Advancements in the Regulatory Role of microRNAs in Childhood Acute Lymphoblastic Leukemia: Mechanisms and Clinical Implications

Abstract

microRNAs (miRNAs), tiny, non-coding RNA molecules, fine-tune the expression of target genes through interacting with mRNAs. These miRNAs are involved in a wide range of biological processes, encompassing cell division, death, blood cell production, and tumor development. When these miRNAs become dysfunctional, they can promote the invasion and spread of cancer cells in various human malignancies, including leukemia. Acute lymphoblastic leukemia (ALL), the preeminent malignancy affecting children, is a blood cancer marked by the uncontrollable growth of immature lymphoid cells that displace healthy blood precursors in the bone marrow. Despite a decline in ALL mortality rates over the past two decades, a significant proportion of deaths still results from a lack of effective diagnostic and prognostic markers that can guide treatment decisions and overcome drug resistance. The analysis of miRNA expression patterns in ALL could lead to more precise disease classification, earlier diagnosis, and better prognostic outcomes in the near future. The connection between miRNA dysfunction and the biology of ALL suggests that these molecules could represent promising therapeutic targets. Therefore, this review delves into the regulatory mechanisms of miRNAs in pediatric ALL, exploring how miRNA-based diagnostic, prognostic, and therapeutic strategies offer unique advantages and hold promise for clinical applications.

Introduction

Acute lymphoblastic leukemia (ALL) is one of the most common malignancies in childhood, with a complex pathogenesis involving the regulation of multiple genes and molecules. ALL is characterized by the aberrant proliferation of immature lymphoid progenitors and clinically classified into three types: B-cell precursor (BCP-ALL, 80-85% of cases), T-cell (T-ALL 10-15%), and mature B-cell (mature B-ALL, < 5%). This clonal expansion of lymphoid cells disrupts normal hematopoiesis, leading to the characteristic clinical manifestations of ALL. ¹ ALL is characterized by chromosomal irregularities and genetic variations which cause the disrupted maturation, arrested differentiation, as well as abnormal multiplication of lymphoid precursor cells. ² ALL develops in both pediatric and adult populations, with the highest frequency observed among individuals aged between 1 year and 4 years. ² Over the past four decades, there has been a significant improvement in outcomes for ALL, with the 5-year overall survival rate increasing substantially from 31% in 1975 to approximately 70% in 2009. ³ Nonetheless, there remain significant disparities among individuals at different ages. For example, the 5-year overall survival rate for pediatric ALL can attain 90%, whereas only 25% of patients aged over 50 years survive 5 years post-diagnosis. This poses a significant challenge due to the adverse disease biology and comorbidities that hinder the administration of curative treatment regimens.^2,4

It is widely recognized that genetic mutations underlie the initial manifestation of ALL. Nevertheless, these genetic alterations alone prove inadequate for the full manifestation of the disease, requiring additional epigenetic modifications as well. Recently, regulatory roles played by non-coding RNAs, particularly microRNAs (miRNAs), have been identified in ALL.^5–7 Notable variations in the expression patterns of miRNAs have been witnessed, leading to modifications of the transcriptional profiles of their corresponding gene targets. ⁸ These epigenetic alterations play a pivotal role during the process of malignant transformation, causing the dysregulation of oncogenes and tumor suppressor genes, thereby disrupting essential cellular functions and potentially triggering the development of ALL. ⁸ Therefore, miRNA alterations, as a major type of epigenetic modification, exert a profound influence on both the onset and advancement of ALL.

miRNAs also display distinct expression patterns during different stages of lymphopoiesis. They bind to specific messenger RNAs, regulate their expression post-transcriptionally and exert an impact on the maturation process lymphoid precursors. ⁹ During oncogenic lymphopoiesis, miRNAs are aberrantly expressed, and these abnormalities can serve as signatures for different ALL subtypes. ¹⁰ Alterations in the expression profiles of miRNAs show functional significance in leukemogenesis or drug resistance, and reversing these miRNA expressions could counteract the disease progression and enhance clinical responses.^9,11 In addition, the expression profiles of miRNAs in acute leukemia exhibit collaborative interactions throughout the development of the disease, displaying a potential as valuable biomarkers for the diagnosis, prognosis, and treatment of ALL. ¹² For example, miRNA-100, miRNA-196a, and miRNA-146a play crucial roles in the leukemogenesis of pediatric ALL, potentially serving as valuable biological molecular indicators for diagnosis. ¹³ The expression levels of both miRNA-155a and miRNA-181a were found to be notably elevated in pediatric ALL patient group, with both miRNAs being associated with elevated levels of minimal residual disease (MRD) and an unfavorable prognosis. ¹⁴ The combination of miR-125b and B-cell lymphoma-2 (Bcl-2) serves as a powerful prognostic indicator in pediatric ALL, thus making them potential therapeutic targets for ALL treatment. ¹⁵ Moreover, miR-155 stands out as a pivotal miRNA and is regarded as a potential therapeutic target in the treatment of pediatric leukemic patients infected with hepatitis C virus. ¹⁶

In recent years, miRNAs, a significant class of non-coding RNAs, have garnered attention in tumor research due to their pivotal roles in cell proliferation, differentiation, and apoptosis. Therefore, a thorough exploration of miRNAs in childhood ALL is crucial for elucidating ALL pathogenesis, discovering therapeutic targets, and enhancing prognosis. Studying miRNA expression profiles in childhood ALL can identify disease-related miRNAs and their specific mechanisms in cell signaling and gene expression. This not only enhances our understanding of ALL pathogenesis but also lays the foundation for miRNA-targeted therapeutic strategies. Comparing miRNA expression profiles among different ALL subtypes and stages can further reveal miRNA-clinical associations, prognosis, and diagnosis, offering new clinical references. This review focuses on miRNAs’ roles and regulatory mechanisms in childhood ALL, offering novel insights for precision treatment.

