Mitochondrial microRNA (MitomiRs) in cancer and complex mitochondrial diseases: current status and future perspectives

Abstract

Mitochondria are not only important for cellular bioenergetics but also lie at the heart of critical metabolic pathways. They can rapidly adjust themselves in response to changing conditions and the metabolic needs of the cell. Mitochondrial involvement as well as its dysfunction has been found to be associated with variety of pathological processes and diseases. mitomiRs are class of miRNA(s) that regulate mitochondrial gene expression and function. This review sheds light on the role of mitomiRs in regulating different biological processes—mitochondrial dynamics, oxidative stress, cell metabolism, chemoresistance, apoptosis,and their relevance in metabolic diseases, neurodegenerative disorders, and cancer. Insilico analysis of predicted targets of mitomiRs targeting energy metabolism identified several significantly altered pathways (needs in vivo validations) that may provide a new therapeutic approach for the treatment of human diseases. Last part of the review discusses about the clinical aspects of miRNA(s) and mitomiRs in Medicine.

Electronic supplementary material

The online version of this article (10.1007/s00018-020-03670-0) contains supplementary material, which is available to authorized users.

Introduction

Mitochondria are dynamic double-membrane organelles that produce power to the cell’s biochemical reactions. Their role in other cellular processes such as calcium signaling, detoxification, autophagy, and apoptosis makes them master regulators of the cell. Healthy mitochondria are prerequisites for a cell to survive [1] and its dysfunction is seen in several human pathologies that include cardiovascular disease, obesity, diabetes, neurodegeneration, and cancer [2–4]. Hence, mitochondria represent an attractive target to treat complex human pathophysiologies.

Several studies have shown the involvement of microRNAs (miRNAs) in the regulation of mitochondrial processes in normal versus diseased conditions [5–7]. Thus miRNA based strategies hold significant promise as therapeutics in complex diseases.

MicroRNAs are small, ~ 22-nucleotides non-coding RNAs expressed in a wide range of eukaryotic organisms. They are transcribed from DNA sequences by RNA polymerase II or RNA polymerase III into primary miRNAs (pri-miRNAs) and then processed by RNase III Drosha and a double stranded-RNA binding protein called DGCR8 (DiGeorge syndrome critical region 8 gene) into precursor miRNAs (pre-miRNAs, ~ 70 nt) in the nucleus [8, 9]. Double-stranded stem looped pre-miRNAs are then exported to the cytoplasm by an exportin 5 (XPO5)/RanGTP complex (Fig. 1). Pre-miRNAs are then processed by the RNase III endonuclease Dicer to generate an ~ 20-bp RNA duplex with 2-nt 3′ overhangs called mature miRNA [10]. The regulatory functions of microRNAs are accomplished by associating with argonaute (AGO) and GW182 (glycine-tryptophan protein of 182 kDa) proteins, which are the main components of the miRNA-induced silencing complex (miRISC) [11]. In most cases miRNA interacts with 3′UTR of messenger RNA (mRNA) and negatively regulates mRNA expression. The complementarity between 8-nucleotidemiRNA seed sequence and its target mRNA determines which silencing mechanism will be employed: cleavage of target mRNA or translational inhibition [12, 13]. The perfect complementarily elicits the Ago2 mediated target mRNA decay in processing bodies (P-bodies) [14], whereas mismatch in two or more nucleotides in the middle of the target site leads to translational inhibition of target mRNA [16–22]. However, under certain conditions miRNAs have also been shown to activate gene expression [15]. Interaction of miRNA with 5′UTR, coding sequence and gene promoters have also been reported [16]. Very recent reports by Sheu-Gruttadauria et al. suggest that apart from miRNA seed sequence, complementarity between 13 and 16 nucleotides in the guide region of mature miRNA sequence enhances miRNA activity by > 20 fold [17].

Fig. 1.

miRNA biogenesis and functioning: miRNA(s) are transcribed and after post-transcriptional processing in nucleus are transported to the cytoplasm in the form of pre-miRNA(s). Further, mature-miRNA is generated upon cytoplasmic processing of pre-miRNA. mitomiRs gets transported into mitochondria through lesser known mechanism (might involve AGO and PNPase proteins). miRNA–mRNA interactions in RISC complex elicits mitomiR-mediated silencing of mitochondrial as well as cytosolic targets and regulates mitochondrial functions

The emerging evidence indicates that mature miRNA localizes in multiple subcellular compartments (mitochondria, endoplasmic reticulum, and exosomes, etc.) [18]. MiRNAs that regulate mitochondrial functions are regarded as mitomiR (mitochondrial miRNA). They are of two major types: mitomiRs that bind to mRNA and repress the gene expression in the cytoplasm to modulate mitochondrial function (Table 1, Fig. 1) and the other type of mitomiRs are those that are imported into mitochondria as part of either RISC or pre-RISC and target mitochondrial encoded mRNAs (Table 2, Fig. 1). Majority of mitomiRs are known to be nuclear-encoded; however, human mtDNA also seems to harbor mitomiR sequences (namely, miR-1974, miR-1977, and miR-1978) [19]. It remains to be ascertained whether miRNA(s) are actually transcribed from mitochondrial genome. The regulation of different signalling pathways by mitomiRs is an unexplored niche and needs detailed investigation.

Table 1.

