MicroRNA-99 family in cancer: molecular mechanisms for clinical applications

Oral, head and neck cancer

Members of the miR-99 family play diverse roles in the development and progression of oral, head, and neck cancer. In oral squamous cell carcinoma (OSCC), the ectopic expression of miR-99b-3p suppresses the p65 (RelA) and G1 regulators (cyclin D1, CDK4, and CDK6) and inhibits cell proliferation by targeting glycogen synthase kinase-3β (GSK3β) (He et al., 2015). In OSCC, miR-99a-5p has been shown to target mTOR and impair cancer cell proliferation (Yan et al., 2012). Ectopic expression of miR-99a-5p markedly reduces cell migration and invasion by targeting IGF1R and myotubularin-related protein 3 (MTMR3) (Kuo et al., 2014; Yen et al., 2014). Suppression of IGF1R expression by miR-99a-5p reduces lung colonization by oral cancer cells in vivo. Isoprenyl cysteine carboxymethyltransferase (ICMT), responsible for enhanced invasiveness, is a target of miR-99a-5p (Sun & Yan, 2021).

miR-100-5p inhibits the proliferation of nasopharyngeal carcinoma by targeting HOXA1 (He et al., 2020). miR-100-5p represses migratory and invasive abilities by targeting IGF1R and RASGRP3 (Peng et al., 2020; Sun et al., 2018). miR-100-5p targets polo-like kinase 1 (PLK1) to reduce CDC25C levels, thereby enhancing the cytotoxicity of radiation treatment (RT) (Shi et al., 2010). miR-100-3p targets ATG10 and activates the PI3K/AKT signaling pathway to attenuate autophagy, which leads to the suppression of proliferation and migration (Peng et al., 2022).

Various studies have reported the multifaceted roles of miR-99a in head and neck squamous cell carcinoma (HNSCC). miR-99a-5p, miR-99b-5p, and miR-100-5p commonly target both IGF1R and mTOR, thereby inducing cell proliferation and migration and enhancing apoptosis (Chen et al., 2012). STAMBP, targeted by miR-99a-3p, facilitates the migration and invasiveness of HNSCC cells (Okada et al., 2019). The levels of miR100-3p (targeting the tumor suppressor LKB1) and miR-100-5p are elevated in HNSCC tissues (Figueroa-González et al., 2020).

Lung cancer

Members of the miR-99 family are frequently downregulated in lung cancer (Feliciano et al., 2017; Han et al., 2021; Mizuno et al., 2020; Ye et al., 2023). miR-99a-5p, miR-99b-5p, and miR-100-5p co-target FGFR3 and sequentially repress Erk1/2 and AKT, thereby delaying lung cancer progression (Du et al., 2018; Jing et al., 2018; Kang et al., 2012; Oneyama et al., 2011). miR-99a-5p induces apoptosis and inhibits proliferation, migration and invasion in non-small cell lung cancer (NSCLC) by targeting IGF1R, AKT1 and HS3ST3B1 (Chen et al., 2015; Chen et al., 2023b; Yu et al., 2015; Zhai, Li & Lin, 2024). Moreover, miR-99a-5p reduces ROS accumulation by targeting NOX4 and inhibits the MAPK pathway by targeting mTOR, which is also targeted by miR-99a-5p in lung adenocarcinoma (LUAD) (Gu et al., 2013; Oneyama et al., 2011; Song et al., 2014; Sun et al., 2016). miR-99a-5p targets the oncogenic proteins E2F2 and EMR2, thereby preventing epithelial-mesenchymal transition (EMT) and repressing pluripotency in the cancer stem-like cell (CSC) population (Feliciano et al., 2017). In LUAD tissue, the potential anti-tumor gene MIR99AHG and its embedded miRNAs are downregulated, partly owing to the copy number deletion of MIR99AHG. Consequently, miR-99a-5p and MIR99AHG synergistically promoted autophagy and delayed LUAD progression by targeting mTOR (Han et al., 2021). Another oncogenic target, FAM64A, is regulated by miR-99a-5p and miR-99a-3p (Mizuno et al., 2020).

