In various T cell types, miR-29s play distinct regulatory roles, particularly by modulating different transcription factors and cytokines, thereby participating in the differentiation, maturation and functional regulation of T cell subsets (31,32). The importance of miRNAs in T cells was first noted in T cells with a Dicer deficiency, which displayed a preference for T helper (Th)1 polarization (33,34). Subsequent stages of immune function and development have confirmed the critical roles of miRNAs in T cells (7).

Upon antigen stimulation, thymic epithelial cells induce production of naive T cells, which then differentiate into various subtypes, most notably Th1 and Th2 cells (27). These cells are crucial for the regulation of immune responses to intracellular (Th1) and extracellular (Th2) attacks. A study published in 2011 revealed that miR-29a-deficient mice exhibit premature thymic involution and hypersensitivity to pathogen-associated signals, which is closely related to the direct targeting of interferon-α receptor (IFNαR) by miR-29a (35). Further studies reported that miR-29s are novel regulators of Th1 differentiation by targeting the transcription factors T-bet and EOMES of IFN-γ, thus influencing Th1 polarization (32,36-38).

miR-29s also play a critical role in the Th1 differentiation. In miRNA-deficient CD4⁺ T cells, reduced expression of T-bet and EOMES, key transcription factors for T cell development, have been linked to the direct targeting of these factors by miR-29b (31,36,39). Additionally, miR-29s can target the IFN-γ mRNA and suppress the differentiation of Th1 cells. IFN-γ signalling can also promote the expression of miR-29s, forming a negative feedback loop that regulates IFN-γ expression (37). This suggests that miR-29b is essential for proper T cell maturation and function, and its dysregulation may impair immune responses.

Furthermore, miR-29s appear to play distinct roles in the differentiation and function of CD8⁺ T cells, and are essential for the normal memory response of mature CD8⁺ T cells; whereas cells deficient in miR-29s prompt the differentiation of naive CD8⁺ T cells into short-lived effector cells with minimal stimulation, while simultaneously reducing differentiation of memory precursor effector cells (39).

NK cells, a type of innate lymphocyte, are crucial for early host defences against infectious pathogens and have been used to monitor malignant transformation, including acute myeloid leukaemia and lymphomas (40-42). Similarly to T cells, miR-29b influences the maturation and function of NK cells by regulating key transcription factors, such as T-bet and EOMES, which are crucial for NK cell development. Dysregulation of miR-29b leads to the downregulation of these transcription factors, thereby impairing the normal development and function of NK cells. Particularly in leukemic mice, the direct targeting of T-bet and EOMES by miR-29b highlights its importance in maintaining NK cell homeostasis (43).

miR-29s are involved in maturation of NK cells through IFN-γ and related transcription factors, such as T-bet, EOMES and IFN-γ (43-45). Regulation of miR-29b has been shown to restore the population of intermediate CD27⁻CD11b⁺ NK cells in vivo (43). In acute myeloid leukaemia, activation of the aryl hydrocarbon receptor pathway upregulates miR-29b expression, which then inhibits the development and function of NK cells, thereby allowing evasion of the innate immune system in both mice and humans (46,47).

In cancer biology, the role of miR-29s in regulating cell cycle proteins has been noted in B-cell lymphoma, particularly mantle cell lymphoma (MCL). miR-29s target the 3′-UTR of CDK6 mRNA, inhibiting the expression of CDK6, a key regulator of cell proliferation (48). Downregulation of miR-29s in MCL leads to frequent overexpression of CDK6, contributing to uncontrolled cell proliferation and the progression of the lymphoma (48). This highlights the tumour-suppressive role of miR-29s in preventing excessive cell growth in MCL. The use of a transgenic mouse model of chronic lymphocytic leukaemia (CLL) demonstrated that miR-29s play key roles in the production of B cells. In this model of overexpressing miR-29s in mouse B cells, 85% (34/40) of the mice exhibited an expanded CD5⁺ B cell population, which is a hallmark of B-cell CLL (49). Subsequent studies have reported additional roles of miR-29s in B cell differentiation. The loss of miR-29ab1 leads to a global loss of cells in the spleen and thymus, which has profound effects on B cell differentiation (50). While mice deficient in miR-29a exhibit congenital defects in B cell activation and germinal centre formation (50).

