come biomarcatori nella diagnosi e nella sorveglianza sanitaria delle malattie professionali: caso del mesotelioma maligno

La sorveglianza sanitaria sui lavoratori a rischio è finalizzata alla formulazione di un giudizio di idoneità specifico per la mansione svolta, attraverso un procedimento che comprenda l’esame clinico effettuato in fase di avviamento al lavoro, nonché accertamenti sanitari preventivi richiesti in base alla valutazione dei rischi. Per tale motivo, la validazione di biomarcatori come rilevatori precoci della patologia potrebbe migliorare la valutazione del rischio per la salute e contribuire a politiche efficaci per la prevenzione di malattie causate da esposizioni ambientali e/o professionali. Il Mesotelioma Maligno della Pleura (MPM) è un tumore strettamente legato all’esposizione professionale all’asbesto. Migliorare le tecniche per la diagnosi è essenziale per una diagnosi precoce e per il trattamento terapeutico, per cui esiste la reale necessità di identificare marcatori tumorali che possano essere utilizzati per una diagnosi non invasiva del MPM. Livelli anomali di microRNAs (miRNA) sono stati ritrovati in molti tipi di cancro. La loro de-regolazione è associabile a diversi tipi di tumori, quindi il profilo di espressione dei miRNA può identificare una neoplasia. L’obiettivo di questo studio è investigare le potenzialità dei miRNA come biomarcatori di esposizione,diagnosi precoce e prognosi. A questo scopo, è stato analizzato il profilo di espressione di 88 miRNA coinvolti nel cancro estratti da biopsie di tessuto fresco. I campioni di MPM e quelli di controllo sono stati analizzati tramite qRT-PCR che ha rilevato una marcata differenza nel profilo di espressione dei miRNA tra tessuto patologico e sano. La maggior parte dei miRNA sono risultati sottoespressi ed in particolare 8 miRNA sottoregolati sono risultati statisticamente significativi. La loro de-regolazione risulta più evidente nel tumore allo stadio avanzato. Tre fra i miRNA significativi (miRNA-335, miRNA-126 emiRNA-32) sono stati confermati in un numero maggiore di campioni paraffinati; solo il miRNA-126 ha mantenuto una de-regolazione significativa e la capacità di discriminare il tessuto normale dal patologico e la stadiazione del tumore. La performance del miRNA-126 come biomarker è stata valutata anche in campioni di siero di soggetti ex-esposti all’amianto e soggetti con MPM e comparata con soggetti di controllo. Il miRNA-126 discrimina significativamente soggetti ex-esposti e soggetti affetti da MPM dai controlli. Pertanto in questo lavoro è stato identificato uno specifico profilo di espressione dei miRNA per il MPM ed è stato proposto il miRNA-126 come potenziale biomarker di esposizione e di diagnosi precoce del MPM.The monitoring of the health of workers at risk is aimed at formulating a fitness certificate specific to their occupational task. Clinical examination is first performed in start-up and then subsequent periodic medical examinations are carry out according to risk. Therefore, the use of biomarkers that could determine the early stages of the disease may improve the assessment of risk to health and contribute to effective policies for preventing diseases caused by environmental and occupational exposures. Malignat pleural Mesothelioma (MPM) is well known to be linked to asbestos exposure at the workplace. Improved detection methods for diagnosis of this type of neoplastic disease are essential for an early and reliable detection and treatment. Thus, focus has been on finding tumour markers used for non-invasive detection of MPM. There is emerging evidence of abnormal levels of microRNAs (miRNAs) in tumours. While some miRNAs commonly exhibit altered amounts across tumours, often, different tumour types produce unique patterns of miRNAs, related to their tissue of origin. In this study, miRNA isolated from MM fresh-frozen biopsies was profiled for the expression of 88 miRNAs involved in cancer. MPM samples and normal tissues were analysed by quantitative reverse transcription-PCR miRNA array. A marked difference in miRNA expression pattern was observed between normal tissue and tumours. Most of miRNA were down-regulated in tumours, and among them 8 miRNA were significantly under expressed. Their down-regulation was more evident in advanced tumours. Three of these miRNAs (miRNA-335, miRNA-126, miRNA-32) were validated in the larger sample series, and only miR-126 showed significant differential expression, distinguish MPM samples from normal tissues and tumour staging. Furthermore, the performance of the miRNA-126 as biomarkers was evaluated in serum samples of asbestos-exposed subjects, MPM patients and compared with age-matched healthy subjects. The miRNA-126 significantly distinguish asbestos-exposed subjects and MPM patients from age-matched controls. We first identify a specific miRNA profile for MPM and we propose miRNA-126 as potential biomarker of exposure and for early diagnosis and prognosis of MPM

