GC ranks fifth worldwide in both incidence and mortality, with ~980,000 new cases and 642,000 deaths estimated globally in 2024 (32). Evidence indicates elevated miR-324-5p expression in both GC tissues and cell lines (15). Mechanistically, inhibition of miR-324-5p suppresses GC cell proliferation and invasive behavior while promoting apoptosis, potentially through relief of miR-324-5p-mediated suppression of PTEN, a downstream target negatively regulated by miR-324-5p (15). Moreover, circ_0049447 modulates GC progression through sequestration of miR-324-5p, potentially involving a protein tyrosine phosphatase receptor Δ (PTPRD)-related mechanism (40). miR-324-5p has been identified as a component of a plasma small extracellular vesicle (sEV)-miRNA signature associated with distant lymph node metastasis (41). However, another study found that miR-324-5p expression is lower in five paired clinical GC than in adjacent normal tissues (14). Mechanistically, inhibition of miR-324-5p reverses the antitumor effects induced by circ_0091994 knockdown (14).

PTC is the predominant histological subtype of thyroid cancer. In 2024, ~959,000 new thyroid cancer cases and 48,000 deaths were estimated worldwide (32). Increased miR-324-5p expression in PTC tissue is associated with unfavorable clinicopathological characteristics, including lymph node metastasis (LNM), greater tumor size and advanced clinical stages (19). Molecular studies indicate that miR-324-5p promotes PTC cell proliferation, migration and invasion (18,19). Mechanistically, miR-324-5p directly targets PTPRD and suppresses its protein expression, leading to CCAAT enhancer-binding protein δ (CEBPD) upregulation (18). Elevated CEBPD expression enhances the migratory and invasive capacities of human umbilical vein endothelial cells within the tumor microenvironment and promotes macrophage polarization toward an M2-like phenotype (18). Additionally, enhanced CEBPD levels reinforce M2 macrophage polarization by increasing IL-4 and IL-13 production (18), thereby contributing to PTC progression. Collectively, these observations highlight the contribution of miR-324-5p to PTC development and progression.

PC is characterized by an unfavorable prognosis. In 2024, ~531,000 new cases and 491,000 deaths from PC were estimated worldwide (32), reflecting its high mortality burden. miRNAs participate in PC development by regulating molecular targets involved in tumor proliferation and apoptosis, including KLF3 (48,49). Evidence has implicated miR-324-5p in PC progression (48,49). Increased miR-324-5p expression is detected in PC cell lines and patient-derived tumor samples (49). Inhibition of miR-324-5p suppresses PC cell proliferation and induces apoptosis, whereas restoration of miR-324-5p expression enhances proliferative activity and attenuates apoptotic responses (49). Mechanistically, Krüppel-like factor 3 (KLF3) is a direct downstream target of miR-324-5p. By suppressing KLF3 expression, miR-324-5p promotes PC cell proliferation and decreases apoptosis (49). Collectively, these data demonstrate that miR-324-5p contributes to PC progression through regulation of the KLF3-mediated molecular pathway. In addition, a recent study implicates miR-324-5p in trace element metabolism (50,51). In pancreatic ductal adenocarcinoma (PDAC) specimens, miR-324-5p abundance is negatively associated with selenium, manganese and zinc levels, suggesting a potential role for miR-324-5p in trace element homeostasis (50).

Bladder cancer is a common malignant neoplasm of the urinary system. In 2024, ~635,000 new bladder cancer cases and 228,000 deaths were estimated worldwide (32). Elevated urinary miR-324-5p levels have been detected in patients with bladder cancer (52), while increased miR-324-5p abundance has also been observed in surgically resected bladder cancer tissues compared with matched adjacent tumor tissues (53). Mechanistic investigations have implicated miR-324-5p in bladder cancer progression through regulatory associations involving regulator of calcineurin 1 (RCAN1), FosB proto-oncogene, AP-1 transcription factor subunit (FOSB) and eyes absent homolog 4 (EYA4) (52,53). In particular, suppression of RCAN1 and EYA4 expression was associated with enhanced bladder cancer cell proliferation, colony formation and invasive behavior (53). Taken together, these findings indicate that miR-324-5p may contribute to bladder cancer progression by promoting oncogenic processes.