Additionally, miR-29s have been found to regulate proliferation and apoptosis of mature B cells by targeting the PTEN mRNA, thereby controlling the PTEN-PI3K axis (51,52). ERK and MAPK are activated downstream in BCR signal transduction, which is crucial for the proliferation and survival of B cells during development and differentiation. Upon BCR engagement, miRNA-29s modulate the survival and proliferation of B cells through the NF-κB and RAS-MAPK signalling pathways (53,54). Moreover, miR-29c targets the RAG1 mRNA, which influences the variable, diversity and joining genes of pre-B cells, thereby controlling B cell differentiation (55,56).

Notably, miR-29s also play crucial roles in the differentiation of monocytes into macrophages and macrophage polarization. They regulate key transcription factors and signalling pathways to control monocyte differentiation, modulate macrophage subtype balance, and contribute to immune regulation and tissue repair. A study conducted in 2013 revealed that miR-29s facilitate the differentiation of monocytes into macrophages (57). Additionally, miR-29s can downregulate mRNA expression of nuclear factor 1 A-type (NFIA), CD93 and G protein-coupled receptor 85, potentially impacting differentiation of macrophage lineages. Moreover, miR-29s promote polarization of macrophages towards the M2 subtype. Further studies have shown that miR-29a mediates macrophage autophagy via the PI3K/AKT/mTOR pathway (58,59).

In inflammatory diseases such as atherosclerosis, miR-29s may alleviate inflammation and enhance anti-inflammatory effects by regulating macrophage polarization. miR-29a amplifies M2-like macrophage polarization and inhibits polarization of M1-like macrophages within atherosclerotic plaques. Additionally, suppressor of cytokine signalling 1 (SOCS-1), the target of miR-29a and a negative regulator of STAT6, appears to be crucial for macrophage polarization (60,61). Inhibition of the STAT6 pathway significantly suppresses polarization of macrophages into the M2 subtype, highlighting the complex regulatory roles of miR-29s in immune effector cell function and inflammation modulation.

Toll-like receptors (TLRs) are pivotal in recognizing pathogen-associated molecular patterns (PAMPs), such as bacterial LPS and viral double-stranded RNA. This recognition activates signalling pathways that initiate immune responses, particularly through MyD88-dependent pathways, which drive inflammation (63,64). Although all TLRs can engage MyD88 to trigger inflammatory responses, they also elicit distinct immune responses tailored to different pathogens, thus bridging the innate and adaptive immunities (65,66). This process culminates in the activation of transcription factors, such as NF-κB, which promotes the expression of pro-inflammatory cytokines (63,66).

More and more findings have revealed that serum miR-29a can directly bind to TLR7 and TLR8, initiating the activation of dendritic cells and triggering the NF-κB pathway, which leads to the secretion of pro-inflammatory cytokines such as TNF-α and IL-6 (67,68). As TLR7 and TLR8 are key upstream components of the TLR signalling pathway, the interaction of miR-29a with these receptors places it at a critical juncture in immune activation (Fig. 1A).

miR-29s influence NF-κB signalling through various mechanisms. By inhibiting DNA methyltransferase (DNMT) activity, miR-29s promote the expression of cyclooxygenase-2 (COX2) and prostaglandin E2 (PGE2), which enhance NF-κB binding to the IFN-λ1 promoter, leading to increased IFN-λ1 expression. This highlights the involvement of miR-29s in both inflammation and antiviral responses (69,70).

Notably, miR-29s also target TNF receptor-associated factor 4 (TRAF4), a critical regulator in NF-κB signalling (71). This finding suggests that miR-29s downregulation in CLL can promote tumour progression by amplifying NF-κB activity through TRAF4. In macrophages, miR-29a promotes NF-κB activation by targeting Akt1, modulating the inflammatory response to bacterial LPS. This illustrates the role of miR-29a in regulating inflammation during bacterial infections (72-74).

In marine organisms, miR-29a also plays a role in immune regulation in marine organisms. In the pearl oyster, miR-29a targets the neuropeptide Y receptor type 2, resulting in the upregulation of IL-17 and NF-κB expression (75,76). This suggests that miR-29a can modulate immune responses in invertebrates, paralleling its role in vertebrates.

In the TLR signalling pathway, miR-29a regulates the immune-suppressive molecule B7-H3, which is highly expressed in tumours. By targeting the 3′UTR of B7-H3, miR-29a downregulates its expression, potentially enhancing the efficacy of immunotherapies for solid tumours (77-79). miR-29s influence the secretion of cytokines IL-12 and IL-23 in dendritic cells co-stimulated by NOD2 and TLR2, further emphasizing their broad role in immune modulation across various contexts (80).

