Radiol Oncol 2021; 55(4): 379-392. doi: 10.2478/raon-2021-0042 379 review MitomiRs: their roles in mitochondria and importance in cancer cell metabolism Andrej Rencelj1,2, Nada Gvozdenovic1, Maja Cemazar1,3 1 Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia 2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Faculty of Health Sciences, University of Primorska, Izola, Slovenia Radiol Oncol 2021; 55(4): 379-392. Received 10 August 2021 Accepted 28 September 2021 Correspondence to: Prof. Maja Čemažar, Ph.D., Institute of Oncology Ljubljana, Ljubljana, Slovenia. E-mail: MCemazar@onko-i.si Disclosure: No potential conflicts of interest were disclosed. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Background. MicroRNAs (miRNAs) are short non-coding RNAs that play important roles in almost all biological path- ways. They regulate post-transcriptional gene expression by binding to the 3’untranslated region (3’UTR) of messenger RNAs (mRNAs). MitomiRs are miRNAs of nuclear or mitochondrial origin that are localized in mitochondria and have a crucial role in regulation of mitochondrial function and metabolism. In eukaryotes, mitochondria are the major sites of oxidative metabolism of sugars, lipids, amino acids, and other bio-macromolecules. They are also the main sites of adenosine triphosphate (ATP) production. Conclusions. In the review, we discuss the role of mitomiRs in mitochondria and introduce currently well studied mito- miRs, their target genes and functions. We also discuss their role in cancer initiation and progression through the regu- lation of mRNA expression in mitochondria. MitomiRs directly target key molecules such as transporters or enzymes in cell metabolism and regulate several oncogenic signaling pathways. They also play an important role in the Warburg effect, which is vital for cancer cells to maintain their proliferative potential. In addition, we discuss how they indirectly upregulate hexokinase 2 (HK2), an enzyme involved in glucose phosphorylation, and thus may affect energy metabo- lism in breast cancer cells. In tumor tissues such as breast cancer and head and neck tumors, the expression of one of the mitomiRs (miR-210) correlates with hypoxia gene signatures, suggesting a direct link between mitomiR expression and hypoxia in cancer. The miR-17/92 cluster has been shown to act as a key factor in metabolic reprogramming of tumors by regulating glycolytic and mitochondrial metabolism. This cluster is deregulated in B-cell lymphomas, B-cell chronic lymphocytic leukemia, acute myeloid leukemia, and T-cell lymphomas, and is particularly overexpressed in several other cancers. Based on the current knowledge, we can conclude that there is a large number of miRNAs present in mitochondria, termed mitomiR, and that they are important regulators of mitochondrial function. Therefore, mitomiRs are important players in the metabolism of cancer cells, which need to be further investigated in order to develop a potential new therapies for cancer. Key words: microRNAs; mitomiR; mitochondria; cancer; cancer cell metabolism Introduction MicroRNAs (miRNAs) are short non-coding RNAs (ncRNAs) of ~18-25 nucleotides that are present in all eukaryotic cells and play important roles in al- most all biological signaling pathways.1–4 Since the discovery of the first miRNA (lin-4) in C. elegans5, approximately 2000 miRNAs have been annotated in the human genome.6 Data from genomic stud- ies show that most miRNAs are highly conserved, making them very interesting targets for study- ing various disease states.7 They regulate post- transcriptional gene expression by binding to the 3’UTR of messenger RNAs.8–14 A single miRNA Radiol Oncol 2021; 55(4): 379-392. Renčelj A et al. / MitomiRs, mitochondria and cancer metabolism380 can regulate many mRNA targets, and conversely, a single mRNA target can be regulated by many miRNAs.15–17 Therefore, by regulating these fun- damental target genes, miRNAs have been impli- cated in signaling pathways to modulate a large set of important biological processes such as cell proliferation12, metastasis18, apoptosis19, senes- cence12, differentiation20, autophagy21, and immune response22. Moreover, miRNAs have been found to be dysregulated in many pathological conditions, such as neurodegenerative diseases23, cardiovascu- lar diseases24, and cancer.25–28 More recently, miRNAs have been found to be specifically present in mitochondria. These mito- chondrial miRNAs were named “mitomiR”.7,29–32 Most of them have a nuclear origin, but some mi- tomiRs originate from mRNA molecules derived from the mitochondrial genome. The association of mitomiRs with mitochondria is species- and cell type-specific.7,33 They have been found in mi- tochondria in various tissues and cells and are thought to have different thermodynamic proper- ties than miRNAs.7,34 Mitochondria have a discrete and unique pool of mitomiRs, which has been demonstrated with various experiments.29 For the first time, in 2011, Barrey and co-workers