**2.1. Long noncoding RNAs are generated in the mitochondria**

Both strands of the mtDNA are entirely transcribed but the light strand carries only genes for seven tRNAs and the ND6 protein. Therefore, large noncoding sequences are thus generated and released upon transcript processing. Three lncRNAs generated from mtDNA transcription have been proposed. Their presence was authenticated by Northern blot and qRT-PCR analysis. These transcripts are complementary to MTND5, MTND6 and MTCYTB genes. These molecules form intermolecular duplexes that resist RNase 1 digestion, suggesting regulation of their complementary coding mRNA. Therefore, pairing of these lncRNAs with their mRNA targets might, for instance, control translation [33].

Recently, a novel mitochondrial long noncoding RNA (mtlncRNA) was identified in the plasma of patients with left ventricular (LV) remodeling post-myocardial infarction. Levels of LIPCAR (long intergenic noncoding RNA predicting cardiac remodeling) decline in early stages after myocardial infarction, but increase in late stages, coinciding with LV remodeling. Therefore, high levels of LIPCAR associate with identified patients with high risk of heart failure, even death, suggesting that this lncmtRNA as a potential biomarker for patients with recent episodes of acute myocardial infarction [34].

In the year 2000, our laboratory described a novel chimeric mitochondrial RNA present in mouse testis and sperm cells. This transcript contained an inverted repeat of 120nt joined to the 5′ end of the 16S mitochondrial rRNA. The presence of this novel mitochondrial RNA in sperm, testis, and somatic tissues was demonstrated by RT-PCR.As to the origin of this novel RNA, one possibility was that it arose from transcription altered mtDNA which contained an insert of 121bp between the tRNAVal and the 16S rRNA genes. However, PCR of sperm, testis, liver, and blood cell mtDNA between these two genes yielded a fragment of 342bp consistent with a normal mtDNA lacking the putative insertion of 121bp. The most surprising result was the localization of this novel RNA in the sperm nucleus. In situ hybridization (ISH) demonstrated that the sequence of the 16S rRNA and that of the inverted repeat were localized in the sperm nucleus [35].

Nuclear localization of this mitochondrial transcript is not specific to mouse since, by ISH, we found that at least the sequence of the 16S rRNA was also localized in the nucleus of human sperm. To determine when during spermatogenesis the mitochondrial RNA is localized in the nucleus, ISH of mouse and human testis was carried out. The nuclei of spermatogonia, spermatocytes and round and elongated spermatids were all positively stained. In human spermatocytes, the nuclear staining pattern was fibrillar, suggesting an association of the mitochondrial transcript with the meiotic chromosomes [36].

The class of long noncoding RNAs (lncRNAs) has been recently recognized, and is defined as transcripts longer than 200 nucleotides. The size cutoff is arbitrary and many functional lncRNAs are considerably longer than 200 nucleotides, including X-inactive specific transcript (XIST) [26], its antisense form Tsix [27], and Hox transcript antisense intergenic RNA

LncRNAs are able to interact with DNA, RNA, and proteins. In doing so, they regulate several processes including chromatin dynamics, gene transcription, splicing, and translation [29]. Their involvement in these processes implicates lncRNAs in various aspects of human physiology and disease, which include cancer. Aberrant lncRNA expression has been associated to various cancer types [30]. Moreover, deregulated lncRNA expression patterns can modulate several hallmarks of cancer [31], including sustained growth signaling, repressed growth inhibition,

Both strands of the mtDNA are entirely transcribed but the light strand carries only genes for seven tRNAs and the ND6 protein. Therefore, large noncoding sequences are thus generated and released upon transcript processing. Three lncRNAs generated from mtDNA transcription have been proposed. Their presence was authenticated by Northern blot and qRT-PCR analysis. These transcripts are complementary to MTND5, MTND6 and MTCYTB genes. These molecules form intermolecular duplexes that resist RNase 1 digestion, suggesting regulation of their complementary coding mRNA. Therefore, pairing of these lncRNAs with their

Recently, a novel mitochondrial long noncoding RNA (mtlncRNA) was identified in the plasma of patients with left ventricular (LV) remodeling post-myocardial infarction. Levels of LIPCAR (long intergenic noncoding RNA predicting cardiac remodeling) decline in early stages after myocardial infarction, but increase in late stages, coinciding with LV remodeling. Therefore, high levels of LIPCAR associate with identified patients with high risk of heart failure, even death, suggesting that this lncmtRNA as a potential biomarker for patients with

In the year 2000, our laboratory described a novel chimeric mitochondrial RNA present in mouse testis and sperm cells. This transcript contained an inverted repeat of 120nt joined to the 5′ end of the 16S mitochondrial rRNA. The presence of this novel mitochondrial RNA in sperm, testis, and somatic tissues was demonstrated by RT-PCR.As to the origin of this novel RNA, one possibility was that it arose from transcription altered mtDNA which contained an insert of 121bp between the tRNAVal and the 16S rRNA genes. However, PCR of sperm, testis, liver, and blood cell mtDNA between these two genes yielded a fragment of 342bp consistent with a normal mtDNA lacking the putative insertion of 121bp. The most surprising result was the localization of this novel RNA in the sperm nucleus. In situ hybridization (ISH) demonstrated that the sequence of

the 16S rRNA and that of the inverted repeat were localized in the sperm nucleus [35].