miR-100-5p exhibits a tumor-suppressive function similar to that in NSCLC. It is under-expressed and consequently no longer suppresses SMARCA5, a gene that promotes cell invasion (Li et al., 2021). Similarly, miR-100-5p inhibits lung-cancer-derived brain metastasis by downregulating ACKR3 and blocks EMT and Wnt/β-catenin signaling by targeting HOXA1 (Han et al., 2020a; Ma et al., 2019; Zhang, Song & Zeng, 2023). Conversely, SCLC cell lines resistant to cisplatin, etoposide, and adriamycin exhibit upregulated miR-100-5p expression, which maintains the resistant phenotype by targeting HOXA1 to prevent apoptosis and cell cycle arrest (Han et al., 2020a; Xiao et al., 2014). In cisplatin-resistant NSCLC cells, miR-100-5p is downregulated, and mTOR is upregulated (Qin et al., 2017). In docetaxel-resistant LUAD cells, miR-100-5p inhibits PLK1 to modulate resistance (Feng, Wang & Chen, 2012); this also in NSCLC, miR-100-5p targets PLK1 to attenuate growth, arrest the G2/M phase, and enhance apoptosis (Liu et al., 2012). miR-100-5p is upregulated in TNF-related apoptosis-inducing ligand (TRAIL) NSCLC cells. In conjunction with miR-21 and miR-30c, miR-100-5p further strengthens NF-κB signaling, establishing a positive-feedback loop that leads to TRAIL resistance and EMT, thus promoting cell survival after TRAIL treatment (Jeon et al., 2015). miR-100-5p confers resistance to ALK tyrosine kinase inhibitors in EML4-ALK NSCLC cells (Lai et al., 2019). However, many drug resistance mechanisms of miR-100-5p have not yet been examined, and further research is needed.

Breast cancer

Due to its high heterogeneity, the diagnosis of BC remains challenging. MiR-99a-5p induces apoptosis, inhibits cell migration and invasion, and reduces sphere formation by targeting mTOR and FGFR3 (Hu, Zhu & Tang, 2014; Long et al., 2019; Yang et al., 2014). Moreover, it reduces the ability of the ATP-binding cassette subfamily G member 2 (ABCG2) to perform doxorubicin efflux and elevates the sensitivity to doxorubicin by targeting COX-2 (Qin & Liu, 2019). In BC, miR-99a-3p and miR-99a-5p co-target FAM64A, affecting cell migration and invasion (Shinden et al., 2021). Both miR-99a-5p and miR-100-5p are induced after irradiation, further validating that miR-99a-5p reduces SNF2H expression and prevents BRCA1 recruitment to sites of DNA damage (Mueller, Sun & Dutta, 2013). BRCA1 reactivation induces the transcription of miR-99a and miR-99b, which then target TRAF2, a key regulator of the NF-κB and MAPK pathways (Tanic et al., 2012). In contrast, based on the genomic profiling of BC tissues, miR-99b also functions as an oncomiR in BC and is associated with high homologous recombination deficiencies and intra-tumor heterogeneity (Oshi et al., 2022). miR-99b-3p is highly expressed in paclitaxel-resistant BC cells and induces cell migration and paclitaxel resistance by targeting PPP2CA directly or promoting M2 polarization of macrophages (Mao et al., 2024).

miR-100 is commonly downregulated in BC owing to the hypermethylation of its host gene, MIR100HG. In BC, miR-100-5p represses proliferation, migration, and invasion by targeting FOXA1 and FZD8, abolishes trastuzumab resistance and CSC-like properties by targeting DLG5, inhibits proliferation and induces apoptosis by targeting IGF2, affects EMT and suppresses tumorigenesis, cell motility, and invasiveness by targeting SMARCA5 and HOXA1 (Bai et al., 2022; Chen et al., 2014; Gebeshuber & Martinez, 2013; Jiang et al., 2016; Xie et al., 2021).

The expression of miR-100 is related to estrogen and progesterone receptor positivity. Ectopic expression of miR-100 promotes luminal differentiation and renders basal-like BC stem cells responsive to hormonal therapy (Mattie et al., 2006). In the luminal-A BC population receiving adjuvant endocrine therapy, miR-100 levels were positively correlated with better overall survival (OS). Further molecular analysis revealed that miR-100 expression was inversely correlated with the expression of various genes, including PLK1, FOXA1, mTOR, and IGF1R, which are involved in resistance to hormonal therapy (Pidíkova, Reis & Herichova, 2020).

Esophageal cancer

ESCC is the predominant subtype of esophageal cancer, accounting for approximately 90% of all esophageal cancer cases worldwide. MiR-99a-5p blocks CSC persistence and sensitizes ESCC cells to radiotherapy by first targeting TRIB2 and blocking HDAC2 activation via the mTOR signaling pathway (Liu et al., 2021b; Sun et al., 2013). Similar to most cancer types, IGF1R is targeted by miR-99a-5p and miR-100-5p in ESCC, resulting in the suppression of tumor cell proliferation, migration, invasion, and SLUG-induced EMT (Chen et al., 2023a; Mei et al., 2017). miR-100-5p prevents cell migration and invasion by targeting mTOR and CXCR7 (Shi et al., 2019; Sun et al., 2013; Zhang et al., 2014a; Zhou et al., 2016b). miR-99b-5p represses ARID3A (a target of miR-125a and let-7e) to inhibit cell invasion and migration (Ma et al., 2017).

NGS-based profiling has revealed that miR-99a is upregulated in cisplatin-resistant esophageal cancer cells (Pandey et al., 2023). In patients with esophageal adenocarcinoma, high miR-100-3p expression predicts poorer survival, and patients who fail to achieve a pathological complete response have higher miR-99b expression (Feber et al., 2011; Skinner et al., 2014). Thus, the role of miR-99 family members in esophageal cancer may be subtype-dependent.

