**4. Circulating microRNA in glioma patients**

**3.9. Evading immune destruction and microRNAs**

46 Neurooncology - Newer Developments

suppressive cytokine IL-10, which promotes tumor growth (142).

and IL-10+ NKT cells via miR-92a.

glioma cells (143).

pathway.

In the past, the infiltration of innate and adaptive immune cells into the tumor microenviron‐ ment was considered an immune attack against tumors. However, now, it is widely accepted that immune cells also promote tumor initiation, progression, and metastasis (138). Tumorassociated macrophages (TAMs) control the majority of immunological processes within tumors exerting both regressive (M1) and progressive (M2) effects on tumor development (139). However, the majority of TAMs exhibit an M2-like phenotype. Remarkably, miRs-29b, miR-125a, miR-146a, and miR-155 are involved in the differentiation of TAMs (140). Overex‐ pression of miRNA-155 was shown to attenuate the production of cytokines (IL-6 and TNFα) by suppressing C/EBPβ expression, which led to an inversion of M2 into M1 macrophages (141). M2 TAMs are capable of releasing anti-inflammatory cytokines such as the immuno‐

Natural killer T (NKT) cells are a subfraction of T cells. A large number of published studies have demonstrated that NKT cells have miscellaneous functions in immune regulation, one of which is that NKT cells are tumor cell killers, based on the production of antitumor cytokines. In addition to contributing to immune protection, NKT cells are involved in immune tolerance in the body. Tang et al. showed that glioma cells can induce immune-tolerant IL-6+

Another study revealed miR-221 and miR-222 as possible regulators of IFN pathways. Type-I IFN receptor activation triggers the JAK-mediated tyrosine phosphorylation of STAT family proteins. Zhang et al. found that STAT1 and STAT2 expression and phosphorylation were upregulated after repression of miR-221/222 in U251 cells (143). Upregulation of STAT pathway is controlled by the IFN-α activation after knockdown of miR-221/222 cluster in U251

NKG2D is one of the major activating receptors of natural killer (NK) cells and binds to several ligands (NKG2DL). NKG2D recognizes different MHC class I-homologous ligands (NKG2DL), including the MHC class I-chain-related molecules A (MICA) and B (MICB) and the UL16- binding proteins (ULBP)1-6 (144), which are also present on the surface of glioma cells (145). Codo et al. reported that miR-20a, miR-93 or miR-106b regulates NKG2DL expres‐ sion in glioma cells (146), suggesting that the expression of miRNA-targeting NKG2DL may contribute to the immune evasion of glioma cells at the level of the NKG2D recognition

In addition, miRNA regulates immunocheckpoint molecules. miR-138 could bind the 3′-UTR of CTLA-4 and PD-1, and transfection of human CD4+ T cells with miR-138 suppressed expression of CTLA-4 and PD-1 (147). *In vivo* treatment in immunocompetent mice using miR-138 revealed marked tumor regression and prolonged survival time. Moreover, inocu‐ lated tumors showed decrease in intratumoral regulatory T cell, CTLA-4, and PD-1 expression (147). miR-138 exerts antiglioma efficacy by targeting immune checkpoints that may have

On the other hand, miRNA expression is transcriptionally regulated by various cytokines. Ohno et al. analyzed the effect of IFN-β treatment on miR-21 expression in glioma cells and

rapid translational potential as novel immunotherapeutic agents.

In 2008, Chim et al. firstly demonstrated the existence of placental miRNAs in maternal plasma (149). In the same year, several miRNAs were detected in the serum of the tumor patients. Quantitative real-time polymerase chain reaction (RT-PCR) analyses revealed sera levels of miR-155, miR-210, and miR-21 are higher in diffuse large B-cell lymphoma patient sera than healthy controls (150). To prevent degradation in the circulation, miRNAs are released by cells in both exosomes and miRNA/protein complexes. Exosomes are lipid vesicles ranging between 50 and 100 nm in size and contain a range of molecules, including mRNA, miRNA, DNA, and proteins. The detection of biomarkers within serum is attractive because of the relatively noninvasive process of collection (**Figure 1**).

**Figure 1.** Cell-cell communication through circulating microRNAs. MicroRNAs contained in exosomes are released from glioma cells where they can enter the blood-stream or CSF-stream and circulate through the body to distant sites. These exosomal miRNAs are taken up by recipient cells, where the miRNAs can then suppress target genes in the re‐ cipient cells. Circulating miRNAs released by glioma cells may involve in growth signal, angiogenesis, anti-apoptosis, tumor metabolism, and immunoregulation.