List of mitomiRs that bind to mRNA and repress the gene expression in the cytoplasm to modulate mitochondrial function

mitomiR	Target gene	Associated disease	PMIDs
miR-17*	Mn-SOD2, GPX2, TRXR2	Prostate cancer	21,203,553
miR-17-3P	Mn-SOD2, GPX2, TRX R2	Prostate cancer	30,240,971
miR-499	DRP1, PPP3CA	Myocardial infarction	21,186,368
miR-140	MFN-1	Cardiomyoocyte apoptosis	24,615,014
miR-214 miR-761 miR-106a MiR-195	MFN-2	Huntington's disease Hepatocellular carcinoma Cardiac hypertrophy Breast cancer	26,307,536 26,845,057 27,565,029 30,932,749
miR-27 a/b miR-27b	PINK1, PRKN BAX	Parkinson disease Type II diabetes	27,456,084 28,698,281
miR-484 miR-483-5p	FIS1	Myocardial infarction Tongue squamous cell carcinoma	22,298,638 25,843,291
MiR 378& miR378*	MED13	Obesity and metabolic syndrome	22,949,648
miR-210-5p	COX10	Colon cancer	20,498,629
miR-338-5p	COXIV	Neurological disorder	19,020,050
miR-761 miR-27	MFF	Myocardial infarction Liver disease	23,867,156 25,431,021
miR-195	SIRT3 ARL2	Heart failure Bladder cancer	29,330,215 29,130,995
miR-30e	UCP2	Kidney fibrosis	23,515,048
miR-145	BNIP3	Cardiovascular disease	23,028,672
miR-324-5p	MTFR1	Cardiac disease	26,633,713
miR-17	ADPKD	Polycystic kidney disease	28,205,547
miR-4331	RB1	Retinoblastoma	29,217,619
miR-668	MTP18	Ischemic acute kidney injury	30,325,740
miR-98	LASS2	Bladder cancer	30,463,687
miR-509-5p	SOD2	Breast cancer	28,925,482
miR-548b-3p	CIP2A	Hepatocellular carcinoma	30,671,469
miR-29a	MCL 1	Intracranial aneurysm	30,015,903
miR-34-a	CYC	Cardiovascular and neurodegenerative disease	26,661,155
miR-101-3p miR-127-5p	ATP5	Viral infection Liver disease	21,291,913 22,433,606
miR-570	ATP5L	Platelet disease	27,561,077
miR-210	SDHD ISCU1/2	Lung cancer Lung disease	20,885,442 19,808,020
MiR-338-5p	ATP5G1	Metabolic disease	22,773,120
miR-155	BAG5/PINK1	Aging	31,948,758
miR-29b	BAX PGC1α	Cardiac fibrosis Heart failure	30,025,410 30,346,946
miR-34	NOTCH-1	Age related cataract	29,299,142
miR-122	Mitochondrial metabolic gene network	Hepato-cellular carcinoma	20,739,924
miR-21	BAX/BCL2 ratio	Keloid fibrosis	29,190,966
miR-31	SIRT3	Oral carcinoma	31,055,111
miR-26	ATF2	Laryngeal cancer	29,108,284
miR-181	PINK1, Parkin, BCL2	Neurodegenerative disease	27,281,615 21,958,558
miR-130b-p miR-761	PGC1α	Metabolic disease	28,433,632 26,408,907
miR-149	PARP-2	Metabolic disease	24,757,201
miR-494 miR-155-5p	TFAM	Metabolic disease	23,047,984 25,869,329
miR-499	CALNA3	Myocardial infarction	21,186,368
miR-7 miR-320	VDAC-1	Neurodegenration Cervical cancer	26,801,612 26,472,185

Table 2.

List of mitomiRs which are imported into mitochondria as part of either RISC or pre-RISC and target mitochondrial encoded mRNA

mitomiR	Target gene	Associated Disease	PMIDs
miR-151a-5p	MT-CYB	Asthenozoospermia	26,626,315
miR-181-c	MT-CO1	Colon Cancer	28,383,996
MiR-378	MT-ATP6	Diabetes	28,709,769
miR-2392	ND4, CYTB, COX1	Chemo-resistance	30,659,020
miR-5787	MT-CO3	Cisplatin-resistant tongue squamous cell carcinoma	31,534,516
miR-92a	MT-CYB	Diabetic heart	31,319,246

Mitochondrial localization of microRNA

Mitochondrial genome encodes for 37 genes out of which 13 are protein-encoding, 22 are tRNA encoding, and 2 are rRNA encoding. Although mitochondria have their own genome and translation machinery, their function is highly dependent upon cargo imported from the cytoplasm. The cargo import into mitochondria is mediated by outer membrane transporter (TOM complex) and inner membrane transporters (TIM complex). The miRNA import into mitochondria was largely unknown until the first report came more than two decades ago, which suggested that the functional mitochondrial protein translocation machinery is a prerequisite for tRNALysCUU import into mitochondria in yeast [20]. It was then believed that miRNAs might also be imported into mitochondria along with the protein by utilizing the same protein import pathways. Maniataki et al. were the first who suggested that AGO2 protein might be involved in miRNA import into mitochondria wherein mitochondrial tRNA^met was reported to interact with human AGO2 protein [21]. Further, Mukherjee et al. reported that the RNA import into mitochondria is facilitated by multi-subunit RNA import complex (RIC) by protein import independent pathway in Leishmania [22]. Co-localization of AGO2 and AGO3 to mitochondria confirmed the presence of RNAi component in the mitochondrial sub-compartment [19]. Recently, AGO2 protein has been found to interact with another protein PNPase (poly nucleotide phosphorylase) that has been previously shown to import small RNAs into mitochondria [23, 24], although there is no direct evidence showing whether miRNAs are part of the pool of small RNAs imported into organelle by PNPase [23, 24]. Localization of PNPase in distinct mitochondrial RNA decay foci known as degradosome suggests that PNPase must be a part of RNAi component in the mitochondrial sub-compartment [25]. Danielle L. Shepherd et al. in their study showed that over-expression of PNPase is responsible for the increased translocation of miR-378 into the mitochondrial sub-compartment to regulate bioenergetics [23]. Collectively, all these studies suggest that Ago and PNPase are two major players that facilitate the mitochondrial miRNA import into the organelle (Fig. 1); however, the exact mechanism and what are the other components of miRNA mitochondrial import machinery are largely unknown.