miRNAs’ Biogenesis and Function

miRNAs are short endogenous nucleic acid molecules, typically comprising 22 nucleotides in length. The biosynthesis of miRNAs commences within the nucleus, where the miRNA gene is transcribed by RNA polymerase II, and generating long primary miRNA (pri-miRNA) molecules containing hairpin structures (Figure 1). These pri-miRNAs undergo processing by the ribonuclease Drosha, yielding precursor miRNA (pre-miRNA) molecules.The Exportin-5 protein facilitates the transportation of pre-miRNAs from the nuclear compartment to the cytosolic domain. Once in the cytoplasm, Dicer ribonuclease cleaves the pre-miRNA, forming brief duplex RNA molecules, which are incorporated into the RNA-induced silencing complex (RISC), a molecular machinery that consists of Dicer and Argonaut (Ago) proteins. Within the RISC, the passenger strand of the miRNA duplex, a non-functional component, is degraded, while the functional guide strand assumes the crucial task of regulating gene expression.^17–18

Figure 1.

A schematic drawing shows the biogenesis process of miRNAs.

miRNAs play a crucial role in post-transcriptional gene expression regulation by repressing the translation of mRNAs or triggering the deadenylation and degradation of mRNAs through the RISC mechanism. ¹⁹ miRNAs bind to miRNA response elements (MREs), primarily situated within the 3’-untranslated regions (3′-UTR) of their intended mRNA targets. ¹⁹ In animals, including humans, the interaction between miRNAs and mRNAs does not necessitate full sequence complementarity. miRNAs serve as integral components of intricate regulatory networks governing gene expression. Typically, a given target mRNA harbors numerous MREs, allowing its regulation by a diverse array of distinct miRNAs. ²⁰ Conversely, a single miRNA can target a range of distinct mRNAs. ²⁰ miRNAs function as essential regulators that modulate virtually all intracellular processes, displaying remarkable temporal and context-specificity, orchestrating cell division, proliferation, differentiation, cell death, exocytosis, antiviral defense, and phenotypic adjustments in response to diverse internal and external stimulators. ²¹ miRNAs’ significance in maintaining normal cellular function cannot be overstated, however, aberrant expression can lead to various diseases, including cancer, indicating their integral role in disease development. Recently, miRNAs have been established as significant regulators in both normal and malignant hematopoiesis. A number of miRNAs have been identified to be either enhanced or suppressed in individuals diagnosed with hematological cancers. However, the comprehensive understanding of the impact of these miRNAs on the pathogenesis of pediatric ALL, specifically their potential utility in disease stratification and prognostic assessment, remains elusive.

Clinical Features in Different Types of Pediatric ALL

The symptoms of pediatric ALL are usually nonspecific and diverse, including persistent fever, bone pain, enlarged lymph nodes, petechiae, and difficulty breathing due to mediastinal enlargement. ²² The diagnosis of ALL is primarily based on morphological identification, specifically, the proportion of leukemic bone marrow blasts exceeding 25%. From the perspective of immunophenotype, most pediatric ALL cases are clinically classified as BCP-ALL, T-ALL, or mature B-ALL, originating from precursor stage of B cells, T lymphocytes, or mature B lymphocytes, respectively.

BCP-ALL typically has a rapid onset and a fast disease progression. Since BCP-ALL involves abnormal proliferation of B-cell precursors, patients may also exhibit enlargement of lymph nodes, liver, and spleen. Symptoms related to central nervous system (CNS) involvement may be more common in BCP-ALL, such as headache, vomiting, and visual impairment. The prognosis of pediatric BCP-ALL depends on various factors, such as the high heterogeneity at the molecular and clinical levels. Despite this significant heterogeneity, all BCP-ALL patients receive the same chemotherapy regimen. Therefore, there is a need for precision medicine approaches aimed at improving cure rates while reducing side effects in BCP-ALL patients. ²³

The clinical features of T-ALL include older age, male preponderance, high frequency of mediastinal masses, and higher leukocyte counts at diagnosis. The prognosis of T-ALL is poor, especially due to the higher risk of relapse involving CNS. In the past, children with T-ALL were normally treated as part of the higher risk group in clinical trials, using the same treatment regimen as for BCP-ALL. However, given the characteristics of T-ALL, recent clinical trials have adopted modifications specific for T-ALL, such as intensification of CNS-directed therapy and more intensive treatment using L-asparaginase and methotrexate. ²⁴

Since mature B-ALL involves abnormal proliferation of mature B cells, patients may exhibit symptoms related to abnormal B-cell function. For instance, skin symptoms may be more prominent, such as skin hardening, purpura, or ecchymosis. Additionally, bone or joint pain may also be more common, which may be related to the infiltration of leukemic cells in the bone marrow. Mature B-ALL exhibits nearly identical immunophenotypic and molecular genomic features to those of mature B-cell lymphoma and thus is treated as a form of lymphoma using short-course and intensive chemotherapy. ²⁵