MicroRNA come biomarcatori nella diagnosi e nella sorveglianza sanitaria delle malattie professionali: caso del mesotelioma maligno

La sorveglianza sanitaria sui lavoratori a rischio è finalizzata alla formulazione di un giudizio di idoneità specifico per la mansione svolta, attraverso un procedimento che comprenda l’esame clinico effettuato in fase di avviamento al lavoro, nonché accertamenti sanitari preventivi richiesti in base alla valutazione dei rischi. Per tale motivo, la validazione di biomarcatori come rilevatori precoci della patologia potrebbe migliorare la valutazione del rischio per la salute e contribuire a politiche efficaci per la prevenzione di malattie causate da esposizioni ambientali e/o professionali. Il Mesotelioma Maligno della Pleura (MPM) è un tumore strettamente legato all’esposizione professionale all’asbesto. Migliorare le tecniche per la diagnosi è essenziale per una diagnosi precoce e per il trattamento terapeutico, per cui esiste la reale necessità di identificare marcatori tumorali che possano essere utilizzati per una diagnosi non invasiva del MPM. Livelli anomali di microRNAs (miRNA) sono stati ritrovati in molti tipi di cancro. La loro de-regolazione è associabile a diversi tipi di tumori, quindi il profilo di espressione dei miRNA può identificare una neoplasia. L’obiettivo di questo studio è investigare le potenzialità dei miRNA come biomarcatori di esposizione,diagnosi precoce e prognosi. A questo scopo, è stato analizzato il profilo di espressione di 88 miRNA coinvolti nel cancro estratti da biopsie di tessuto fresco. I campioni di MPM e quelli di controllo sono stati analizzati tramite qRT-PCR che ha rilevato una marcata differenza nel profilo di espressione dei miRNA tra tessuto patologico e sano. La maggior parte dei miRNA sono risultati sottoespressi ed in particolare 8 miRNA sottoregolati sono risultati statisticamente significativi. La loro de-regolazione risulta più evidente nel tumore allo stadio avanzato. Tre fra i miRNA significativi (miRNA-335, miRNA-126 emiRNA-32) sono stati confermati in un numero maggiore di campioni paraffinati; solo il miRNA-126 ha mantenuto una de-regolazione significativa e la capacità di discriminare il tessuto normale dal patologico e la stadiazione del tumore. La performance del miRNA-126 come biomarker è stata valutata anche in campioni di siero di soggetti ex-esposti all’amianto e soggetti con MPM e comparata con soggetti di controllo. Il miRNA-126 discrimina significativamente soggetti ex-esposti e soggetti affetti da MPM dai controlli. Pertanto in questo lavoro è stato identificato uno specifico profilo di espressione dei miRNA per il MPM ed è stato proposto il miRNA-126 come potenziale biomarker di esposizione e di diagnosi precoce del MPM.The monitoring of the health of workers at risk is aimed at formulating a fitness certificate specific to their occupational task. Clinical examination is first performed in start-up and then subsequent periodic medical examinations are carry out according to risk. Therefore, the use of biomarkers that could determine the early stages of the disease may improve the assessment of risk to health and contribute to effective policies for preventing diseases caused by environmental and occupational exposures. Malignat pleural Mesothelioma (MPM) is well known to be linked to asbestos exposure at the workplace. Improved detection methods for diagnosis of this type of neoplastic disease are essential for an early and reliable detection and treatment. Thus, focus has been on finding tumour markers used for non-invasive detection of MPM. There is emerging evidence of abnormal levels of microRNAs (miRNAs) in tumours. While some miRNAs commonly exhibit altered amounts across tumours, often, different tumour types produce unique patterns of miRNAs, related to their tissue of origin. In this study, miRNA isolated from MM fresh-frozen biopsies was profiled for the expression of 88 miRNAs involved in cancer. MPM samples and normal tissues were analysed by quantitative reverse transcription-PCR miRNA array. A marked difference in miRNA expression pattern was observed between normal tissue and tumours. Most of miRNA were down-regulated in tumours, and among them 8 miRNA were significantly under expressed. Their down-regulation was more evident in advanced tumours. Three of these miRNAs (miRNA-335, miRNA-126, miRNA-32) were validated in the larger sample series, and only miR-126 showed significant differential expression, distinguish MPM samples from normal tissues and tumour staging. Furthermore, the performance of the miRNA-126 as biomarkers was evaluated in serum samples of asbestos-exposed subjects, MPM patients and compared with age-matched healthy subjects. The miRNA-126 significantly distinguish asbestos-exposed subjects and MPM patients from age-matched controls. We first identify a specific miRNA profile for MPM and we propose miRNA-126 as potential biomarker of exposure and for early diagnosis and prognosis of MPM