Gastric cancer

miR-99 is dysregulated in gastric cancer (GC). miR-99a-3p acts as a TS miRNA in GC, inducing apoptosis by targeting BMI1 and preventing proliferation by targeting MMP8 (Jiang et al., 2022b; Liu et al., 2018). miR-99b-3p and -5p induce cell cycle arrest in the S phase by targeting HoxD3 and IGF1R, respectively (Chang et al., 2019; Wang et al., 2018b). miR-100-3p represses tumor growth by targeting BMPR2, whereas miR-100-5p inhibits cell growth by targeting CXCR7, represses invasion and metastasis by targeting ZBTB7A (Shi et al., 2015), and activates the autophagic pathway by targeting mTOR (Cao et al., 2018; Chen et al., 2018b; Peng et al., 2019).

One of the leading causes of GC is Helicobacter pylori infection, which is associated with the expression of the miR-99 family. In cancer tissues, miR-99b-3p and -5p levels are higher when H. pylori is present (Chang et al., 2015). miR-99b-5p induces GC cell death via autophagy by targeting mTOR and eliminating intracellular H. pylori (Yang, Li & Jia, 2018).

Although miR-99 members counteract GC, they also promote its progression. In GC with poor prognosis, miR-99a-3p is overexpressed and promotes cell proliferation, migration, invasiveness, and EMT by targeting TRIM21 (He et al., 2024). Similarly, in cisplatin-resistant GC cells, miR-99a-5p is upregulated, whereas its target gene calpain small subunit 1 (CAPNS1) is downregulated. Silencing miR-99a-5p activates the catalytic subunits of CAPNS1 (calpain1 and calpain2), leading to GC cell apoptosis (Zhang et al., 2016). In cisplatin-resistant GC cells, high expression of miR-99a 5p silences MTMR3, which fails to repress autophagy, thus maintaining resistance (Sun et al., 2020). Yang et al. (2015, 2017) demonstrated the oncogenic effects of miR-100-5p. They targeted HS3ST2 to inhibit the Notch signaling pathway and apoptosis, attenuating cisplatin sensitivity. Upregulation of miR-100-5p is associated with primary tumorigenesis and progression of GC; miR-100-5p targets RNF144B, an E3 ubiquitin ligase. RNF144B interacts with pirh2, another p53 E3 ubiquitin ligase, to accelerate ubiquitin-mediated p53 degradation (Yang et al., 2017, 2015). This finding suggests that miR-100-5p is associated with genomic instability (Xu et al., 2023).

Colorectal cancer

The miR-99 family plays an important role in intestinal cancer. miR-99a-5p and miR-99b-5p impair the proliferation, invasion, and migration of intestinal cancer cells by targeting mTOR and FGFR3 (Ning et al., 2023; Zhu et al., 2019). In CRC cells, miR-100-5p targets mTOR, contributing to their proliferation, migration, and invasion of CRC cells (Fujino et al., 2017; Jahangiri et al., 2022). miR-100-5p has been identified to target Lgr5 and RAP1B (Peng et al., 2014; Zhou et al., 2015). miR-100-5p, miR-125b, and their host, MIR100HG, are overexpressed in cetuximab-resistant CRC and HNSCC cells (Liu et al., 2022a). Lu et al. (2017) further examined the potential mechanism, reporting that miR-100 and miR-125b coordinately repress five Wnt/β-catenin negative regulators, resulting in increased Wnt signaling. In contrast, Wnt inhibition restored cetuximab responsiveness in cetuximab-resistant cells. Interestingly, miR-99a-5p was associated with blood sugar levels. Advanced glycation end-products (AGEs) effectively reduce miR-99a-5p levels in vitro. In CRC tissue, the expression of miR-99a-5p was lower in patients with diabetes mellitus (DM) than in those without DM (Zhu et al., 2019). It has been found that insulin downregulates miR-99a-5p (Li et al., 2013b). Therefore, it is worth examining the risk of using insulin for glycemic control in patients with CRC and DM.

