Genomic and Epigenomic Biomarkers of Toxicology and Disease. Группа авторов

these miRNAs were located in abnormal chromosomal positions of MM patients and that their target genes, such as CDKN2A, NF2, JUN, HGF, and PDGFA, were most likely to affect the occurrence and development of MM. This pioneering research has opened up a new kind of approach in the field. Since its publication in 2009, more studies have discussed the role of miRNA in MM in detail (Truini et al. 2014).

      Methods for Detecting the Expression of miRNAs

      As a biomarker of human diseases, ideally the detection method of miRNA should not need expensive reagents and instruments and be easy to operate. Such a method should have good specificity for distinguishing miRNAs with similar sequences. At the same time, it should have sufficient sensitivity for quantitative analysis, even for micro clinical samples, and it should be able to detect multiple samples in parallel (de Planell-Saguer and Rodicio 2011; Van Roosbroeck, Pollet, and Calin 2013).

      The Gold Standard Conference believes that the method for meeting these requirements and for detecting miRNA in clinical laboratories is quantitative reverse transcription polymerase chain reaction (PCR) (qRT-PCR). miRNA microarray is more expensive than qRT-PCR and is normally used in the discovery stage of biomarkers.

      However, these methods all need to extract miRNAs from tumor samples of patients, which include non-tumor matrix and inflammatory cells in addition to malignant tumor cells. If the goal is to detect the specific expression of miRNA in malignant tumor cells, it is recommended to use flow cytometry to sort liquid tumors or laser capture microscopy to cut solid tumors. In situ hybridization technology (Sempere and Korc 2013) can also be used; it can detect target miRNA in different types of cells (malignant tumor cells or microenvironment cells) that constitute tumors. This method provides additional miRNA subcellular localization information. At present, the latest method for detecting miRNA expression is next-generation sequencing. This technique is highly sensitive and specific, can be used for high-throughput analysis, and can discover new miRNAs. Next-generation sequencing produces a large amount of complex data, which need to be analyzed by a trained bioinformatician; besides, the cost of single RNA sequencing is too high, so this method is not suitable for diagnosis but can still be considered as a screening method for miRNAs of interest (Berindan-Neagoe et al. 2014).

      Early Screening for Malignant Mesothelioma

      In view of the high degree of malignancy and difficult treatment of MM, early screening is particularly important in the prevention and treatment of the disease. At present, there have been studies designed to improve the detection rate of early MM patients by identifying the expression level of specific miRNAs in serum or plasma (Mujoomdar et al. 2010).

Santarelli et al. (2011) found that miR-126 is downregulated in the tissues of MM patients. They noticed that one of the target genes of miR-126 is vascular endothelial growth factor (VEGF), which encodes a protein that stimulates angiogenesis and increases the VEGF content in the serum of MM patients. In addition, miR-126 expression is downregulated and VEGF expression is upregulated in lung cancer patients, which suggests that miR-126 plays a role in tumor inhibition (Liu et al. 2009). Moreover, there is a correlation between miR-126 and the serum MM-specific marker soluble mesothelin-related protein (SMRP); and the level of SMRP is related to the high risk of MM development. These kinds of evidence indicate that the correlation between miR-126 and SMRP can be used as an early detection marker of MM (Santarelli et al. 2015). Further research on serum samples from forty-five untreated MM and twenty non-small cell lung cancer (NSCLC) patients showed that the circulating miR-126 level in MM patients decreased and could be significantly different from the level in non-small cell lung cancer and in healthy control group samples. Although miR-126 can be considered as a marker of MM, it lacks tumor specificity owing to its low expression in various tumors, which indicates that miR-126 will be used only to bind to other MM-specific biomarkers such as mesothelin (Tomasetti et al. 2012).

      Weber et al. (2014) verified the expression level of miR-103 in the plasma of MM patients and of an asbestos-exposed control population, with sensitivity and specificity of 85% and 63% respectively. However, when combined with mesothelitin, sensitivity reached 95% and specificity reached 81%.