Importance of mitomiRs in the pathophysiology

Role of mitomiRs in metabolic disorders

Mitochondria serve as a metabolic hub of the cell and play a pivotal role in diverse pathological conditions [26]. Tricarboxylic acid cycle (TCA) and fatty acid oxidation (FAO) are two major metabolic processes that take place exclusively in the mitochondria (Fig. 2). The other metabolic pathways such as cardiolipin synthesis, steroid biosynthesis, and urea cycle also involve mitochondria. The acetyl Co-A generated from fatty acid oxidation is used as a precursor molecule to produce energy via NADH and FADH during the tricarboxylic acid cycle (TCA cycle) in the mitochondria (Fig. 2). These molecules act as an initiator of electron transport chain (ETC) by donating an electron to ETC complexes and produce ATP by OXPHOS (oxidative phosphorylation). ETC is a string of five multimeric complexes (Complex 1 to V) that allows anelectron to flow across the inner mitochondrial membrane (IMM) to generate a proton gradient across IMM (Fig. 2).

Fig. 2.

Schematic diagram showing different processes of mitochondria being regulated by various mitomiRs (mitomiRs involved in metabolic disorders are labeled with blue color, mitomiRs involved in neurodegenration are labeled with purple color, and mitomiR involved in different cancers are labeled with black color)

Diabetes, cardiovascular disease, and obesity are three major metabolic disorders in which mitochondrial dysfunction and aberrant miRNA expression have been reported [27–30]. Several independent studies have identified compromised oxidative metabolism, altered mitochondrial structure, impaired biogenesis, and altered gene expression in insulin resistance type 2 diabetes (T2DM) [31, 32]. Mitochondrial ATP production has been found to be decreased in Type-I diabetes, wherein withdrawal of insulin has led to reduced expression of muscle mitochondrial genes involved in oxidative phosphorylation [33]. Several miRNA(s) have been found to alter the activity of ETC complexes by targeting their subunits: miR-181c and miR-338-5p alter mitochondrial complex IV activity by targeting different subunit of cytochrome oxidase [34, 35]. The ATP synthase (complex V) has been found to be a potential target of miRNA(s) in variety of diseases: miR-101-3p and miR-127-5p target ATP5B whereas mitomiR-378 down regulates mitochondrial encoded ATP6 following the diabetic insult [36–38]. In addition to these, miR-570 targets ATP5L, a subunit of F1-F0 complex, and promotes ATP loss in platelets [39]. The presence of miRNA-mRNA regulatory network in platelets during storage suggests mitochondrial bioenergetics play an important role in platelet activation [40, 41]. Future studies are warranted to validate these observations.

Further during diabetes, all the pathways that utilize glucose as an energy source get perturbed and energy is produced primarily by FAO in mitochondria. The metabolic switch from glucose oxidation to excess mitochondrial fatty acid oxidation leads to diabetic cardio-myopathy [42]. Xiaoxia Wang et al. in their study have shown differential expression of miRNAs in heart failure, wherein miR-696, miR-532, miR-690, and miR-345-3p have been found to be enriched in mitochondria during the early stage of heart failure [43]. Their study suggests the association of mitochondrial-enriched miRNA(s) with energy metabolism during heart failure. Another mitochondrially localized miR-181c has been shown to attenuate complex IV activity by targeting the MT-CO1 gene in rat heartsuggesting its possible role in heart failure [34]. The relation of miR-145 in myocardial infarction was established by showing its protective effects in the disease, wherein miR-145 restores mitochondrial membrane potential and attenuates cardiomyocyte apoptosis [44]. Carrer M et al. further reported that miR-378 /miR-378* knockout mice have shown resistance to high-fat diet-induced obesity by enhancing FAO [45]. In the same study, they have shown that miR-378 and miR-378* target mitochondrial carnitine O-acetyltransferase (CRAT) and MED13, crucial components of FAO [45]. In addition to these, Azzoui et al. observed that miR-199a∼214 cluster represses cardiac peroxisome proliferator-activated receptor δ (PPARδ) post-transcriptionally and facilitates the metabolic shift from FAO to glycolysis under hypoxic conditions [46]. Taken, together, all these above independent findings points towards important role of mitomiRs in regulating mitochondrial gene expression/function in metabolic disorders (Figs. 2, 3).

Fig. 3.

Upper panel shows important mitomiRs playing a role in neurodegenerative diseases, whereas bottom panel shows involvement of important mitomiRs in metabolic disorders

Role of mitomiRs in Neurodegenerative disorders

Mutation in the mitochondrial genome and mitochondrial abnormalities has been observed in age-related neurodegenerative disorders [47]. Mitochondria regulate complex apoptotic signaling pathways in a ROS (reactive oxygen species) dependent and independent manner, which contributes towards neuronal death and disease pathogenesis [48, 49]. The excess ROS is removed by mitochondrial antioxidant enzymes (superoxide dismutase and peroxidases) which serve a protective role in neuronal cell death [2].

Accumulating evidence suggests that miRNA(s) are critical for neuronal function and their deregulation leads to mitochondrial oxidative stress and neuronal apoptosis [50]. Alzheimer's and Parkinson’s are two major neurodegenerative diseases where miRNA(s) have been found to be implicated [51, 52] (Fig. 3).

