Abstract

   Gastric cancer (GC) is a significant global health issue, characterized
   by poor prognosis due to its metastatic nature. This study investigates
   the role of microRNA-146a-5p (miR-146a-5p) in regulating migration and
   invasion of KATO III GC cells by targeting vimentin and matrix
   metalloproteinase-9 (MMP-9), critical mediators of
   epithelial–mesenchymal transition (EMT). miR-146a-5p expression was
   analyzed in GC cell lines, and functional assays were performed after
   transfection with a miR-146a-5p mimic. Results showed that miR-146a-5p
   expression was significantly reduced in GC cells, particularly in KATO
   III. Transfection with the mimic reduced cell viability, increased
   apoptosis, and inhibited migration, as shown by wound-healing assays.
   Additionally, miR-146a-5p downregulated vimentin and MMP-9 mRNA
   expression. These findings suggest that miR-146a-5p may influence
   metastatic behavior in GC cells through regulation of EMT-related
   genes. However, further studies, including protein-level validation and
   direct target confirmation, are needed. These results provide a basis
   for future investigation into the potential therapeutic role of
   miR-146a-5p in GC.

Supplementary Information

   The online version contains supplementary material available at
   10.1007/s12672-025-02929-x.

   Keywords: MiR-146a-5p, Gastric cancer (GC), Migration, Invasion,
   Vimentin, MMP-9, Epithelial-mesenchymal transition (EMT)

Introduction

   Gastric cancer (GC) is one of the most frequent types of cancer which
   remains a critical international health concern, constituting the fifth
   most prevalent malignancy and one of the leading causes of
   cancer-related deaths [[38]1]. A recent evaluation of GC patients
   evidenced that the prevalence of this cancer is steadily rising in
   youth [[39]2]. The absence of recognizable clinical manifestations in
   the preliminary stages of this disease leads to delayed diagnosis and
   treatment [[40]3]. Despite advances in early identification and
   treatment options, GC’s prognosis remains poor with a low survival rate
   in upcoming years [[41]4]. This is largely due to high rates of
   metastasis of GC cells to the other organs which is a key factor in
   determining the cancer’s outcome [[42]5].

   The metastasis cascade is a sequence of complex processes, including
   invasion, intravasation into the circulatory system, survival,
   maintenance, extravasation, and outgrowth in the distant organ [[43]6].
   One of the crucial steps in this process is the epithelial–mesenchymal
   transition (EMT), an evolutionarily conserved developmental program,
   that plays a pivotal role in carcinogenesis by endowing cancer cells
   with metastatic potential. This process enhances cellular motility,
   invasiveness, and resistance to apoptotic signals. During this process,
   cancer cells lose their connections between themselves and the cell’s
   polarity, while gaining more motility and the ability to degrade the
   extracellular matrix, which allows cancer cells to spread away and
   metastasize from the primary cancer site [[44]7].

   One of the key regulators of the EMT process is an important type III
   intermediate filament protein named vimentin, expressed in mesenchymal
   cells [[45]8]. Vimentin plays a critical role in remodeling the
   cytoskeleton, interlinking the cytoskeleton with the Extracellular
   matrix, and the acquisition of cancer cell migration and invasion
   [[46]9]. Moreover, matrix metalloproteinases (MMPs), such as MMP-9,
   have emerged as potential factors for cancer cell invasion. MMPs are
   zinc-dependent endopeptidases which can degrade the extracellular
   matrix and play a pivotal role in cancer progression [[47]10].

   Recently microRNAs (miRNAs) have become as important regulators in gene
   expression, with the ability to serve as a tumor-suppressive agent in
   multiple tumors [[48]11]. One miRNA which is particularly noteworthy in
   the context of cancer is miRNA-146a-5p which has been shown to adjust
   cell migration and invasion in different cancers [[49]12, [50]13].
   MiR-146a-5p is a member of the microRNA-146 family that can target and
   impede the expression of various genes in the EMT process, such as
   SOX5, Notch2, etc [[51]14–[52]16]. Furthermore, miRNA-146a-5p has been
   proven to suppress MMP-9 expression, reducing cancer cells’ invasion
   [[53]17].

