MiR-5100 promotes the malignant phenotype of triple-negative breast cancer cells by targeting SMAD4 and predicts poor prognosis

Abstract

Background

Triple-negative breast cancer (TNBC) is a notoriously aggressive subtype with poor prognosis. MicroRNAs (miRNAs) critically regulate epithelial-mesenchymal transition (EMT), a central driver of cancer metastasis. Nevertheless, the function of hsa-miR-5100 in TNBC was poorly understood. This study investigated the effects of miR-5100 on the malignant behaviors of TNBC cells, its correlation with patient prognosis, and underlying molecular mechanisms.

Methods

Plasma levels of miR-5100 were assessed in a cohort of 80 TNBC patients and 80 healthy controls. Based on miR-5100 expression levels, patients were categorized into high and low groups, with 5-year survival as the primary endpoint for Kaplan-Meier and Cox regression analyses. MiR-5100 was assessed for its effects on proliferation, migration, invasion, and EMT in TNBC cell lines (MDA-MB-231 and MDA-MB-468), with its direct interaction with SMAD4 confirmed by dual-luciferase reporter assay.

Results

Upregulation of miR-5100 was observed in TNBC patients. Elevated miR-5100 levels correlated with advanced TNM stage, lymph node metastasis, high Ki-67 proliferation index, and reduced survival. Overexpression of miR-5100 promoted the proliferation, migration and invasion of TNBC cells. MiR-5100 overexpression mediated the reciprocal regulation of E-cadherin (down) and N-cadherin (up) at both mRNA and protein levels in TNBC cells. Mechanistically, the oncogenic effects of miR-5100 on TNBC cell proliferation, migration, invasion, and EMT were mediated through its direct targeting of SMAD4.

Conclusions

MiR-5100 was upregulated in TNBC patients and was associated with a poor prognosis. By targeting SMAD4, miR-5100 promoted the malignant phenotypes of TNBC cells in vitro, suggesting its potential research value as a prognostic biomarker and therapeutic target.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12957-026-04271-0.

Background

Globally, breast cancer (BC) constitutes nearly one-third of all female malignancies, causing roughly 15% of cancer mortality in this population [1]. Different subtypes of BC exhibit significant differences in clinical features and therapeutic strategies [2], among which triple-negative breast cancer (TNBC)—defined by the absence of estrogen receptor, progesterone receptor, and HER2 expression—is notably aggressive and heterogeneous. This subtype poses a severe challenge to clinical diagnosis and treatment due to its significant molecular heterogeneity and the absence of clear therapeutic targets [3]. Approximately 90% of deaths from BC (including TNBC) are caused by metastasis, and elucidating the molecular mechanisms of metastasis and developing corresponding therapeutic strategies have become key directions in current research [4, 5].

The epithelial-mesenchymal transition (EMT) is a fundamental cellular differentiation process wherein epithelial cells shed their adhesive properties and gain a motile, mesenchymal phenotype [6]. This conversion is critically implicated in facilitating cancer invasion and metastasis [7]. MicroRNAs (miRNAs) are known to regulate EMT and influence cancer development [8, 9]. For instance, long non-coding RNAs such as MALAT1 can promote EMT through specific miRNA-mediated axes, underscoring the complexity of the regulatory network [10]. MiR-138 acts as a tumor suppressor by targeting EZH2; its downregulation due to promoter hypermethylation promotes proliferation, metastasis, and EMT in cervical cancer [11]. MiR-199a-5p inhibits EMT and BC metastasis by negatively regulating the transcription factor SP1, thereby suppressing its binding and activation of the LOXL2 promoter [12]. MiR-34a and miR-34b/c modulate chemosensitivity in colorectal cancer cells through coordinated regulation of EMT and autophagy [13]. Notably, hsa-miR-5100 has been reported to promote EMT in pulmonary epithelial cells by targeting TOB2 and activating the Smad2/3 pathway, suggesting its potential role in fibrosis-associated EMT processes [14]. In lung cancer and melanoma, miR-5100 has been found to promote cancer cell metastasis by facilitating the EMT process [15, 16].

However, a comprehensive understanding of the function of miR-5100 in TNBC remains limited. Mechanistic studies have validated specific miRNA-target interactions, such as miR-584-5p targeting MSMO1 in BC, highlighting the importance of delineating precise regulatory pathways [17]. This study sought to examine the influence of miR-5100 on TNBC cell malignancy, including proliferation, migration, and invasion, and to analyze its correlation with clinical prognosis. Furthermore, the present study aimed to validate the underlying molecular mechanism involving its targeting of SMAD4. Our results provided preliminary evidence supporting the role of miR-5100, which could be explored in future studies on TNBC prognosis and treatment.