The release of the pro-apoptotic signals upon oxidative stress is mediated through mitochondrial permeability transition pore (mPTP) to initiate cell death [53]. miR-7 has been shown to down regulate VDAC1 (crucial component of mPTP), deplete ROS, and maintain mitochondrial membrane potential to protect the cell against neuronal apoptosis [54]. Further miR-7 has also been reported to enhance the levels of glucose transporter (GLUT3) by targeting RelA in the cellular model of Parkinson's disease [55]. The increased GLUT3 expression by miR-7 augments glycolysis and shows a protective role in Parkinson’s disease with defective oxidative phosphorylation [55]. Similarly, miRNA-34b and miRNA-34c target alpha-synuclein (a-SYN) by binding to its 3′UTR and down regulation of these miRNA(s) contributes to Parkinson's disease pathogenesis [56]. The levels of miR-34b/c have been reported to decrease by low-frequency magnetic field and this in-turn enhances mitochondrial ROS accumulation to give rise to the diseased phenotype [57]. The decreased expression of miR-34b/c has been accompanied by altered mitochondrial function and dynamics, oxidative stress,and reduced ATP in the advanced stage of Parkinson's disease [58]. Further, reduced levels of miR-34b/c have been correlated with increased neuronal death and decreased expression of DJ1 and parkin in Parkinson disease brain tissues [58]. Mitochondrial dynamics (fission and fusion) and mitophagy maintain a healthy pool of mitochondria. The unrepaired excess oxidative stress is known to elicit mitochondrial fission-mediated mitophagy of damaged mitochondria (Fig. 2). The perturbation in mitophagy is the major reason for enhanced oxidative stress in Parkinson’s disease (PD). The pink1-parkin pathway of mitophagy is found to be compromised in PD by various mutations [59]. Therefore, enhancing miR34b/c in PD patients may impose neuroprotective function by enhancing neuronal viability via damaged mitochondrial degradation and reducing oxidative stress. miR34a along with miR-146a is known to promote TNFα dependent mitochondrial dysfunction in Alzheimer's disease [60]. MicroRNA-98 has been shown to reduce amyloid β-protein production and improve mitochondrial oxidative stress through the Notch signaling pathway in mice model of Alzheimer's disease [61]. In addition to this, neuron-specific miR-124 regulates mitochondrial function and neuronal reshaping by targeting intermediate filament vimentin [62]. Kim et al. in their study demonstrated miR-27a and miR27b as potential therapeutic targets in PD [63]. miR-27a and miR27b inhibit mitophagy by down-regulating pink-1 in the PD model [63].

Mitochondrial quality control by mitophagy is coupled with mitochondrial biogenesis. Mitochondrial biogenesis is a synthesis process essential to replenish the healthy pool of mitochondria that otherwise leads to decreased mitochondrial mass and diseased phenotype. In Parkinson's disease pathogenesis, decreased mitochondrial function has been improved by boosting miR-144-3p levels, wherein it targets APP (amyloid precursor protein) and augments mitochondrial biogenesis by elevating the levels of PGC1α, NRF1, and TFAM [64]. In addition to this, up-regulation of miR-590-3p has also been observed to improve mitochondrial function by enhancing mitochondrial biogenesis in the Parkinson disease model [65].

MiRNA-181 family has been found to play a role in various complex diseases including neurodegeneration. MiR-181 enhances apoptosis in astrocytes by downregulating BCL2 family of proteins and mitochondrial depolarization [66]. Consequently, miR-181 plays a critical role in neuronal cell death. Haixia Ding et al. observed decreased expression of miR-181a along with other 3 miRNAs in the serum of PD patients [67]. On the contrary, Hoss et al. observed enhanced expression of miR-181a in the prefrontal cortex of PD brains [68]. These observations hint toward a potential role of miR-181 in Parkinson disease development and progression. The regulation of mitophagy by miR-181a has been shown by Cheng M et al. in neuroblastoma cells, wherein miR-181a has been found to downregulate mitophagy by directly targeting parkin E3 ubiquitin ligase and thereby contributing towards overall oxidative stress accumulation [69].

Recently, Indrieri et al. have unveiled an interesting role of miR-181a/b in mitochondrial disease [7, 70], wherein it downregulates mitophagy and mitochondrial biogenesis concurrently [7]. Therefore, Inhibition of miR-181a/b will augment the degradation of damaged mitochondria and replenish them with healthy mitochondria by enhancing mitochondrial biogenesis [7]. Hence mitochondrial miR-181 not just serves as a potential biomarker but its down-regulation can also protect against oxidative injury and neuronal apoptosis in neurodegenerative disease.

Collectively all these reports suggest a potential role of mitomiRs in oxidative stress management and mitochondrial biogenesis in neurodegenerative disorders (Fig. 3). Especially, miR-7, miR-34, and miR-181 are key mitomiRs that target several closely related pathways responsible for disease development (Table 1). Future studies are warranted to confirm/validate these observations using model systems.

Role of mitomiRs in cancer

Cancer is the result of functional abnormalities in an array of physiologically relevant pathways. The hallmarks of cancer include the following: resistance to apoptosis, enhanced proliferation, epithelial to mesenchymal transition, invasion, metastasis, adaptation to hypoxia, and metabolic rewiring (Fig. 4). The induction of intrinsic pathway of apoptosis involves mitochondria primarily, and cancer cells evade apoptosis either by reducing the death signals (ROS, calcium overload) or by enhancing the expression of anti-apoptotic proteins. Perturbation in major mitochondrial pathways such as oxidative stress repair, mitochondrial metabolism, and adaptation to stress leads to cancer (Fig. 4).

Fig. 4.

Different hallmarks of cancer governed by mitomiRs. (*Represents our own studies)

mitomiRs in mitochondrial oxidative stress and apoptosis

The signaling pathways of apoptosis and mitochondrial oxidative stress play a vital role in cancers [71]. Cell death via apoptosis in response to enhanced oxidative stress serve as a tumor-suppressive mechanism [72], whereas mitochondrial death by mitophagy is a pro-survival mechanism (by diminishing oxidative stress) that allows cells to proliferate [72, 73]. Several mutations in mitochondrial DNA have been observed in cancer [74]. miRNA(s) have been found to regulate all signaling circuits within a cell and their dysregulation plays an important role in the cancer development and progression [75]. The unrepaired ROS is a source of apoptosis induction and miRNAs have been shown to impair the process of ROS removal to suppress the tumorigenicity of various cancers [75]. MiR-17* and miR-17-3p have been reported to inhibit mitochondrial antioxidant enzymes such as mn-SOD2, GPX2, and TRXR2 in prostate cancer [76, 77]. Further miR-509-5p has been shown to render the anti-oncogenic effect in breast cancer by targeting SOD2 [78]. Hence, down-regulation of mitochondrial antioxidant machinery and the accumulation of oxidative stress augments apoptosis by miRNA(s) in cancer.