   While several studies demonstrated miRNA-146a-5p’ tumor suppressive
   effect in GC inducing cell cycle arrest and triggering apoptosis in GC
   cells [[54]18], the exact molecular mechanisms mediating these effects
   Have not yet been completely clarified. Therefore, in this research, we
   aimed to evaluate miRNA-146a-5p impact on the capacity of the migration
   of human KATO III cancer cells and to explain the probable mechanisms
   involved, with a particular emphasis on the control of vimentin and
   MMP-9 production. miRNA-146a-5p could be a promising biomarker for
   early GC treatment. By understanding the role of miRNA-146a-5p in
   gastric cancer metastasis, our study may contribute to a novel
   therapeutic strategy targeting this malignancy.

Materials and methods

Bioinformatic analysis

   To investigate the potential regulatory mechanisms of miR-146a-5p, we
   conducted an in silico analysis using several widely used databases.
   Predicted mRNA targets of miR-146a-5p were retrieved from miRDB
   ([55]http://mirdb.org). These targets were compiled and subjected to
   KEGG pathway enrichment analysis using the Enrichr tool
   ([56]https://maayanlab.cloud/Enrichr/). Pathways with a p-value less
   than 0.05 were considered significantly enriched. Additionally,
   miRTarBase ([57]https://mirtarbase.cuhk.edu.cn/) and TargetScan
   ([58]http://www.targetscan.org) were queried to determine whether
   vimentin (VIM) or MMP-9 are predicted or experimentally validated
   direct targets of miR-146a-5p.

Cell culture

   Human GC cell lines AGS, MKN-45, and KATO III were obtained from the
   Pasteur Institute of Iran. They were grown in RPMI media with 10% fetal
   bovine serum (FBS) (Gibco, USA), 100 IU/mL penicillin, and 100 µg/mL
   streptomycin. The cultures were stored in a 37 °C incubator (Memmert,
   Schwabach, Germany) in a humidified atmosphere containing 5% CO[2] and
   used throughout the logarithmic phase of cell growth.

RNA isolation and cDNA synthesis

   To evaluate the expression levels of miR-146a-5p and its potential
   target genes, vimentin and MMP-9, total RNA was extracted from the
   three GC cell lines using TRIzol reagent (RiboEx). cDNA synthesis was
   performed using 1 µg of RNA and a cDNA synthesis kit (Biofact, South
   Korea). qRT-PCR was carried out on a LightCycler 96 system (Roche
   Diagnostics, Mannheim, Germany) following standard protocols. Data
   analysis was performed using the 2^−∆∆CT method. U6 and β-actin were
   used as internal controls for miRNA and housekeeping genes,
   respectively. The primer sequences for both the target genes and
   internal controls are provided in Table [59]1.

Table 1.

   Primer sets for quantification of target genes’ mRNA expression
   Genes    Chain   Sequences
   MMP-9    Forward 5’-ATTTCTGCCAGGACCGCTTCTAC-3’
            Reverse 5’-ATCCGGCAAACTGGCTCCTTC-3’
   Vimentin Forward 5’-AATCGTGTGGGATGCTACCT-3’
            Reverse 5’-CAGGCAAAGCAGGAGTCCA-3’
   β-Actin  Forward 5’-TCCCTGGAGAAGAGCTACG-3’
            Reverse 5’-GTAGTTTCGTGGATGCCACA-3’
   U6       Forward 5’-CTTCGGCAGCACATATACTAAAATTGG-3’
            Reverse 5’-TCATCCTTGCGCAGGGG-3’
   [60]Open in a new tab

MiRNA transfection

   Following an initial evaluation of miR-146a-5p expression across the
   three GC cell lines, the cell line exhibiting the least miRNA
   expression was chosen for further experimentation. The miR-146a-5p
   mimic and negative control miRNA (miR-NC) were obtained from Microcynth
   (AG, Switzerland). The chosen cell line was maintained in an
   antibiotic-free medium at a density of 3 × 10^5 cells/well in six-well
   plates. Transfection was performed when cells reached approximately 80%
   confluency using varying concentrations of the miRNA mimic (50 nM, 75
   nM, and 100 nM) and the jetPEI reagent (PolyPlus, France), as per the
   manufacturer’s instructions. The optimal concentration of 100 nM
   miR-146a-5p mimic, which resulted in the highest expression levels, was
   used for subsequent assays. After 6 h of incubation, the medium was
   replaced with RPMI containing 20% FBS, and the cells were incubated for
   an additional 48 h before proceeding with the MTT assay, wound healing
   assay, and qRT-PCR.