In 2024, ~843,000 new liver cancer cases and 732,000 deaths were estimated worldwide (32). HCC represents the predominant histological subtype of primary liver cancer (61). Recent studies have highlighted miR-324-5p-associated regulatory networks involved in HCC progression, immune regulation and prognosis (62,63). Notably, miR-324-5p expression is reduced in hepatitis C virus (HCV)-infected HCC cells but is restored following IFN-α treatment, indicating its involvement in the molecular interplay between HCV infection and the response to IFN-α therapy (64). Similarly, HCC tissue and cell-based models exhibit diminished miR-324-5p expression (27). Conversely, increasing miR-324-5p expression inhibits the invasive capacity and metastatic properties of HCC cells (27). Mechanistically, miR-324-5p regulates ETS1 and SP1, resulting in the downregulation of MMP2 and MMP9, and decreased HCC cell invasion and migration (27). In addition to direct target regulation, miR-324-5p activity in HCC is modulated by multiple lncRNAs (27). For example, LINC00491 serves as a ceRNA that binds miR-324-5p, thereby increasing ROCK1 expression and promoting liver cancer progression (22). Similarly, lnc RP11-85G21.1, is abundantly expressed in HCC cells and cell-derived exosomes, where it promotes malignant phenotypes by modulating the functional availability of miR-324-5p (65). LINC01134 interacts with miR-324-5p and attenuates its suppressive effect on insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) (13). Through stabilization of Yin Yang-1 (YY1) transcripts, IGF2BP1 increases YY1 expression, which promotes LINC01134 transcription and creates a positive feedback mechanism contributing to HCC progression (13). Moreover, evidence suggests an association between C-type lectin domain family 4s (CLEC4s) member expression in HCC tissue and infiltration of immune cells (66). miR-324-5p, a potential regulator of CLEC4s, may modulate the HCC immune microenvironment by targeting CLEC4s expression (66). In HCC, exosomal circGSE1 interacts with miR-324-5p and modulates the transforming growth factor β receptor 1 (TGFBR1)/Smad3 pathway, resulting in enhanced regulatory T cell (Treg) expansion and immune evasion (21). In addition, (1E,4E)-1,5-bis(2-bromophenyl) penta-1,4-dien-3-one (GL63), a synthetic derivative of curcumin, displays antitumor activity, partially by modulating circZNF83, a ceRNA within the miR-324-5p regulatory network, thereby attenuating malignant phenotypes and promoting apoptosis in HCC cells (25). Collectively, these data elucidate the intricate molecular pathways involving miR-324-5p in HCC pathogenesis and therapy.

In 2024, ~604,000 new CC cases and 280,000 deaths were estimated worldwide (32). Accumulating evidence implicates miR-324-5p in the regulation of CC progression and treatment response through diverse molecular pathways (11,67). Decreased expression of miR-324-5p is reported in CC tissue, indicating its potential involvement in tumor development (11). Mechanistically, LINC00511 attenuates miR-324-5p activity through competitive binding, resulting in DRAM1 dysregulation and altered proliferative and migratory behavior of CC cells (11). Moreover, re-expression of miR-324-5p increases the radiosensitivity of CC cells by enhancing apoptosis and limiting migratory capacity (67). This radiosensitizing effect is mediated, at least partially, by directly targeting ELAVL1, followed by modulation of apoptosis-associated proteins, including Bcl-2 and Bax (67). Therefore, miR-324-5p may be a critical regulator of CC pathogenesis and treatment-associated responses.