Methods

Study population

The peripheral blood was collected from 80 TNBC patients and 80 healthy controls. The control subjects were age- and sex-matched to the patient group. The sample size was calculated using GPower (3.1.9.7). Total sample size of 126 was sufficient to detect large effects, assuming an alpha error of 0.05 and a power of 0.8. Plasma miR-5100 levels were quantified, and patients were subsequently divided into low- and high-expression groups according to the median value. The 5-year overall survival, defined as time from diagnosis to death, was evaluated by retrospectively reviewing medical records and telephone interviews, and analyzed using Kaplan-Meier and Cox regression analyses. The median follow-up time was 36 months (range: 3–60 months).

Clinical parameters, including age, BMI, tumor size, TNM stage, lymph node status, and Ki-67 proliferation index were recorded. All TNBC patients in this cohort underwent surgical resection of the primary tumor. Adjuvant chemotherapy for TNBC was primarily divided into two categories based on the drugs used: the standard doxorubicin/cyclophosphamide followed by a taxane regimen (AC-T) and the intensified AC-T plus capecitabine regimen (AC-T + Cap). This study was performed in line with the principles of the Declaration of Helsinki. The protocol was approved by the Ethics Committee of Shanxi Cancer Hospital, with written informed consent acquired from all participants. The inclusion criteria for TNBC patients were histologically diagnosed as BC, with negative expressions of ER, PR and HER2. The exclusion criteria were non-primary BC or coexistence of other malignant tumors.

Plasma sample collection and RNA isolation

Plasma samples were retrospectively obtained from de-identified TNBC cases and healthy controls with ethical approval and informed consent. Following an overnight fast from 20:00, blood was collected by venipuncture between 08:00 and 09:00 into EDTA tubes (BD, 366643). To minimize miRNA degradation, plasma was separated via a standardized two-step centrifugation protocol: first at 2,000 × g for 15 min at 4 °C to remove cells, followed by centrifugation of the supernatant at 2,500 × g for 15 min at 4 °C to remove platelets and debris. The processed plasma was aliquoted within 30 min of collection and stored at -80 ℃ until analysis.

For miRNA detection, total RNA was isolated from 200 µL of plasma. To monitor isolation efficiency and ensure reproducibility, a known quantity of synthetic cel-miR-39 spike-in control was added to each sample prior to extraction using TRIzol™ LS Reagent (ThermoFisher, 10296028), followed by the miRNeasy Serum/Plasma Kit (Qiagen, 217204). Samples exhibiting outlier recovery rates of the spike-in control were re-extracted to maintain technical consistency. RNA concentration and purity were determined using a spectrophotometer. All RNA extractions and subsequent analyses were performed in three independent experimental replicates.

Reverse transcription and quantitative PCR (RT‑qPCR)

Total RNA was isolated from cell cultures with TRIzol™ Reagent (ThermoFisher, 15596-018, and Qiagen, 217184). The concentration and purity of RNA were determined using a nanoparticle spectrophotometer. Subsequently, using the reverse transcription kits (KeyGEN, KGF2205-200, and ThermoFisher, 4366597), cDNA was synthesized from 1 µg of total RNA. The expression of target mRNAs was quantified via SYBR Green-based qPCR (Solarbio, SR1110, and ThermoFisher, 4440040) using the 2^−∆∆Ct method. β-actin (for mRNA), U6 snRNA (for miRNA), and cel-miR-39 (for plasma miRNA) were used as internal reference genes. The primer sequences were shown in Supplementary Table 1.

Dataset collection

The miRNA expression profiles GSE144534 and GSE235355 were obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The GSE144534 dataset included 15 BC tissue samples and 3 reduction mammoplasty-derived normal breast tissue samples. The GSE235355 dataset contained serum samples from 5 BC patients and 2 healthy individuals.