Apoptosis assay

   To assess the effects of miR-146a-5p on apoptosis, cells were subjected
   to flow cytometry (FCM) using an Annexin V/PI double-staining kit
   (EXBIO, Czech Republic). KATO III cells were seeded in six-well plates
   at 2 × 10^5 cells/well and treated with either 100 nM miR-146a-5p mimic
   or miR-NC. After 48 h, cells were stained according to the
   manufacturer’s protocol, and apoptotic cells were analyzed using an FCM
   instrument (FACSQuant; Milteny Biotec, Germany). The apoptotic rates
   were determined, and the data were analyzed using FlowJo software (Tree
   Star, Inc., San Carlos, CA).

Cell viability assay

   The viability of the cells was assessed using the MTT assay. KATO III
   cells were plated at a density of 15,000 cells per well in 96-well
   plates and incubated for a period of 24 h. The cells were then treated
   with 100 nM miR-146a-5p mimic or miR-NC for 48 h. Following treatment,
   the culture medium was discarded, and the cells were incubated with
   2 mg/mL MTT (Sigma, Germany) for 4 h at 37 °C. The resulting formazan
   crystals were solubilized using 200 µL of dimethyl sulfoxide (DMSO),
   and absorbance was measured at 570 nm using a Sunrise™ microplate
   reader (Tecan, Switzerland).

Migration assay

   Migration of the cells was assessed using the scratch assay. KATO III
   cells were plated in 24-well plates at 2 × 10^5 cells/well and cultured
   for 24 h to achieve confluence. A wound was created by scratching the
   surface of the monolayer with a micropipette tip. Following the removal
   of cell debris, the cells were treated with 100 nM miR-146a-5p mimic or
   miR-NC and incubated for 48 h. Images of the wound gap were captured at
   0, 24, and 48 h post-treatment. The migration rate was quantified by
   measuring the closure of the wound gap using ImageJ software.

Statistical analysis

   Statistical assessments were carried out utilizing GraphPad Prism 6
   software (San Diego, CA, USA). One-way analysis of variance (ANOVA) was
   conducted to compare differences between experimental groups, followed
   by Tukey’s post-hoc test for multiple comparisons. A p-value of less
   than 0.05 was considered statistically significant.

Results

Bioinformatic analysis

   Database searches using miRDB, miRTarBase, and TargetScan revealed that
   neither vimentin nor MMP-9 were predicted or validated as direct
   targets of miR-146a-5p. No seed sequence binding sites or experimental
   evidence of direct interaction were identified for these genes.

   However, KEGG pathway enrichment analysis of the predicted targets of
   miR-146a-5p revealed significant associations with several
   cancer-related signaling pathways. These included the NF-κB signaling
   pathway, PI3K-Akt signaling pathway, TGF-β signaling pathway, and cell
   adhesion molecules. These pathways are known to regulate critical
   processes involved in cancer progression, including inflammation, cell
   proliferation, migration, invasion, and epithelial–mesenchymal
   transition [[61]19–[62]22]. Notably, vimentin and MMP-9 are
   well-established markers and functional mediators of EMT and cancer
   cell invasion, which are key steps in tumor metastasis.

   Based on these findings, we hypothesize that miR-146a-5p may influence
   the expression of vimentin and MMP-9 indirectly, through modulation of
   upstream signaling pathways involved in EMT and metastatic progression.

   .

miR-146a-5p downregulation in GC cell lines

   Among the cell lines evaluated (AGS, MKN-45, and KATO III), KATO III
   exhibited the lowest expression level of miR-146a-5p. Based on this
   observation, the KATO III cell line was selected for subsequent
   functional experiments (Fig. [63]1).

Fig. 1.

   [64]Fig. 1
   [65]Open in a new tab

   Relative expression of miR-146a-5p in GC cell lines (AGS, MKN-45, and
   KATO III) analyzed by qRT-PCR. Among the tested cell lines, KATO III
   showed the lowest expression level of miR-146a-5p

miR-146a-5p upregulation after the KATO III GC cells transfection

   miR-146a-5p mimic transfection carried out for a duration of 24, 48,
   and 72 h and the results indicated that the optimal upregulation was at
   48 h. Following dose optimization, GC cells were transfected with 50 nM
   and 100 nM concentrations of the miR-146a-5p mimic (P < 0.0001)
   (Fig. [66]2). Based on the findings, a concentration of 100 nM
   miR-146a-5p mimic was determined to be the optimal condition for
   subsequent experiments.

Fig. 2.