The interferon (IFN) signalling pathway is a core component of the innate immune system, consisting of type I, II and III interferons, which plays critical roles in antiviral defence, immune regulation and cell cycle control (32,81,82). Upon binding to their specific receptors, interferons initiate the JAK-STAT signalling pathway, leading to the activation of transcription factors, particularly STATs, which subsequently induce the expression of interferon-stimulated genes (83,84). This process is essential for the proper execution of antiviral responses, immune regulation and the maintenance of homeostasis (Fig. 1B).

In immune responses, IFN-γ serves as a key factor in the differentiation of Th1 cells (36,37,85). Previous studies have shown that the expression levels of miR-29s are significantly downregulated in mice infected with Listeria monocytogenes or vaccinated with Bacillus Calmette-Guérin (32). miR-29s directly target IFN-γ mRNA, thereby suppressing IFN-γ production, which suggests that miR-29s play a crucial role in regulating Th1-mediated immune responses.

Furthermore, miR-29s exert regulatory effects at the interferon receptor level (35,86). For instance, miR-29a-deficient mice exhibit premature thymic involution and hypersensitivity to pathogen-associated signals, which is closely related to the direct targeting of interferon-α receptor (IFNAR) by miR-29a (35). Notably, in human host cells infected with respiratory syncytial virus (RSV), the RSV non-structural protein 1 (NS1) protein enhances miR-29a expression. Elevated miR-29a subsequently downregulates IFNAR1 expression, facilitating viral immune evasion (86).

At the downstream level of interferon signalling, several members of the JAK-STAT pathway are also regulated by miR-29s (60,87,88). In certain patients with oral cancer, elevated levels of miR-29a are observed in both cancer tissues and exosomes. miR-29a indirectly modulates STAT6 signalling by targeting the negative regulator SOCS-1, further influencing immune responses (60). In hepatocytes infected with hepatitis C virus (HCV), the downregulation of miR-29c increases the expression of its target gene, STAT3, contributing to antiviral effects (87). However, it is important to note that the upregulation of STAT3 is not solely due to miR-29c downregulation; factors such as the HCV core protein and oxidative stress also contribute to STAT3 induction.

These findings highlight that miR-29s play a critical role in the interferon signalling pathway at multiple levels, from interferon production and receptor regulation to downstream signalling.

HBV is a hepatotropic DNA virus that has been identified as a key risk factor for hepatocellular carcinoma (HCC) in epidemiological studies (95,96). As compared to controls, miR-29c is significantly downregulated in HBV-related HCC cell lines and HBV-infected transgenic mice. In HBV-infected HCC cells, miR-29c targets A20, a critical regulator of inflammation and immunity, thereby exerting a tumour-suppressive effect (97). In HepG2.2.15 cells, overexpression of miR-29c significantly inhibits HBV DNA replication, suppresses cell proliferation and induces apoptosis. In chronic HBV infection, serum levels of miR-29s are negatively correlated to the stage of liver fibrosis and necroinflammatory grading (98). In the context of HBV infection, miR-29c targets the HBV S gene and inhibits the expression level of S protein significantly in sperm embryos of patients with HBV (99).

Conversely, miR-29a expression is upregulated in HepG2.2.15 cells infected with HBV and directly regulates SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1 (SMARCE1) to promote HBV replication. SMARCE1 is known to inhibit HBV replication by binding to the core promoter of the virus (92,100). Moreover, high expression of miR-29a-5p (the passenger strand of miR-29a-3p) is significantly associated with shorter time to recurrence and overall survival of patients with HBV-related HCC after resection (101).

HIV is a single-stranded RNA virus that primarily targets CD4⁺ T cells. As of 2022, ~39 million people globally were living with HIV (https://aidsinfo.unaids.org/). A growing body of research suggests that miRNAs, particularly miR-29a, inhibit HIV-1 replication both in vivo and in vitro (93,102-105). Both miR-29a and miR-29b directly target the HIV-1 Nef mRNA, thereby inhibiting translation of the Nef protein, a critical component of HIV (103). Additionally, miR-29a enhances binding of HIV-1 gag mRNA to endogenous P-bodies, which facilitates mRNA degradation (104). IL-21 produced by CD4⁺ T cells is a significant immune modulator and facilities the production of miR-29s during HIV infection through the STAT3 pathway. Serum levels of IL-21 are reduced in patients with HIV-1, suggesting that the IL-21-miR-29s axis may be involved in resistance to HIV-1 infection (102,106). However, during the latent period following HIV infection, which can be as brief as several months or up to 15 years, the virus is not cleared from the host cell. Mediation of RNA interference against HIV-1 by cellular miRNAs is limited. The extensive secondary structure of HIV RNA can resist binding by host miRNAs, thereby facilitating viral replication (107).