Cancer cells often show resistance to apoptosis. BCL2 is up regulated in various cancers that contribute significantly towards apoptosis resistance [79–83]. In a previous report from our laboratory we have shown that down regulation of BCL2 using miRNA(s) augments apoptosis as well as enhances mitochondrial depolarization in breast cancer cells [84]. The depolarized mitochondria undergo mitochondrial fission during apoptosis. However, it has not been well explored that mitochondrial fission and apoptosis are interdependent or independent events in the cancer cell [85]. Amongst this ambiguity, our recent findings (Purohit PK et al.) showed that mitomiR-195 is an anticancer microRNA having potential to enhance mitochondrial fragmentation in apoptotic breast cancer cells [86]. We further revealed that pro-apoptotic role of mitomiR-195 is independent of mitochondrial fission in breast cancer cells [86].

mitomiRs in cancer cell metabolism in the tumor microenvironment

Cancer cells require extra energy and nutrient supply in order to sustain proliferation. They are not dependent on the TCA cycle, rather they have reprogrammed their metabolism toward aerobic glycolysis to meet their energy demand under hypoxic (low oxygen) tumor microenvironment [87]. Cancer cell accelerates glucose uptake by modulating GLUTs for rapid glycolysis [88]. Various miRNAs have been unveiled to target glucose import by down-regulating different isoforms of GLUT in different cancers [89–91]. The Hif1α is a master regulator of hypoxic cell survival, and it up regulates various pathways (Cancer cell stemness, Angiogenesis, Erythropoiesis, and Glycolysis) responsible for cancer cell proliferation and survival under hypoxic tumor microenvironment [92]. Further, the localization of HIF1α in mitochondria also suggests the association of the organelle in hypoxic cancer cell survival [93]. Hif1α showed the pro-survival effects by augmenting mitochondrial autophagy to eliminate ROS under hypoxia. It also confers resistance to cell death by repressing the mitochondrial electron transport chain. The miR-210 (hypoxamiR- upregulates in response to hypoxia) is observed to be up-regulated in the late stage of lung cancer and is known to deplete SDHD (succinate dehydrogenase subunit D) levels to affect complex II activity [94, 95]. This mitomiR also modulates complex IV activity by down-regulating COX10 and ISCU [96]. miR-18a has been shown to downregulate HIf1α to diminish metastasis in breast cancer cells [97]. Similarly, MiR-199 is a major Hif1α targeting miRNA that has been unveiled to induce apoptosis in different cancers [98]. The mutation in the miR-199 binding site of Hif1α leads to cancer cell proliferation and poor clinical outcomes [99]. Recently, Zhuang X et al. have shown that mitomiR-181a-5p reduced multiple genes encoding for ETC subunits and concurrently enhances the expression of hexokinase 2 (HK2) and GLUT1 [100]. The metabolic reprogramming by miR-181a-5p promotes lung cancer cell proliferation and metastasis [100]. MiR-199 and miR-181a-5p along with miR-210 seem to be major modulator(s) of tumor microenvironment and metabolism through Hif1α and altering mitochondrial function (Fig. 4). Future studies in this direction may provide conclusive answers.

mitomiRs in chemoresistance and tumor recurrence

The chemoresistance and tumor recurrence are major hurdles in effective cancer treatment. The development of cancer stem cell-like cells (CSC) in a heterogeneous population of cancer play a major role in chemo-resistance, therapeutic failure,and tumor recurrence [101, 102]. There is a significant body of literature describing mitochondria as important determinants of several aspects of cancer development and progression—metabolic reprogramming, acquisition of metastatic capability, and response to chemotherapeutic drugs [103–105] (Fig. 4). Cancer cell attains resistance to chemotherapeutic agents via multiple mechanisms—up regulated anti-apoptotic signals, down regulated pro-apoptotic signals, faulty apoptotic signaling, etc. [106]. The BCL2 family of proteins that regulate the mitochondrial apoptotic pathway play an important role in tumor maintenance, as well as the response of cancers to chemotherapies / targeted therapies [107]. In addition, HAX1 is another antiapoptotic protein that inhibits cell death and metastasis in different cancers [108]. The expression level of HAX1 has been found to increase during breast cancer relapse and contribute toward poor prognosis [109, 110]. The antiapoptotic pathways that are observed to be upregulated in cancer stem cells are targeted by multiple miRNAs. Decreased expression of miR-125 in Hep-2-CSCs and laryngeal cell carcinoma contributes to poor clinical outcomes [111]. On the contrary, the over expression of miR-125a in Hep-2-CSCs enhances sensitivity to cisplatin, vincristine, vinblastine, and doxorubicin [111]. Hence, miR-125a down regulates HAX1 to induce apoptosis in Hep-2-CSCs by mitochondrial pathways to revert chemo resistance [111]. Further, miR-125b has also been found to enhance apoptosis through mitochondrial pathways by inhibiting HAX1 expression in doxorubicin-resistant breast cancer cells [112]. Contrastingly, Shi et al. have shown that miR-125b levels are augmented and confer resistance to apoptosis in temozolomide-resistant glioblastoma stem cells, responsible for poor clinical outcome [113]. HAX1 protein has also been found to be a direct target of miR-100 in the breast cancer [114]. The overexpression of miR-100 down regulates HAX-1 and enhances apoptosis in cisplatin-resistant breast cancer cells [114]. Apart from these, few miRNAs have also been reported to modulate hypoxia-induced chemoresistance in gastric cancer [115]. Oridonin, a natural agent with potent anticancer activity, upregulates BIM-S (BCL-2 interacting mediator of cell death, variant 11) by downregulating miR-17 and miR-20a miRNAs sensitize leukemia cells resistant to chemotherapy-induced apoptosis [116]. MiR-24 expression has been found to be upregulated in osteosarcoma patient’s serum as well as tissues, and down regulation of miR-24 in osteosarcoma cell lines has been shown to improve the sensitivity towards doxorubicin by augmenting BIM mediated apoptosis [117]. Independently, Qing Xie et al. in their study demonstrated reduced levels of miR-519d in breast cancer stem cells [118]. And over-expression of miR-519d enhanced the sensitivity of cisplatin-resistant breast cancer stem cells by down-regulating MCL1 protein (another member of the antiapoptotic BCL2 family) via mitochondria-mediated apoptosis [118].