Mechanisms of miRNAs Involved in Pediatric ALL Pathogenesis

miRNAs are the master regulators of gene expression, while genes also significantly impact miRNA expression, creating a complex relationship between them. Understanding the intricate mechanisms of how miRNAs regulate crucial cell processes like apoptosis, proliferation, invasion, migration, as well as drug resistance is paramount in developing effective miRNA-based ALL treatments. A thorough comprehension of the regulation of abnormal miRNA expression and mechanism on cell behavior is the key to devising innovative therapeutic strategies for miRNA-targeted therapies.

miRNAs Regulate the Cellular Proliferation and Apoptosis in Pediatric ALL

The regulation of ALL cell proliferation and apoptosis constitutes a pivotal mechanism mediated by miRNAs. Several miRNAs fulfill this role through up-regulation. For instance, GATA3-AS1-mediated up-regulation of miR-515–5p expression inhibited the multiplication and induced cell death of T-ALL Jurkat cells. ²⁶ The upregulation of miR-653-5p expression enhanced T-ALL cell autophagy and apoptosis by suppressing the activation of Reelin-driven phosphatidylinositide 3-kinase (PI3 K)/ protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) signaling (P < .001). ²⁷ Up-regulation of miR-486-5p led to a reduction in the viability of ALL cells and an augmentation in apoptotic processes. ²⁸ miR-1294 was shown to target and inhibit SOX15 expression, thus stimulating the Wnt/β-Catenin signaling cascade to enhance the proliferative capacity of ALL cells, repress apoptotic mechanisms, and ultimately affect disease progression. ²⁹ miR-146a was found to increase in ALL pediatric patients, and enhance the proliferation and suppress the apoptosis of ALL Jurkat cells through the activation of janus kinase 2 (JAK2)/ signal transducer and activator of transcription 3 (STAT3) signaling. ³⁰ The expression of miR-149* was significantly upregulated in both T-ALL cell lines and bone marrow samples obtained from T-ALL patients. miR-149* mimics was found to promote T-ALL cell proliferation, decrease the percentage of cells residing in the G1 phase of the cell cycle, and reduce apoptosis, whereas inhibitors of miR-149* abrogated these influences, demonstrating that miR-149* could potentially function as a tumorigenic modulator in T-ALL. ³¹ miR-155 level was also elevated in the bone marrow of children with B-ALL, which might be related to the activation of Wnt/β-Catenin signaling that promotes B-ALL cell proliferation and inhibits their apoptosis. ³²

miRNAs can also fulfill this role through their downregulation. For example, boric acid-mediated miR-21 downregulation was associated with the increased caspase activity and apoptotic cell death in ALL Jurkat cell lines. ³³ miR-582 was downregulated and functioned as a suppressive modulator in BCP-ALL cells. Overexpression of miR-582 artificially suppressed the proliferative and survival rates of BCP-ALL cells, yet conferred resistance to NK cell-induced cytotoxicity. ³⁴ Decreased miR-652-5p expression impeded the growth and promoted apoptosis of T-ALL cells in vitro, while simultaneously enhancing overall survival rates in vivo. ³⁵ miR-29a-3p was low expressed in serum of patients with ALL and leukemia cells, its overexpression inhibited ALL cell growth and promoted the apoptosis by regulating expression of hepatoma-derived growth factor (HDGF) (P < .05). ³⁶

In addition, miRNAs derived from extracellular vesicles (EVs) or their genetic variants can also regulate ALL cell proliferation and apoptosis. For example, bone marrow mesenchymal stem cell (BMSC)-EVs-derived miR-29b-3p was found to suppress the growth and induce cell death in ALL cells via suppressing the mitogen-activated protein kinase (MAPK) pathway. ³⁷ The rs2292832 mutation in miR-149 similarly suppressed the proliferative capacity of ALL cells and triggered apoptotic processes, offering a conceivable pathway through which this genetic alteration might affect ALL susceptibility. ³⁸

miRNAs Regulate the Cellular Invasion and Migration in Pediatric ALL

miRNAs have also actively participated in the regulation of ALL cell invasion and migration. For example, miR-181b-5p is abundant within the exosomes and vesicles secreted by blood cells. These EVs carrying miR-181b-5p can be internalized by ALL cells, leading to an elevation in the expression of miR-181b-5p within these cells. This upregulation of miR-181b-5p has been observed to affect the malignant progression of ALL by promoting migratory and invasive capabilities of ALL cells. ³⁹ Also, miR-146a was found to promote the migratory and invasive abilities of ALL Jurkat cells through the downregulation of ciliary neurotrophic factor receptor (CNTFR) (P < .05). ³⁰ miR-590 promoted the migratory and invasive behaviors of T-ALL cells by upregulating E-cadherin and suppressing matrix metalloproteinase (MMP)-9, thus presenting itself as a potential therapeutic candidate for the intervention of T-ALL. ⁴⁰ miR-139 exhibited low expression levels in both T-ALL cell lines and patient samples. Upon introduction, miR-139 effectively impeded the invasive capacity of T-ALL cells in vitro and attenuated pulmonary dissemination in vivo. ⁴¹ miR-101 was downregulated in T-ALL, and the introduction of its mimic suppressed the migratory and invasive abilities of T-ALL cells by modulating PI3 K/AKT pathway. ⁴² The expression of miR-125b peaked in early-stage T cells and was elevated in the T leukemia homeobox 3 (TLX3)-positive subtype of T-ALL, where it contributed to the invasive nature of the disease in vivo. ⁴³

