miR-548t-3p impairs nuclear mechanosensitivity and focal adhesion via lamin A/C downregulation

Abstract

Lamin proteins are essential structural elements of the nuclear envelope, critically involved in maintaining nuclear shape and mechanical stability. Lamin A/C, specifically, acts as a mechanotransducer that senses extracellular mechanical cues and transmits them into intracellular biochemical signals, thereby influencing cell adhesion, motility, and differentiation. Although microRNAs (miRNAs) have emerged as key regulators of cellular mechanotransduction pathways, the precise roles of miRNAs in modulating lamin A/C at the single-cell level remain poorly understood. Here, we utilized advanced biosensors based on fluorescence resonance energy transfer (FRET) and traction force microscopy to elucidate the impact of miRNA-548t-3p-induced lamin A/C downregulation on nuclear mechanical properties in single cells. Our findings demonstrate that miRNA-548t-3p specifically reduces lamin A/C levels, resulting in decreased nuclear tension and compromised focal adhesion dynamics. Furthermore, miRNA-548t-3p significantly diminishes the ability of cells to sense and respond to variations in extracellular matrix stiffness, leading to reduced cellular traction forces. These results underscore the pivotal role of lamin A/C in cellular mechanosensitivity and highlight miRNA-548t-3p as a critical modulator of nuclear mechanotransduction and mechanical homeostasis at the single-cell level. This study provides new insights into the complex interplay between miRNAs, nuclear mechanics, and cell-environment interactions, suggesting potential avenues for therapeutic intervention in diseases associated with disrupted mechanotransduction.

Significance

This study elucidates the essential role of lamin A/C proteins in preserving nuclear structural integrity and mechanotransduction and identifies microRNA-548t-3p as a key regulator of these processes. By employing advanced single-cell visualization techniques, we demonstrate that miRNA-548t-3p-mediated downregulation of lamin A/C significantly decreases nuclear tension and compromises focal adhesion dynamics. Importantly, miRNA-548t-3p impairs cellular responses to changes in extracellular matrix stiffness, leading to diminished mechanosensitivity and reduced traction force generation. Our findings provide critical insights into the molecular mechanisms underlying cellular adaptation to mechanical environments and highlight the potential of targeting miRNA-548t-3p-lamin A/C axis in mechanotransduction-related disorders.

Introduction

Lamins are integral structural proteins of the nuclear envelope, serving critical functions in maintaining nuclear morphology, mechanical stability, and genome organization (1,2). Classified as type V intermediate filaments, lamins form dynamic networks that maintain nuclear integrity and act as a mechanical interface between the nuclear interior and the cytoskeletal framework. Lamin A/C, a major component of the nuclear lamina, serves as a central nuclear mechanotransducer by interfacing with the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex to physically bridge the cytoskeleton and the nucleus (3). This connection transmits mechanical forces from the extracellular matrix—via focal adhesions and actin stress fibers—directly to the nuclear envelope (4). As a result, applied forces induce nuclear deformation and increase nuclear envelope tension, stretching nuclear pore complexes. These deformations regulate nucleocytoplasmic transport, for example, by reducing the mechanical resistance of nuclear pores and promoting the nuclear entry of mechanosensitive transcription factors such as YAP/TAZ (5). Simultaneously, lamin A/C’s tethering to chromatin (e.g., lamin-associated domains) allows force transmission into the nucleus to reorganize chromatin architecture and alter the epigenetic landscape (6,7)—changes that can modulate gene expression in response to mechanical stimuli. Moreover, these mechanotransductive functions of lamin A/C are dynamically fine-tuned by posttranslational modifications (such as force-sensitive lamin A/C phosphorylation and acetylation), which alter lamin assembly, nuclear stiffness, and lamin-chromatin interactions to modulate how mechanical cues are converted into biochemical signals (8,9). This conversion facilitates essential cellular processes, including gene expression regulation, DNA replication, cellular differentiation, and proliferation (10). Central to this mechanotransduction process is the LINC complex, a multiprotein assembly that directly connects cytoplasmic actin filaments and focal adhesions at the cell periphery to the nuclear lamina. This structural linkage enables efficient mechanical signal transduction, critically influencing cellular functions such as adhesion, migration, and mechanosensing (8,11,12).

MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression, primarily through complementary binding to target mRNAs, thereby repressing translation or promoting downregulation of the transcript (13). They play critical roles in numerous cellular processes, including development, differentiation, apoptosis, and the maintenance of tissue homeostasis. Accumulating evidence demonstrates that specific miRNAs respond dynamically to mechanical cues such as changes in matrix stiffness, shear stress, and mechanical loading (14,15,16). These mechanosensitive miRNAs can modulate the expression of genes associated with the cytoskeleton, cell adhesion molecules, and components of the extracellular matrix (ECM), thus enabling cells to adapt to mechanical challenges in their microenvironment (15,16,17). Despite considerable interest in the mechanobiological functions of miRNAs, their precise regulatory roles, particularly in the context of nuclear mechanotransduction, remain inadequately understood.

In this study, we specifically focus on miRNA-548t-3p and investigate its potential role in regulating lamin A/C expression, thereby influencing nuclear mechanical properties and cellular responses to mechanical stimuli. miRNA-548t-3p was selected based on in silico predictions using the miRDB database (https://mirdb.org/), which ranked it as the top candidate (Target Rank 1) for targeting the LMNA gene. This high ranking is determined by the Target Score, a computational metric reflecting the predicted binding strength, sequence complementarity, and conservation of the miRNA-mRNA interaction. A higher Target Score indicates greater confidence in the likelihood and efficacy of target regulation. Thus, the strong prediction that miRNA-548t-3p targets LMNA with high specificity and affinity motivated its selection for further experimental validation. To test this hypothesis, we employed fluorescence resonance energy transfer (FRET)-based biosensors to visualize miRNA-mediated mechanotransduction at the single-cell level (18,19,20,21). These single-cell imaging approaches complement traditional biochemical and transcriptomic methods, offering detailed spatial and temporal insights into miRNA-related mechanical signaling. Although mechanosensitive miRNAs have been implicated in maintaining mechanical homeostasis (22,23), detailed mechanisms by which these miRNAs modulate nuclear mechanosensitivity remain to be fully elucidated.

Previous studies have demonstrated that certain miRNAs, such as miR-9 and miR-124-3p, regulate lamin A/C expression by targeting its mRNA or associated processing enzymes, thereby influencing nuclear architecture and cellular fate decisions in a context-dependent manner (24,25,26). Although these studies have established that miRNAs can regulate lamin A/C expression through direct or indirect mechanisms, their functional relevance in the context of nuclear mechanics and mechanical homeostasis has not been thoroughly investigated.

Our findings reveal a novel and significant role for miRNA-548t-3p in directly targeting lamin A/C, consequently influencing nuclear tension, focal adhesion dynamics, and cellular mechanosensitivity in response to changes in ECM stiffness. By elucidating the complex relationship between miRNA-548t-3p and lamin A/C-mediated mechanotransduction, this study expands our fundamental understanding of how cells integrate mechanical signals at the nuclear level to maintain mechanical homeostasis. The insights gained from this work contribute valuable knowledge to the broader field of mechanobiology and suggest new therapeutic avenues for treating disorders associated with impaired mechanotransduction pathways.

