**4. Non-coding RNA molecules**

Protein-coding genes comprise only about 1.5% of the genome. At the same time, it was shown that more than 90% is transcriptionally active [49]. Transcription of this so-called junk DNA leads to creation of thousands of RNA molecules that are not capable of coding proteins. Surprisingly, it was shown that the more complex the organism, the higher number of non-coding RNA (ncRNA) molecules it contains [50]. These molecules have many different functions in the most important cell processes, such as differentiation, proliferation, apoptosis and many others. They are involved in tumorigenesis as well.

Based on their length, ncRNA are divided into two groups: short (sncRNA) and long (lncRNA). SncRNA are smaller than 200 nucleotides (nt), while lncRNA are longer than 200 nt. The first ncRNA molecules that were identified more than 50 years ago were ribosomal RNA (rRNA) and transfer RNA (tRNA) [4, 5, 51]. While there are many classes of ncRNA, the most studied and well known are microRNA (miRNA) and long non-coding RNA (lncRNA).

### **4.1. Definition and biogenesis of microRNA**

MiRNA are short, non-coding, single-stranded RNA molecules about 21–23 nt long. They are involved in regulation of gene expression and influence various cell processes, such as proliferation, differentiation, apoptosis and tumorigenesis. MiRNA genes account for 1–2% of the human genome, and mature miRNA regulate around 50% of protein-coding genes [49].

Based on the canonical model of miRNA biogenesis, miRNA genes are transcribed by RNA polymerase II or III into primary precursors, stem-loop structures (pri-miRNA) that contain 5′ end cap and polyA on the 3′ end. Pri-miRNA are cleaved in the nucleus by RNAse II enzyme Drosha and Pasha leading to pre-miRNA [52]. Pre-miRNA are exported into cytoplasm by transport protein exportin 5 [53]. In the cytoplasm, the pre-miRNA molecule is processed by the RISC complex that contains RNAse III Dicer and protein Argonaute 2 (Ago2). RISC complex cuts the molecule into 20–23 nt long double-stranded miRNA duplex with 2 nt overhang on 3′ ends [54]. One of the strands is the so-called guide strand and is complementary to the mRNA sequence. The other ('passenger' strand) is degraded. Which one of these strands is degraded is based on the stability of pairing on the 5′ end of the miRNA duplex [55]. Based on the level of miRNA/mRNA complementarity, the target mRNA is either silenced translationally in case of non-complete complementarity or degraded in case of 100% complementarity [56, 57].

MiRNA regulate a large spectrum of physiological and pathological processes including oncogenesis; they can act as oncogenes or tumor suppressors. Several mechanisms of miRNA role in tumorigenesis have been described: increased expression levels, amplification, epigenetic silencing or loss of miRNA gene that regulates expression of a tumor suppressor gene [58]. On the other hand, deletion and epigenetic silencing of miRNA gene expression that silences oncogene expression have been described as well [59]. Moreover, mutations in target sequences of mRNA lead to failed translational repression or degradation of target mRNA [60].

In a pilot study of miRNA expression in malignant transformation of PC, increased expression of miR-181a/b, cluster miR-106b-25 (miR-93, miR-106b, miR 25) and miR-21 in MGUS and MM patients in comparison to healthy donors (HD) was found. Interestingly, MM patients showed increased expression of cluster miR-17-92a suggesting a possible role of this cluster in disease progression [61].

### **4.2. Circulating miRNA**

The last study was published by Mithraprabhu et al., and the subject of this study was mutational characterization of MM. Paired DNA samples of BM PC and plasma derived cfDNA were analyzed for the presence of activating mutations of four oncogenes—*KRAS*, *NRAS*, *BRAF* and *TP53* by NGS. In total, 48 MM patients (33 relapsed/refractory and 15 newly diagnosed) and 21 healthy donors (HD) enrolled in the study. Overall, 128 different mutations were detected in MM patients (cfDNA = 31, BM = 59 and both = 38), while none were found in HD. Interestingly, almost a quarter of all found mutations were detected only in cfDNA samples. These findings proved spatial heterogeneity of MM and showed that cfDNA molecules are derived from multiple tumor sites within a patient's body. This was supported also by majority of cfDNA-specific mutations found in relapsed/refractory patients (30 mutations) in contrast to newly diagnosed patients (1 mutation) as they are more prone to have multiple focal lesions. Moreover, sequences of cfDNA were evaluated by ddPCR in seven patients throughout their treatment, and changes in fractional abundance were discovered, reflecting progression of disease. This proof-of-concept study confirmed the presence of mutations only in cfDNA and proved that genetic composition of MM is complex and evolves during progression of disease. In addition, it proposed that cfDNA analysis could be used as an adjunct to standard BM biopsy for disease monitoring to enable obtaining more complex results [47]. So far, not many cfDNA studies in MM have been conducted; however, the data are exciting