In 2024, ~331,000 new ovarian cancer cases and 204,000 deaths were estimated worldwide (32). Dysregulated miRNA profiles contribute to OC progression and therapeutic response (68). Survival analyses of serous OC cohorts reveal that patients with lower miR-324-5p levels exhibit poorer OS (69,70) and, in one cohort, with shorter progression-free intervals (69). Notably, although miR-324-5p downregulation is associated with olaparib resistance, inhibition of the ataxia telangiectasia and Rad3-related/checkpoint kinase 1 (ATR/CHK1) pathway suppresses miR-324-5p expression and enhances olaparib sensitivity in BRCA2-mutant olaparib-resistant ovarian cancer cells (PEO1-OR) (69). Computational network analysis has identified G2/M checkpoint-associated genes, including tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein θ, as potential downstream effectors of miR-324-5p, suggesting miR-324-5p may influence cell cycle regulation during the development of olaparib resistance (69). Furthermore, circZNF609 promotes OC progression by sequestering miR-324-5p and relieving miR-324-5p-mediated repression of voltage-dependent anion channel 1 expression (71). Conversely, enforced miR-324-5p expression impairs OC cell proliferative and invasive capacity and decreases xenograft tumor growth, highlighting its tumor-suppressive function in OC (71).

MM is a hematological malignancy driven by the uncontrolled expansion of clonal plasma cells in the bone marrow microenvironment, which is associated with clinical complications, including renal dysfunction, hypercalcemia, anemia and osteolytic bone lesions (72). Increased miR-324-5p expression may attenuate MM progression by suppressing cell proliferation, viability and cancer stem-like characteristics (73,74). miR-324-5p enhances the therapeutic response to bortezomib (BTZ) by regulating multidrug resistance-associated proteins and apoptosis-associated members of the Bcl-2 family (73). At the molecular level, circATIC participates in a ceRNA regulatory network by sequestering miR-324-5p, thereby relieving miR-324-5p-mediated suppression of hepatocyte growth factor (HGF) expression. This regulatory pathway enhances proliferation, migration, invasion and glycolytic reprogramming while attenuating apoptosis in MM cells (74). Collectively, these results identify miR-324-5p as a key target downstream of circATIC and regulator of MM biology.

GBM is a highly aggressive primary brain malignancy (75). Despite multimodal treatment, the median overall survival is ~15 months, and the 5-year survival rate remains ~5.8% (76). In GBM tissue, decreased miR-324-5p abundance is observed with upregulation of circ_0079593 and X-box binding protein 1 (XBP1) (77). Mechanistically, miR-324-5p directly binds XBP1 and suppresses its expression, thereby limiting GBM cell proliferation (77). By contrast, circ_0079593 promotes GBM progression by competitive binding with miR-324-5p, thereby increasing XBP1 expression (77). Furthermore, lncRNA NEAT1 contributes to GBM progression through a ceRNA mechanism, in which it binds miR-324-5p and prevents miR-324-5p-mediated suppression of potassium channel tetramerization domain-containing 20 (KCTD20). Restoration of KCTD20 expression promotes GBM cell proliferation and attenuates apoptosis (78). circSERPINE2 contributes to GBM progression by competitively binding miR-324-5p and miR-361-3p, thereby relieving miRNA-mediated suppression of Bcl-2 and promoting malignant cell survival (79). Conversely, miR-324-5p exerts inhibitory effects on GBM by targeting enhancer of zeste homolog 2 (EZH2), thereby limiting tumor growth and increasing temozolomide (TMZ) responsiveness (23). Therefore, miR-324-5p appears to function as a negative regulator of GBM progression.