Identification of differentially expressed miRNAs (DEMs) and downstream target genes in BC

DEMs were identified in GSE144534 and GSE235355 with a threshold of |log₂FC| > 1.0 and p < 0.05, resulting in the identification of a single overlapping DEM: miR-5100. Potential downstream target genes of miR-5100 were predicted using miRDB (https://mirdb.org/), miRWalk (http://mirwalk.umm.uni-heidelberg.de/), and TargetScan (https://www.targetscan.org/vert_80/), and intersected with genes related to BC disease predicted by GeneCards (https://www.genecards.org/), yielding 389 potential target genes.

Enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)

GO and KEGG enrichment analyses of the 389 candidate target genes were performed using the DAVID online database (https://david.ncifcrf.gov/) identified significantly enriched functions and pathways.

Cell culture and transfection

The TNBC cell lines MDA-MB-231 (ATCC, HTB-26) and MDA-MB-468 (ATCC, HTB-132) were cultured in high-glucose DMEM supplemented with 10% FBS, whereas the normal breast epithelial cell line MCF-10 A (ATCC, CRL-10317) was maintained in standard DMEM with 10% FBS. All cells were maintained at 37 °C in an incubator with 5% CO₂.

MiR-5100 mimic, miR-5100 inhibitor, and their corresponding negative controls (NC mimic and NC inhibitor) were constructed by MedChemExpress (USA). The final concentrations used for NC mimic, NC inhibitor, miR-5100 mimic, and miR-5100 inhibitor were 50 nM, 100 nM, 50 nM, and 100 nM, respectively. The SMAD4 overexpression plasmid (oe-SMAD4) and its negative control (oe-NC), supplied by RiboBio (Guangzhou, China), were transfected at a concentration of 2 µg per well into MDA-MB-231/468 cells using Lipofectamine™ 3000 (Invitrogen, L3000015). Subsequent assays were carried out 48 h after transfection.

Cell viability assay

Transfected MDA-MB-231/468 cells were plated in 96-well plates (3000 cells per well), and viability was monitored at 0, 24, 48, and 72 h with a CCK-8 assay kit (tsbiochem, C0005). Absorbance at 450 nm was measured following a 4-hour incubation at 37 °C in the dark after the addition of 10 µL CCK-8 reagent.

Transwell-based assays

MDA-MB-231/468 cells were placed in the upper chamber (1 × 10⁵ per well) of Transwell chambers (Corning, 3422) for migration and invasion assays. For the invasion assay, Matrigel was used to pre-coat the inserts. Serum-free medium was applied to the upper chamber, whereas the lower compartment was filled with medium supplemented with 10% FBS. Following a 48-hour incubation, transmigrated cells were fixed, stained, and quantified.

Western Blot (WB)

Cellular proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (Invitrogen, PB5310). After blocking, the membranes were incubated with primary antibodies against E-cadherin (CST, 3195T), N-cadherin (CST, 13116T), SMAD4 (CST, 46535T), and β-actin (CST, 4967 S), followed by HRP-conjugated secondary antibodies (ThermoFisher, 31460) for 2 h at room temperature. Protein bands were detected using an enhanced chemiluminescence substrate.

Dual-luciferase reporter assay

GenePharma generated both wild-type and mutant SMAD4 reporter constructs. Both reporter constructs were co-transfected with either NC mimic or miR-5100 mimic into MDA-MB-231/468 cells. Measured 48 h post-transfection, dual-luciferase activity was quantified using a commercial assay kit (Solarbio, D0010).

Statistical methods

Data are expressed as mean ± SD from a minimum of three independent replicates. GraphPad Prism 10.1.2 was used for statistical analysis and graph generation. Group differences were analyzed by Student’s t-test (two-group comparisons) or two-way ANOVA with Tukey’s multiple comparisons test (multi-group comparisons). Significance was defined as p < 0.05.

Results

Identification of miR-5100 as an upregulated and clinically significant miRNA in TNBC

By analyzing miRNA expression profiles from datasets GSE144534 and GSE235355 using the GEO2R tool, DEMs were discerned between BC patients and healthy controls (Fig. 1A-B). Venn diagram analysis further revealed one common DEM across both datasets: miR-5100 (Fig. 1C).

Fig. 1.