demonstrated the presence of pre-miRNAs (precur- sor-miRNAs) in mitochondria and postulated that some pre-miRNA sequences could be processed into mature miRNAs that could immediately be- come active on mitochondrial transcripts or ex- ported to the cytosol to disrupt genomic mRNA.35 Barrey’s group screened for 742 miRNAs using qRT-PCR and showed that 243 miRNAs had sig- nificant expression in mitochondrial RNA samples isolated from human myotubes by in situ hybridi- zation. This study was the first to provide evidence that pre-miRNAs can be localized in mitochondria. Subsequently, a number of studies have identified “signatures” of miRNAs localized to mitochon- dria through various experimental approaches. Mercer et al.15 examined the human mitochondrial transcriptome and demonstrated that 3 miRNAs (miR-146a, miR-103, and miR-16) have quite high expression in the intermembrane region compared to the matrix. Latronico and Condorelli36 found 15 nuclear-encoded miRNAs in mitochondria isolated from rat liver, 20 miRNAs from mouse liver mi- tochondria, and 13 miRNAs from HeLa cells (iso- lated from human cervical cancer) by microarray. Some other groups identified novel mitomiRs from HEK293 cells (isolated from human embryonic kid- neys)37, 143B cells (isolated from human bone mar- row)38, mouse heart39 and HeLa cells.37,40 MitomiRs have been shown to be important regulators of mitochondrial function.35,38,41 The reg- ulation of mitochondria by mitomiRs influences the development of many diseases caused by mi- tochondrial dysfunction, which is responsible for the pathophysiology of numerous diseases, such as cardiovascular and neurodegenerative diseases, diabetes, obesity, and cancer.42 In the first part of this review article, we de- scribe the biosynthesis of mitomiRs and the trans- port mechanisms from mitomiRs to mitochondria. The next part is dedicated to the role of these small molecules in mitochondria and the presentation of some important mitomiRs, their target genes and functions. In the last part of the review, we discuss the functions of mitomiRs in cancer cell metabo- lism and introduced mitomiRs in the context of cancer. Biosynthesis of miRNA/mitomiRs Most miRNAs/mitomiRs are produced via the canonical biosynthetic pathway, which involves transcription by RNA polymerase II (Pol II) to pro- duce a primary transcript (pri-miRNA/mitomiR). The primary transcript is first cleaved in the nu- cleus by the nuclear heterodimer Drosha/DGCR8 (DiGeorge syndrome chromosomal region 8), which cleaves the pri-miRNA/mitomiR and pro- duces a pre-miRNA/mitomiR with a hairpin struc- ture that is much more stable than the pri-miRNA/ mitomiR due to its characteristic hairpin loop struc- ture.43 Exportin 5 (EXP5) and GTP-binding nuclear protein (RANGTP) then form a transport machin- ery to export the pre-miRNA from the nucleus to the cytoplasm. After export to the cytoplasm, the pre-miRNA/mitomiR is further cleaved by the enzyme Dicer to form a double-stranded RNA (dsRNA) duplex (Figure 1). Only a single strand of the dsRNA duplex forms the mature miRNA/mi- tomiR and is incorporated into the RNA-induced silencing complex (RISC), which directs the bind- ing of Argonaute (AGO) proteins in the RISC to the 3’UTR of the target mRNA to either repress protein translation or promote mRNA degradation.43–45 After incorporation into RISC, mature miRNA/mi- tomiRs are transported into mitochondria, back to nucleus by importin 8 (IPO-8) or extracellular envi- ronment (Figure 1).46,47 In addition to the canonical miRNAs/mitomiRs biosynthesis pathway, there are also non-canon- ical, Drosha/DGCR8-independent and Dicer- independent biosynthesis pathways. Prominent Radiol Oncol 2021; 55(4): 379-392. Renčelj A et al. / MitomiRs, mitochondria and cancer metabolism 381 classes of Drosha/DGCR8-independent miRNAs/ mitomiRs are the “mirtrons” derived from introns that, once spliced, function as pre-miRNAs and thus do not require cleavage by Drosha/DGCR8 and can be immediately exported to the cytoplasm for processing by Dicer. MiRNAs/mitomiRs can also be processed from hairpins generated directly by Pol II at specific transcription start sites. These pre-miRNAs are capped and exported via the ex- portin 1 (EXP1) pathway. The Dicer-independent miRNAs/mitomiRs biosynthesis pathway involves the unusually short hairpin of miR-451, which is directly cleaved by argounaute 2 (AGO2).45 MitomiRs transport to mitochondria The discovery of mitomiRs raised the question of elucidating the underlying molecular mechanisms of their transport into mitochondria. Due to their size and charged nature, mitomiRs are unlikely to cross membranes under their own power. The mo- lecular mechanisms of mitomiR transport into mi- tochondria may vary between species and are not well understood.29 Some proposals have been published on AGO2 as a potential mitomiR import protein.7,29,48 Due to its RNA-binding ability and dual localization in the cytosol and mitochondria, AGO2 might be in- volved in the