Materials and methods

Cell culture and transfection

HeLa cells and MCF7 cells were maintained in Dulbecco’s Modified Eagle Medium (GenDEPOT, CM002-050), supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, 16000-044) and 1% (v/v) penicillin-streptomycin (P/S; GenDEPOT, CA005-010), containing 100 units/mL penicillin and 100 μg/mL streptomycin. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂. For transfection experiments, cells were seeded and allowed to reach 70%–80% confluency. Subsequently, cells were transfected with either a miRNA mimic (hsa-miR-548t-3p; Accutarget, SMM-003), a miRNA inhibitor (hsa-miR-548t-3p inhibitor; Accutarget, SMI-003), or expression plasmids encoding Nesprin-TS (Addgene #68127), Lyn-FAK (Addgene #78299), mCherry–Lamin A/C (Addgene #65568), or mCherry–NLS (Addgene #58476). Transfections were carried out using Lipofectamine 2000 (Invitrogen, 11668027) in accordance with the manufacturer’s instructions. Successful transfection of miRNAs was confirmed by gel electrophoresis. The sequence of the miR-548t-3p mimic was 5′-CAAAAGUGAUCGUGGUUUUG-3′, and that of the inhibitor was 5′-AAAAACCACAAUUACUUUUUGCACCA-3′. The miRNA inhibitor is a chemically synthesized, single-stranded oligonucleotide designed to bind complementarily to endogenous miR-548t-3p and prevent its interaction with target mRNAs, thereby effectively suppressing its posttranscriptional regulatory function. After transfection, cells were incubated for an additional 24 h prior to further analysis.

Gel electrophoresis

Total RNA was extracted from cultured cells using TransZol reagent (Invitrogen, Cat. 15596018), following the manufacturer’s protocol. For each sample, approximately 300 ng of total RNA was reverse-transcribed into complementary DNA (cDNA) using the SmartGene Compact cDNA Synthesis Kit (SmartGene, SG-cDNAC100). The resulting cDNA served as a template for reverse transcription– polymerase chain reaction (RT-PCR), which was performed using Q5 DNA polymerase (New England Biolabs, M0491S) according to the manufacturer’s instructions. Primer sequences used for amplification are provided below. PCR products were analyzed via electrophoresis on a 2% agarose gel. A DNA ladder was loaded in parallel to assess product size. After electrophoresis, gel images were acquired and quantified using ImageJ software (National Institutes of Health, https://imagej.nih.gov/ij). Table 1 lists the primer sequences used for RT-PCR amplification of GAPDH, LMNA, and LMNB, along with their respective amplicon sizes. Gene expression levels were normalized against GAPDH, which was used as the internal reference control.

Table 1.

Primer Sequences Used for RT-PCR and Corresponding Amplicon Sizes

Gene	Sequence	Amplicon (Bp)
GAPDH	F: 5′-GTCATCCCTGAGCTGAACGG-3′	187
	R: 5′-CCACCTGGTGCTCAGTGTAG-3′
LMNA	F: 5′-GAGGAGGGCAAGTTTGTCCG-3′	82
	R: 5′-CATTCTGGCGCTTGATCTGC-3′
LMNB	F: 5′-AAGCATGAAACGCGCTTGG-3′	152
	R: 5′-AGTTTGGCATGGTAAGTCTGC-3′

Immunofluorescence staining

Immunofluorescence staining was performed on HeLa cells seeded onto confocal dishes. Cells were fixed with 4% paraformaldehyde (PFA) (Biosesang, Cat. No. PC2031-050-00) in phosphate-buffered saline (PBS) and subsequently permeabilized using 0.1% Triton X-100 (Sigma, Cat. No. STBG3972V) in PBS. To prevent nonspecific binding, cells were blocked with 3% bovine serum albumin (BSA) (MP Biomedicals, Cat. No. 152401) for 1 h at room temperature. Between each step, cells were washed three times with PBS. After blocking, cells were incubated overnight at 4°C with primary antibodies, including mouse anti-Lamin A/C (ABclonal, Cat. #A19524) and rabbit anti-Emerin (Invitrogen, Cat. #PA5-79201). The next day, cells were incubated for 1 h at room temperature with secondary antibodies, including rhodamine (TRITC)-conjugated goat anti-rabbit IgG (H + L) (1:100, ABclonal, AS040) and ABflo 488-conjugated goat anti-mouse IgG (H + L) (1:100, ABclonal, AS037). After incubation with secondary antibodies, cells were washed five times with PBS. Samples were then mounted using VECTASHIELD mounting medium containing DAPI (VECTOR Laboratories, H-1200) to stain the nuclei and stored at 4°C in the dark until imaging.