Hematological malignancy comprise neoplastic disorders involving molecular alterations and exhibiting substantial variability in treatment response. A circulating miRNA study in diffuse large B cell lymphoma (DLBCL) have linked miR-324-5p to responsiveness to rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (80). Tumor miRNA profiling demonstrated an association between miR-324-5p expression and survival outcomes in patients with DLBCL (81). Circulating and tissue-derived miR-324-5p display divergent expression patterns in DLBCL, with elevated serum levels observed in refractory patients but decreased expression detected in tumors from non-responsive cases (80). Re-expression of miR-324-5p impairs DLBCL cell survival through thioredoxin reductase 1 regulation and ferroptosis induction, providing functional evidence for its context-dependent antitumor activity (80). Bioinformatics suggests a potential role for miR-324-5p in acute myeloid leukemia (AML) (82). A miRNA profiling study identified miR-324-5p among the miRNAs upregulated in primary AML samples compared with normal bone marrow (83). A bioinformatic analysis predicted a hsa_circ_0058058/miR-324-5p regulatory axis involving adaptor-associated protein complex 1 γ1 subunit (AP1G1) and SP1, suggesting this network may contribute to AML progression (82). Collectively, the aforementioned studies indicate that miR-324-5p has context-dependent roles in hematological malignancy with its biological effects varying across disease entities and regulatory networks. The expression patterns, regulatory mechanisms and biological functions of miR-324-5p across different cancer types are summarized in Table I.

The major tumor-associated signaling pathways involving miR-324-5p are summarized in Fig. 2.

TGF-β signaling is extensively involved in tumor biology and influences multiple cell programs, including differentiation, cell motility, EMT and metastatic spread (84). In prostate cancer, lncRNA PCAT7 promotes TGFBR1 expression by competitively interacting with miR-324-5p, thereby increasing TGF-β pathway activity. This PCAT7/miR-324-5p/TGFBR1 regulatory pathway contributes to prostate cancer bone metastasis (85). Conversely, exosome-derived circGSE1 in HCC decreases miR-324-5p availability, resulting in TGFBR1 upregulation, Smad3-dependent signaling activation and increased Treg infiltration, thereby promoting tumor immune escape (21). The influence of miR-324-5p on TGF-β signaling is shaped by tumor-specific ceRNA networks, resulting in distinct functional outcomes across cancer types.

Hh signaling is key to developmental patterning and the maintenance of tissue homeostasis in adulthood, whereas aberrant pathway activity is associated with tumor progression (86). In MM, the miR-324-5p locus resides within a chromosomal region typically affected by 17p deletion. Patients with del(17p) frequently exhibit decreased miR-324-5p expression, resulting in enhanced Hh pathway activity (73). Experimental findings show miR-324-5p negatively regulates the Hh pathway by targeting Gli family zinc finger 1 and Smoothened, with consequent inhibition of MM cell proliferation, survival and stem-like behavior (73). Moreover, restoration of miR-324-5p expression disrupts the supportive interactions between MM and bone marrow stromal cells (73). Increased miR-324-5p levels also regulate multidrug resistance-associated proteins and Bcl-2 family members via Hh pathway inhibition, thereby improving the therapeutic response to BTZ in MM (73). Overall, miR-324-5p influences MM progression and treatment sensitivity through Hh pathway regulation, suggesting this miRNA may represent a candidate for therapeutic intervention in MM.

JAK2/STAT3 signaling coordinates cellular activities and has been implicated in both tumorigenesis and malignant progression (87). Zhao et al (25) reported that GL63, a curcumin derivative, suppresses circZNF83 expression, thereby restoring miR-324-5p availability and relieving circZNF83-mediated sequestration of miR-324-5p. Increased miR-324-5p activity suppresses cyclin-dependent kinase 16 (CDK16), leading to decreased JAK2/STAT3 signaling activity and suppression of malignant phenotypes in HCC (25). Thus, GL63 may inhibit JAK2/STAT3 signaling in HCC via the circZNF83/miR-324-5p/CDK16 regulatory pathway (25).