MiR-5100 was elevated in TNBC patients and correlates with an unfavorable prognosis. (A, B) Volcano plots displaying DEMs from the (A) GSE144534 and (B) GSE235355 datasets. DEMs were identified with |log₂FC| > 1.0 and p < 0.05. (C) Venn diagram showing the overlap of commonly DEMs between the two datasets. (D) Relative expression level of plasma miR-5100 in 80 TNBC patients and 80 healthy controls, measured by RT‑qPCR. (E) Kaplan-Meier survival curves for TNBC patients stratified by plasma miR-5100 expression (high vs. low). Survival differences were assessed by the log‑rank test. Statistical significance was determined by Student’s t-test (D). (p < 0.001***)

Plasma miR-5100 levels were quantified in peripheral blood samples from 80 TNBC patients and 80 matched healthy controls. MiR-5100 expression was markedly elevated in the plasma of TNBC patients relative to healthy controls (Fig. 1D). Clinical characteristics, including age, BMI, tumor size, adjuvant chemotherapy, and lymph node status, were documented for the 80 TNBC patients. We stratified the 80 patients into low and high miR-5100 expression groups, dichotomized by the median plasma level. Elevated plasma miR-5100 correlated with high Ki-67 proliferation index (p = 0.013), advanced TNM stage (p = 0.012), and lymph node metastasis (p = 0.025) (Table 1). In contrast, this miRNA showed no association with patient age, BMI, tumor size, or adjuvant chemotherapy as detailed in Table 1. Kaplan-Meier analysis revealed significantly improved overall survival in patients with low versus high miR-5100 expression (Fig. 1E). Cox regression further confirmed high miR-5100 expression (HR = 3.326, 95% CI: 1.060–10.438, p = 0.039), advanced TNM stage (HR = 2.777, 95% CI: 1.043–7.392, p = 0.041), and Lymph node status (HR = 2.654, 95% CI: 1.047–6.730, p = 0.040) as risk factors for poor prognosis (Table 2).

Table 1.

The correlation between serum hsa-miR-5100 and clinical characteristics

Characteristics	n	miR-5100 expression		p* value
		low	high
Age				0.500
< 50 years	36	16	20
≥ 50 years	44	24	20
BMI				0.822
< 24	44	23	21
≥ 24	36	17	19
Tumor size				0.066
< 5 cm	49	29	20
≥ 5 cm	31	11	20
Ki-67 proliferation index				0.013
< 30%	40	26	14
≥ 30%	40	14	26
TNM stage				0.012
Ⅰ + Ⅱ	46	29	17
Ⅲ +Ⅳ	34	11	23
Lymph node status				0.025
Negative	39	25	14
Positive	41	15	26
Adjuvant chemotherapy				0.593
AC-T	52	26	26
AC-T + Cap	28	14	14

BMI Body Mass Index, TNM Tumor, Node, Metastasis Staging System, AC-T standard doxorubicin/cyclophosphamide followed by a taxane regimen, AC-T + Cap the intensified AC-T plus capecitabine regimen, p*: Chi-square test

Table 2.

Cox regression analysis results

	Hazard Ratio	95% CI	p value
Age	1.098	0.431-2.800	0.844
BMI	1.191	0.440–3.221	0.731
Tumor size	1.735	0.709–4.244	0.227
Ki-67 proliferation index	2.130	0.664–6.837	0.204
TNM stage	2.777	1.043–7.392	0.041
Lymph node status	2.654	1.047–6.730	0.040
Adjuvant chemotherapy	1.159	0.443–3.027	0.764
miR-5100	3.326	1.060-10.438	0.039

BMI Body Mass Index, TNM Tumor, Node, Metastasis Staging System

MiR-5100 promoted malignant phenotypes in TNBC cells

To investigate the functional role of miR-5100 in TNBC cells, miR-5100 basal expression levels were examined across several cell lines. Compared with MCF-10 A, miR-5100 was significantly up-regulated in MDA-MB-231/468 cells, with higher expression observed in MDA-MB-231 cells (Fig. 2A), suggesting a potentially important role of miR-5100 in TNBC.

Fig. 2.

MiR-5100 promoted malignant phenotypes of TNBC cells. (A) Relative expression of miR-5100 in MCF‑10 A, MDA‑MB‑231, and MDA‑MB‑468 cells. (B) Relative expression levels of miR-5100 after transfection. (C, D) Cell proliferation assessed by CCK‑8 assay in MDA-MB-231(C) and MDA-MB-468 (D). (E, F) Cell migrated (E) and invasive (F) number. (G, H) Relative mRNA expression levels of E-cadherin (G) and N-cadherin (H) after miR-5100 modulation. (I-K) Western blot images (I) and corresponding quantitative results of the E‑cadherin (J) and N‑cadherin (K). Statistical significance was determined by one-way ANOVA (A) and two-way ANOVA (B-H, and J-K). Independent replicates: n = 4 for experiments in A-H; n = 3 for experiments in I-K. (ns: not significant, p < 0.05*, p < 0.01**, p < 0.001***; p < 0.01&&, p < 0.001&&&, for NC mimic versus miR-5100 mimic; p < 0.05#, p < 0.001###, for NC inhibitor versus miR-5100 inhibitor)

