The Role of Mitochondrial MicroRNAs in Hepatocarcinogenesis: Mechanisms, Implications, and Therapeutic Prospects

1. Introduction

Hepatocellular carcinoma (HCC) represents a significant global health challenge, ranking as the sixth most prevalent cancer and the third leading cause of cancer-related mortality worldwide [1]. Despite advances in diagnosis and treatment, the prognosis for HCC remains poor due to late diagnosis and limited therapeutic options. Recent research has uncovered the critical roles of non-coding RNAs, particularly microRNAs (miRNAs), in cancer biology. miRNAs are small, non-coding RNA molecules, typically 17–25 nucleotides in length, that post-transcriptionally regulate gene expression by targeting messenger RNAs (mRNAs) for degradation or translational repression [2].

While traditionally studied in the cytoplasm and nucleus, emerging evidence indicates that miRNAs also localize within mitochondria, termed mitochondrial microRNAs (mt-miRNAs), where they regulate mitochondrial gene expression and influence mitochondrial functions critical to cellular metabolism and apoptosis [3,4]. Given the centrality of mitochondrial dysfunction in cancer pathogenesis, understanding the role of mt-miRNAs in hepatocarcinogenesis may reveal novel mechanisms and therapeutic targets for HCC.

2. Cancer Overview

Liver cancer primarily manifests as hepatocellular carcinoma (HCC), which accounts for approximately 75–85% of all liver cancer cases, followed by intrahepatic cholangiocarcinoma and other rare histological subtypes [5]. The development of HCC is strongly associated with several well-characterized etiological factors, including chronic viral hepatitis B (HBV) and C (HCV) infections, chronic alcohol consumption, and metabolic conditions such as non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) [6]. These factors promote chronic liver inflammation, fibrosis, and cirrhosis, providing a pro-tumorigenic microenvironment.

3. Genetic Causes of Liver Cancer

Genetic and epigenetic alterations are central to HCC pathogenesis. Frequent mutations have been identified in key tumor suppressors and oncogenes, including TP53, CTNNB1 (encoding β-catenin), and TERT promoter mutations, which collectively disrupt cellular proliferation, apoptosis, and senescence pathways [7]. Additionally, non-coding RNAs, especially miRNAs, contribute to HCC by regulating gene expression networks involved in tumor initiation and progression [8]. Altered miRNA expression profiles have been correlated with disease stage, metastasis, and patient survival, indicating their functional and clinical relevance.

4. MicroRNAs: Biogenesis and Localization

miRNAs are transcribed by RNA polymerase II as long primary transcripts (pri-miRNAs), which are processed in the nucleus by the Microprocessor complex, comprising Drosha and DGCR8, into precursor miRNAs (pre-miRNAs) (~70 nucleotides) [9]. These pre-miRNAs are exported to the cytoplasm via Exportin-5, where they are further processed by Dicer into mature miRNAs, which assemble into the RNA-induced silencing complex (RISC) to regulate target mRNAs [10].

Recent studies have revealed that a subset of miRNAs localize within mitochondria, suggesting a specialized role in mitochondrial gene regulation. This mitochondrial localization has been confirmed through mitochondrial fractionation and RNA sequencing, revealing a distinct mitochondrial miRNA repertoire [3,11]. These mt-miRNAs are believed to regulate mitochondrial DNA-encoded transcripts and nuclear-encoded mitochondrial genes, impacting mitochondrial metabolism and apoptosis.

5. Mitochondrial MicroRNAs (mt-miRNAs)

Mitochondrial microRNAs differ from their cytoplasmic counterparts by their subcellular localization and potential targets. Although the origin of many mt-miRNAs remains debated—whether they are transcribed from mitochondrial DNA or imported from the nucleus—they are functionally distinct and have been shown to regulate mitochondrial biogenesis, dynamics (fission and fusion), and apoptosis [4]. For instance, miR-181c has been shown to localize in mitochondria and target mitochondrial cytochrome c oxidase subunit 1 (MT-COX1), affecting respiratory chain function [12].

6. Physiological Role of mt-miRNAs

mt-miRNAs are involved in the fine-tuning of mitochondrial functions essential for cellular homeostasis. They regulate genes critical for mitochondrial biogenesis, such as PGC-1α and TFAM, influencing the replication and transcription of mitochondrial DNA [13]. Furthermore, mt-miRNAs modulate apoptosis by targeting pro- and anti-apoptotic proteins, including members of the BCL-2 family, thereby affecting mitochondrial membrane permeability and cytochrome c release [14]. mt-miRNAs also participate in metabolic regulation by influencing oxidative phosphorylation and fatty acid oxidation pathways, thereby controlling cellular energy balance [15].

To better understand the regulatory landscape of mt-miRNAs, Table 1 outlines their roles across key mitochondrial functions, including biogenesis, dynamics, metabolism, and apoptosis.

7. mt-miRNAs in Disease Pathogenesis

Dysregulation of mt-miRNAs has been implicated in various diseases characterized by mitochondrial dysfunction. In cancer, aberrant mt-miRNA expression leads to altered mitochondrial bioenergetics, promoting tumor cell survival, proliferation, and resistance to apoptosis [4]. Similarly, neurodegenerative diseases like Parkinson's and Alzheimer's have shown characteristic changes in mt-miRNA profiles, which contribute to neuronal death via mitochondrial impairment [16]. Metabolic disorders, including obesity and type 2 diabetes, also demonstrate disrupted mt-miRNA-mediated regulation of mitochondrial metabolism [17].