Raw and thermally treated cement asbestos exerts different cytotoxic effects and Nitric Oxide production in A549 cells.

Raw cement asbestos (RCA) undergoes a complete solid state transformation when heated at high temperatures (1200 °C). The secondary raw material produced after treatment (HT-CA) is composed of newly-formed crystals in place of the original asbestos fibres present in RCA. Our previous studies (1) showed that HT-CA treatment exerts cytotoxic effects of lower grade compared to RCA. In this study we found that RCA treatment of A549 epithelial cells increase inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release in the culture media in RCA cells compared to control and HT-CA cells. It is already known that the asbestos-induced inflammatory status may drive lung cells to the carcinogenesis (2), events sustained by iNOS expression and NO production that play a role in cell-mediated immune response in lung cells. Interestingly, HT-CA cell treatment reveals Fe content in cristallized instead of soluble form found in RCA that can be related to the significantly lower toxicity for inertized cement-asbestos. Our findings confirm that HT-CA can be considered a transformed phase exerting direct lower cytotoxic and inflammatory potential compared to RCA on biological systems. High-temperature RCA treatment could represent a safe approach for storing or recycling asbestos materials. Further studies are in progress to exclude any residual carcinogenicity in HT-CA matrix

Stability of mineral fibres in contact with human cell cultures. An in situ \u3bcXANES, \u3bcXRD and XRF iron mapping study

Relevant mineral fibres of social and economic importance (chrysotile UICC, crocidolite UICC and a fibrous erionite from Jersey, Nevada, USA) were put in contact with cultured diploid human non-tumorigenic bronchial epithelial (Beas2B) and pleural transformed mesothelial (MeT5A) cells to test their cytotoxicity. Slides of each sample at different contact times up to 96 h were studied in situ using synchrotron XRF, \u3bc-XRD and \u3bc-XAS (I18 beamline, Diamond Light Source, UK) and TEM investigations. XRF maps of samples treated for 96 h evidenced that iron is still present within the chrysotile and crocidolite fibres and retained at the surface of the erionite fibres, indicating its null to minor mobilization in contact with cell media; this picture was confirmed by the results of XANES pre-edge analyses. \u3bc-XRD and TEM data indicate greater morphological and crystallinity modifications occurring in chrysotile, whereas crocidolite and erionite show to be resistant in the biological environment. The contact of chrysotile with the cell cultures seems to lead to earlier amorphization, interpreted as the first dissolution step of these fibres. The formation of such silica-rich fibre skeleton may prompt the production of HO in synergy with surface iron species and could indicate that chrysotile may be much more reactive and cytotoxic in vitro in the (very) short term whereas the activity of crocidolite and erionite would be much more sluggish but persistent in the long term

Stability of mineral fibres in contact with human cell cultures. An in situ mXANES, mXRD and XRF iron mapping study