Transfection with miR-5100 mimic and inhibitor effectively up- and down-regulated miR-5100 expression, respectively, in MDA-MB-231/468 cells (Fig. 2B). Overexpression of miR-5100 significantly promoted cell proliferation. CCK-8 assays showed markedly enhanced proliferative activity at 48 h and 72 h after transfection (Fig. 2C-D). Conversely, inhibition of miR-5100 expression notably suppressed cell proliferation (Fig. 2C-D).

Overexpression of miR-5100 also enhanced cell migration and invasion capabilities after transfection for 48 h, whereas its inhibition significantly suppressed these malignant behaviors (Fig. 2E-F), indicating that miR-5100 is involved in regulating metastatic processes in TNBC cells.

To investigate EMT modulation, miR-5100 overexpression in MDA-MB-231/468 cells lowered E-cadherin and elevated N-cadherin levels (Fig. 2G-H). Inhibition of miR-5100 produced the opposite effects (Fig. 2G-H). The protein changes of E-cadherin and N-cadherin detected by WB were similar to the mRNA expression trends in MDA-MB-231/468 cells (Fig. 2I-K). These results suggest that miR-5100 may facilitate invasion and metastasis in TNBC by inducing EMT.

SMAD4 is a direct target of miR-5100 in TNBC

Bioinformatic prediction via miRDB, miRWalk, and TargetScan identified potential downstream targets of miR-5100 in TNBC. By integrating genes associated with BC from GeneCards, 389 potential miR-5100 targets implicated in BC progression were identified (Fig. 3A). Functional enrichment of these candidates was assessed through GO and KEGG analyses.

Fig. 3.

SMAD4 is a direct target of miR-5100. (A) Venn diagram of miR-5100 target genes (from miRDB, miRWalk, and TargetScan) and BC-associated genes from GeneCards. (B, C) GO Biological Process (B) and KEGG pathway (C) enrichment analysis of the 389 overlapping genes. (D) The putative binding sequence between miR-5100 and SMAD4. (E) Schematic diagram of the SMAD4 3’UTR reporter construct and the mutant sequence alignment. (F, G) The relative luciferase activity in MDA-MB-231 (F) and MDA-MB-468 (G) cells. (H) Relative mRNA expression levels of SMAD4 by RT-qPCR. (I, J) SMAD4 protein Western blot images (I) and corresponding quantitative results (J). Statistical significance was determined by two-way ANOVA (F-H, and J). Independent replicates: n = 4 for experiments in F-H; n = 3 for experiments in I-J. (ns: not significant, p < 0.05*, p < 0.01**, p < 0.001***)

GO analysis revealed significant involvement of the predicted targets in key biological processes (Fig. 3B), including intracellular signal transduction, negative regulation of protein catabolic processes, and positive regulation of DNA-templated transcription. Notably, SMAD4 was predicted to be involved in several of these functional modules, suggesting that miR-5100 may target SMAD4 to modulate broad cellular processes like signal transduction and transcriptional regulation.

KEGG pathway analysis further revealed enrichment in multiple oncogenic pathways (Fig. 3C), such as pathways in cancer, signaling pathways regulating pluripotency of stem cells, and hormone-related signaling pathways. SMAD4 was also identified as a key candidate molecule in these significantly enriched pathways. Particularly in “signaling pathways regulating pluripotency of stem cells” and “pathways in cancer”, SMAD4, as a core component of the TGF-β pathway, is closely associated with oncogenic mechanisms.

Accordingly, SMAD4 was prioritized as a principal candidate target of miR-5100 for subsequent functional verification. The potential binding site between miR-5100 and SMAD4 was predicted using Starbase (Fig. 3D). Dual-luciferase assays showed that miR-5100 mimic markedly inhibited wild-type SMAD4 reporter activity, while the mutant reporter remained unaffected, verifying direct miR-5100–SMAD4 binding in MDA-MB-231/468 cells (Fig. 3E-G). Moreover, overexpression of miR-5100 significantly down-regulated the expression of SMAD4 mRNA and protein (Fig. 3H-J).