trafficking of mitomiRs.7 Shepherd et al.49 showed that the exoribonuclease polyribonu- cleotide nucleotidyltransferase (PNPT1/ PNPase) has a major role in the import of mitomiRs. Therefore, PNPase could be part of an alternative, AGO2-independent, uptake pathway of mitochon- drial miRNA. Furthermore, a possible mechanism could involve the voltage-dependent anion-se- lective channel protein (VDAC).34 Several studies have suggested that the instability of RISC in the FIGURE 1. Canonical biosynthesis of miRNAs/mitomiRs (adopted from 29,43,45). Mature miRNA can be transported into any part of the cell; but miRNA/mitomiR regulation is possible only after incorporation into RISC. (AGO2 = argonaute 2; DGCR8 = DiGeorge critical region 8; EXP5 = exportin 5; GTP = guanosine triphosphate; IMP8 = importin 8; mRISC = RNA induced silencing complex loaded with mature miRNA; POLII = DNA polymerase II; RANGTP = binding nuclear protein RAN; RISC = RNA-induced silencing complex; TRBP2 = RISC-loading complex subunit TRBP2). Radiol Oncol 2021; 55(4): 379-392. Renčelj A et al. / MitomiRs, mitochondria and cancer metabolism382 cytoplasm promotes miRNA translocation to mito- chondria, but the molecular components that facili- tate this translocation process are not fully under- stood. Furthermore, the concept that mammalian mitochondria can import cytosolic ncRNAs may facilitate research in another exciting area, long ncRNAs. Clearly, these translocation mechanisms and the identification of pathway components for mitochondrial targeting require further studies.7 Roles of mitomiRs in mitochondria Mitochondria are semi-autonomous cell orga- nelles with their own DNA (mtDNA) encoding 22 tRNAs, 2 rRNAs, and 13 polypeptides. These polypeptides and those encoded by nuclear genes, form 4 protein complexes of the electron transport chain (ETC). Mitochondria are constantly dividing and fusing, and the balance between mitochon- drial fission and fusion influences mitochondrial morphology, whose dynamics and turnover are critical for cellular homeostasis and differentia- tion.50 Several proteins are involved in the regu- lation of mitochondrial dynamics. Deregulation of mitochondrial dynamics is not only associated with deregulation of mitochondrial function, but is also closely related to several biological pro- cesses such as proliferation, cell death, apoptosis and production of reactive oxygen species (ROS), since mitochondria are the major sites of oxidative metabolism of sugars, lipids, amino acids and ATP production.1,51–53 It’s also worth noting that the mitochondrial matrix has its own set of environmental variables. Because of its thioester bond, acetyl-coenzyme A (acetyl-CoA) is a very abundant metabolite in mi- tochondria and functions as a powerful acetylation reagent. Protein lysine acetylation and succinyla- tion are caused by acetyl-CoA and mitochondrial matrix pH concentrations. Non-enzymatic acety- lation occurs often in mitochondria.54 The most of mitochondrial proteins have acetyl groups, which is consistent with this hypothesis. Non-enzymatic acetylation of RNA molecules, including miRNAs, is a logical possibility for mitochondrial modifica- tion. An acetyl group covalently attached to a miR- NA might change its mRNA recognition behavior. If it happens at the 2 OH group of ribose needed for the cleavage process, it could inhibit spontane- ous bond cleavage and therefore increase the half- life of mRNA. Furthermore, post-transcriptional alterations can result in structural changes55 as well as changed interactions with other RNA molecules or proteins.56 As stated, mitomiRs are regulators of mitochon- drial function, as shown in the following examples. In silico analysis identified miR-378, miR-24, and miR-23b in liver mitochondria (Table 1) and these mitomiRs have been shown to regulate systemic energy homeostasis, oxidative capacity, ROS, and mitochondrial lipid metabolism.35,57–62 Several re- ports have indicated that miRNAs such as miR- 1291, miR-138, miR-150, miR-199a, and miR-532- 5p can alter the expression of some important gly- colytic enzymes (Table 1).4,63–70 miR-29a, miR-29b and miR-124 (Table 1) regulate the expression of monocarboxylate transporter 1 (SLC16A1) in pan- creatic beta cells.71 miR-33a/b has been shown to regulate lipid metabolism by targeting the choles- terol transporter ATP-binding cassette transporter (ABCA1).72 miR-143 and miR-24 have also been shown to regulate mitochondrial lipid metabolism (Table 1).73,74 On the other hand, miR-204 acceler- ates fatty acid oxidation by inhibiting acetyl-coen- zyme A carboxylase (ACC).75 Ahmad et al. (2011) showed that miR-200 is associated with the regula- tion of phosphoglucose isomerase (PGI), which is an important factor in glycolysis and glucogenesis. Overexpression of miR-338 leads to downregula- tion of the protein level of cytochrome c oxidase IV and reduces mitochondrial oxygen consumption and ATP production.77,78 Similarly, overexpres- sion of miR-181c decreases mt-COX1 