The downregulation of miRNAs have also take part in ALL cell invasion and migration. For instance, miR-146b-5p expression was reduced in both T-ALL patients and cell lines, suggesting its involvement within the hematological system by bioinformatics analysis; reduced miR-146b-5p expression resulted in the enhanced expression of interleukin (IL)-17A (P < .05), thereby facilitating the migratory and invasive capabilities of T-ALL cells. ⁴⁴ miR-3173 was downregulated in B-ALL patients, and its expression suppressed migratory and invasive abilities in B-ALL cell lines. ⁴⁵ T-cells from leukemia patients displayed reduced expression of miR-146b-5p, which served as a crucial functionally relevant gene whose suppression enhanced leukemia cell migration and ALL progression. ⁴⁶

miRNAs Regulate the Drug Resistance in Pediatric ALL

Failure to effectively treat leukemia is primarily attributed to refractory disease and relapse. Despite advancements in risk assessment, therapeutic regimens, and timely identification of residual disease, survival outcomes for leukemia patients remain unsatisfactorily poor due to drug resistance. There is growing evidence of miRNAs’ impact on treatment results in lymphoid cancers and their interaction with drug response. ¹¹ For instance, the levels of miR-324-3p and miR-508-5p were significantly lower in ALL group compared to non-cancer control (P < .0001 and P = .005, respectively) and exhibited a negative correlation with ATP-binding cassette transporter A3 (ABCA3), an ABC transporter causing multidrug resistance (MDR) across multiple cancer types, and was strongly linked to elevated resistance to chemotherapy in childhood ALL, confirming their regulatory role in drug resistance through interaction with ABCA3. ⁴⁷ miR-503 was reduced in glucocorticoid (GC)-resistant ALL patients and cell lines, overexpressing miR-503 promoted the sensitivity of ALL cells to dexamethasone by inactivating Wnt/β-catenin signaling. ⁴⁸ The BCP-ALL cell line that has acquired resistance to dexamethasone displays elevated expression levels of the miR-142-3p and miR-17∼92 clusters, suggesting that targeting these two miRNAs may effectively overcome resistance to GCs in BCP-ALL cells. ⁴⁹ miR-331-3p was found to reverse GC resistance through rapamycin treatment via directly targeting mitogen-activated protein kinase kinase 7 (MAP2K7) in vitro. ⁵⁰ Remarkably, it was discovered that miR-331-3p expression also correlates with GC resistance in a cohort of pediatric ALL patients. Ex vivo evaluation revealed that patientsexhibiting elevated miR-331-3p levels showed increased sensitivity to methylprednisolone (MP), reinforcing the significance of this miRNA in modulating GC responsiveness. ⁵⁰

In drug-resistant ALL cell lines and patients, a notable decrease in miR-326 levels was observed, suggesting miR-326 might potentially play a role in the resistance to therapeutic agents. ⁵¹ Compared to healthy controls, the expression of miR-101 was significantly reduced in blood samples from patients with T-ALL, with Notch1 as it direct target (P < .01). ⁵² Furthermore, overexpression of miR-101 increased the responsiveness of Jurkat cells to the anticancer drug adriamycin, suggesting its potential as a suppressor of tumorigenesis in T-ALL. ⁵² By focusing on the miR-29a-involved network, an apoptotic response in doxorubicin-resistant B-ALL cells was induced and the sensitivity to doxorubicin was restored by modulating PI3 K/AKT/mTOR signaling; additionally, the synergistic effects when combined with inotuzumab ozogamicin treatment were also observed. ⁵³ Together, the use of miRNA mimics, anti-miRs, and other molecular strategies targeting miRNAs shows promise as therapeutic approaches, which may overcome the drug resistance in ALL, and enhance patient responses and survival rates.

The Clinical Implication of miRNAs in Pediatric ALL

miRNAs as Diagnostic Biomarkers for Pediatric ALL

miRNAs have shown diagnostic values for ALL. For instance, miR-146a expression was upregulated in both pediatric and adult patients in comparison to healthy individuals. Also, miR-146a expression was independent of age, gender, as well as clinical and hematological features. Therefore, the expression level of plasma miR-146a could potentially serve as a non-invasive biomarker for diagnosing ALL in both pediatric and adult patients. ⁵⁴ The concentration of miR-32 in the plasma of T-ALL patients was notably elevated (P < .001), while its target gene F-box and WD repeat domain containing 7 (FBXW7) was significantly downregulated (P < .001), suggesting the potential of miR-32 as a noninvasive indicator for identification and distinguish T-ALL patients with reasonable sensitivity and specificity. ⁵⁵ In another study, ALL group showed notably elevated expressions of serum miR-922 and miR-506 compared to the control. According to the receiver operating characteristic (ROC) curve analysis, it was revealed that the optimal cut-off values for miR-922 and miR-506 in diagnosing pediatric ALL were determined to be 1.46 and 2.17, respectively. ⁵⁶ The high miR-922/miR-506 expression group had a significantly higher incidence of lymph node enlargement, leukocytosis (≥50 × 109/L), medium-high risk stratification, mixed-lineage leukemia gene rearrangement, and karyotype abnormality, suggesting that the expression levels of miR-922 and miR-506 possess significant diagnostic value in the assessment of childhood ALL. ⁵⁶ A recent study performed in Egyptian children recommended the elevated plasma miRNA 92a and miRNA 638 expressions to be used as potential predictive and follow-up markers in children with ALL remitted and relapsed cases. ⁵⁷