Relevant mineral fibres of social and economic importance (chrysotile UICC, crocidolite UICC and a fibrous erionite from Jersey, Nevada, USA) were put in contact with cultured diploid human nontumorigenic bronchial epithelial (Beas2B) and pleural transformed mesothelial (MeT5A) cells to test their cytotoxicity. Slides of each sample at different contact times up to 96 h were studied in situ using synchrotron XRF, m-XRD and m-XAS (I18 beamline, Diamond Light Source, UK) and TEM investigations. XRF maps of samples treated for 96 h evidenced that iron is still present within the chrysotile and crocidolite fibres and retained at the surface of the erionite fibres, indicating its null to minor mobilization in contact with cell media; this picture was confirmed by the results of XANES pre-edge analyses. m-XRD and TEM data indicate greater morphological and crystallinity modifications occurring in chrysotile, whereas crocidolite and erionite show to be resistant in the biological environment. The contact of chrysotile with the cell cultures seems to lead to earlier amorphization, interpreted as the first dissolution step of these fibres. The formation of such silica-rich fibre skeleton may prompt the production of HO in synergy with surface iron species and could indicate that chrysotile may be much more reactive and cytotoxic in vitro in the (very) short term whereas the activity of crocidolite and erionite would be much more sluggish but persistent in the long term

Raw and thermally treated cement asbestos exerts different cytotoxicity effects on A549 cells in vitro

Raw cement asbestos (RCA) undergoes a complete solid state transformation when heated at high temperatures. The secondary raw material produced, high temperatures-cement asbestos (HT-CA) is composed of newly-formed crystals in place of the asbestos fibers present in RCA. Our previous study showed that HT-CA exerts lower cytotoxic cell damage compared to RCA. Nevertheless further investigations are needed to deepen our understanding of pathogenic pathways involving oxidative and nitrative damage. Our aim is to deepen the understanding of the biological effects on A549 cells of these materials regarding DNA damage related proteins (p53, its isoform p73 and TRAIL) and nitric oxide (NO) production during inducible nitric oxide synthase (iNOS)-mediated inflammation. Increments of p53/p73 expression, iNOS positive cells and NO concentrations were found with RCA, compared to HT-CA and controls mainly at 48 h. Interestingly, ferrous iron causing reactive oxygen species (ROS)-mediated DNA damage was found in RCA as a contaminant. HT-CA thermal treatment induces a global recrystallization with iron in a crystal form poorly released in media. HT-CA slightly interferes with genome expression and exerts lower inflammatory potential compared to RCA on biological systems. It could represent a safe approach for storing or recycling asbestos and an environmentally friendly alternative to asbestos waste

Angiogenic effect induced by mineral fibres

Due to the toxic effect of asbestos, other materials with similar chemical-physical characteristics have been introduced to substitute it. We evaluate the angiogenic effect of certain asbestos substitute fibres such as glass fibres (GFs), ceramic fibres (CFs) and wollastonite fibres (WFs) and then compare angiogenic responses to those induced by crocidolite asbestos fibres (AFs). An in vitro model using human endothelial cells in small islands within a culture matrix of fibroblasts (Angio-Kit) was used to evaluate vessel formation. The release of IL-6, sIL-R6, IL-8. VEGF-A and their soluble receptors, 5vEGFR-1, sVEGFR-2, was determined in the conditioning medium of Angio-Kit system after fibre treatment. ROS formation and cell viability were evaluated in cultured endothelial cells (HUVEC). To evaluate the involvement of intracellular mechanisms, EGFR signalling, ROS formation and nuclear factor-kappa B (NF kappa B) pathway were then inhibited by incubating HUVEC cells with AG1478, NAC and PDTC respectively, and the cytokine and growth factor release was analyzed in the culture medium after 7 days of fibre incubation. Among the mineral fibres tested, WFs markedly induced blood vessel formation which was associated with release of IL-6 and IL-8. VEGF-A and their soluble receptors. ROS production was observed in HUVEC after WFs treatment which was associated with cell cytotoxicity. The EGFR-induced ERK phosphorylation and ROS-mediated NF kappa B activation were involved in the cytokine and angiogenic factor release. However, only the EGFR activation was able to induce angiogenesis. The WFs are potential angiogenic agents that can induce regenerative cytokine and angiogenic factor production resulting in the formation of new blood vessels. (C) 2011 Elsevier Ireland Ltd. All rights reserved