Hepatocellular carcinoma

Dysregulation of the miR-99 family has been linked to hepatocellular malignancies. The expression of miR-99a-5p in cancerous liver tissues was lower than in normal tissues. miR-99a-5p suppresses the invasion and migration of HCC cells by targeting HOXA1 (Tao et al., 2019). Furthermore, miR-99a-5p induces G1/S arrest and suppresses cell proliferation by targeting IGF1R and mTOR (Li et al., 2011). Specifically, the activation of mTOR induces PKM2 and HIF-1α expression, subsequently promoting glucose consumption and lactate production and thus promoting glycolysis (Li et al., 2013b). miR-100-5p exhibits effects similar to those of miR-99a-5p and functions as a TS miRNA in HCC cells. The dysregulation of miR-100 and PLK1 is closely associated with carcinogenesis, and miR-100-5p targets PLK1 to reduce HCC growth and enhance apoptosis (Chen, Zhao & Ma, 2013; Petrelli et al., 2012). miR-100-5p targets IGF2 to repress the AKT/mTOR pathway, thereby abolishing the maintenance of CSC properties and attenuating invasive and proliferative abilities by targeting CXCR7 (Ge et al., 2021; Seol et al., 2020). Angiopoietin 2 (Angpt2), essential for forming vessels encapsulating tumor clusters (VETCs), facilitates the entry of the entire tumor cluster into the bloodstream in an invasion-independent manner. miR-100-5p reduces the protein levels of Angpt2 by blocking the mTOR-p70S6K pathway, thereby decreasing VETC formation and metastasis (Zhou et al., 2016a). miR-100-3p reduces mTOR levels, thereby triggering autophagy by targeting SNRPD1 (Wang et al., 2022b). Both miR-99a and miR-100 target mTOR, suggesting that mTOR-autophagy signaling is a core pathway targeted by the miR-99 family.

Although miR-99a and miR-100 exhibit suppressive functions in HCC, miR-99b is oncogenic (Yang et al., 2015; Yao et al., 2019). miR-99b-3p induces proliferation, invasion, and migration by targeting PCDH19, whereas miR-99b-5p and miR-100-5p promote invasion and migration by targeting CLDN11 (Wang et al., 2023; Yang et al., 2015; Yao et al., 2019).

Pancreatic adenocarcinoma

There are multifaceted findings regarding the role of miR-100-5p in PDAC; some studies have reported that miR-100-5p is downregulated in cancerous tissues, whereas others have noted the opposite (Dobre et al., 2021; Panarelli et al., 2012). Contrary to the findings for miR-99a-5p in CRC, the expression of miR-100-5p is higher in patients with PDAC and DM than in those without DM, and the expression of miR-100-5p may be associated with high HbA1c (Hara et al., 2023). The levels of miR-100-5p and E-cadherin are negatively correlated, implying that miR-100-5p reduces overall survival by inducing robust EMT and motility (Hara et al., 2023; Ottaviani et al., 2018).

miR-99a, miR-100, and miR-125b are upregulated in gemcitabine-resistant PDAC cells (Dhayat et al., 2015). The impairment of miR-100 or miR-125b activity can reduce CSC marker expression and sensitize cells to gemcitabine treatment (Ottaviani et al., 2018). Similarly, the miR-99b/let-7e/miR-125a cluster inhibited cell proliferation, invasion, and metastasis by targeting NR6A1 (Li et al., 2024). In PDAC, miR-99b-5p induces radiation resistance by targeting mTOR (Wei et al., 2013).

Urological cancer

The role of the miR-99 family in genitourinary cancers depends on the tumor subtype and progression stage. In clear cell renal cell carcinoma (ccRCC), miR-99a is overexpressed (Oliveira et al., 2017); however, its expression is reduced in bladder cancer (BCa) and prostate cancer (PCa) (Borkowska et al., 2023; Sun et al., 2011). miR-100-5p, which represses migration, invasion, EMT, and stemness by targeting AGO2, is under expressed in PCa, whereas it promotes migration and prevents apoptosis in RCC (Chen et al., 2017b; Wang et al., 2014b). Interestingly, although miR-100 expression decreased during the transition from localized to metastatic PCa, biochemical recurrence was associated with high levels of miR-100. This discrepancy suggests that miR-100 is a context-dependent miRNA, sometimes acting as an oncomiR and sometimes as a TS miRNA (Leite et al., 2011). While miR-100 is expressed as a biomarker in all representative lesions during carcinogenesis in PCa, it exhibits progressive downregulation during the progression from precancerous to advanced metastatic cancer (Leite et al., 2013). In patients with bladder urothelial carcinoma, miR-100-5p shows heterogeneous expression and its role depends on the cancer stage. Dip et al. (2012) suggested that under expression of miR-100 in low-grade pTa specimens may increase FGFR3 levels, thus facilitating mutations by increasing cell turnover and the selection of mutant cells. In contrast, in invasive tumors, miR-100 overexpression may induce THAP-2 silencing and lead to the dysregulation of cell proliferation (Dip et al., 2012).

miR-99a-5p suppresses growth, migration, and invasion by targeting mTOR in RCC and bladder cancer and FGFR3 in PCa, and inhibits tumor growth by targeting IGF1R (Cui et al., 2012; Liu et al., 2019; Sun et al., 2014; Wu et al., 2015). miR-100-5p and miR-99b-5p exhibit similar antitumor effects by targeting mTOR, NOX4, HS3ST3B1, and IGF1R (Jiang et al., 2022a; Li et al., 2019; Liu et al., 2022c; Xu et al., 2013; Ye, Li & Wang, 2020).