Microscopy and FRET image analysis

For fluorescence imaging, HeLa cells were cultured in confocal imaging dishes (SPL, 200350). Before imaging, the cells were washed twice with PBS (Welgene, LB004-02), and the culture medium was replaced with CO₂-independent medium (Gibco, 18045–088) supplemented with 0.5% FBS, 1% (v/v) penicillin-streptomycin (GenDEPOT, CA005), and 1X GlutaMAX (Gibco, 35050–061). Fluorescence imaging was performed using a Leica DMi8 fluorescence microscope equipped with an LED8 light source, a K5-sCMOS camera, and an HC PL APO 100×/1.40 oil immersion objective. To maintain optimal imaging conditions, the microscope was fitted with a CO₂/37°C incubation chamber, ensuring physiological conditions throughout the imaging process. To visualize Nesprin-TS and Lyn-FAK biosensors, a filter set consisting of a 436/28-nm excitation filter, a CYR71010 filter cube (11525416, Leica), a 460-nm dichroic mirror (shared for both channels), and a 473/22-nm emission filter was used to detect enhanced cyan fluorescent protein (ECFP). For FRET imaging, a similar excitation filter (436/28 nm) and filter cube (CYR71010) were used, but the emission was detected using a 535-nm dichroic mirror and a 539/24-nm emission filter. In immunofluorescence experiments using fluorescein isothiocyanate (FITC), excitation was achieved with a 479/33-nm filter, whereas emission was collected using a DFT51010 filter cube (11525418, Leica), a 460-nm dichroic mirror, and a 519/25-nm emission filter. For rhodamine detection, cells were excited at 554/24 nm, and fluorescence was captured through a 594/32-nm emission filter using the DFT51010 filter cube (11525418, Leica) and a 460-nm dichroic mirror. To ensure accurate fluorescence quantification, background fluorescence intensities were measured and subtracted from the region of interest using LAS X software. Processed fluorescence intensity data were then analyzed using GraphPad Prism 10.0 (GraphPad Software, San Diego, CA, USA) for statistical evaluation.

Bis-acrylamide-polyacrylamide gel fabrication

To investigate cellular responses to mechanical stimuli, polyacrylamide (PA) hydrogels with varying stiffness were fabricated by modulating the concentrations of acrylamide and bis-acrylamide cross-linkers. Specifically, PA solutions were prepared to achieve stiffness levels of 1 kPa, 5 kPa, and 40 kPa by adjusting the relative concentrations of these reagents. The PA solution was prepared by mixing ammonium persulfate (APS) (TransLab, Lot. 050224) and N,N,N′,N′-tetramethylethylenediamine (TEMED) to initiate polymerization. The polymerization reaction was carried out using 40% (w/v) acrylamide (Bio-Rad, Cat. #161-0140) and 2% (w/v) bis-acrylamide (Bio-Rad, Cat. #161-0142) stock solutions. After polymerization, the surface of the PA gel was functionalized using N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate (sulfo-SANPAH) (Thermo Fisher Scientific, Cat. No. 22589) to enable protein attachment. To activate cross-linking, the sulfo-SANPAH-treated PA gel was exposed to ultraviolet light, followed by three sequential washes with 10 mM HEPES buffer (Dojindo Laboratories, Lot. No. WQ858) to remove unreacted reagents. Finally, 200 μL of collagen solution (0.2 mg/mL, Sigma) was applied to the sulfo-SANPAH-coated PA gels and incubated at 37°C for 16 h to facilitate cell adhesion.