      Therefore miRNA can be used as a biomarker in the early detection of MM, especially in combination with other specific protein markers. It can also be used as a serum-monitoring indicator for asbestos-exposed population.

      Diagnosis of Malignant Mesothelioma

      Studies have shown that the expression of some miRNAs in cancer tissues or body fluids of patients with MM is dysregulated and affects the expression of target genes, which may play an important role in the occurrence and development of malignant mesothelioma. In addition, some differentially expressed miRNAs have a certain specificity, which can distinguish MM from lung adenocarcinoma and other tumors. In consequence, these miRNAs can be used as biomarkers of MM for clinical diagnosis and differential diagnosis (Kirschner et al. 2012).

      Differential Expression of miRNAs in Cancer Tissues

      At present, the diagnosis of MM is mainly based on histopathology, but it is difficult to distinguish some cases from metastatic carcinoma only by pathology. Some scholars have found that the expression of miR-145 (Casarsa et al. 2011; Cioce et al. 2014; Schramm et al. 2010) and miR-16 (Reid et al. 2013) in MM tissues is significantly downregulated; and in vitro experiments have also found that the expression of these two miRNAs in MM cell lines is significantly downregulated. Further study found that miR-16 can, specifically, bind to the 3 UTR of BCL-2 and CCND-1, induce apoptosis of MM cells, and inhibit cell proliferation and clone formation by inhibiting the expression of its target genes BCL-2 and CCND-1. This indicates that miR-16 may have the effect of inhibiting the growth of MM.

Significant downregulation of miR-126, miR-143, miR-145, and miR-652 can be used to distinguish malignant pleural mesothelioma (MPM) from reactive mesothelial hyperplasia. Logistic regression analysis has shown that it had high sensitivity and specificity (0.96 area under the curve) to distinguish MPM from non-tumor mesothelium, and the overall accuracy reached 94% (Andersen et al. 2014).

      This being the case, the expression of miRNAs may become a new clinical diagnostic method, to be used as a supplement to the current immunohistochemical methods, and may improve the diagnosis rate.

      Differential Expression of MicroRNAs in Serum and Plasma

      A large number of studies have shown that free miRNAs in serum may be an important marker for cancer diagnosis (Santarelli et al. 2011). Tomasetti et al. (2012) found that miR-126 in the serum of patients with MM was significantly downregulated, and patients with MM could be distinguished from the control population according to the expression level of miR-126. Studies by Kirschner et al. (2012) show that serum miR-625-3p in patients with MM is significantly increased. Receiver operating curve (ROC) analysis shows that the accuracy rate of distinguishing patients with MM from controls by serum miR-625-3p expression level is 82.4%; sensitivity and specificity are 73.33% and 78.58% respectively. When the digital methylation-specific PCR (MSP) method was used to detect quantitatively the methylation level of miR-34b/c in circulating serum from MPM patients, from benign asbestos pleurisy patients, and from healthy people, it was found that the methylation level of miR-34b/c in the serum of MPM patients was significantly increased. Through ROC analysis it was found that the accuracy of detecting the methylation of miR-34b/c in the serum to diagnose MPM was 77%, and the sensitivity and specificity were 67% and 77% (Muraoka et al. 2013) .

      The expression level of circulating miR-132-3p in the plasma of MM patients and asbestos-exposed people is different. Its sensitivity and specificity for MM diagnosis are 86% and 61%, and it is not affected by age and smoking. The sensitivity and specificity of miR-126 combined with miR-132-3p in the diagnosis of malignant mesothelioma were 86% and 77%, respectively (Weber et al. 2017). Plasmatic extracellular vesicles-associated miR-103a-3p and miR-30e-3p are able to discriminate MPM subjects from past asbestos exposure (PAE) subjects (Cavalleri et al. 2017).

      Serum or plasma miRNA is a rapid, convenient, and relatively non-invasive marker. If multiple miRNAs can be used in combination