In PCa, an androgen analog has been found to repress miR-99a-5p and miR-100-5p expression, thereby partly reducing the tumor-suppressive effects of the miR-99 family; furthermore, the miR-99 family inhibits androgen-receptor activity by targeting SMARCA5, SMARCD1, and mTOR, thus reducing prostate-specific antigen levels. However, inhibiting androgen-independent cell growth by the miR-99 family requires the presence of an androgen receptor (Sun et al., 2011). miR-99b-5p inhibits AR-mediated mTOR translocation and reduces mTOR expression, enhancing docetaxel-induced cytotoxicity (Gujrati et al., 2022). Likewise, miR-99a-5p inhibits the expression and nuclear translocation of mTOR, SMARCD1, and AR, and miR-99a-5p participates in metabolic reprogramming by recruiting the AR/mTOR complex to its target genes, thereby inhibiting EMT-mediated metastasis and elevating the cytotoxicity of enzalutamide (Enz) and abiraterone acetate (Waseem, Gujrati & Wang, 2023; Waseem & Wang, 2024). Androgen deprivation induces the expression of miR-100-5p, which is necessary for the survival and proliferation of PCa cells in a hormone-independent manner (Nabavi et al., 2017).

The expression of miR-99a-5p in gemcitabine-resistant BCa cells is lower than that in parental BCa cells, and its ectopic expression induces cellular senescence by targeting SMARCD1, thereby restoring sensitivity to gemcitabine (Tamai et al., 2022). In sunitinib-resistant ccRCC cells, miR-99a-3p is similar in targeting RRM2 to repress proliferation and induce apoptosis (Osako et al., 2019).

Gynecological cancers

Cervical, ovarian, and endometrial cancers are common gynecological tumors that impose a significant burden on women. In endometrial cancer, miR-99a-5p induces apoptosis and represses cancer cell proliferation and invasion via dual suppression of AKT and mTOR (Li et al., 2016). In cervical cancer, miR-99a-5p enhances apoptosis, represses glycolysis by targeting RRAGD, suppresses migration and invasion, and promotes apoptosis by targeting CDC25A (Gu & Bao, 2022; Wang et al., 2022a). In epithelial ovarian cancer (EOC), miR-99a-5p inhibits proliferation by targeting FGFR3 (Jiang et al., 2014). In cervical cancer, miR-99a-5p and miR-99b-5p suppress cell proliferation and invasion by targeting mTOR (Wang et al., 2014a). miR-99b-3p inhibits ovarian cancer cell viability by targeting SRPK1 (Xu et al., 2022).

Low miR-100-5p expression in cervical cancer is associated with unfavorable clinical outcomes (Yao et al., 2022). miR-100-5p inhibits proliferation by targeting PLK1 and represses migration and invasion by targeting SATB1 and mTOR (Huang et al., 2020; Peng et al., 2012; Yao et al., 2022). Consistently, in cisplatin-resistant EOC cells, miR-100-5p is downregulated, and re-expression of miR-100-5p reduces mTOR and PLK1 protein levels and induces apoptosis and cell cycle arrest in the G1 phase (Guo et al., 2016). In contrast, miR-100-5p expression is elevated in ovarian cancer, which promotes cell migration and invasion by targeting EPDR1 (Liu et al., 2021a).

EOC-derived exosomes increased fibronectin and vitronectin expression by transporting miR-99a-5p in human peritoneal mesothelial cells. Treating EOC cells with HPMCs promoted their invasiveness, suggesting that cancer cell-derived exosomal miRNAs modulate the tumor microenvironment (TME) to provide feedback to the cancer phenotype (Yoshimura et al., 2018).

Neurological cancers

miR-99 family members are essential for reducing the malignant phenotype of glioblastoma (GBM). Overexpression of miR-99a inhibits FGFR3 and PI3K/AKT signaling, thereby augmenting the repression of proliferation and induction of apoptosis via photofrin-based photodynamic therapy (Chakrabarti, Banik & Ray, 2013). The tumorigenic FGFR3–TACC3 fusion has been consistently detected in GBM. This fusion promotes cell proliferation and tumor progression by allowing GBM cells to escape recognition by miR-99a-5p (Parker et al., 2013). Studies examining the anti-GBM mechanism of miR-100 revealed that the miR-100-5p guide strand represses STAT3 by targeting SMARCA5 and SMRT, whereas miR-100-3p reduces AKT and ERK phosphorylation by targeting ErbB3 (Alrfaei et al., 2020; Alrfaei, Vemuganti & Kuo, 2013). miR-100-5p sensitizes GBM cells to ionizing radiation by targeting ATM, repressing their growth and migration, and enhancing their chemosensitivity by targeting FGFR3 (Luan et al., 2015; Ng et al., 2010). Neuroblastoma (NB) is a malignant tumor derived from immature neuronal cells of the sympathetic nervous system that is frequently reported in children. In NB, miR-99b-5p acts as a chemosensitizing miRNA and enhances DOX cytotoxicity by targeting PHOX2B (Holliday et al., 2022).