Measurement of Fourier transform traction force

The contractile forces exerted by adherent cells on the substrate were analyzed using a Leica DMi8 fluorescence microscope. HeLa cells were seeded onto PA elastic gel substrates with Young’s moduli of 5 kPa and 40 kPa, which were coated with type I collagen (0.2 mg/mL). Cells were allowed to adhere and stabilize for 5 h, with approximately 1500 cells per gel. To visualize traction forces, red fluorescent beads (Invitrogen, Cat. No. F8810, 580/605 nm) embedded within the gel matrix were used as fiducial markers. Fluorescence imaging of these beads was performed using a 578/24-nm excitation filter, a 598-nm dichroic mirror, and a 641/78-nm emission filter. Traction force measurements were conducted using an analysis software based on fast Fourier transform developed by Prof. Ning Wang’s lab at the University of Southern California (27,28). Fluorescence images of the beads were acquired before and after cell detachment using 1× trypsin-EDTA (GenDEPOT, CA015-010). The displacement of the beads, induced by cell-generated forces, was analyzed using a custom MATLAB program, and the average traction forces were calculated accordingly.

Nesprin-TS biosensor ratio image analysis

FRET images were processed and analyzed using ImageJ software (http://fiji.sc/). To facilitate ratio imaging, the multi-channel image stack was first split into two separate stacks, each corresponding to a specific fluorescence channel. To minimize noise and improve image clarity, a Gaussian blurring filter was applied. For nuclear segmentation, an intensity threshold was applied to the blurred image, generating a binary mask in which pixels exceeding the threshold were classified as nuclear regions. This process effectively distinguished nuclei from the background. To refine nuclear morphology, the binary mask was further processed using morphological operations. Specifically, the original image underwent erosion, which slightly contracted the nuclear regions, whereas a duplicate image was subjected to dilation, expanding these regions. The difference between the dilated and eroded images was then computed, allowing for precise identification of the nuclear boundary. The segmented nuclear region was subsequently overlaid onto the FRET ratio image, enabling the extraction of FRET signals specifically from the nuclear membrane. This approach ensured accurate quantification of the Nesprin-TS biosensor FRET efficiency at the nuclear envelope.

Immunoblotting

HeLa and MCF-7 cells were seeded in six-well culture plates (SPL, Cat. No. 30006) and transfected with miR-548t-3p mimic or inhibitor for 48 h. Cells were then lysed using RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Cat. No. 89901) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (100×, Thermo Fisher Scientific, Cat. No. 78442). All lysis procedures were carried out on ice to prevent protein degradation. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Reagents A and B; Thermo Fisher Scientific, Cat. Nos. 23228 and 23224), and equal amounts of protein (15 μg per lane) were loaded for electrophoresis. Tris-Glycine SDS-PAGE gels were prepared using the following reagents: 1 M Tris-HCl (pH 6.8) (TransLab, Cat. No. 261022-10), 1.5 M Tris-HCl (pH 8.8) (TransLab, Cat. No. TLP-107), 10% SDS (TransLab, Lot No. SDS241122-2), 10% APS (TransLab, Cat. No. TLP-109.1), and TEMED (Thermo Fisher Scientific, Cat. No. 17919). Proteins were separated at a constant voltage of 50 V for 10 min through the stacking gel, followed by 100 V for 1.5 h in the resolving gel. After electrophoresis, proteins were transferred to 0.45-μm nitrocellulose membranes (Bio-Rad, Cat. No. 1620115) for 1.5 h. Membranes were blocked with 5% nonfat dry milk (BD Difco, Cat. No. 232100) in TBST for 1 h at room temperature and incubated overnight at 4°C with primary antibodies. HRP-conjugated secondary antibodies (Santa Cruz, sc-2357, sc-516102) were then applied, and signals were visualized using enhanced chemiluminescence. The following primary antibodies were used: anti-Lamin A/C (Santa Cruz Biotechnology, sc-7293), anti-Lamin B1 (Invitrogen, PA5-19468), and anti-GAPDH (TransGen Biotech, HC301-01) as a loading control. Band intensities were quantified using ImageJ software.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 10.0. Data were expressed as the mean ± standard error of the mean (SEM), and statistical evaluation was performed by unpaired t-test using GraphPad Prism 10.0. Significant differences were determined by p-values (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001).