Protein-coding genes comprise only about 1.5% of the genome. At the same time, it was shown that more than 90% is transcriptionally active [49]. Transcription of this so-called junk DNA leads to creation of thousands of RNA molecules that are not capable of coding proteins. Surprisingly, it was shown that the more complex the organism, the higher number of non-coding RNA (ncRNA) molecules it contains [50]. These molecules have many different functions in the most important cell processes, such as differentiation, proliferation, apoptosis

Based on their length, ncRNA are divided into two groups: short (sncRNA) and long (lncRNA). SncRNA are smaller than 200 nucleotides (nt), while lncRNA are longer than 200 nt. The first ncRNA molecules that were identified more than 50 years ago were ribosomal RNA (rRNA) and transfer RNA (tRNA) [4, 5, 51]. While there are many classes of ncRNA, the most studied

MiRNA are short, non-coding, single-stranded RNA molecules about 21–23 nt long. They are involved in regulation of gene expression and influence various cell processes, such as proliferation, differentiation, apoptosis and tumorigenesis. MiRNA genes account for 1–2% of the human genome, and mature miRNA regulate around 50% of protein-coding genes [49].

and well known are microRNA (miRNA) and long non-coding RNA (lncRNA).

and strongly suggest the future role of cfDNA in MRD monitoring.

and many others. They are involved in tumorigenesis as well.

**4.1. Definition and biogenesis of microRNA**

**4. Non-coding RNA molecules**

72 Liquid Biopsy

Essentially, all human body fluids (PB, saliva, urine, breast milk, etc.) contain the so-called circulating miRNA [62]. Circulating miRNA are quite stable and resistant to RNases as they are part of protein (Ago2) or lipoprotein (high-density lipoprotein (HDL)) complexes or they are bound inside exosomes—small transport vesicles [63]. It seems that circulating miRNA are involved in cell-to-cell communication as they are exported outside of cells based on biological stimuli. These molecules can also take part in cell processes, such as communication, proliferation, differentiation and in case of tumors also metastases [64]. Specific profiles of circulating miRNA are diagnostic markers differentiating HD from patients, but they also correlate with progression and staging of the tumor [65, 66]. A major advantage of these molecules as potential biomarkers is their simple structure, easy access and measurability by standard laboratory techniques [64].

### *4.2.1. Circulating microRNA in monoclonal gammopathies*

In MM, circulating miRNA were first described in 2012. In a study by Jones et al., PB serum samples of MGUS and MM patients were analyzed in comparison to HD. They found that miR-720, miR-1246 and miR-1308 could serve as potential markers of MG [67].

The success of this study led to other studies, especially in MM. However, different approaches lead to varying results. The main differences were type of samples (serum or plasma of PB), design of experiments (patients vs. HD) and used methods and platforms.

miR-16-5p, miR-15a-5p, miR-20a-5p and miR-17-5p were significantly decreased in MM

Liquid Biopsies in Multiple Myeloma http://dx.doi.org/10.5772/intechopen.78630 75

As for miRNA in other body fluids, our group performed analysis of circulating miRNA in urine of MM patients in comparison with HD. Unfortunately, we did not find any miRNA

While a lot of work was done on circulating miRNA in MG, so far no clear biomarkers of the diseases have been identified. It is possible that MM as a heterogeneous disease will not have a single circulating miRNA as a biomarker. Further studies and standardization of sample

In our opinion, miRNA have a large potential for diagnostics of monoclonal gammopathies. Most studies were done on diagnostics samples; however, the data are not consistent and more standardization and optimization is needed. The possibility of using miRNA as prog-

LncRNA are an abundant class of RNA between 200 nt and 100 kb long [80, 81]. To date, approximately 16,000 lncRNA have been identified in the human genome (http://www.gencodegenes.org/). On the other hand, the functional characterization of most of them has not

Genes encoding for lncRNA are present in many types of organisms, including animals [82], plants [83], yeast [84], prokaryotic organisms [85] and viruses [86]. LncRNA do not possess protein-coding capacity due to the absence of open reading frames (ORFs) or because of insufficient length of ORFs [87–90]. They can be classified according to their genomic localization into three major groups: long intergenic non-coding RNA (lincRNA), long intronic RNA and

The expression of lncRNA genes is developmental and tissue-specific, and they have been associated with a large spectrum of biological processes, for example, alternative splicing, modulation of protein activity, alternation of protein localization, epigenetic regulation and generally regulation of gene expression. These molecules can be precursors of small RNA and even tools for miRNA silencing [91–96]. LncRNA play an important role both in physiological and pathological processes. The deregulated expression levels of these molecules were identified in a large variety of tumor diseases: breast cancer [97], small-cell lung carcinoma [98] and also in MM [22]. It was shown that alterations in lncRNA can influence regulation of cancer progression [99]. Interestingly, lncRNA seem to have higher tissue specificity even in comparison with proteincoding mRNA and miRNA. Thus, they are even more interesting as new specific biomarkers [88]. Function of lncRNA can also be derived from their localization within the cell. These molecules can be found in the nucleus and in the cytoplasm. LncRNA are preferably located in

The classification of lncRNA can be based on their influence on the DNA sequence. From this perspective, there are two classes of lncRNA: cis-lncRNA (cis-acting lncRNA) and translncRNA (trans-acting lncRNA). Cis-IncRNA can positively or negatively regulate expression

processing, types of samples and analytical methods need to be performed.

nostic or monitoring markers needs to be further validated.

long non-coding RNA transcribed from specific genomic regions.

the cell nucleus, deriving their significant effect on the DNA sequence [88].

patients resistant to bortezomib [79].