In addition, patients with ALL exhibited decreased expression of serum miR-16-2-3p, which could aid ALL diagnosis. ⁵⁸ The expression of serum miR-217 was significantly reduced in ALL　patients in comparison to controls，while miR-367 was up-regulated. Furthermore, serum miR-217 and miR-367 could clearly distinguish　patients with ALL from healthy subjects by ROC analysis, providing initial indications that circulating miR-217 and miR-367 could potentially serve as powerful diagnostic indicators. ⁵⁹ Exosomal miR-181b-5p underwent dynamic changes during the course of petridic ALL, making it a potential biomarker for discriminating ALL subtypes or monitoring disease status. ⁶⁰ The expression level of exosomal miR-181b-5p was notably reduced in both the newly diagnosed and relapsed groups, in contrast to the remission group and the control group, its level was elevated in children with T-ALL in comparison to those with B-ALL, and higher in male children compared to female children. ⁶⁰ A retrospective case-control study has explored the correlation between four miRNA single nucleotide polymorphisms (miR-SNPs) and the modified risk of ALL, and found that the polymorphisms of miR-146a (rs2910164) and miR-149 (rs2292832) serve as biomarkers for individualized diagnosis of ALL. ⁶¹ To assess the discriminatory capacity of differentially expressed miRNAs and their association with AML/ALL, ROC curve, area under curve (AUC), and 95% confidence interval (CI) were analyzed. ⁶² Specifically, miR-30a, miR-101, miR-132, miR-143, miR-124, and miR-129 exhibited outstanding AUC with high sensitivity and specificity, holding promise as candidates for early AML/ALL detection; miR-124 demonstrated strong discrimination between AML and non-cancer group, with an outstanding AUC of 0.9706 for AML and 0.9856 for ALL; miR-137 showed high AUC of 0.8961 for ALL but lacked discriminatory power for AML; miR-199 had moderate potential for ALL detection with AUC of 0.5161 but showed significance for AML; miR-25, with AUCs of 0.7739 and 0.6972, had limited diagnostic potential for AML and ALL. ⁶²

miRNAs as Prognostic Biomarkers for Pediatric ALL

miRNAs are drawing growing attention as prospective indicators of prognosis, either alone or in conjunction with traditional prognostic factors. For example, overexpression of miR-155 enhanced proliferation and suppressed apoptosis in ALL cells, and higher miR-155 levels were associated with poorer outcomes in ALL patients. ⁶³ ALL patients showed downregulated serum miR-16-2-3p (P < .01) with an AUC of 0.837 and a cut-off value of 0.745 (67.92% sensitivity, 96.94% specificity), indicating miR-16-2-3p as a standalone prognostic factor for ALL patient survival. ⁵⁸ Minimal residual disease (MRD) stands as one of the most significant independent prognostic indicators in ALL, however, bone marrow (BM) aspiration is a procedure that involves intrusive techniques to obtain a sample, making it an invasive medical process. A recent study discovered the positive correlations between alterations in miR-128-3p expression in exosome-enriched fraction (EEF) and platelet free plasma fraction (PFP) on day 8 of chemotherapy and flow cytometry (FC) MRD on day 15 (r_EEF= 0.99, p_EEF= 1.13 × 10⁻9; r_PFP= 0.99, p_PFP= 4.75 × 10⁻⁹) ⁶⁴ ; Also, a positive correlation was noted between the decrease in miR-128-3p in EEF on day 15 and FC MRD on the same day (r_EEF= 0.96; p_EEF= 4.89 × 10⁻⁵), indicating the potential of circulating miR-128-3p as a blood-derived liquid biopsy biomarker for MRD in childhood ALL. ⁶⁴ Caserta et al identified a dormant and chemotherapy-resistant subset of human B-ALL cells that was related to MRD, elevated miR-126 level and demethylation of the corresponding locus at the time of diagnosis were linked to an inferior response to induction chemotherapy and a less favorable prognosis (MRD > 0.05% at day +33 or MRD + at day +78). ⁶⁵

miRNA signatures also play a crucial role in predicting ALL prognosis. A study on the consensus clustering of miRNA expression data identified a miR-low cluster (MLC) characterized by miRNA downregulation. ⁶⁶ Strikingly, MLC patients exhibited significantly shorter event-free survival (median 21 vs 33 months; log-rank P = 3 × 10⁻⁵), including in hyperdiploid ALL. Notably, non-MLC profiles at diagnosis often transitioned to MLC at relapse. ⁶⁶ Therefore, this prognostic MLC signature offers unbiased molecular stratification for pediatric BCP-ALL, potentially improving clinical outcomes. The diversity of ALL, with numerous genetic variations detected in individual patients as well as across diverse cases, poses significant challenges in effectively navigating intricacies of the disease and developing personalized therapeutic strategies accordingly. Novel, dependable prognostic factors that can enhance risk-based stratification of therapy are also required in miRNA research. The genetic variations of rs2114358 G > A within pre-hsa-miR-1206 after multiple logistic regression (P = .010), and rs56103835 T > C within pre-hsa-mir-323b (P = .000) demonstrated the potential to influence hematological toxicities associated with high-dose methotrexate administration. These markers might potentially emerge as promising clinical indicators for predicting severe hematological toxicities (Grade 3/4) in children diagnosed with ALL. ⁶⁷