   [67]Fig. 2
   [68]Open in a new tab

   A Time-course analysis of miR-146a-5p expression in KATO III cells
   following mimic transfection at 24, 48, and 72 h, showing maximum
   upregulation at 48 h.B Dose-dependent qRT-PCR analysis of miR-146a-5p
   expression after transfection with 50 nM and 100 nM mimics, with
   significant upregulation observed compared to the control group (***P <
   0.0001)

Transfection with miR-146a-5p resulted in a significant impact on apoptosis
and cell viability

   The MTT test was employed to assess the miR-146a-5p mimic’s
   transfection impact on the viability of KATO III cells. Unlike other
   doses, treatment with 100 nm miR-146a-5p exerted a significant impact
   on the viability of KATO III cells, with significant proliferative
   changes (P < 0.05) (Fig. [69]3A). Moreover, FCM test revealed that
   miR-146a-5p mimic significantly impacted apoptosis in KATO III cells
   (**P < 0.01) (Fig. [70]3B). Given that the KATO III cells are a
   metastatic cell line, we concentrated the rest of the research on
   determining the impacts of miR-146a-5p mimic on their migration rates.

Fig. 3.

   [71]Fig. 3
   [72]Open in a new tab

   Effects of miR-146a-5p transfection on cell viability and apoptosis in
   KATO III cells. A MTT assay results showing that 100 nM miR-146a-5p
   mimic significantly reduced cell viability compared to the control
   group (*P < 0.05), while 50 nM and 75 nM concentrations had no
   significant effect. B Flow cytometry analysis (Annexin V/PI staining)
   demonstrating a significant increase in apoptosis following
   transfection with 100 nM miR-146a-5p mimic compared to the control
   (*P < 0.01)

Suppression of migration in the KATO III cell line after the overexpression
of miR-146a-5p

   A scratch assay was used to compare the cells’ rate of migration of the
   KATO III cell line in the miRNA-treated group compared to the control
   group. The wound area was measured at 0, 24, and 48 h. As shown in
   Fig. [73]4, miR-146a-5p transfection in KATO III cells caused a
   considerable decrease in cell migration over 48 h compared to the
   control cells. After transfection, we assessed the expression of genes
   associated with migration to evaluate miR-146a-5p’s impact on
   migration.

Fig. 4.

   [74]Fig. 4
   [75]Open in a new tab

   Wound healing assay to evaluate the migration ability of KATO III cells
   following miR-146a-5p transfection. Cells treated with miR-146a-5p
   mimic showed significantly reduced migration at 24 and 48 h compared to
   cells transfected with the negative control miRNA

miR-146a-5p transfection changes the expression of genes related to
metastasis

   qRT-PCR was used to analyze the impact of miR-146a-5p mimic
   transfection on vimentin and MMP-9 mRNA expression, which are key
   metastatic genes (Fig. [76]5A and B). Transfection with miR-146a-5p
   mimic sequences significantly inhibited MMP-9 and vimentin production
   (P < 0.0001).

Fig. 5.

   [77]Fig. 5
   [78]Open in a new tab

   qRT-PCR analysis of MMP-9 (A) and vimentin (B) mRNA expression in KATO
   III cells following transfection with miR-146a-5p mimic. A significant
   reduction in the expression levels of both genes was observed in
   miR-146a-5p–transfected cells compared to the control group
   (***P < 0.0001). Cells transfected with control miRNA were used as the
   baseline

Discussion

   Gastric cancer is one of the leading causes of cancer-related deaths
   worldwide. Its poor prognosis is mainly due to late diagnosis and the
   presence of metastasis. Identifying molecular mechanisms involved in GC
   progression is essential for improving treatment strategies. This study
   focused on miR-146a-5p, a microRNA that has been implicated in cancer
   development and metastasis.

   Our findings show that miR-146a-5p expression is reduced in KATO III
   cells. Transfection with miR-146a-5p mimic increased its expression and
   led to reduced cell viability, increased apoptosis, and decreased
   migration. These results are in agreement with previous studies.
   miR-146a-5p was reported to suppress cell proliferation and promote
   apoptosis in GC by targeting CDC14A, a regulator of cell cycle
   transition [[79]18].Wang et al. also showed that miR-146a-5p reduces
   invasiveness in colorectal cancer cells [[80]23].

   After transfection, miR-146a-5p expression was highest at 48 h. The
   most effective concentration was 100 nM, which produced consistent
   biological effects. These findings are in line with earlier work
   indicating that microRNA mimic concentration and timing are important
   for cellular uptake and gene modulation [[81]24].