Recently, Zhang et al. in their study showed that miR-1overexpression caused mitochondrial damage and induced mitophagy in CSCs whilst it did not affect mitochondrial function of cancer non-stem cells [119]. miR-1 targets MINOS1(mitochondrial inner membrane organizing system (1) and GPD2 (glycerol-3-phosphate dehydrogenase (2) genes by directly binding to their 3′UTR; it also interacts with mitochondrial localized LRPPRC (leucine-rich pentatricopeptide-repeat containing) protein in CSCs [119]. In HCC cells, Morita et al. showed the relevance of increased miR-18a and decreased miR-199 in hepatocellular carcinoma recurrence post-liver transplantation. They also observed that miR-18a regulated the expression of tumor necrosis factor alpha-induced protein 3 (TNFAIP3), and miR-199a-5p regulated the expression of hypoxia-inducible factor 1 alpha (HIF1α), vascular endothelial growth factor A (VEGFA), insulin-like growth factor 1 receptor, and insulin-like growth factor 2 in HCC [120]. He et al. in their study showed association of miR-944 with cisplatin resistance by down regulating Bnip3 in breast cancer [121]. miR-98 is also found to promote chemoresistance in bladder cancer by targeting LASS2 and regulating mitochondrial dynamics [122]. Recently, miR-5787 has been reported to reprogramme cancer cell metabolism and contribute towards cisplatin resistance in tongue squamous cell carcinoma [123]. Lately, Song Fan et al. for the first time revealed the role of mitomiR in mitochondrial DNA transcription and its contribution to tumor cell metabolism and chemoresistance [5]. Taken together, all these data suggest the potential role of mitomiRs in CSCs and tumor recurrence. (Please see Table 3, Fig. 4).

Table 3.

List of mitomiRs involved in resistance to chemotherapy and cancer reoccurrence

microRNA	Associated genes	Associated chemoresistant cancer/cancer reoccurrence	PMIDs
miR-125a	HAX-1	Cisplatin-resistant laryngeal cell carcinoma	27,880,721
miR-125b	HAX-1	Doxorubicin-resistant breast cancer	29,434,858
miR-100	HAX-1	Cisplatin-resistant breast cancer	29,975,932
miR-17, miR-20	BIM-S	Chemo-resistant leukemia	24,872,388
miR-24	BIM	Doxorubicin-resistant osteosarcoma	27,681,638
miR-519d	MCL-1	Cisplatin-resistant breast cancer	28,423,543
miR-944	BINP3	Cisplatin-resistant breast cancer	26,298,722
miR-5787	MT-CO3	Cisplatin-resistant tongue squamous cell carcinoma	31,534,516
miR-2392	ND2, ND4, ND5, CYTB, COX1, COX2	Chemoresistant tongue squamous cell carcinoma	30,659,020
miR-1	MINOS1 GPD2, LRPPRC	Breast cancer stem cells and melanoma cancer stem cells	31,765,945
miR-18a miR-199	TNFAIP3 HIF1α, VEGFA, IGF1R, IGF2	Hepato-cellular carcinoma reoccurrence	26,783,726
miR-125b	BCL-2, BAX	Temozolomide-resistant glioblastoma	21,879,257

Bioinformatics prediction of mitomiRs and their associated pathways

The electron transport chain (ETC) comprises of transmembrane protein complexes (I-IV) and electron transporters, ubiquinone and cytochrome c. The electron flow through electron transport pathways is coupled with the generation of a proton gradient across the inner membrane and the energy accumulated is used by complex V (ATP synthase) to produce ATP, the main source of energy in eukaryotic cells. It is now well recognized that miRNAs control metabolism [124]. Hence, it is extremely important to understand the role of mitomiRs in affecting energy metabolism. To explore the involvement of mitomiRs, we did insilco analysis. Complex-I is the largest amongst all ETC components and serves as a major electron entry site of the OXPHOS system. Interestingly, we observed that the mitochondrial gene (s) MT-NDL4 (encodes a protein called NADH dehydrogenase 4L) and MT-ND5 (a protein called NADH dehydrogenase 5), which are part of the large mitochondrial enzyme complex-I, are predicted to be targeted by 587 and 196 different miRNAs, respectively. Out of these, 50 miRNAs were found to target both genes thereby suggesting that they might have a regulatory role on maintenance of energy homeostasis and oxidative stress management by affecting mitochondrial Complex-I activity (Fig. 5a). Complex V (mitochondrial ATP synthase) is made up of two functional domains: F(1), situated in the mitochondrial matrix, and F(o), located in the inner mitochondrial membrane. Subunits of the ATP synthase F(o) component, ATP8 and ATP6, are encoded by mitochondria and are predicted to be targeted by 203 miRNA(s) and 307 miRNA(s), respectively (Fig. 5a). Out of these 9 miRNAs were found to target both subunits of complex-V thereby suggesting that they might perturb the process of oxidative phosphorylation (Fig. 5a) as it is highly dependent upon the expression and assembly of all the subunits including ATP8 and ATP6. The over-expression of both subunits showed significant restoration of complex-V assembly and activity in cells having depletion of ATP8 and ATP6 levels [125]. Surprisingly, we observed that two miRNAs (miR-5004-3p and miR-3591-5p) out of 1293 predicted mitomiRs appear to affect the activity of both ETC complexes (Complex-I and Complex-V) by targeting MT-NDL4, MT-ND5, ATP8, and ATP6 genes (Fig. 5a). Taken together, our insilico data suggest that miR-5004-3p and miR-3591-5p might regulate oxidative stress and energy homeostasis by regulating expression and assembly of multiple subunits of mitochondrial respiratory complex-I and complex-V.