protein and causes remodeling of the complex IV (in vitro)48 and a dysfunctional complex IV (in vivo)79, along with increased production of ROS. It has also been reported that miR-210 modulates the function of the complex IV by targeting the nuclear-encoded mRNA, COX10.80,81 It has also been reported that miR-15b, miR-16, miR-195 and miR-338 (Table 1) regulate ATP production by targeting several nu- clear genes that play important roles in ETC.77,82,83 miR-101-3p regulates the expression of ATP syn- thase subunit beta (ATP5B) in ETC (Table 1).84 In addition, miR-210-5p reduces the expression of iron-sulfur cluster assembly enzyme (ISCU) under hypoxic conditions, which affects the proteins con- taining iron-sulfur clusters (Fe-S).85 It has also been reported that miR-29a-3p86 is involved in ß-oxida- tion of lipids (Table 1) and that miR-19b negatively regulates mitochondrial fusion by downregulating mitofusin 1 (MFN1).87 The microRNAs listed in Table 1 significantly affect mitochondrial regulation and function, which is why they are classified in the group of mitomiRs, which are crucial regulatory molecules Radiol Oncol 2021; 55(4): 379-392. Renčelj A et al. / MitomiRs, mitochondria and cancer metabolism 383 TABLE 1. Summary of microRNAs and their roles in mitochondria miR miR accession number Target genes Gene accession number Function Functional pathway Location Species References miR-378 MI0000795 Crat ENSMUSG00000026853 Downregulation Mitochondrial oxidative metabolism Mitochondria in liver cells Mouse Carrer et al., 2012 59 miR-24 MI0000080 H2ax ENSMUSG00000049932 Downregulation Insulin signaling pathway Mitochondria in liver cells Human Jeong et al., 2017 61 miR-23b MI0000439 GLS ENSG00000115419 Downregulation Glutamine metabolism LMitochondria in liver cells Human Gao et al., 2009 60 miR-1291 MI0006353 SLC2A1 ENSG00000117394 Downregulation Mitochondrial metabolism Mitochondria in renal cells Human Yamasaki et al., 2013; Chen et al., 2020, Tu et al., 202063–65 CPT1C ENSG00000169169 Downregulation ESRRA ENSG00000173153 Downregulation ASS1 ENSG00000130707 Downregulation GLUT1 ENSG00000117394 Downregulation miR-138 MI0000455 PDK1 ENSG00000152256 Downregulation Glucose metabolism Mitochondria in cardiac cells Human Zhu et al., 2017 66 miR-150 MI0000920 Slc2a4 ENSRNOG00000017226 Downregulation Metabolism Mitochondria in cardiac cells Rat Ju et al., 202067 MI0000479 SLC2A1 ENSG00000117394 Downregulation Human Li et al., 201768 miR-199a MI0000941 Slc2a4 ENSRNOG00000017226 Upregulation Expression of glucose transporters Mitochondria in muscle cells Rat Esteves et al., 2018, Yan et al., 2014, Guo et al., 20154,69,70 Hk2 ENSRNOG00000006116 Upregulation MI0000242 HK2 ENSG00000159399 Upregulation Mitochondria in liver cells Human miR-532-5p MI0006154 Slc2a4 ENSRNOG00000017226 Upregulation Expression of glucose transporters Mitochondria in muscle cells Rat Esteves et al., 2018 70 Hk2 ENSRNOG00000006116 Upregulation miR-29a MI0000576 Slc16a1 ENSMUSG00000032902 Downregulation Mitochondrial oxidative metabolism Mitochondria in pancreatic beta- cells Mouse Pullen et al., 201171 miR-29b MI0000143 Slc16a1 ENSMUSG00000032902 Downregulation Mitochondrial oxidative metabolism Mitochondria in pancreatic beta- cells Mouse Pullen et al., 201171 miR-124 MI0000716 Slc16a1 ENSMUSG00000032902 Downregulation Mitochondrial oxidative metabolism Mitochondria in pancreatic beta- cells Mouse Pullen et al., 201171 miR-33a/b a-MI0002684, b-MI0007603 CROT ENSANAG00000028065 Downregulation Lipid metabolism Mitochondria in liver cells Monkey Rayner et al., 2011 72 CPT1A ENSANAG00000017356 Downregulation HADHB ENSANAG00000027802 Downregulation PRKAA1 ENSANAG00000032687 Downregulation ABCA1 ENSANAG00000033387 Downregulation SREBF1 ENSANAG00000021477 Upregulation FASN ENSANAG00000032055 Upregulation ACLY ENSANAG00000036009 Upregulation ACACA ENSANAG00000035253 Upregulation miR-143 MI0000916 Map2k5 ENSRNOG00000007926 Downregulation Adipogenesis Mitochondria in adipose cells Rat Chen et al., 2014 73 MI0000459 APOL6 ENSG00000221963 Downregulation Adpiogenesis Mitochondria in adipose cells Human Ye et al., 2013 74 miR-204 MI0000284 ACACB ENSG00000076555 Downregulation Lipid metabolism Mitochondria in adipose cells Human Civelek et al., 2013 75 miR-200 MI0000737 ZEB1 ENSG00000148516 Upregulation Lipid metabolism Mitochondria in breast cells Human Ahmad et al., 2011 76 ZEB2 ENSG00000169554 Upregulation miR-338 MI0000618 COXIV ENSRNOG00000007827 Downregulation Mitochondria oxidative metabolism Mitochondria in neural cells Rat Aschrafi et al., 2008 77 miR-181c MI0000924 COX1 ENSRNOG00000034234 Downregulation Mitochondria oxidative metabolism Mitochondria in cardiac cells Rat Das et al., 2012 88 miR-210 MI0000268 ISCU ENSG00000136003 Downregulation Mitochondria oxidative metabolism Mitochondria in placenta cells Human Colleoni et al., 2013; Qiao et al., 201381,85 miR-15b MI0000843 Arl2 ENSRNOG00000021010 Downregulation ATP production Mitochondria in cardiac cells Rat Nishi et al., 2010 