EVs, nanosized particles emitted by all cells, are particularly intriguing as they circulate throughout the body irrespective of the location of the cells from which they originate. Compared to controls, patients exhibited a significant elevation in plasma-derived exosomal miR-326 (P < .05, AUC = 0.7500). ⁶⁸ Furthermore, a comparative analysis between sensitive and drug-resistant patients indicated a prognostic significance of exosomal miR-326 (P < .05, AUC = 0.7755), hinting at its potential as a prognostic biomarker and a novel therapeutic target for drug-resistant ALL. ⁶⁸ Certain miRNAs have been linked to leukemogenesis and have the ability to disrupt oncogenic or tumor-suppressor pathways, ultimately affecting the patient outcomes. A recent study revealed that T-ALL-derived EVs contained miRNAs dependent on Notch1 pathway, primarily consisting of members of the miR-17-92a cluster and their paralogues. These miRNAs regulate oncogenic pathways, serving as autocrine stimuli and ultimately facilitating the growth and survival of rapidly dividing cellular subpopulations within human T-cell leukemias. ⁶⁹

Therapeutic Implications of miRNAs in Pediatric ALL

The reduced expression of tumor suppressor miRNAs or the increased expression of tumor promoter miRNAs can promote leukemia cell growth, indicating that restoring the expression of these miRNAs might be a promising therapeutic approach for ALL. A recent small RNA-seq investigation revealed the resemblance in the miRNA transcriptome between children and young adults suffering from T-ALL, highlighting miR-143-3p as a potential novel tumor suppressor candidate with fibroblast growth factor (FGF)1 (P < .05), FGF9 (P < .05), and Kirsten rat sarcoma viral oncogene (KRAS) (P < .001) as its target genes. ⁷⁰ The reduced expression of miR-143-3p in patients with T-ALL contributed to the increased growth and survival of leukemic cells. ⁷⁰ miRNA-181b-5p was highly and differentially expressed in ALL with synovial sarcoma, X breakpoint 2 interacting protein (SSX2IP) as its downstream target (P = 4.743 × 10⁻34). ⁷¹ It was found to control ALL cell proliferation, cell cycle, apoptosis, and facilitate tumor growth in vivo, suggesting miRNA-181b-5p as a promising target for intervention against the malignant behaviors of ALL. ⁷¹ The discovery that miR-27a simultaneously influences four critical factors driving blast transformation in t(4;11) ALL cell lines, including 14-3-3θ (Pearson's R = −0.961 ± 0.032, P < .05), runt-related transcription factor 1 (RUNX1) (P < .01), ALL1-fused gene from chromosome 4 protein (AF4) (P < .01), and mixed lineage leukemia (MLL)-AF4 (P < .01), suggests its potential as a innovative and potentially effective therapeutic candidate for this aggressive and refractory subtype of leukemia. ⁷²

Because of the diversity of leukemia cells, monotherapy frequently leads to the development of drug resistance. Hence, combing chemotherapy with miRNA modulation holds the potential to be an extremely efficient strategy for treating ALL. For instance, the combination of miR-34a (with Bcl-2 as its target, P < .00001) and doxorubicin as a therapeutic approach displayed a synergistic effect to trigger apoptosis and reduce the survival rate of T-ALL cells, while also enhancing their responsiveness to doxorubicin. ⁷³ miRNA-411-3p negatively modulated Yin-yang 1 (YY1) (P < .05), and was found to boost the cellular uptake and cytotoxicity of methotrexate in ALL cells. ⁷⁴ Moreover, the miR-126 inhibitor miRisten, either as a monotherapy or in conjunction with tyrosine kinase inhibitors, exhibited remarkable anti-leukemic activity, leading to a full remission and leukemia-free survival rate of 90% in Ph⁺ ALL mice and 75% in patient-derived xenografts of Ph⁺ ALL. ⁷⁵ These findings suggest that targeting miR-126 might offer an additional therapeutic option for patients with poor prognosis, reinforcing the potential of miRisten as a promising treatment for these otherwise difficult-to-treat patients. ⁷⁵ A table listing the expression, target genes, pathways and functions of miRNAs in pediatric ALL has been shown in Table 1.

Table 1.

miRNA Expression, Target Genes, Pathways and Functions in Pediatric ALL Have Been Listed.