Nuclear localization of this mitochondrial transcript is not specific to mouse since, by ISH, we found that at least the sequence of the 16S rRNA was also localized in the nucleus of human

apoptosis evasion, stimulated proliferation, and the promotion of angiogenesis [32].

(HOTAIR) [28], which are several kilobases (kb) in length.

182 Mitochondrial DNA - New Insights

**2.1. Long noncoding RNAs are generated in the mitochondria**

mRNA targets might, for instance, control translation [33].

recent episodes of acute myocardial infarction [34].

These results suggested that the nuclear localization of the 16S mitochondrial rRNA in spermatogenic cells is the result of an intriguing process of translocation of the transcript from the organelle to the nucleus. This hypothesis leads to several important questions; for example, how does the organelle regulate the exit of the 16S mitochondrial rRNA without affecting the number of copies needed to assemble mitoribosomes for normal mitochondrial translation? Or what is the mechanism by which this RNA is exported from mitochondria? At present these questions remain unanswered.

However, the extramitochondrial localization of the 16S mitochondrial rRNA described by us is not unique, since the same transcript has been found consistently in the cytoplasm of Drosophila and Xenopus embryos [37, 38]. Moreover, injection of an anti-16S rRNA ribozyme into cleavage embryos of Drosophila demonstrated that the rRNA is actively involved in the generation of pole cells, progenitors of the germ line [39].

As mentioned above, ISH revealed that this lncRNA is over-expressed in human sperm and precursor cells [36]. These results suggest that human cells might contain a transcript with structural features similar to the mouse RNA. We found that the human RNA is over-expressed in several human proliferating cells but not in resting cells. The structure of this transcript of 2374 nt, which we designated sense noncoding mitochondrial RNA or SncmtRNA, revealed the presence of an inverted repeat (IR) of 815 nt linked to the 5′ end of the 16S mtrRNA. The expression of this transcript can be induced in resting lymphocytes stimulated with phytohaemagglutinin (PHA), together with DNA synthesis and the expression of the proliferation markers proliferating cell nuclear antigen (PCNA), Ki-67 and phosphohistone H3. On the other hand, treatment of DU145 cells with aphidicolin reversibly blocks cell proliferation as well as the expression of the ncmtRNA. These results suggested that the ncmtRNA is a new marker of cell proliferation [40].

Afterwards, we described 2 mitochondrial transcripts (ASncmtRNAs) in human cells containing stem-loop structures similar to that of the previously described SncmtRNA.Regarding expression of the SncmtRNA and the ASncmtRNAs, 3 different phenotypes of human cells can be defined. Normal proliferating cells express both families of transcripts; in striking contrast, tumor cells express the SncmtRNA and down-regulate the ASncmtRNAs. Finally, neither of these transcripts is expressed in nondividing cells. Down-regulation of the ASncmtRNAs was observed in 15 different tumor cell lines and in tumor cells present in 273 cancer biopsies corresponding to 17 different cancer types [41]. SncmtRNA is expressed in all proliferating cells, independently whether we are dealing with a regulated or a dysfunctional cell cycle. The fact that the ASncmtRNAs are always down-regulated in tumor cells suggests that, hypothetically, the ASncmtRNAs might function as a unique mitochondria-encoded tumor suppressor. **Figure 1** shows a panel of ISH for S and ASncmtRNA in non-proliferating tissue (liver), tumor tissue (cervix carcinoma) and normal proliferating tissue (normal cervix epithelium), representing the concept of differential expression mentioned. For in situ hybridization, tissue sections were incubated with hybridization mixture

transcripts might play a role in retrograde signaling. Down regulation of the ASncmtRNAs seems to be an important step in neoplastic transformation and cancer progression [42].

Long Noncoding Mitochondrial RNAs (LncmtRNAs) as Targets for Cancer Therapy

http://dx.doi.org/10.5772/intechopen.75453

185

The study of RNA and DNA oncogenic viruses has proven valuable in the discovery of key cellular pathways that are rendered dysfunctional during cancer progression. Because of this, we studied human foreskin keratinocytes (HFK) immortalized with HPV in order to gain insight on the role of the lncmtRNAs in cell proliferation. We showed that immortalization of HFK with HPV-16 or 18 causes downregulation of the ASncmtRNAs and induces the expres-

Transduction of HFK with both E6 and E7 oncoproteins is sufficient to induce expression of SncmtRNA-2. On the other hand, the E2 oncogene is involved in downregulation of the ASncmtRNAs. Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-viral-induced oncogenesis [43]. Our results suggest that a fraction of SncmtRNA-1 is processed outside of the organelle, to give rise to SncmtRNA-2 and a 63-nt fragment released from the IR. In silico analysis of this sequence revealed that the 63-nt fragment is highly complementary to hsa-miR-620. Using the TargetScan algorithm (www.targetscan.com), we found >100 predictive targets for hsa-miR-620 [44]. An interesting example is the mRNA of promyelocytic leukemia (PML) protein, which is a core component of PML nuclear bodies found in tumor cells, important structures involved in HPV replication. Several reports indicate that the E6 and E7 oncoproteins are localized in these nuclear structures [45].