Hematological malignancies

In leukemia, regulating the miR-99 family and its clusters is complex and specific to the leukemia type. miR-99a, miR-100, and let-7 negatively regulate pro-proliferative genes, partially counteracting the hyperproliferation of hematopoietic stem cells induced by miR-125b, thereby conferring a steady-state growth advantage and preventing the exhaustion of miR-125b-transduced hematopoietic stem cells (Emmrich et al., 2014a). In AML, miR-100-5p inhibits apoptosis by targeting ATM, arrests the differentiation of human granulocytes and monocytes, and promotes cell survival by targeting RBSP3 (Sun et al., 2020; Zheng et al., 2012). miR-99a-5p inhibits differentiation of hematopoietic and AML stem cells, promoting self-renewal by targeting HOXA1. It promotes proliferation and inhibits apoptosis in AML and chronic myeloid leukemia (CML) cells by targeting CTDSPL and TRIB2 (Khalaj et al., 2017; Zhang et al., 2013). Contrary to their role as oncomiRs in AML, miR-99a-5p, and miR-100-5p were downregulated in childhood ALL tissues, especially in high-risk groups. Furthermore, they activate glucocorticoid receptors to increase dexamethasone sensitivity and suppress the IGF1R/mTOR pathway to induce apoptosis by targeting FKBP51, IGF1R, and mTOR (Li et al., 2013a). In AML, miR-99b and miR-125a induce proliferation and maintain LSC function (Uebbing et al., 2021).

In diffuse large B-cell lymphoma, miR-100-5p functions as a TS miRNA and suppresses the proliferation, migration, and invasion of cancer cells (Shu et al., 2022). Multiple myeloma (MM), the most prevalent malignant plasma cell disease, is characterized by the abnormal proliferation of bone marrow plasma cells. Wei et al. (2023) detected the upregulation of miR-100-5p in MM tissues and found that the inhibition of miR-100-5p induced targets, such as CLDN11, ICMT, MTMR3, RASGRP3, and SMARCA5, thus reducing metastasis and inducing apoptosis.

Other cancers

In thyroid cancer tissues, especially in the advanced stage, miR-100-5p r suppresses proliferation, induces apoptosis, and inactivates Wnt/β-catenin signaling by targeting FZD8 and RBSP3; moreover, it is under expressed in these cancers (Ma & Han, 2022; Zhang et al., 2014a). By targeting GRP94, miR-99a-3p disrupts anoikis resistance and inhibits the cytoplasmic relocation of ITGA2, thereby suppressing EMT (Gao et al., 2021). In cholangiocarcinoma in vivo, the miR-99a/let-7c/miR-125b cluster reduces STAT3 activity and further suppresses migration and invasiveness, as well as suppressing CSC-like mammosphere generation and tumorigenicity by targeting IGF1R and IL-6, respectively (Lin et al., 2016).

In melanoma, miR-99b-3p targets CYLD, thereby preventing RIPK1 polyubiquitination and inducing apoptosis (La et al., 2020). The levels of miR-99a/-100 are significantly higher in nevi than in malignant lesions and are negatively associated with IGF1R expression; restoration of miR-99a/-100 reduces IGF1R expression and melanoma cell proliferation (Damsky et al., 2015). Notably, IGF1R levels were lower in PTEN-silenced cells than in CDKN2A-silenced cells, indicating that IGF1R depletion by miR-100 was greater with PTEN silencing than with CDKN2A silencing, which is consistent with other findings (Majewska et al., 2022).

In osteosarcoma, miR-99a-5p induces cell death and cell cycle arrest by targeting TNFAIP8 (Xing & Ren, 2016). Chordoma is a malignant mesenchymal tissue bone tumor in which miR-100-5p represses proliferation and EMT and induces apoptosis by targeting IGF1R (Zhang et al., 2020). In epithelial cells, miR-100-5p targets HOXA1, thus reducing BCL-2 expression and inducing apoptosis. Similarly, in cutaneous squamous cell carcinoma cells, inhibition of miR-100 enhances radiation resistance (Fahim Golestaneh et al., 2019).

In invasive pituitary adenomas, miR-99a-3p is under expressed, and its expression is negatively correlated with invasiveness. The ectopic expression of miR-99a-3p inhibits cell growth, metastasis, and tube formation in endothelial cells by targeting NOVA1, DTL, and RAB27B (Zhao et al., 2021). miR-99a-5p and miR-100-5p are downregulated in childhood adrenocortical tumors and repress the proliferation of both adrenocortical tumors and pediatric adrenocortical carcinoma cells by targeting mTOR, RAPTOR, and IGF1R (Doghman et al., 2010).