HCV, an RNA virus belonging to the Flaviviridae family, is a leading cause of chronic liver diseases and cirrhosis. In most patients with acute HCV infection, miR-29s are consistently downregulated. An in vitro study showed that levels of miR-29s were decreased in HCV-infected Huh7.5 cells, while overexpression of miR-29s reduced HCV RNA levels (94). The HCV NS2 protein has been linked to steatosis and increased transcription of the lipogenic transcription factor sterol regulatory element-binding protein 1-c (SREBP-1c). Targeting SREBP-1c by miR-29a contributes to lipid metabolism and inhibits HCV replication (108). Furthermore, downregulation of miR-29c in Huh7 cells infected with the HCV isolate JFH-1 was associated with upregulation of mRNA and protein expression of the transcription factor STAT3, which is directly targeted by miR-29c (87). This interaction enhanced the I-IFN pathway and suppressed viral replication.

IAV is an enveloped single-stranded negative-sense RNA virus that primarily targets respiratory epithelial cells, causing symptoms ranging from mild upper respiratory infections to severe pneumonia. The miR-29 family plays an antiviral role in IAV infection by targeting the 3′-UTR of BCL2L2 mRNA. BCL2L2 is an anti-apoptotic factor that inhibits translation and promotes IAV-mediated apoptosis of the host cell (109). Epigenetic modifications mediated by miR-29s have been reported in various diseases. Moreover, miR-29s can induce the expression of COX2 and PGE2 by inhibiting DNMT activity. COX2 enhances IFN-λ1 expression by facilitating the binding of NF-κB to enhancers in the IFN-λ1 promoter (69). Notably, inhibition of miR-29c significantly accelerates viral replication, while overexpression has no effect (110). Additionally, miR-29s target Frizzled-5, a positive regulator of the non-classical Wnt-Ca²⁺ signalling pathway, to inhibit IAV infection (111). Activation of the Wnt-Ca²⁺ pathway increases expression of IAV mRNA. During IAV infection, members of the miR-29 family, particularly miR-29c, are upregulated. Also, miR-29s enhance the abundance of A20, a negative regulator of the NF-κB signalling pathway, thus inhibiting RIP1-mediated NF-κB activation, thereby exerting an antiviral effect. Intriguingly, miR-29s can act as an RNA decoy to stabilize A20 mRNA independent of the seed sequence, prevent binding of human antigen R to the A20 3′-UTR and recruit the RNA-induced silencing complex, thereby protecting A20 mRNA (112).

In addition, changes in miR-29s have been reported during other viral infections. In COVID-19 infection, the levels of miR-29a exhibit significant variations at different stages of infection, although these changes differ across various studies (113,114). In patients recovering from Japanese encephalitis virus infection with sequelae, the expression level of miR-29b is significantly elevated (62,115). In the case of human T-lymphotropic virus 1 (HTLV-1), the upregulation of miR-29c in serum may serve as a novel potential biomarker for HTLV-1 diagnosis (116). Notably, in human host cells infected with RSV, the RSV NS1 protein enhances the expression of miR-29a. The subsequent elevation of miR-29a downregulates IFNAR1 expression, thereby facilitating viral immune evasion (86). Following Epstein-Barr virus infection, miR-29a is upregulated, which targets and reduces B-cell lymphoma and Burkitt's lymphoma levels (117). Apart from roles in viral infections in humans, miR-29s also exert antiviral effects in other animals. Porcine reproductive and respiratory syndrome virus (PRRSV), a positive-strand RNA virus of the Arteriviridae family, infects pigs. Transcript levels of miR-29a are increased in pig peripheral blood mononuclear cells following PRRSV infection both in vitro and in vivo (118). In the early stages of viral infection, miR-29ab might promote PRRSV replication through AKT3 (119). Bovine viral diarrhoea virus (BVDV) causes an endemic viral disease of cattle in North America and is considered a major pathogen worldwide. In Madin-Darby bovine kidney cells infected with BVDV strain NADL, miR-29b directly targets the 3′-UTR of genes associated with apoptosis (caspase-7 and NAIF1) and autophagy (ATG14 and ATG9A), thereby inhibiting BVDV replication. Additionally, during BVDV infection, decreased methylation of the promoter region of host miR-29b leads to upregulation of miR-29b expression and subsequent inhibition of BVDV replication (120-122).

The results of these studies emphasize the wide-range of antiviral effects of miR-29s in different animal models through multiple pathways, suggesting significant potential for therapeutic exploitation.