Over the past few years, researchers have investigated the capability of using miRNAs as blood biomarkers for the diagnosis of tumors. Circulating miRNAs can be isolated from serum or plasma, but recent studies have indicated plasma to be a more adequate source for miRNA extraction (150). However, depending on plasma preparation, the level of circulating miRNA can be altered. Cheng et al. showed that processing differences resulted in a variation in residual platelet contamination in plasma and significant differences in miRNA abundance (151). Consequently, supplementary centrifugation, the refusal of samples with platelet counts above a certain limit and the quantification of hemolysis are mandatory in accurately deter‐ mining miRNA levels in plasma.

Cerebrospinal fluid (CSF) is another useful biofluid and is in direct contact with the extracel‐ lular fluid of the brain. CSF has various functions, such as protecting the brain, transporting biological substances, and excreting toxic and waste substances. Although the composition of CSF reflects that of the blood plasma, active transport and secretions from the brain tissues contribute to the composition of CSF. Therefore, the analyses of CSF can suggest biological brain processes and is indispensable for understanding disorders of brain (152).

Clinically, histopathological examinations have been widely used to diagnose glioma, but they are invasive because microsurgical resection or stereotactic biopsy is needed to acquire specimens. In this context, it is of great interest to develop novel biomarkers for glioma. Currently, several miRNAs have been identified as noninvasive biomarkers for the diagnosis of cancers, including breast, lung, and gastric cancer. Previous studies have reported that miRNAs can be detected in circulating exosomes in the serum or CSF of glioma, suggesting that miRNAs might be useful biomarkers for glioma diagnosis. However, to effectively apply these findings to clinical detection, further studies must be conducted (**Table 2**).


#### The Roles of MicroRNAs in Glioblastoma Biology and Biomarker http://dx.doi.org/10.5772/63243 49


**Table 2.** Circulating microrna in glioma patients.

above a certain limit and the quantification of hemolysis are mandatory in accurately deter‐

Cerebrospinal fluid (CSF) is another useful biofluid and is in direct contact with the extracel‐ lular fluid of the brain. CSF has various functions, such as protecting the brain, transporting biological substances, and excreting toxic and waste substances. Although the composition of CSF reflects that of the blood plasma, active transport and secretions from the brain tissues contribute to the composition of CSF. Therefore, the analyses of CSF can suggest biological

Clinically, histopathological examinations have been widely used to diagnose glioma, but they are invasive because microsurgical resection or stereotactic biopsy is needed to acquire specimens. In this context, it is of great interest to develop novel biomarkers for glioma. Currently, several miRNAs have been identified as noninvasive biomarkers for the diagnosis of cancers, including breast, lung, and gastric cancer. Previous studies have reported that miRNAs can be detected in circulating exosomes in the serum or CSF of glioma, suggesting that miRNAs might be useful biomarkers for glioma diagnosis. However, to effectively apply

brain processes and is indispensable for understanding disorders of brain (152).

these findings to clinical detection, further studies must be conducted (**Table 2**).

20 (Healthy controls)

10 (Healthy controls)

123 (Healthy controls)

(Healthy controls)

(Healthy controls)

(Healthy

**Number of control**

**microRNA**

miR-128(↑), miR-342-3p(↓)

miR-19b, miR-21, miR-26a, miR-27a, miR-92, miR-93, miR-320, miR-20

miR-21(↑), miR-128(↓), miR-342-3p(↓)

miR-548b-5p(↓)

miR-454-3p(↑)

miR-576-5p(↑), miR-340(↑), miR-626(↑), miR-320(↓), let-7g-5p(↓), miR-7-5p(↓)

miR-320(↑), miR-574-3p(↑)

miR-15b\*(↓), miR-23a(↓), miR-133a(↓), miR-150\*(↓),miR-197(↓), miR-497(↓),

none let-7a, miR-15b, miR-16,

mining miRNA levels in plasma.

48 Neurooncology - Newer Developments

**No. Authors Year Source Number**

1 Skog 2008 serum 2

2 Roth 2011 blood 20

3 Wang 2012 plasma 50

4 Yang 2013 serum 122

5 Dong 2014 serum 3 (GBM) 3

6 Manterola 2014 serum 25 (GBM) 25

7 Shao 2015 plasma 70 (Glioma) 70

**Blood**

**of glioma**

(GBM)

(GBM)

(GBM)

(Astro cytoma)