Results

miR-548t-3p induces morphological and structural alterations in the nucleus by targeting lamin A/C

To identify miRNAs capable of regulating lamin A/C (LMNA), we performed bioinformatic predictions using TargetScan 8.0 and the MicroRNA Target Prediction Database (miRDB). Among the candidate miRNAs identified (Figs. 1 A and S1), miR-548t-3p was previously suggested to target LMNA according to miRDB. To experimentally verify this interaction, we conducted RT-PCR analysis followed by gel electrophoresis. Our results clearly showed that treatment with miR-548t-3p significantly reduced LMNA mRNA expression levels, whereas LMNB mRNA expression remained unchanged (Fig. 1 B). Moreover, co-transfection with a specific miR-548t-3p inhibitor restored LMNA mRNA levels, confirming the specificity of miR-548t-3p-induced LMNA downregulation.

Figure 1.

miR-548t-3p induces downregulation of LMNA. (A) Putative binding sites of miR-548t-3p within LMNA 3′ UTR, as predicted by TargetScan and miRDB. (B) Reverse transcription-polymerase chain reaction analyses showing LMNA and LMNB mRNA levels in HeLa cells. The graph values were normalized by the signal from the GAPDH mRNA level. All error bars represent SEM (black, gray, and charcoal; n = 3, ^∗∗: p < 0.01, Student's t-test). (C) Representative images of HeLa cells (Hoechst, blue), brightfield image, and merged image with brightfield image. Hoechst 33342: λ ex = 360 nm; λ em = 460 nm (blue). Treatment of cells with miR-548t-3p increases the size of the nucleus and deforms the shape of the nucleus. All images were acquired using a 100× objective lens. All images share the same scale bar. (D) Quantification of nuclear circularity. The treatment with miRNA resulted in a decrease in circularity. All error bars represent SEM (black, gray, and charcoal; n = 171, 136, and 134, respectively, ^∗∗∗∗: p < 0.0001, Student's t-test). (E) Quantification of nuclear areas. The downregulation of LMNA increases the size of the nucleus. All error bars represent SEM (black, gray, and charcoal; n = 171, 136, and 134, respectively;^∗: p < 0.05,^∗∗∗∗: p < 0.0001, Student's t-test). (F) Left, representative immunofluorescence images showing lamin A/C (green), emerin (red), and DAPI staining in HeLa cells. Right, quantification of the fluorescence intensity of lamin A/C and emerin along a line across the nucleus. Scale bar, 10 μm.

Next, we assessed the impact of LMNA downregulation on nuclear morphology. Hoechst staining analysis demonstrated that miR-548t-3p treatment led to a significant decrease in nuclear circularity (Fig. 1 C and D). Additionally, nuclear size was increased markedly upon LMNA depletion induced by miR-548t-3p treatment, and this phenotype was partially reversed by co-treatment with the inhibitor treatment (Fig. 1 E). These observations align well with previous studies reporting that reduced lamin A/C expression correlates with increased nuclear size (29,30).

To further elucidate the structural consequences of miR-548t-3p-mediated LMNA downregulation, we performed immunostaining to visualize the localization of lamin A/C and emerin, critical components of the nuclear envelope and the LINC complex. Immunofluorescence results indicated that miR-548t-3p treatment significantly disrupted lamin A/C and emerin colocalization at the nuclear envelope and reduced their fluorescence intensity. These structural disruptions were consistently observed not only in HeLa cells but also in MCF-7 cells, which exhibit relatively high levels of endogenous lamin A/C (Figs. 1 F and S2). Importantly, co-transfection with the miRNA inhibitor effectively reversed these alterations in both cell lines. To determine whether miR-548t-3p also affects lamin A/C expression at the protein level, we performed immunoblotting analyses in both HeLa cells and MCF-7 cells, the latter of which exhibits relatively high endogenous lamin A/C expression. As shown in Fig. S3, miR-548t-3p treatment led to a marked reduction in lamin A/C protein levels in both cell lines, whereas lamin B1 expression remained unchanged. These findings further confirm that miR-548t-3p specifically downregulates lamin A/C expression at both the mRNA and protein levels. Collectively, our data demonstrate that miR-548t-3p specifically targets LMNA, leading to reduced expression of lamin A/C at both the mRNA and protein levels. This downregulation results in pronounced morphological changes in nuclear shape and disrupted localization of nuclear envelope proteins essential for maintaining nuclear integrity and mechanotransduction.