Fig. 5.

Bioinformatic analysis of predicted mitomiRs targeting energy metabolism. a mitomiR prediction and analysis to find out common mitomiRs. b Analysis of processes affected by common mitomiR (miR-3591-5p and miR-5004-3p) using panther gene list analysis tool. c Pathway analysis of miR-3591-5p targets (top 10 pathways). d Pathway analysis of miR-5004-3p targets (top 10 pathways). Red bar indicates statistically significant pathways with adjusted p value < 0.05

We further retrieved the list of genes that were predicted to be targeted by both mitomiRs using target scan software. Using the panther pathway analysis tool and target scan we have generated the major biological processes that are predicted to be affected by both mitomiRs (miR-5004-3p and miR-3591-5p) (Fig. 5b). Biological regulation, metabolic process, and cellular process were found to be the most affected processes by both mitomiRs (Fig. 5b). Further digging into the pathway analysis using the GeneEnricher pathway analysis tool we unveiled that 18 different pathways might be significantly affected (adjusted p value < 0.05) by miR-3591-5p (please see supplementary data for detailed analysis). The insulin signaling pathway and pathways affecting insulin-like growth factor (IGF1)-Akt signaling are observed to be the most significantly altered pathway by miR-3591-5p (Fig. 5c). The role of mitochondria in insulin signaling cannot be circumvented due to its extensive involvement in metabolism. Other significantly altered pathways by miR-35915p that have been widely studied in cancer include TGFβ signaling, PI3-Akt signaling, VEGFA-VEGFR2 signaling, and ErbB signaling pathway. The Brain-derived neurotrophic factor (BDNF) signaling pathway is also observed to be significantly altered by miR-3591-5p; this hints the potential role of miR-3591-5p in neurological disorders. In addition to this, miR-3591-5p and miR-5004-3p both seem to affect the ectoderm differentiation pathway significantly (Fig. 5c, d). Mitochondrial functional suppression during ectoderm differentiation into neural precursor cells suggests a potential role of both mitomiRs in metabolic remodeling [126]. Our preliminary in-silico finding on miR-3591-5p and miR-5004-3p suggests that both miRNA(s) have the potential to affect several pathways that are differentially regulated in multiple complex mitochondrial disorders (please see supplementary data for detailed analysis). Further validation of these mitomiRs in diseases context is essential to characterize them as a therapeutic.

Clinical aspects of miRNA(s) and mitomiRs in medicine: As biomarkers, prognostics, and therapeutics

miRNA(s) are small, endogenous, non-coding RNAs that regulate gene expression at post-transcriptional or translational levels. They function as master regulators of gene expression and their aberrant expression has been associated with many human diseases such as cancers, diabetes, viral infections, cardiovascular diseases, neurodegenerative diseases, etc. Till date, more than 2000 miRNA(s) have been found to regulate up to 30% of the protein-coding genes in the human genome. Single miRNA can bind and regulate the expression of several different transcripts and single transcripts can be regulated by multiple miRNAs. Identification and functional characterization of miRNAs, miRNA-target interactions, and miRNA-disease associations provided clues that they will have immense potential for the treatment of various human diseases. Clinical trials on the use of miRNAs as biomarkers for diagnostic, prognostic, and therapeutics have started emerging. Several miRNA-targeted drug(s) have now entered different phases of clinical trials. The first miRNA based therapeutics Miravirsen (SPC3649) is a DNA phosphorothioate antisense oligonucleotide and is under Phase-II clinical trial. It targets the stability and proliferation of the Hepatitis-C virus (HCV) by inhibiting the liver expressed miR-122 level. Simultaneously, another miRNA, MRX34 entered into a phase-I clinical trial (Table 4). MRX34 is a miR-34 mimic that has been reported to show anticancer properties by inhibiting oncogenic transcription, proliferation, metastasis, chemo resistance, and cancer cell self-renewal. Tumor suppressor properties of miR-34 make MRX-34 a complete anti-cancer therapeutic molecule. Interestingly, miR-122 and miR-34 both have been shown to affect mitochondrial functions in different diseases (Table 1). The potential of miR-122 and miR-34 to implicate mitochondrial pathways in hepatocellular carcinoma and neurodegenerative diseases puts them into mitomiR class of miRNAs.

Table 4.

List of miRNA or miRNA-associated molecules as a therapeutics in clinical trial

Compound	Associated miRNA	Disease	Government identifier or PMIDs	Stage of clinical trial
MRG-106	miR-155	Blood Cancer	NCT02580552	Phase I
MRG-201	miR-29b	Pathologic Fibrosis	NCT03601052	Phase II
MRG-110	miR-92	Ischemic Condition	NCT03603431	Phase I completed
MRX-34	miR-34	Advanced Solid Tumor	NCT01829971	Withdrawn after Phase I
Miravirsen	miR-122	Hepatitis C	NCT01200420	Phase II
RG-101	miR-122	Hepatitis C	–	On hold
RG012	miR-21	Alport Syndrome	NCT03373786	Phase I completed
MGN-1374	miR-208, miR-15/195	Myocardial Infraction	21,900,086	Preclinical
MGN-4893	miR-451	Polycythemia Vera	20,679,397	Preclinical
MGN-2677	miR-143, miR-145	Vascular Disease	28,918,016	Preclinical
MGN-4220	miR-29	Cardiac Fibrosis	28,918,016	Preclinical
MGN-5804	miR-378	Cardiometabolic Disese	28,918,016	Preclinical
MGN-6114	miR-92	Peripheral Arterial Disease	28,918,016	Preclinical
MGN-9103	miR-208	Chronic Heart Failure	22,541,436	Preclinical
Cetuximab/ FOLFOX	miR-31-3p, miR-31-5p	Colorectal Cancer	NCT03362684	Phase III
interferon-alpha (IFN-alpha)	miR-26	Hepato-cellular Carcinoma	NCT01681446	Phase III
miRNA-210	miR-210	Peripheral Arterial Disease	NCT04089943	–

Later, miRagen Therapeutics introduced MRG-106 and MRG-201 that modulate the miR-155 and miR-29b activity in blood cancer fibrosis (Table 4). Currently, MRG106 is undergoing Phase-I and MRG201 is undergoing Phase-II clinical trials. Interestingly, MRG-110 had completed Phase-I clinical trials for wound healing via inhibition of miR-92 expression. In addition, a variety of miRNA(s) are in different phases of preclinical trial for different diseases (Table 4).