#### **4.1. Serum and plasma**

The first analysis of exosomes in serum identified the presence of 11 miRNAs in the samples from two different patients with primary GBM in 2008 (153). However, the levels were generally lower in exosomes but correlated well with the tumor profile (153). In 2011, Roth et al. analyzed miRNA profiles from the blood of 20 patients with GBM and 20 matched healthy controls (154). Among 1158 tested miRNAs, 52 were significantly deregulated, and of these, two candidates, miR-128 (upregulated) and miR-342-3p (downregulated), remained the most significant miRNAs. The altered expression of these two miRNAs was confirmed in a valida‐ tion cohort by RT-PCR. In this model, the discrimination between blood samples of patients with GBM and healthy controls reached an accuracy of 81%, specificity of 79%, and sensitivity of 83%. In 2012, Wang et al. determined the plasma miRNA levels of 50 patients with glioma and 10 healthy donors using RT-PCR (155). The plasma level of miR-21 was increased and the levels of miR-128 and miR-342-3p were significantly decreased in the patients with glioma compared with those in normal controls. These miRNAs were able to discriminate patients with glioma from healthy controls with high specificity and sensitivity. However, there were not significant differences between patients with glioma and other brain tumors such as meningioma or pituitary adenoma. Yang et al. performed genomewide serum miRNA analysis using serum samples of 122 untreated astrocytoma patients and 123 normal controls (156). The seven-miRNA panel (miR-15b\*, miR-23a, miR-133a, miR-150\*, miR-197, miR-497, and miR-548b-5p) demonstrated a high sensitivity (88.00%) and specificity (97.87%) for malignant astrocytoma prediction (156). These identified miRNAs also exhibited a global decrease in tumor tissues relative to normal tissues. Interestingly, these miRNAs in serum were markedly elevated after tumor removal.

In miRNA microarray analysis of the serum of patients with GBM and normal controls, 115 miRNAs were upregulated in the GBM group and 24 miRNAs were downregulated (157). In these microRNAs, a six-membered serum miRNA expression profile (upregulated miRs; miR-576-5p, miR-340, and miR-626, downregulated miRs; miR-320, let-7g-5p, and miR-7-5p) could serve as a noninvasive biomarker for GBM diagnosis (157). Manterola et al. found that the serum expression levels of miR-320 and miR-574-3p were significantly altered in the patients with GBM (158). In addition, small noncoding RNA (RNU6-1) was an independent predictor of a diagnosis of GBMs. Shao et al. compared the expression levels of miR-454-3p between preoperative plasmas from 70 patients with glioma and 70 healthy controls and between these preoperative and postoperative plasmas (159). The expression levels of miR-454-3p in plasma in patients with glioma were significantly higher, and the area under receiver operating characteristic (ROC) curve (AUC) of the expression of miR-454-3p for glioma diagnosis was 0.9063. In addition, the expression levels of miR-454-3p in the postop‐ erative plasmas were significantly downregulated relative to the preoperative plasmas. Wu et al. analyzed serum from 83 patients with glioma and 69 healthy controls and evaluated the availability of the serum miR-29 family in the screening of glioma (160). The predictive value of the serum miR-29 family for glioma was moderate (AUC = 0.74), but that in high-grade glioma detection was sufficient (AUC = 0.81). Another study also showed that the expressions of miR-29b in blood were significantly different compared with those of a healthy control (161). miR-125b is widely considered a tumor suppressor-miRNA and an ideal biomarker for clinical diagnosis in various human cancers. The study of serum miR-125b from 33 gliomas and 33 healthy controls revealed that the serum miR-125b level was significantly lower in patients with glioma (162). The ROC curve analysis yielded an AUC value of 0.839 (162). Furthermore, a meta-analysis was conducted to assess the diagnostic accuracy of miR-125b in cancer diagnosis, and the results revealed that employing miR-125b as a biomarker for cancer detection achieved a sensitivity of 82% and a specificity of 77% (162).

miR-210 is reported to be another potentially useful biomarker in the serum of patients with glioma. Lai et al. analyzed blood samples collected from patients with glioma (*n*=136) and healthy controls (*n*=50) and revealed that an approximately sevenfold increase in miR-210 expression was detected in serum samples from patients with GBM relative to healthy controls (163). miR-210 has been found to be upregulated in a variety of other solid tumor types and potentially influences cellular function through diverse pathways; the miRNA has also been correlated with hypoxia (164). A number of targets of miR-210 have been reported, including VEGF (165), BCL2 (166), and E2F transcription factor 3 (167). Sun et al. analyzed the expression levels of miR-128 in serum samples from 151 gliomas, 52 meningiomas, and 53 normal donors and showed that miR-128 expression was significantly decreased in glioma preoperative serum compared with others. ROC analyses showed that serum miR-128 levels were reliable in distinguishing patients with glioma from normal controls and patients with meningioma, with AUC values of 0.9095 and 0.8283, respectively (168). Although the mechanism has not yet been clarified, the study demonstrated that serum miR-128 expression was significantly elevated after surgery. miR-128 functions as a vital suppressor of tumorigenesis in glioma cells and is reported to downregulate p70S6K1 and its downstream signaling molecules, including VEGF and HIF-1. Another study showed that serum miR-205 expression was significantly lower in patients with glioma than in healthy controls (p < 0.001) (169). Interestingly, serum miR-205 expression levels were inversely correlated with pathological grades (169).