miRNAs	Expression	Target genes	Pathways	Functions
miR-515-5p miR-653-5p miR-486-5p miR-1294 miR-146a miR-149* miR-155 miR-21 miR-582 miR-652-5p miR-29a-3p miR-29b-3p miR-181b-5p miR-590 miR-139 miR-101 miR-125b miR-146b-5p miR-3173 miR-146b-5p miR-324-3p miR-508-5p miR-503 miR-142-3p miR-17∼92cluster miR-331-3p miR-326 miR-101 miR-29a miR-146a miR-32 miR-922 miR-506 miR-92a miR-638 miR-16-2-3p miR-217 miR-367 miR-181b-5p miR-155 miR-128-3p miR-126 miR-326 miR-17-92a miR-143-3p miR-181b-5p miR-27a miR-34a miR-411-3p	Down Down Down Up Up Up Up Up Down Down Down Down Up Up Down Down Up Down Down Down Down Down Down Up Up Up Down Down Down Up Up Up Up Up Up Down Down Up Down Up Up Up Up Up Down Up Down Down Down	/ Reelin MAML3 SOX15 CNTFR JunB MDR1 / PPTC7 TIGAR HDGF GDF15 / RB1 CXCR4 / Ets1/CBFβ IL-17A PTK2 / ABCA3 ABCA3 WNT3A / / MAP2K7 BAALC Notch1 PIK3CA / FBXW7 / / / / / / / / ZNF238 / / / FGF1/9 SSX2IP 14-3-3θ Bcl-2 YY1	/ PI3 K/AKT/mTOR / Wnt/β-Catenin JAK2/STAT3 / Wnt/β-Catenin / / PFKFB3 / MAPK / / / PI3 K/AKT / NF-κB / / / / Wnt/β-catenin / / JNK/MAPK / / PI3 K/AKT/mTOR / / / / / / / / / / / / / Notch1 FGFR / / / /	its up-regulation inhibited proliferation and induced apoptosis of childhood ALL cells 26 its elevation enhanced autophagy and apoptosis in T-ALL cells 27 its upregulation decreased cell viability and increased ALL cell apoptosis 28 it promoted the proliferation of ALL cells, inhibited apoptosis, and affected the disease progression 29 it promoted the proliferation, migration, and invasion and inhibited the apoptosis of ALL Jurkat cell 30 it promoted cell proliferation and suppressed apoptosis, serving as an oncogenic regulator 31 it promoted the proliferation of B-ALL cells and inhibited apoptosis, leading to chemotherapy resistance 32 it promoted ALL cell proliferation, inhibited apoptosis, and enhanced the amounts of anti-oxidants in cells 33 it overexpression reduced proliferation and survival, but protected cells from NK cell-mediated cytotoxicity 34 its impair repressed glycolysis, thus to slow the growth of T-ALL cells 35 its overexpression inhibited the proliferation and promoted the apoptosis of ALL cells 36 its upregulation inhibited proliferation and promoted apoptosis of ALL cells 37 it promoted the proliferation, migration, invasion abilities of ALL cell lines 39 it promoted proliferation by increasing G1/S transition, migration and invasion of T-ALL cells 40 it inhibited cell proliferation and invasion in vitro, and suppressed tumor growth and lung metastasis in vivo 41 its mimic inhibited viability, migration and invasion of T-ALL cells 42 its activation might impact T-cell differentiation in part 43 its downregulation promoted T-ALL cell migration and invasion 44 it could inhibit proliferation and invasion of B-ALL cell lines 45 its downregulation contributed to disease progression by modulating cell motility and disease aggressiveness 46 it could serve as a potential diagnostic and drug-resistant biomarker in pediatric ALL 47 a diagnostic and drug-resistant biomarker, and a promising relapsed indicator in childhood ALL 47 its overexpression increased sensitivity of ALL-resistant cells 48 its interference might overcome the resistance of BCP-ALL to GCs 49 its interference might overcome the resistance of BCP-ALL to GCs 49 its expression was associated with GCs-resistance in patient leukemia cells taken at diagnosis 50 a novel, independent prognostic biomarker, and contributed to the development of drug resistance 51 a tumor suppressor in T-ALL, and enhanced chemotherapeutic sensitivity 52 its upregulation could induce an apoptotic effect on Dox-resistant B-ALL and restore Dox chemosensitivity 53 it might be utilized as non-invasive marker to diagnose and predict prognosis in ALL 54 it is considered as a novel oncomir and might have a role in the etiology or progression of T-ALL 55 its expression level is of good value in the diagnosis and prognostic assessment of childhood ALL 56 its expression level is of good value in the diagnosis and prognostic assessment of childhood ALL 56 a potential predictive and follow-up marker in children with ALL remitted and relapsed cases 57 a potential predictive and follow-up marker in children with ALL remitted and relapsed cases 57 an independent prognostic factor for ALL patient survival 58 a potent diagnostic biomarker and therapeutic goal in ALL 59 a potent diagnostic biomarker and therapeutic goal in ALL 59 it changed dynamically in the course of ALL, and could be used as a marker to monitor disease status 60 it promoted ALL cell proliferation and inhibited apoptosis, and associated with poor prognosis 63 a blood-based liquid biopsy biomarker for ALL minimal residual disease (MRD) 64 it identified a quiescent and chemo-resistant human B-ALL cell subset that correlated with MRD 65 a prognostic biomarker and novel candidate for treatment of drug resistant pediatric ALL 68 it promoted the expansion/survival of highly proliferative cell subsets of human T-ALL 69 its downregulation enhanced the growth and viability of T-ALL cells, and served as a novel tumor suppressor 70 it regulated cell proliferation, the cell cycle, and apoptosis, and facilitated tumor growth in vivo 71 its overexpression decreased viability, proliferation, and clonogenicity of t(4;11) cells, whereas enhanced apoptosis 72 its combination with doxorubicin synergistically induced the apoptosis in T-ALL 73 it motivated methotrexate's cellular uptake and cytotoxicity in leukemia cells 74

Abbreviations: BAALC, brain and acute lymphoblastic leukemia; CBFβ, core binding factor-beta; CNTFR, ciliary neurotrophic factor receptor; CXCR4, C-X-C chemokine receptor type 4; Dox, doxorubicin; FBXW7, F-box and WD repeat domain containing 7; FGFR, fibroblast growth factor receptor; GDF15, growth differentiation factor-15; HDGF, hepatoma-derived growth factor; IL-17A, interleukin-17A; MAML3, mastermind like transcriptional coactivator 3; MDR1, multidrug resistance protein 1; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3; PIK3CA, phosphoinositide-3-kinase, catalytic alpha; PPTC7, peptidyl-prolyl cis-trans isomerase T family member 7; PTK2, protein tyrosine kinase 2; RB1, retinoblastoma protein 1; SOX15, SRY (sex determining region Y)-box 15; SSX2IP, synovial sarcoma, X breakpoint 2 interacting protein; TIGAR, YY1, Yin-yang 1; ZNF238, zinc finger protein 238.