82 Bcl2 ENSRNOG00000002791 Downregulation miR-16 MI0000844 Bcl2 ENSRNOG00000002791 Downregulation ATP production Mitochondria in cardiac cells Rat Nishi et al., 2010 82 Arl2 ENSRNOG00000021010 Downregulation miR-195 MI0000939 Arl2 ENSRNOG00000021010 Downregulation ATP production Mitochondria in cardiac cells Rat Nishi et al., 2010 82 miR-29a-3p MI0000576 Foxa2 ENSMUSG00000037025 Upregulation Lipid metabolism Mitochondria in liver cells Mouse Kurtz et al., 2014 86 miR-19b MI0000074 MFN1 ENSG00000171109 Downregulation Apoptosis Mitochondria in bone cells Human Li et al., 2014 87 miR-101-3p MI0000103 ATP5B ENSG00000110955 Silencing Mitochondria metabolism Mitochondria in heLa cells Human Zheng et al., 2011 84 Radiol Oncol 2021; 55(4): 379-392. Renčelj A et al. / MitomiRs, mitochondria and cancer metabolism384 of mitochondrial function and regulation of me- tabolism. In the figures (Figure 2 and Figure 3), we have shown how these mitomiRs are linked to their target genes in primates (Figure 2) and rodents (Figure 3). In primates, there is no regulation of the same genes by different mitomiRs from Table 1 (Figure 2). Moreover, most mitomiRs target one gene and only a few mitomiRs target a larger num- ber of genes and in most cases mitomiRs down- regulate genes. In contrast to primates, in rodents, some genes are regulated by different mitomiRs (Figure 3). The mitomiRs miR-15b and miR-16 both regulate the Arl2 gene82, which is a nucleotide-binding gene, and the Bcl2 gene, which regulates apoptosis. In addition, the mitomiRs miR-199a69,70 and miR-532- 5p70 both regulate the Hk2 gene, which has an im- portant function in regulating glucose metabolism, and the Slc2a4 gene, which is a glucose transmem- brane transporter. It can be concluded that there is a greater overlap of mitomiRs in rodents than in primates. In most cases, mitomiRs downregulate genes. From the figures (Figure 2 and Figure 3), we can summarize that some mitomiRs and their target genes are related in primates and rodents. MitomiR miR-199a69,70 regulates the same gene in both primates and rodents (Figure 3), the gene Hk2, which has an important function in regulating glu- cose metabolism. MiR-14373,74 regulates the same gene MAP2K5 (Figure 3), which has an important FIGURE 2. The network of the mitomiRs and their target genes (grey rectangle) in primates (data from Table 1). Blue arrows present downregulation, green arrows present upregulation and black T-line present silencing. Purple octagon shape presents monkey miRNA and cyan hexagon presents human miRNAs (figure constructed with Cytoscape Network Data Integration, Analysis, and Visualization in a Box V3.8.2). Radiol Oncol 2021; 55(4): 379-392. Renčelj A et al. / MitomiRs, mitochondria and cancer metabolism 385 function in signal cascade involved in growth fac- tor stimulated cell proliferation and muscle cell dif- ferentiation. MitomiRs in cancer Traditional cancer traits include ten biological capabilities gained during the multistage devel- opment of human tumors.89 These ten traditional cancer traits include resistance to cell death, induc- tion of angiogenesis, maintenance of proliferative signaling, evasion of growth suppressors, acti- vation of invasion and metastasis, facilitation of replicative immortality, altered metabolism, eva- sion of destruction by the immune system, tumor- promoting inflammation, and genome instability (Figure 4).89,90 An important feature of cancer is the presence of the Warburg effect. Under aerobic conditions, normal cells generate ATP primarily in the mi- tochondrial oxidative phosphorylation process (OXPHOS), which utilizes the products of glyco- lysis and the Krebs cycle. Under anaerobic condi- tions, relatively little pyruvate, the end product of glycolysis, is added to the Krebs cycle and is in- stead converted to lactate. However, this metabolic conversion of glucose appears to be energetically detrimental. In tumor cells, ATP deficiency can be compensated to some extent by upregulation of glycolysis.91 Interestingly, it has been observed that many cancer cells prefer glycolysis over OXPHOS even in the presence of an adequate amount of oxy- gen. This abnormal energy metabolism is known as the Warburg effect. Reduced OXPHOS and en- hanced aerobic glycolysis are the main manifesta- tions of reprogramming of glucose metabolism in tumor cells.1,92 Albeit the specific causes and utili- tarian outcomes of this metabolic switch are as yet unclear, there is a developing agreement that the impact of Warburg effect is certifiably not an incon- sequential result of carcinogenesis, yet is impera- tive for cancer cells to keep up with their prolifera- tive potential and is driven by a few elements.92–94 It has been confirmed that abnormal expression of mitomiRs in mitochondria is related to the oc- currence of cancer features.95 Moreover, mitomiRs play an essential role in the control of cancer cell metabolism by regulating mRNA expression. They regulate several oncogenic signaling pathways