Roles in diagnosis and progression

miRNAs have been reported to play crucial roles in tumorigenesis and development, and their expression is associated with clinical outcomes. Their roles as potential diagnostic and prognostic biomarkers in various cancers have also been examined. The expression of miR-99 varies between cancerous and normal tissues and sera. Consequently, miR-99 members have been verified as biomarkers for cancer diagnosis, individually and as components of miRNA signatures (Holubekova et al., 2020; Kaba et al., 2023). Interestingly, tissues and serum often exhibit contrasting miRNA expression patterns. The expression of miR-99a-5p was significantly lower in BC tissues than in healthy tissues, while the opposite pattern was observed in the plasma of these patients (Garrido-Cano et al., 2020). Receiver operating characteristic (ROC) curve analysis revealed that miR-99a-5p has good diagnostic potential, even for detecting early BC, suggesting that circulating miR-99a-5p is a novel and promising non-invasive biomarker for BC detection. However, miRNA signatures comprising several miRNAs appear more accurate than those comprising individual miRNAs. For example, miRNA signatures can be used to classify endometrial cancer tissue (miR-99a/-100/-199b) and plasma (miR-99a/-199b) samples with a higher accuracy than single miRNAs (Torres et al., 2012). In addition to being measured in plasma, miRNAs can be measured in other body fluids, such as urine, for urological cancer diagnosis (Liu et al., 2024; Pospisilova et al., 2016). Salido-Guadarrama et al. (2016) identified a miR‑100/200b signature that discriminates between patients with PCa and benign hyperplasia, achieving greater accuracy than prostate-specific antigen.

The expression of miR-100 is lower in ESCC tissues than in non-cancerous esophageal tissues, and its dysregulation is associated with an advanced clinical stage, distant metastasis, increased depth of invasion, and poor survival probability, suggesting that miR-100 could serve as a biomarker for ESCC prognosis (Zhou et al., 2014). In GC, miR-100 expression increases with the progression stage, making it a marker of tumor advancement (Ueda et al., 2010). Similarly, miR-100-5p is a marker of tumorigenic progression in cervical cancer, and its expression decreases with progression from low-grade cervical intraepithelial neoplasia (CIN) to high-grade CIN and then to cancer (Li et al., 2011). In patients with OSCC, serum miR-99a-5p levels are higher after tumor resection than before (Chen et al., 2018a). Conversely, in patients with ESCC, serum miR-100 levels are lower after surgery, suggesting that miR-99 levels reflect tumor burden and serve as an auxiliary indicator of surgical effectiveness (Wu et al., 2014). Recurrence, another important indicator of cancer progression, has been reported to be associated with the miR-99 family. MiR-99a is highly expressed in pediatric AML and CML at the time of diagnosis and relapse; however, its expression is significantly reduced during complete remission (Zhang et al., 2013).

RNA editing of miR-99a/-99b occurs more frequently in cancers (most cancers) than in normal tissues and is correlated with survival probability (Pinto et al., 2018). For example, patients with LUAD with a loss of A-to-I miR-99a-5p editing exhibit reduced overall survival (Maemura et al., 2018). miR-99a editing is associated with different molecular drivers and signaling pathways in different cancers, such as the TP53 pathway in BC and HNSCC and the HRAS and NRAS pathways in thyroid carcinoma (Wang et al., 2017).

Overall, the miR-99 members exhibit substantial potential as diagnostic and prognostic biomarkers. They can be used to distinguish between cancerous and non-cancerous tissues, sera, and other body fluids of patients with malignant tumors and the normal population. In particular, circulating miR-99 members are attractive markers for early non-invasive cancer detection and can be easily analyzed in large batches of clinical samples.

Roles in predicting therapeutic responses

miR-99 expression varies dynamically during anticancer therapy, and its dysregulation may reverse treatment efficiency. Therefore, its expression is potentially helpful in predicting the treatment response. MiR-99b-5p is downregulated in imatinib-resistant CML patients. Among patients with high-risk myelodysplastic syndromes or AML with myelodysplasia-related changes, miR-100-5p levels are higher in azacitidine-responsive patients than in non-responders (Krejcik et al., 2018, Yap et al., 2017). In patients with BC, serum miR-100-5p expression was significantly lower in those who responded to initial dovitinib treatment than in those with treatment-resistant metastatic BC. This helps clinicians decide whether to continue planned treatment (Shivapurkar et al., 2017).

MiRNA signatures display impressive response-predictive ability. For example, a signature comprising circulating miR-100, miR-92a, miR-16, miR-30e, miR-144-5p, and let-7i can distinguish patients with oxaliplatin-based chemo resistant CRC from those with chemo sensitive CRC (Han et al., 2020b). The circulating miR-21/-99b/-375 panel is an effective indicator of the preoperative chemoradiotherapy response in locally advanced rectal cancer (Campayo et al., 2018). In esophageal adenocarcinoma, miR-99b and three other miRNAs form a signature that predicts a pathological complete response to neoadjuvant chemoradiation, suggesting the potential of miR-99 members as indicators of neoadjuvant chemotherapy efficacy (Skinner et al., 2014). Among patients with melanoma, higher miR-100-5p expression predicts greater clinical benefit from PD-1-inhibitor treatment, whereas the opposite has been reported for miR-100 in myeloid-derived suppressor cells (Huber et al., 2018; Sloane et al., 2021). In BCa tissues, miR-100-5p expression is inversely correlated with that of PD-L1 and PD-L2 (El Ahanidi et al., 2021). Although miR-100 plays a role in immunotherapy, this requires further validation in future studies.