Many investigations reveal that differential miRNA-expression profiles can be used as diagnostic/ prognostic biomarkers for multiple human diseases. Table 5 lists miRNAs that are being used as a biomarker in disease diagnosis in clinical trial. The circulatory miR-126 is used as a biomarker and is under clinical trial for myocardial infarction (Table 5). miR-25 is being used as a potential biomarker for pancreatic cancer (Table 5). miR-155 is being used as a diagnostic marker for bladder cancer (Table 5).

Table 5.

List of miRNA as a biomarker in disease diagnosis in clinical trial

microRNA	Disease	Government Identifier
miR-126	Myocardial infarction	NCT01875484
miR-25	Diagnostic test for carcinoma, pancreatic dutal	NCT03432624
MiR-155	Bladder cancer	NCT03591367

In addition to miR-34 and miR-122, several miRNA(s) currently under clinical trials (Table 4) which directly affect mitochondrial functions are being regarded as mitomiRs (Table 1). Mitochondrial localization of miR-92, miR-122, and miR-155 (under clinical trials) also points toward their unexplored role as mitomiRs and further studies are warranted in this area [127–129].

Conclusions and future perspectives

In the current study, we have described various mitomiRs, which directly or indirectly alter critical functions of mitochondria. The mitomiRs that alter mitochondrial function upon localization into the organelle (Table 2) are very less in number in comparison to those that target nuclear gene(s) (Table 1). Metabolic diseases (such as diabetes, cardiovascular disease, and obesity), Neurodegenerative disease (such as Alzheimer's disease, Parkinson's diseases), and Cancer (cancer initiation, metastasis, and recurrence) are major mitochondrial pathologies and mitomiRs have emerged as novel players in them.

miRNA(s) and mitomiRs have immense potential in clinical medicine as prognostics and diagnostics as they regulate more than one pathway. miRNA mimics and antimiRs can be used to manipulate altered miRNA expression or function and they may serve as potential therapeutic strategies for different diseases.

Lot needs to be understood regarding the import of miRNAs into mitochondrial sub-compartments and miRNA regulatory network within the organelle. There is no concrete evidence which suggests that mitochondrial genome can also encode miRNA(s). Although the sequence of miR-1974, miR-1977, and miR-1978 have been observed in mitochondrial DNA but much needs to be explored before they can enter clinics. The detailed and better understanding of biogenesis, import, and the function of mitomiR is still in infancy but might provide a novel strategy to deal with complex mitochondrial-associated human diseases.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

MitomiR

Mitochondrial microRNA

miRISC

MiRNA inducing silencing complex

TOM

Outer membrane transporter

TIM

Inner membrane transporters

P-bodies

Processing bodies

RNAi

RNA interference

AGO

Argonaute

PNPase

Polynucleotide phosphorylase

TCA

Tricarboxylic acid

ETC

Electron transport chain

T2DM

Type 2 diabetes mellitus

CRAT

Carnitine O-acetyltransferase

PPARδ

Peroxisome proliferator-activated receptor δ

ROS

Reactive oxygen species

VDAC

Voltage-dependent anion channel

GLUT

Glucose transporter

TNFα

Tumor necrosis factor-alpha

APP

Amyloid precursor protein

TFAM

Mitochondrial transcription factor

NRF

Nuclear respiratory factor

SOD

Superoxide dismutase

GPX

Glutathione peroxidase

TrxR

Thioredoxinreductase

SDHD

Succinate dehydrogenase subunit D

COX

Cytochrome oxidase

HIF

Hypoxia-inducible factor

GalNAc

N-Acetylgalactosamine

HCV

Hepatitis c virus

BDNF

Brain-derived neurotrophic factor

VEGF

Vesicular endothelial growth factor

Funding

No funding was used in generation of manuscript.

Compliance with ethical standards

Conflict of interest

Authors declare no conflict of interest.

Consent to participate

Authors give consent to participate.

Consent for publication

Authors give consent for publication.

Methodology

Web-based prediction tools were used for bioinformatic prediction of potential mitomiRs and the pathways that might get affected by them. Target scan software (https://www.targetscan.org/vert_72/) was used for the prediction of mitomiRs that target MT-NDL4, MT-ND5 (both are an integral part of complex-I of ETC), and ATP8 and ATP6 gene (both are an integral part of complex-V of ETC). All the predicted mitomiRs were compared by using a Venn diagram (https://bioinformatics.psb.ugent.be/webtools/Venn/) (Fig. 5a). The common mitomiRs that were found to affect the functioning of both ETC complexes by targeting all four mitochondrial genes were retrieved. The gene targets of these two common mitomiRs (miR-3591-5p and miR-5004-3p) were predicted using a target scan and the process affected by candidate mitomiRswas analyzed using PANTHER-Gene List Analysis tool (https://www.pantherdb.org/) (Fig. 5b). The pathways, in which the predicted genes of the common mitomiRs involved, were analyzed using GeneEnricher tool (https://amp.pharm.mssm.edu/Enrichr/). The most affected top 10 pathways by both predicted mitomiRs in the wiki-pathway analysis were plotted and pathways with adjusted p value < 0.05 were labeled in red color (Fig. 5c, d).