   The MTT and flow cytometry assays showed that miR-146a-5p reduces cell
   viability and promotes apoptosis in KATO III cells. These findings are
   supported by other studies in different cancers. In breast cancer,
   miR-146a-5p induced apoptosis in vitro [[82]25]. In bladder cancer, it
   contributed to apoptosis in response to melittin treatment [[83]26].
   These effects may involve pathways such as PI3K/Akt, MAPK, and NF-κB,
   which are known to regulate cell survival and apoptosis [[84]27,
   [85]28].

   Our data also showed that miR-146a-5p inhibits migration in KATO III
   cells, as demonstrated by the wound healing assay. This effect is
   relevant because migration is a key step in the metastatic process
   [[86]29]. The reduced migration was accompanied by decreased mRNA
   expression of vimentin and MMP-9, both of which are associated with
   EMT. Vimentin is a mesenchymal marker that enhances cell motility
   during EMT [[87]30]. MMP-9 degrades the extracellular matrix and
   promotes invasion [[88]10]. These findings are consistent with previous
   research showing that suppression of vimentin and MMP-9 reduces the
   metastatic potential of cancer cells [[89]31, [90]32].

   Although in silico tools did not predict vimentin and MMP-9 as direct
   targets of miR-146a-5p, the observed downregulation suggests that they
   may be indirectly regulated. This indicates a possible role for
   miR-146a-5p in modulating EMT-related pathways.

Limitations and future directions

   This study has several limitations. Functional experiments were
   conducted in only one cell line (KATO III), which may limit the
   generalizability of the results. Additional GC cell lines should be
   used in future studies to confirm these findings. Protein-level
   validation of vimentin and MMP-9 was not performed. Although mRNA
   levels were measured using qRT-PCR, Western blot analysis is needed to
   confirm changes at the protein level and is planned in future work.

   Direct regulatory interactions between miR-146a-5p and its potential
   target genes were not evaluated. To address this, future experiments
   will include dual-luciferase reporter assays and functional recovery
   studies to determine whether vimentin and MMP-9 are directly regulated
   by miR-146a-5p. The study also assessed migration using a wound healing
   assay but did not include invasion-specific assays such as the
   Transwell assay. Incorporating this method in future experiments will
   help to better understand the role of miR-146a-5p in GC invasion.

   The gene expression analysis was limited to vimentin and MMP-9. Broader
   transcriptomic profiling using RNA sequencing is necessary to identify
   additional genes and pathways regulated by miR-146a-5p. Furthermore, as
   all experiments were conducted in vitro, in vivo studies using animal
   models are required to validate the biological relevance and
   therapeutic potential of miR-146a-5p.

   The use of synthetic miRNA mimics may not accurately represent
   physiological conditions. Therefore, future studies should consider
   using delivery systems that enhance the stability and specificity of
   miRNAs in vivo. Recent studies have demonstrated that
   nanoparticle-based delivery systems, including metal-organic frameworks
   (MOFs) and electrospun nanofibers, can improve the efficacy of
   chemotherapy agents such as temozolomide, doxorubicin, and
   platinum-gold nanorods [[91]33, [92]34]. These technologies may also
   enhance the delivery and therapeutic application of miR-146a-5p in GC
   and should be investigated further.

   Addressing these limitations in future work will help clarify the
   function of miR-146a-5p in GC and support its development as a
   potential therapeutic target.

Conclusion

   In conclusion, our findings suggest that miR-146a-5p may play a
   functional role in modulating key characteristics of gastric cancer
   cells, including migration, apoptosis, and the expression of
   EMT-associated genes. Overexpression of miR-146a-5p in KATO III cells
   led to reduced cell viability, increased apoptosis, and decreased
   migration, accompanied by significant downregulation of vimentin and
   MMP-9 at the mRNA level. While our results support a potential
   tumor-suppressive role for miR-146a-5p in gastric cancer, we
   acknowledge that the mechanisms underlying this regulation—particularly
   whether these target genes are directly or indirectly affected—require
   further validation. These preliminary findings highlight miR-146a-5p as
   a promising candidate for future investigation and warrant additional
   studies to explore its therapeutic and mechanistic implications in
   gastric cancer.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [93]Supplementary Material 1^ (134.5KB, xlsx)
   [94]Supplementary Material 2^ (48.7KB, xlsx)

Acknowledgements