In summary, the association between miR-99 expression and treatment response can be used to predict resistance. Quantifying miR-99 levels in patients with malignant lesions provides a sensitive, effective, and timely method for detecting resistance, guiding the selection of chemotherapy regimens, and improving prognosis.

Clinical translation potential of the miR-99 family

As our understanding of miRNAs in cancer has improved, they have emerged as attractive tools and targets for novel therapeutic approaches. Two key strategies for miRNA-based cancer therapy are: (1) administration of synthetically derived miRNA mimics to restore the activity of mutated or deleted TS miRNAs or (2) inhibition of endogenous oncomiRs. Considering their simple structure and ease of synthesis, miRNAs exhibit strong competitiveness as treatment options, potentially bypassing expensive medicinal chemistry research.

Many studies have suggested the potential therapeutic use of miR-99 family members, the reintroduction of their synthetic mimics, or the use of their antisense sequences. miRNAs are unable to cross cell surface membranes directly. Extracellular vesicles, particularly exosomes, provide a key pathway for the delivery of extracellular miRNAs into recipient cells, where they alter their genetic profile (Garcia-Martin et al., 2022). Connections between lung cancer cell membranes and mast cells induce the release of extracellular vesicles enriched with miR-100-5p and miR-125b, resulting in accelerated lung cancer cell proliferation (Shemesh et al., 2023). Exosomal miR‑100‑5p from highly invasive HCC cells promotes the migration and invasion of low-invasive HCC cells, further confirming the role of exosomes in miRNA transport (Wang et al., 2023). In contrast, MSC-derived exosomal miR-100 efficiently suppressed CRC cell proliferation and induced apoptosis by reducing the expression of mTOR, cyclin D1, KRAS, and HK2 (Jahangiri et al., 2022).

Synthetically derived miRNAs are rapidly degraded by the plasma. Delivery systems that enhance in vivo delivery and minimize miRNA degradation during systemic circulation have been proposed for the clinical translation of miRNAs. As an effective theranostic antitumor approach, nanomaterials provide an efficient platform for loading miRNAs. Sun et al. (2017) designed and synthesized a nanovector for miR-100 delivery that predominantly targeted and suppressed FGFR3, thereby significantly inhibiting the growth of FGFR3-amplified patient-derived xenografts. Holliday et al. (2022) constructed nanoparticle complex-modified miR-99b-5p mimics with MYCN amplification that increased the susceptibility of neuroblastoma patient-derived xenografts with MYCN amplification. Nanoparticles that deliver miR-99a-5p along with doxorubicin or anti-vascular endothelial growth factor (VEGF) antibody to tumor cells have been successfully developed, and treatment with nanoparticle-loaded combinations was more effective and less toxic than treatment with free doxorubicin or VEGF antibody alone (Cai et al., 2017; Garrido‐Cano et al., 2022). The intranasal nanoparticle-mediated co-delivery of miR-100 and antisense (anti)-miR-21 bypasses the blood-brain barrier and potentiates the effects of systemic temozolomide treatment in GBM (Sukumar et al., 2019). In mice, intranasally delivered nanoparticles carrying tumor-suppressive genes (thymidine kinase, p53, and nitroreductase) along with therapeutic miRNAs (anti-miR-21, anti-miR-10b, and miR-100) predominantly accumulated in the lungs, thus reducing triple-negative BC–lung metastases (Liu et al., 2022b).

In addition to functioning directly in tumor cells, miRNAs can also affect the TME, potentially regulating cancer progression and providing another strategy for cancer treatment. Tumor-associated macrophages (TAMs), the primary immune components of the TME, mediate various tumor-promoting mechanisms such as angiogenesis stimulation, tumor migration enhancement, and antitumor immunity suppression. In BC, high miR-100 expression maintains the TAM phenotype by targeting mTOR and increasing IL-1ra secretion via stat5a-mediated transcriptional regulation, thus enhancing metastasis, stemness, chemoresistance, and features of malignancy (Wang et al., 2018a). In contrast, miR-99b induces the conversion of TAMs into an antitumor phenotype with enhanced immune surveillance. When conjugated to a nucleic acid drug delivery system and then delivered into TAMs, miR-99b promotes M1 macrophage polarization, thus enhancing phagocytosis and antigen presentation by targeting κB-Ras2 or mTOR. Furthermore, it suppresses M2 macrophage polarization by repressing the mTOR/IRF4 axis, suppressing tumor growth in HCC and Lewis lung cancer. Amplification of the M1-like effect by miR-99b overexpression in TAMs causes tumor regression by reprogramming the antitumor immune microenvironment (Wang et al., 2020).