As mentioned above, the ASncmtRNAs are downregulated in tumor cell lines and cells in tumor biopsies, independently of the tissular origin of the tumor analyzed. Therefore, this

Cervical cancer is the fourth most common cancer in women worldwide. In 2012, this disease accounted for 528,000 new cases and 266,000 deaths among females [46]. Cervical cancer is of slow progression and, according to histopathological studies, there are at least three well-defined stages preceding cervical squamous carcinoma, known as cervical intraepithelial neoplasia (CIN). These stages (CIN1, CIN2 and CIN3) correspond to the progressive invasion of the cervical epithelium from the basal cell layer to the surface of the squamous epithelium [47]. Therefore, detection of premalignant lesions is key to preventing disease progression to advanced stages. Therefore, we performed a study in order to evaluate and quantify the differential expression of non-coding mitochondrial RNAs during the progression of the disease. We found down-regulation of the antisense mitochondrial transcripts at early stages of cervical neoplasia (CIN1). Moreover, differential expression of ASncmtRNA v/s S-ncmtRNA showed significant difference, while, as expected, normal proliferating tissues did not display downregulation of ASncmtRNAs. Moreover, downregulation of ASncmtRNAs correlated with the

Bladder cancer (BC) is a significant cause of morbidity and mortality with a high recurrence rate. Early detection of bladder cancer is essential in order to remove the tumor, to preserve

**2.2. Differential expression of lncmtRNAs as a tool for cancer diagnostics**

differential expression might be used for screening of cancer cells.

over-expression of the tumor suppressor protein p16INK-4a [48, 49].

sion of a new sense transcript termed SncmtRNA-2.

**Figure 1.** Representative in situ hybridization assay showing the differential expression pattern of lncmtRNAs in tissues according to proliferative status. Upper panel shows absence of signal for both RNAs in non-proliferating tissues such as liver. Middle panel shows presence of strong punctuate signal, corresponding to nuclei in normal proliferating cervix epithelium. Lower panel shows a strong signal corresponding only to SncmtRNA and complete absence of signal corresponding to ASncmtRNA in a tumor tissue, exemplified by cervix carcinoma. H&E, hematoxylin-eosin staining. Magnification in upper panel, ×200. Magnification in cervix tissues, ×100.

containing probes complementary to sense or antisense ncmtRNAs, previously labeled at the 3′ end with digoxigenin-11-dUTP (Boehringer Mannheim, Germany) as described previously [36]. For detection, sections were incubated with a monoclonal anti-digoxigenin antibody conjugated to alkaline phosphatase, and after color development, positive signal correspond to a blue color, representing the expression of the corresponding RNA (see **Figure 1**).

Regarding subcellular localization, we found that in biopsies of normal and cancer tissues, nuclear localization of these transcripts was frequently observed. The extra-mitochondrial localization of these transcripts was confirmed by electron microscopy ISH. In normal cells, SncmtRNA and the ASncmtRNAs were found in the nucleus associated to chromatin. In tumor cells, SncmtRNA shows similar localization plus association with nucleoli, while the ASncmtRNAs are down-regulated. Although the meaning of the nuclear localization in normal proliferating cells of SncmtRNA and the ASncmtRNAs is unclear, the results suggest that these transcripts might play a role in retrograde signaling. Down regulation of the ASncmtRNAs seems to be an important step in neoplastic transformation and cancer progression [42].

The study of RNA and DNA oncogenic viruses has proven valuable in the discovery of key cellular pathways that are rendered dysfunctional during cancer progression. Because of this, we studied human foreskin keratinocytes (HFK) immortalized with HPV in order to gain insight on the role of the lncmtRNAs in cell proliferation. We showed that immortalization of HFK with HPV-16 or 18 causes downregulation of the ASncmtRNAs and induces the expression of a new sense transcript termed SncmtRNA-2.

Transduction of HFK with both E6 and E7 oncoproteins is sufficient to induce expression of SncmtRNA-2. On the other hand, the E2 oncogene is involved in downregulation of the ASncmtRNAs. Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-viral-induced oncogenesis [43]. Our results suggest that a fraction of SncmtRNA-1 is processed outside of the organelle, to give rise to SncmtRNA-2 and a 63-nt fragment released from the IR. In silico analysis of this sequence revealed that the 63-nt fragment is highly complementary to hsa-miR-620. Using the TargetScan algorithm (www.targetscan.com), we found >100 predictive targets for hsa-miR-620 [44]. An interesting example is the mRNA of promyelocytic leukemia (PML) protein, which is a core component of PML nuclear bodies found in tumor cells, important structures involved in HPV replication. Several reports indicate that the E6 and E7 oncoproteins are localized in these nuclear structures [45].