MiR-5100 promoted malignant phenotypes in TNBC cells via SMAD4

First, SMAD4 expression was examined in MCF-10 A cells and TNBC cell lines. SMAD4 mRNA and protein expression was both markedly reduced in MDA-MB-231/468 cells relative to MCF-10 A controls (Fig. 4A-C). To determine whether miR-5100 mediates malignant phenotypes in TNBC by targeting SMAD4, rescue experiments were conducted in MDA-MB-231/468 cells transfected for 48 h with miR-5100 mimic alone or in combination with oe-SMAD4.

Fig. 4.

Overexpression of SMAD4 rescued the oncogenic effects induced by miR-5100. (A) Relative mRNA expression levels of SMAD4 in MCF‑10 A, MDA‑MB‑231, and MDA‑MB‑468 cells.(B-C) SMAD4 Western blot images (B) and corresponding quantitative results (C) in different cells. (D) Cell proliferation (CCK‑8) after transfection with miR-5100 mimic alone or together with oe‑SMAD4. (E, F) Cell migration (E) and invasion (F) by Transwell assays. (G, H) Relative mRNA expression levels of E-cadherin (G) and N-cadherin (H). (I-K) Western blot images (I) and corresponding quantitative results of the E‑cadherin (J) and N‑cadherin (K). Statistical significance was determined by one-way ANOVA (A, C) and two-way ANOVA (D-H, and J-K). Independent replicates: n = 4 for experiments in A, D-H; n = 3 for experiments in B-C, I-K. (p < 0.05*, p < 0.01**, p < 0.001***)

Restoration of SMAD4 expression via oe-SMAD4 transfection significantly reversed the pro-proliferative effect induced by miR-5100 overexpression (Fig. 4D). Transwell assays showed that simultaneous overexpression of SMAD4 markedly suppressed the enhanced migration and invasion abilities caused by miR-5100 mimic (Fig. 4E-F). Furthermore, miR-5100 overexpression down-regulated E-cadherin and up-regulated N-cadherin at both mRNA and protein levels (Fig. 4G-K). And these effects were significantly reversed upon co-transfection with oe-SMAD4 (Fig. 4G-K).

These results demonstrate that miR-5100 promotes proliferation, migration, invasion, and EMT in TNBC cells by suppressing SMAD4 expression. Exogenous restoration of SMAD4 effectively counteracted the effects of miR-5100, indicating that SMAD4 is a downstream effector in miR-5100-mediated malignant phenotypes in TNBC.

Discussion

This study identified a significant upregulation of miR-5100 in TNBC patients relative to healthy individuals. Elevated miR-5100 levels correlated strongly with high Ki-67 proliferation index, advanced TNM stage, lymph node metastasis, and reduced survival. We analyzed miR-5100 expression in MCF-10 A versus MDA-MB-231/468 cell lines. Compared with normal mammary epithelial cells, miR-5100 was markedly up-regulated in TNBC cells. Functional assays demonstrated that miR-5100 significantly promoted TNBC cell proliferation, migration, invasion, and EMT, supporting an oncogenic role in TNBC.

Our analysis included 80 healthy controls and 80 TNBC patients, with clinical data collected on age, BMI, tumor size, Ki-67 proliferation index, TNM stage, lymph node status, and treatment regimens. Plasma miR-5100 levels, measured across all subjects, were markedly elevated in TNBC patients, suggesting its potential involvement in disease progression. Further evaluation linked higher miR-5100 expression to increased Ki-67 index, advanced TNM stage, and lymph node involvement. The Ki-67 proliferation index, a well-established marker of tumor growth, is routinely applied in clinical prognostic evaluation [18], as are tumor size, TNM stage, and nodal status in TNBC [19]. Critically, Kaplan-Meier and Cox regression analyses indicated that elevated miR-5100 is associated with significantly poorer survival in TNBC patients, suggesting its potential prognostic value.

BC development initiates through the accumulation of genetic mutations and DNA damage [20]. When the damage exceeds the cell’s self-repair capacity, it can lead to the formation of carcinoma in situ, where abnormally proliferating cells are still confined within the ducts or lobules and have not yet broken through the basement membrane [21]. Subsequently, some carcinomas in situ can further progress to invasive cancer. This transformation involves the accumulation of various genetic changes, activation of EMT, and interaction with the tumor microenvironment, ultimately endowing cancer cells with the ability to invade and metastasize [22]. Invasive progression is usually accompanied by an increase in malignancy and a higher risk of distant metastasis. Therefore, early detection and intervention are crucial to prevent disease progression [23]. The promoting effect of miR-5100 on EMT and invasion, and metastasis observed in this study was highly consistent with this process.