NF-κB signaling regulates tumor cell biology, including proliferation, inflammatory activity, survival and immune escape (88). Persistent disturbance of this pathway may promote malignant transformation, invasion and metastatic dissemination. Elevated miR-324-5p levels are detected in NSCLC cells and other malignancies carrying gain-of-function TP53 mutations (89). At the molecular level, mutant p53 enhances miR-324-5p transcription via the c-Myc signaling pathway, resulting in CUEDC2 downregulation and NF-κB activation. This regulatory pathway promotes key cancer stem-like properties, including increased proliferation, survival and chemotherapy resistance (89). Conversely, miR-324-5p silencing restores CUEDC2 expression, suppresses NF-κB signaling activity and attenuates cancer-like phenotypes (89). Available evidence implicates the TP53/c-Myc/miR-324-5p/CUEDC2 pathway in coupling miR-324-5p activity with NF-κB signaling and cancer stem-like properties (89).

Several in vivo studies have evaluated the tumor-regulatory functions of miR-324-5p in cancer models (Table II) (14,22,49,58,59,79,90,91). These studies indicate that miR-324-5p-related interventions can influence tumor growth, metastasis and treatment-associated phenotypes through mechanisms involving direct target gene regulation, EMT-associated changes, ceRNA networks and intestinal barrier-associated pathways. These in vivo findings complement cell-based evidence and provide additional support for the context-dependent role of miR-324-5p in tumor regulation.

Integrin and focal adhesion kinase (FAK) signaling pathways are key regulators of tumor invasion and metastatic progression (92,93). ITGA9 is directly regulated by miR-324-5p through interaction with the ITGA9 3′UTR (94). In rhabdomyosarcoma (RMS), miR-324-5p-mediated suppression of ITGA9 limits proliferative and invasive behavior in vitro and decreases tumor growth in mouse xenograft models (94). Decreased ITGA9 levels are accompanied by reduced FAK phosphorylation, indicating that the miR-324-5p/ITGA9 pathway may regulate RMS progression via FAK-related signaling (94). In a COAD xenograft model, enforced miR-324-5p expression and VPS9D1-AS1 silencing markedly inhibits tumor growth. VPS9D1-AS1 regulates the miR-324-5p/ITGA2 pathway by limiting miR-324-5p activity, thereby favoring ITGA2 expression and COAD progression (59).

Beyond integrin-associated pathways, miR-324-5p is associated with EMT regulation and metastatic dissemination. In gallbladder carcinoma (GBC), elevated miR-324-5p expression decreases cell motility, invasion and EMT-related phenotypes, leading to fewer hepatic metastatic lesions in vivo (90). Conversely, miR-324-5p is downregulated in liver cancer tissue and cells models relative to corresponding non-tumor controls and its restoration suppresses tumor-associated phenotypes (22,27). Mechanistically, miR-324-5p limits tumor expansion and metastatic potential by modulating ROCK1 (22).

miR-324-5p regulates tumor growth by modulating transcriptional factors. In endometrial cancer xenografts, miR-324-5p inhibits subcutaneous tumor formation by suppressing high mobility group AT-hook 1 (HMGA1) expression (91). In a PC xenograft model, miR-324-5p promotes xenograft tumor growth by targeting KLF3. Conversely, inhibition of miR-324-5p markedly attenuates tumor growth (49).

circRNAs and lncRNAs modulate miR-324-5p activity in ceRNA networks, thereby affecting tumor-associated phenotypes. In a GBM xenograft model, loss of circSERPINE2 markedly inhibits tumor growth (79). Mechanistically, circSERPINE2 sequesters miR-324-5p and miR-361-3p, decreasing suppression of Bcl-2 and favoring GBM progression (79). In GC xenografts, higher miR-324-5p abundance is associated with smaller and lighter tumors (14). Silencing hsa_circ_0091994 increases miR-324-5p availability, resulting in decreased HMGA1 expression and suppression of GC progression (14).