8. Role of mt-miRNAs in Liver Cancer

In hepatocarcinogenesis, mt-miRNAs modulate key oncogenes and tumor suppressors, impacting mitochondrial energy metabolism and apoptotic pathways. For example, altered levels of mt-miR-181c and mt-miR-210 have been correlated with HCC progression and poor prognosis, likely through their effects on mitochondrial respiration and hypoxia response [18,19]. Functional studies involving overexpression or knockdown of these mt-miRNAs in HCC cell lines have demonstrated their ability to alter mitochondrial membrane potential, reactive oxygen species (ROS) production, and cell survival, underscoring their direct involvement in tumor cell biology [23,24]. Additionally, mt-miRNAs hold promise as minimally invasive biomarkers, detectable in patient serum or plasma, which could aid early diagnosis and prognosis stratification [20].

Several approaches have been explored to manipulate mt-miRNA levels in cancer cells, including the use of chemically modified miRNA mimics or inhibitors encapsulated in mitochondria-targeted nanoparticles, such as liposomes conjugated with mitochondrial-penetrating peptides [22,23]. Despite promising preclinical results, challenges including off-target effects, delivery specificity, and stability under physiological conditions remain to be fully addressed before clinical application.

Table 2 summarizes key mitochondrial microRNAs implicated in hepatocellular carcinoma, highlighting their molecular targets, mitochondrial functions, and potential clinical significance.

9. Current Research and Therapeutic Implications

Research into the mechanistic roles of mt-miRNAs in HCC is rapidly expanding. Recent studies have utilized CRISPR/Cas9-mediated gene editing, antisense oligonucleotides, and miRNA mimics to elucidate the functional impact of specific mt-miRNAs on tumor growth, metastasis, and chemoresistance in both in vitro and in vivo models [21,23]. These experimental approaches have provided valuable insights into how mt-miRNAs influence mitochondrial dynamics and cellular metabolism, highlighting their potential as therapeutic targets.

Therapeutic strategies to modulate mt-miRNA levels include the use of chemically modified miRNA mimics or inhibitors delivered via nanoparticle-based systems designed to target mitochondria specifically. Mitochondria-targeted liposomes and polymeric nanoparticles conjugated with mitochondrial-penetrating peptides have shown promise in enhancing cellular uptake and mitochondrial localization of therapeutic RNAs [22,25]. Despite these advances, challenges such as achieving delivery specificity to tumor cells, avoiding off-target effects, ensuring miRNA stability in circulation, and minimizing immune responses remain significant hurdles for clinical translation. Additionally, the heterogeneity of HCC tumors may necessitate personalized approaches tailored to individual mt-miRNA expression profiles.

Various therapeutic strategies have been developed to modulate mt-miRNA expression in HCC, including the use of synthetic mimics, antisense inhibitors, and gene editing tools. These approaches are summarized in Table 3, along with their delivery methods, preclinical evidence, and associated challenges.

10. Challenges and Controversies

The study of mt-miRNAs remains an emerging field with unresolved questions. One major controversy is whether mt-miRNAs are transcribed within mitochondria from mitochondrial DNA or imported from the cytoplasm, as current evidence supports both possibilities depending on the specific miRNA and cell type [26]. Technical challenges, including contamination during mitochondrial isolation and difficulties in accurately quantifying mt-miRNAs, complicate the interpretation of experimental data. Rigorous methodological standardization and development of more sensitive mitochondrial RNA detection techniques will be necessary to clarify the origins and functions of mt-miRNAs. Despite these challenges, the unique ability of mt-miRNAs to modulate mitochondrial gene expression positions them as promising regulators in cancer and other diseases.

11. Future Directions

Future research should prioritize comprehensive profiling of mt-miRNAs in large HCC patient cohorts using high-throughput sequencing and multi-omics integration to identify novel candidates and pathways. Functional validation through advanced molecular tools, including mitochondrial-targeted gene editing and real-time imaging of mitochondrial function, will be essential to establish causal roles. Furthermore, development of innovative delivery systems—such as mitochondria-targeted exosomes or synthetic nanocarriers—could improve the specificity and efficiency of mt-miRNA-based therapeutics. Clinical trials assessing the safety and efficacy of these novel interventions in HCC patients will be critical for translating mt-miRNA biology into practical treatment strategies.

12. Conclusions

Mitochondrial microRNAs (mt-miRNAs) represent a novel and increasingly recognized class of post-transcriptional regulators implicated in the modulation of key mitochondrial functions relevant to hepatocarcinogenesis. Their unique subcellular localization, coupled with their ability to influence mitochondrial dynamics, bioenergetics, and apoptosis, highlights their potential utility as both diagnostic biomarkers and therapeutic targets in hepatocellular carcinoma (HCC). Advancing our understanding of mt-miRNA biogenesis, function, and interaction networks will be critical to harnessing their clinical potential and integrating them into precision medicine frameworks aimed at improving prognostic accuracy and therapeutic efficacy in HCC management.