Targeting miRNAs to Improve Clinical Outcomes in Pediatric ALL

Targeting miRNAs to improve clinical outcomes in pediatric ALL patients is a complex and sophisticated process that involves multiple scientific and technological components. Firstly, to gain a deep understanding of the pathogenesis and development of pediatric ALL, particularly the role played by miRNAs. Through high-throughput sequencing, bioinformatics analysis, and functional validation methods, disease-specific miRNA markers or therapeutic targets related to the disease can be identified. Secondly, to design and develop corresponding therapeutic strategies for these specific miRNAs. There are two main approaches: one is to inhibit the expression of miRNAs by using antisense oligonucleotides, miRNA sponges, etc, which can bind to the target miRNAs and prevent them from functioning. The other is to enhance the expression of miRNAs through transfection of miRNA mimics or the use of expression vectors. Thirdly, to effectively deliver these therapeutic agents to the target tissues or cells through the use of specific delivery systems, such as nanoparticles, liposomes, or viral vectors. These delivery systems protect the therapeutic agents from the body's environment and help them reach their target locations accurately. Fourthly, safety and effectiveness are crucial considerations during the treatment process. This includes assessing the potential toxicity of the therapeutic agents on normal cells and monitoring clinical responses and disease progression after treatment. Through regular clinical examinations, biomarker monitoring, and imaging examinations, we can promptly understand the treatment response and prognosis of pediatric ALL patients and adjust the treatment plan accordingly to improve treatment outcomes. Finally, it is important to note that miRNA therapy does not exist in isolation. Instead, it needs to be combined with other treatment methods to form a comprehensive treatment plan. For example, in some complex diseases, it may be necessary to use multiple miRNA therapeutics simultaneously or combine miRNA therapy with drug therapy, surgical treatment, and other methods to achieve better treatment outcomes.

Therefore, targeting miRNAs to improve clinical outcomes in pediatric ALL patients is a multi-step and multi-strategy process that requires comprehensive consideration of disease mechanisms, treatment strategies, delivery systems, safety, and effectiveness. With the continuous advancement of science and technology and the accumulation of clinical experience, miRNA-based therapy will play a greater role in improving clinical outcomes for pediatric ALL patients in the future.

Limitations, Challenges and Future Prospects

miRNAs, as non-invasive blood biomarkers, possess immense potential value in medical research and clinical applications. However, their limitations cannot be overlooked. Firstly, the expression levels of miRNAs in blood are influenced by numerous factors, including individual differences, environmental factors, and disease states, which pose challenges for their application as stable and reliable biomarkers. Secondly, the extraction and detection of miRNAs require highly sensitive and specific techniques, such as RT-PCR, microarrays, and next-generation sequencing. These techniques are costly and complex to operate, limiting their widespread application in routine clinical testing. Thirdly, the functions and mechanisms of miRNAs have not been fully elucidated, and their interactions with other biomolecules, as well as their specific roles in the development and progression of pediatric ALL, require further investigation. Therefore, before the widespread clinical application of miRNAs, it is necessary to overcome these limitations and conduct more thorough scientific research and clinical validation.

miRNAs play an important role in the regulation of gene expression, not only regulating the expression of target genes but also being regulated by other transcription factors. Moreover, the interaction between miRNAs and target genes is not a simple one-to-one relationship, which constitutes a complex miRNA regulatory network. Currently, there are many studies on the miRNA expression profiles in ALL, but further research is still needed on the regulatory mechanisms of miRNAs in the occurrence and development of pediatric ALL, as well as the role of miRNAs in the pathogenesis of pediatric ALL.

The research on miRNAs in pediatric ALL drug resistance is still in its infancy, with most drug-resistant genes yet to be discovered and their functions remaining unclear. At this stage, how to effectively use miRNAs to block or activate relevant signal pathways in order to efficiently reverse clinical drug resistance in pediatric ALL patients and improve their rates of complete remission, long-term survival, and cure remains a difficult aspect of related research. It is necessary to further explore the miRNA expression profiles related to ALL drug resistance, screen miRNAs associated with ALL drug resistance, and study their functions. This will help to investigate ALL drug resistance from different perspectives and provide new directions for the treatment of refractory pediatric ALL. In addition, the application of miRNA/anti-miRNA-based technology in animal models has increasingly become a research hotspot, and miRNAs are expected to open up a new pathway for the treatment of pediatric ALL.

However, miRNA-based therapeutic approaches should be carefully considered with accurate selection of target miRNAs, as some miRNAs can be oncogenic or tumor-suppressive under different conditions. In addition, the regulatory and functional mechanisms of miRNAs have not been fully elucidated. Therefore, the development of miRNA-based treatment strategies should be carefully evaluated to ensure the safe use of miRNA therapy in clinical settings. More efforts are needed to improve the specificity of miRNA-based therapy and test their effectiveness in combination with conventional approaches, while avoiding toxicities and off-target effects.

Conclusion

In summary, this article provides a comprehensive overview of the recent advancements in understanding the pivotal roles of miRNAs in pediatric ALL. Special attention is paid to elucidating their intricate regulatory mechanisms and untapped therapeutic potential. miRNAs play a crucial role in modulating the pathogenesis of pediatric ALL, encompassing cellular processes such as proliferation, apoptosis, invasion, migration, and drug resistance. Their ability to fine-tune gene expression offers insights into the complex molecular interactions underlying this hematological malignancy. Furthermore, miRNAs exhibit promising clinical applications as diagnostic, prognostic, and therapeutic biomarkers, holding the key to more precise disease classification, earlier detection, and improved treatment outcomes in pediatric ALL. This review underscores the significance of miRNAs in ALL research and their potential as targets for novel therapeutic strategies.