**4.3. Long non-coding RNA molecules**

been determined yet.

significantly dysregulated [13].

Plasma of PB was reported to have lower levels of miR-92a in newly diagnosed MM patients in comparison to HD. Moreover, the level of miR-92a fluctuated based on progression of disease and treatment response, which would suggest a possible role of this miRNA as a predictive biomarker [68]. Another study showed increased expression of miR-148a, miR-181a, miR-20a, miR-221 and miR-88b in plasma of PB of MM patients in comparison with HD. Expression level of miR-20a and miR-148a was connected to shorter time to relapse of MM; this study suggested that circulating plasma miR-20a could be a marker of worse prognosis of MM [69]. On contrary, another study showed mostly decreased miRNA expression in MM patients compared to HD. MiR-483-5p and miR-20a were shown to have diagnostic and prognostic potential [70].

So far, most studies were performed using serum miRNA. Our own pilot study showed significantly increased levels of miR-29a, miR-660 and miR-142-5p in MM patients in comparison with HD. We showed that circulating serum miR-29a could be a biomarker for MM patients [71]. In our follow-up study, we showed dysregulation of five serum miRNA, miR-744, miR-130a, let-7d, let-7e and miR-34a in MGUS and MM patients in comparison with HD. Multivariate analysis showed that combination of miR-34a and let-7e distinguishes the patient cohorts with good sensitivity and specificity. Moreover, dynamics of serum miRNA with disease progression was shown [72].

In another study, increased expression of miR-181a/b, miR-221, miR-222 and miR-382 was found in relapsed MM patients and MM cell lines [51]. On contrary, lower expression of miR-15a and miR-16 was described; these miRNA have been described in chronic lymphocytic leukemia and seem to be part of pathogenesis of this disease. The genes for these miRNA are coded in the 13q14 locus; this locus is often deleted also in MM [73]. MiR-15a and miR-16 support apoptosis and decrease proliferation of MM cells by AKT and MAP kinase signaling [51].

In a study by Rocci et al., higher levels of miR-25, miR-16 and miR-30a in MM patients correlated with longer overall survival (OS) [74]. Another study showed that miR-19a and miR-4254 distinguish MM and HD. In addition, decreased level of serum miR-19a was positively correlated with international staging system (ISS) stage, presence of del(13q14) and gain 1q21 and shorter progression-free survival (PFS) and OS. Surprisingly, these patients responded to bortezomib better [75].

Serum miRNA were also analyzed at CR after autologous stem cell transplantation (ASCT). MiR-16, miR-17, miR-19b, miR-20a and miR-660 were decreased in diagnostic samples in comparison with CR samples [76]. Patients with lower levels of miR-19b and miR-331 had shorter PFS after ASCT. Level of miR-19b was significantly lower in samples obtained at relapsed than at CR.

The most common clinical manifestation of MM is osteolytic lesions. Increased levels of serum miR-214 and miR-135b were found in MM patients with osteolytic lesions, and their expression correlated with severity of the symptoms [77]. Moreover, increased level of miR-214 associated with shorter PFS and OS.

Using NGS, miRNA (let-7b a miR-18a) from exosomes isolated from serum of MM patients significantly correlated with PFS and OS in univariate analysis and with ISS and cytogenetic abnormalities in multivariate analysis [78]. Moreover, it was shown that levels of exosomal miR-16-5p, miR-15a-5p, miR-20a-5p and miR-17-5p were significantly decreased in MM patients resistant to bortezomib [79].

As for miRNA in other body fluids, our group performed analysis of circulating miRNA in urine of MM patients in comparison with HD. Unfortunately, we did not find any miRNA significantly dysregulated [13].

While a lot of work was done on circulating miRNA in MG, so far no clear biomarkers of the diseases have been identified. It is possible that MM as a heterogeneous disease will not have a single circulating miRNA as a biomarker. Further studies and standardization of sample processing, types of samples and analytical methods need to be performed.

In our opinion, miRNA have a large potential for diagnostics of monoclonal gammopathies. Most studies were done on diagnostics samples; however, the data are not consistent and more standardization and optimization is needed. The possibility of using miRNA as prognostic or monitoring markers needs to be further validated.