and FIGURE 3. The network of the mitomiRs and their target genes (grey rectangle) in rodents (data from Table 1). Blue arrows present downregulation and green arrows present upregulation. Orange diamond shape presents rat miRNAs, yellow rectangle presents mouse miRNAs and cyan hexaon presents human miRNAs. miR-199a and miR-143 show that this two miRNAs regulate (figure constructed with Cytoscape Network Data Integration, Analysis, and Visualization in a Box V3.8.2). Radiol Oncol 2021; 55(4): 379-392. Renčelj A et al. / MitomiRs, mitochondria and cancer metabolism386 target key transporters or enzymes in cellular me- tabolism. In addition, they may have a function as tumor suppressors that inhibit tumor cell prolifera- tion or as oncogenes that induce tumorigenesis.96–98 MitomiRs can be isolated from any tissue or body fluid of any organism to study the level of expres- sion in the organism in a diseased state, and thus can function as novel prognostic and predictive biomarkers.99 The first evidence of miRNA involvement in hu- man cancers was provided in a study of chronic lymphocytic leukemia (CLL).100 MiR-15a and miR- 16-1 localized to 13q14 were reported to be fre- quently deleted and/or reduced in patients with B-cell chronic lymphocytic leukemia. This finding provided the first evidence that miRNAs may be involved in the pathogenesis of human cancers, as deletion of chromosome 13q14 resulted in the loss of these two miRNAs. MiR-15a induces apoptosis by regulating mitochondrial function and affecting the activity of Bcl-2 and Mcl-1 in human (Table 2). In addition, miR-15a causes mitochondrial dys- function, leading to the release of cytochrome c into the cytoplasm and depletion of mitochondrial membrane potential.101 MiR-15a and miR-16a have been shown to be ATP modulators correlated with FIGURE 4. Traditional cancer traits.89 Radiol Oncol 2021; 55(4): 379-392. Renčelj A et al. / MitomiRs, mitochondria and cancer metabolism 387 TABLE 2. Summary of mitomiRs with roles in cancer miR miR accession number Target genes Gene accession number Function Functional pathway Type of cancer Species References miR-210 MI0000286 HIF-1 ENSG00000258777 Upregulation Hypoxia Breast cancer, neck and head cancer, lung cancer Human Qin et al., 2014; Gee et al., 2010; Puissegur et al., 2011109–111 ISCU ENSG00000136003 Upregulation COX10 ENSG00000006695 Upregulation SDHD ENSG00000204370 Upregulation NDUFA4 ENSG00000189043 Upregulation miR-200a MI0000342 TFAM ENSG00000108064 Downregulation Mitochondrial biogenesis, cancer metabolism Breast cancer Human Yao et al., 2014112 miR-155 MI0000681 HK2 ENSG00000159399 Upregulation Glucose phosphorylation Breast cancer Human Fang et al., 2012; Jiang et al., 2012104 miR-124 MI0000443 PKM ENSG00000067225 Upregulation Glucose metabolism Colorectal cancer Human Sun et al., 2012 105 miR-137 MI0000454 PKM ENSG00000067225 Upregulation Glucose metabolism Colorectal cancer Human Sun et al., 2012 105 miR-340 MI0000802 PKM ENSG00000067225 Upregulation Glucose metabolism Colorectal cancer Human Sun et al., 2012 105 miR-326 MI0000808 PKM2 ENSG00000067225 Downregulation Glucose metabolism Glioblastoma Human Kefas et al., 2010 106 miR-181-5p MIMAT0000256 RASSF6 ENSG00000169435 Downregulation Mitogen-activated protein kinase (MAPK) signaling pathway Gastric cancer, cervical cancer Human Mi et al., 2017; Zhuang et al., 2017108,113INPP5A ENSG00000068383 Downregulation miR-92a-1 MI0000093 BCL2L11 ENSG00000153094 Downregulation Apoptosis Lymphoma Human Mogilyansky and Rigoutsos, 201394 miR-126 MI0000471 PIK3R2 ENSG00000105647 Downregulation Inflammation, angiogenesis Breast cancer cells Human Zhu et al., 2011114 PLK2 ENSG00000260410 Downregulation Acute leukaemia cells Li et al., 2008 115 EGFL7 ENSG00000172889 Downregulation Oral squamous cells Sasahira et al., 2012 116 CRK ENSG00000167193 Downregulation Lung cancer cells Crawford et al., 2008117 ADAM9 ENSG00000168615 Downregulation Melanoma cancer cells Felli et al., 2013 118 HOXA9 ENSG00000078399 Downregulation Acute leukaemia cells Shen et al., 2008 119 IRS1 ENSG00000169047 Downregulation Breast cancer cells Zhang et al., 2008120 SOX-2 ENSG00000242808 Downregulation Gastric cancer cells Otsubo et al., 2011 121 SLC7A5 ENSG00000103257 Downregulation Lung cancer cells Miko et al., 2011122 VEGFA ENSG00000150630 Downregulation Oral squamous cells Sasahira et al., 2012 116 MMP7 ENSG00000137673 Downregulation Melanoma cancer cells Felli et al., 2013 118 miR-15a MI0000069 BCL-2 ENSG00000171791 Downregulation Apoptosis, ATP production B-cell chronic lymphocytic leukemia Human Gao et al., 2010101 MCL-1 ENSG00000143384 Downregulation COX4I2 ENSG00000131055 Downregulation Siengdee et al., 2010102 COX6A2 ENSG00000156885 Downregulation NDUFB7 ENSG00000099795 Downregulation NDUFV1 ENSG00000167792 Downregulation NDUFS4 ENSG00000164258 Downregulation miR-16a MI0000070 COX4I2 ENSG00000131055 Downregulation Apoptosis, ATP production B-cell chronic lymphocytic leukemia Human Siengdee et al., 2010102 COX6A2 