MiR-5100 has been shown to regulate progression in multiple cancer types. MiR-5100 has been proven to regulate the EMT process of head and neck squamous cell carcinoma cells, thereby promoting invasion [24]. In lung cancer, miR-210-3p and miR-5100 were reported to induce EMT and enhance invasiveness via STAT3 pathway activation [15]. Conversely, in oral squamous cell carcinoma, silencing of miR-5100 suppressed proliferation, invasion, and migration by up-regulating SCAI expression [25]. These findings collectively suggest that miR-5100 may exert its oncogenic functions across different cancer types through shared molecular mechanisms.

Through bioinformatic analysis, this study predicted potential downstream target genes of miR-5100. By integrating the GeneCards database, this study identified 389 candidate target genes potentially involved in BC progression. GO and KEGG analyses revealed enrichment of these target genes in key processes, notably intracellular signaling and TGF-β pathways. SMAD4, a central TGF-β pathway transcription factor, was bioinformatically predicted as a miR-5100 target. Subsequent experiments verified that miR-5100 directly binds SMAD4 and represses its expression, thereby regulating malignant phenotypes in TNBC cells. In TNBC, down-regulation of SMAD4 - a critical mediator of TGF-β signaling—has been significantly correlated with poor prognosis [26]. Mechanistically, loss of SMAD4 may not only disrupt normal TGF-β signaling but also activate the mTOR pathway, promoting tumor proliferation, invasion, and metastasis [27, 28]. Therefore, our results suggested a plausible miR-5100/SMAD4 axis that may represent one of the mechanisms influencing the multifaceted TGF-β signaling network in TNBC. Further studies are needed to elucidate how this interaction precisely integrates with other regulators to collectively drive disease progression.

While the present study provides evidence linking miR-5100 to TNBC progression, certain limitations should be considered when interpreting these findings. The cohort, though reasonably sized, was recruited from a single institution. Nevertheless, such studies are also needed for future prospective multicenter studies to independently validate the prognostic utility of miR-5100 in TNBC and confirm its wider applicability, a process that requires harmonized protocols and multicenter ethical approvals. What’s more, the cellular origin of the elevated plasma miR-5100, while correlated with disease, remains to be precisely determined. MiR-5100 may derive from tumor cells, the tumor microenvironment, or a systemic response. Additionally, our functional validation was conducted in two widely used TNBC cell lines (MDA-MB-231 and MDA-MB-468). Although the results were consistent and significant across both models, future studies employing a broader panel of cell lines representing the diverse molecular subtypes of TNBC are warranted to confirm the generalizability of our observations. On the mechanistic level, SMAD4 was validated as a direct target of miR-5100 and holds a central role within its oncogenic function, as one might surmise from rescue experiments. However, miRNAs generally regulate multiple targets. Thus, while SMAD4 is central, the involvement of other targets is not to be ruled out. Further studies of downstream effectors of SMAD4, crosstalk with other pathways, and identification of other relevant targets will yield a more comprehensive view of miR-5100-driven oncogenicity. Addressing these aspects in future investigations will help better define the translational potential of targeting this miRNA in TNBC.

Conclusion

This study demonstrated that miR-5100 is upregulated in TNBC patients and is significantly associated with a poor prognosis. MiR-5100 promoted cell proliferation, migration, invasion, and EMT by targeting SMAD4. Collectively, our findings suggest that miR-5100 may serve as a potential prognostic biomarker and therapeutic target worthy of further investigation in TNBC.

Authors’ contributions

Conceptualization: Y S, H W, J Z, P D; Methodology: Y S, H W, J Z, P D; Formal analysis and investigation: Y S, H W, J Z, P D, L H; Writing - original draft preparation: J Z; Writing - review and editing: Y S, H W, P D, L H.

Funding statement

The authors did not receive support from any organization for the submitted work.

Data availability

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

Declarations

Ethics approval and consent to participate

This study was performed in line with the principles of the Declaration of Helsinki. The protocol was approved by the Ethics Committee of Shanxi Cancer Hospital, with written informed consent acquired from all participants.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.