Altered Dicer expression is implicated in CRC (57), and experimental Dicer deficiency reduces miR-324-5p expression, supporting a role for Dicer-mediated miRNA processing in the regulation of miR-324-5p (58). In a CRC model, Dicer deficiency markedly decreases miR-324-5p expression in mice, resulting in abnormal cytoskeletal alterations in intestinal epithelial cells, compromised barrier function, enhanced inflammatory response and increased tumor formation (58). Conversely, intraperitoneal delivery of miR-324-5p agonist alleviates dextran sulfate sodium-induced intestinal inflammation and tissue injury, restores epithelial barrier integrity and decreases HMGXB3 and WASF-2 expression, thereby suppressing CRC progression (58).

miRNAs, which are detectable in both intracellular and extracellular environments, have attracted attention for their potential utility in cancer detection, prognostic evaluation and assessment of therapeutic response (95). Combined RNA sequencing and reverse transcription-quantitative (RT-q)PCR have detected enrichment of miR-324-5p in plasma-derived exosomes from patients with NSCLC, indicating potential biomarker relevance (33). Similarly, elevated miR-324-5p levels are observed in urine specimens from individuals with bladder cancer and show an inverse association with the expression of the lncRNA miR-497-HG (52). These clinical observations are complemented by a separate functional study showing that miR-324-5p promotes bladder cancer cell proliferation, colony formation, migration and invasion (53). In PTC, higher miR-324-5p levels are linked to LNM, greater tumor burden and more advanced disease stage, supporting its potential utility for patient stratification and risk assessment (19). Gómez-Acebo et al (45) reported higher circulating miR-324-5p levels in patients with breast cancer than in healthy controls and incorporated this miRNA into a seven-miRNA diagnostic signature (OR=1.60, 95% CI: 1.08–2.37), supporting its potential value as a diagnostic marker. Collectively, evidence points to a potential diagnostic application for miR-324-5p, although validation is needed to establish its clinical biomarker utility.

miR-324-5p expression patterns are associated with clinical outcomes across several types of malignancies. In glioma, miR-324-5p has been identified as an independent prognostic indicator (23). In GC, RT-qPCR has revealed elevated miR-324-5p levels in tumor tissue and cell lines, which are associated with lymphatic metastasis, advanced disease stage and poor survival (15). Another study profiled miRNAs in plasma-derived small extracellular vesicles from patients with GC using sEV-miRNA sequencing followed by RT-qPCR validation and found that miR-324-5p abundance is associated with organ-specific metastatic patterns, suggesting potential prognostic relevance (41). By contrast, lower miR-324-5p levels in GBC are linked to adverse clinical outcomes (90). This association may be partly explained by miR-324-5p-mediated regulation of TGFB2, which affects GBC cell migration, invasion and EMT (90). In triple-negative breast cancer, next-generation sequencing of 51 specimens showed that elevated miR-324-5p levels were associated with shorter OS (43). Overall, these observations indicate that miR-324-5p may have prognostic value across multiple tumor types.

lncRNAs interact with miRNAs through regulatory networks involved in tumor initiation and progression (98). Several lncRNAs have been reported to alter the availability or activity of miR-324-5p, with effects on malignant phenotypes (12,59,78). In GBM, lncRNA NEAT1 sequesters miR-324-5p, thereby weakening its inhibitory effect on KCTD20 and facilitating tumor cell proliferation (78). A similar regulatory mechanism operates in COAD, where lncRNA VPS9D1-AS1 overexpression suppresses miR-324-5p activity and elevates ITGA2 expression. The VPS9D1-AS1/miR-324-5p/ITGA2 pathway promotes COAD cell proliferation and decreases apoptosis (59). In melanoma, HLA complex group 18 (HCG18) expression is elevated in both tumor specimens and cultured cells, and modulates the miR-324-5p/CDK16 pathway (12). By decreasing miR-324-5p-mediated suppression of CDK16, HCG18 enhances malignant cell proliferation, motility and invasiveness while decreasing apoptotic activity (12). Although these lncRNAs act in different tumor settings, they converge on miR-324-5p and regulate malignant phenotypes through distinct downstream effectors, including KCTD20, ITGA2 and CDK16 (12,59,78). Whether these networks are independent or share common upstream regulatory features remains unclear. Comparison of their expression patterns, intracellular localization and interaction with miR-324-5p may clarify this issue.