ENSG00000156885 Downregulation NDUFB7 ENSG00000099795 Downregulation NDUFV1 ENSG00000167792 Downregulation NDUFS4 ENSG00000164258 Downregulation Radiol Oncol 2021; 55(4): 379-392. Renčelj A et al. / MitomiRs, mitochondria and cancer metabolism388 cytochrome c oxidase subunit 4I2 (Cox4i2), subu- nit 6A2 (Cox6a2), NADH:ubiquinone oxidore- ductase subunit B7 (Ndufb7), NADH:ubiquinone oxidoreductase core subunit V1 (Ndufv1) and NADH:ubiquinone oxidoreductase subunit S4 (Ndufs4) expression.102 Glycolysis is the initial step in glucose catabo- lism, and occurs outside of the mitochondria in the cytoplasm. In the context of miRNAs affecting cell metabolism, miR-155 (Table 2) was found to indi- rectly upregulate hexokinase 2 (HK2), a glucose phosphorylation enzyme that might affect energy consumption in breast cancer cells. Mir-143 ap- pears to be one of two potential pathways regu- lating miR-155-dependent HK2 regulation.103,104 Alternative splicing of pyruvate kinase isoenzyme (PKM), whose splicing proteins are regulated by miR-124, miR-137, and miR-340, is another path- way regulating glucose metabolism (Table 2). This miRNA-dependent regulation of PKM is able to in- fluence colorectal cancer growth and counteract the Warburg effect.105 In addition, pyruvate kinase (PK) is a direct target of the tumor suppressor miR-326, making it a potential glucose metabolism regula- tor.94,106,107 In hepatocellular carcinoma, reduced mRNA levels were detected in 11 of the 13 genes encoded in the mtDNA, including the genes encoding cy- tochrome B (mt-CYB) and cytochrome C oxidase II (mt-CO2).108 When miR-181a-5p expression was increased, the levels of mt-CYB and mt-CO2 were reduced in hepatocellular carcinoma cells, while mitochondrial membrane potential (MMP) main- tained by electron transfer chain was reduced. In vivo experiments, which were done by Zhuang et al.108, have shown to have caused glucose metabo- lism to reprogram and stimulated tumor growth and early lung metastasis in patients with hepato- cellular carcinoma. Several studies reported that miR-126 has an important role in different human cancers (Table 2) such as breast, lung, gastric cancers, melanoma can- cer and acute leukaemia. Tomasetti et al.83 reported that miR-126 affects mitochondrial energy metabo- lism, resulting in malignant mesothelioma tumor suppression. This mitomiR reduce mitochondrial respiration and promote glycolysis in H28 cells, as- sociated with IRS1 modulate ATP-citrate lyase de- regulation. This leads to an increase in ATP and cit- rate production which is linked with reducing Akt signaling and inhibiting cytosolic sequestration of Forkhead box O1 (FoxO1), which promote the ex- pression of genes involved in gluconeogenesis and oxidative stress defense.83 Hypoxia has previously been related to altered mitomiR expression, with hypoxia-regulated mi- tomiRs being found to play a key role in cell sur- vival in oxygen-depleted settings.123 MiR-210 is one of the mitomiRs that is continuously increased in normal and transformed cells during hypoxia, sug- gesting that miR-210 plays a role in cells’ adaptive response to hypoxia.109 MiR-210 expression cor- responds with hypoxia gene signatures in tumor tissues such as breast and head and neck cancers, demonstrating a direct connection between miR- 210 expression and hypoxia in cancer.110 MiR-210 has been researched extensively and has a number of functionally significant targets in cell cycle con- trol, cell survival, differentiation, angiogenesis, and metabolism.123 Cell metabolism switches from mi- tochondrial OXPHOS to glycolysis under hypoxic environments. HIF-1, a hypoxia-inducible factor that upregulates the expression of most glycolytic enzymes as well as pyruvate dehydrogenase kinase while downregulating mitochondrial respiration, plays a key role in this action. Previous research has looked into how miR-210 regulates mitochon- drial metabolism under hypoxia. MiR-210 target iron-sulfur cluster assembly proteins (ISCU1/2) and inhibit the activity of iron-sulfur proteins that govern mitochondrial metabolism, such as com- plex I and aconitase, resulting in lower OXPHOS.123 It acts directly on cytochrome c oxidase assembly factor heme A:farnesyltransferase (COX10), succi- nate dehydrogenase complex subunit D (SDHD), and NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 (NDUFA4) in regulating mitochon- drial activity.123 Another study found an abnor- mal mitochondrial phenotype in A549 lung cells overexpressing miR-210, and mRNA expression profile analysis connecting miR-210 to mitochon- drial dysfunction.112 Interestingly, HIF is rapidly destroyed upon reoxygenation of hypoxic cells due to miR-210’s high stability, whereas miR-210 stays stable to maintain the glycolytic phenotype. Under normal conditions, this slows mitochondrial metabolism and may contribute to the Warburg ef- fect in cancer cells. This result supports miR-210’s involvement in regulating mitochondrial metabo- lism and promoting cancer cells’ adaptability to hypoxic environments. Another important mitomiR is miR-200, which has been identified as involved in tumor progres- sion.124,125 One of miR-200 targets, is transcription factor mitochondria (TFAM) which is one of the most important proteins regulating mitochondrial biogenesis. TFAM has been described as a func- tional target of miR-200 in breast cancer cells.113 Its Radiol Oncol 2021; 55(4): 379-392. Renčelj A et al. / MitomiRs, mitochondria and cancer metabolism 389 transcription factor activity is required for mtDNA replication and transcription. In addition to its func- tion in replication and transcription, the presence of TFAM is necessary for mtDNA maintenance.126 It has also been implicated as a primary architectural protein of the mitochondrial genome by packaging mtDNA. In addition, TFAM expression has been re- ported to be involved in tumor progression, cancer cell growth, and chemoresistance.127 Regarding the role of miRNAs in cancer and metabolism, the miR-17/92 cluster is one of the best characterized oncogenic miRNAs. This cluster is also known as oncomiR-1, and there is growing evi- dence of its oncogenic potential.93 It has been shown that miR-17/92 suppresses apoptosis and was origi- nally found amplified in B-cell lymphomas, where ectopically overexpressed truncated versions lack- ing miR-92a-1 were shown to possess oncogenic properties.110 The MiR-17/92 cluster is deregulated in B-cell lymphomas, T-cell lymphomas, B-cell chronic lymphocytic leukemia, and acute myeloid leukemia. This cluster is particularly overexpressed in several other cancers, including osteosarcoma, neuroblastoma, cervical, pancreatic, breast, lung, colorectal, ovarian, kidney, and liver cancers.93,105 Izreig et al.128 reported that this miRNA cluster is a key factor in metabolic reprogramming of tumors. If oncomiR-1 is absent in Myc+ tumor cells, there is a global decrease in glycolytic and mitochondrial metabolism. If increased oncomiR-1 expression is present, this is sufficient for increased nutrient utilization by tumor cells. Deletion of miR-17/92 promoted changes in gene expression in Myc+ lym- phoma which results in global decrease in metabol- ic pathways including glycolysis, the Krebs cycle, components of the electron transfer chain, amino acid metabolism, the pentose phosphate pathway, serine biosynthesis and nucleotide biosynthesis.128 Conclusions MiRNAs have been found in the mitochondria of many cell types, as shown by an increasing num- ber of studies and they were named mitomiRs. In general, mitomiR populations differ in various tissues and under different pathological circum- stances, implying that mitomiR populations are regulated by mechanisms that remain to be discov- ered. Based on the available information, we can deduce that there are a significant number of miR- NAs which are present in mitochondria.7,29–33 In our review, we have shown that various mi- tomiRs play a role in the initiation and progres- sion of cancer via the regulation of mitochondria. They are involved in the Warburg effect, which is necessary for cancer cells to maintain their prolif- erative capacity.91 MitomiRs also upregulate HK2, a glucose phosphorylation enzyme, in an indirect manner, which may impact energy consumption in breast cancer cells.103,104 Expression of one of the mi- tomiRs (miR-210) corresponds with hypoxia gene signatures in tumor tissues such as breast cancer and head and neck cancers, demonstrating a clear connection between mitomiR expression and hy- poxia in cancer.108,109,121 MiRNAs have emerged in the last decade as key regulators in cancer-related processes and are classified as either oncogenic or tumor suppressive miRNAs. The miR-17/92 clus- ter was first discovered to be amplified in diffuse cell lymphoma and B-cell lymphoma. This mito- miR cluster suppresses apoptosis and may act as an oncogene in B-cell lymphomas, B-cell chronic lymphocytic leukemia, acute myeloid leukemia, and T-cell lymphomas. It is also overexpressed in numerous other malignancies. This cluster is a key factor in metabolic reprogramming of tumors by regulating glycolytic and mitochondrial metabo- lism. Tumor-targeting treatments based on mito- miRs are emerging as a novel diagnostic and thera- peutic tool.94,106,111,128 Future perspectives We have shown that mitomiRs are important play- ers in mitochondria of cancer cell that need to be further investigated to develop a new potential therapies for cancer. Numerous studies that have been published in recent years give promising pre- dictions that mitomirRs will receive more attention in the context of their role in cancer as possible bio- markers or targets for treatment. Acknowledgement This work was financially supported by the Slovenian Research Agency (ARRS grant # P3-0003). References 1. Bienertova-Vasku J, Sana J, Slaby O. The role of microRNAs in mito- chondria in cancer. Cancer Lett 2013; 336: 1-7. doi: 10.1016/j.can- let.2013.05.001 2. 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