Depletion of lamin A/C regulates nuclear tension and focal adhesion activity

The ability of cells to sense and respond to mechanical cues from the ECM is critical for regulating intracellular signaling and cellular behavior. These processes are mediated by the LINC complex, which facilitates mechanotransduction by physically connecting the cytoskeleton to the nuclear lamina (31,32). Lamin A/C, a key structural component of the nuclear lamina, plays a pivotal role in transmitting mechanical signals from the cytoskeleton to the nuclear interior. To investigate the consequences of lamin A/C depletion induced by miR-548t-3p, we utilized FRET-based biosensors to assess nuclear tension and focal adhesion kinase (FAK) activity (32,33). Nesprin, a major component of the LINC complex, facilitates the transmission of actomyosin-generated forces between actin filaments and the nuclear envelope. The FRET-based biosensor detects variations in nuclear tension by measuring changes in the distance between the fluorescent protein pair, which is inversely correlated with FRET efficiency (34).

Our experimental findings demonstrate a significant reduction in nesprin-mediated nuclear tension after miR-548t-3p treatment (Fig. 2 C and D). Importantly, co-transfection with a miR-548t-3p inhibitor effectively restored nesprin tension, suggesting a direct role for miR-548t-3p in modulating nuclear mechanics. To further test whether the observed mechanical defects were specifically due to reduced lamin A/C levels, we performed a targeted rescue experiment by cotransfecting mCherry-tagged lamin A/C lacking the miR-548t-3p target site in the 3′ UTR. Notably, overexpression of this rescue construct successfully restored nuclear envelope tension in miR-548t-3p-treated cells (Fig. S4), confirming that lamin A/C is a principal effector of miR-548t-3p-induced mechanical alterations. Reduced nuclear tension was transmitted to the actin cytoskeleton through the LINC complex, leading to a decrease in actin stress fiber tension and a concomitant weakening of focal adhesions (35,36). These alterations in nuclear tension suggest a broader mechanotransduction cascade that governs cell adhesion and motility (37,38).

Figure 2.

LMNA is degraded by miRNA, affecting nuclear membrane tension and FAK. (A) Schematic representation of Nesprin-TS biosensor and the inverse relationship between FRET and nuclear tension. (B) Schematic of the Lyn-FAK biosensor and how the biosensor works. Active FAK phosphorylates the substrate peptide in the FAK biosensor and subsequently binds to the SH2 domain, causing a decrease in FRET. (C) Nesprin-TS biosensor localized to the nuclear membrane in HeLa cells. Nuclear membranes were visually masked and processed into FRET ratios. Images are pseudocolored according to the normalized FRET ratio. The FRET at the nuclear envelope was extracted using ImageJ. (D) Normalized CFP/FRET ratio of Nesprin-TS in HeLa cells transfected with miRNA mimic and mimic with inhibitor along with the Nesprin-TS sensor. When treated with miRNA mimic, it exhibits a lower tension (lower FRET ratio) compared with the control. All error bars represent SEM (gray, green, and orange; n = 110, 110, and 90, respectively; ^∗∗∗∗: p < 0.0001, Student's t-test). (E) CFP, FRET, and CFP/FRET ratio images of Lyn-FAK biosensor in HeLa cells; Donor (blue, ECFP), acceptor (yellow, YPet), and CFP/FRET ratio (rainbow) channels. White scale bar represents 10 μm. Image backgrounds were processed using Las X. (F) The representative ECFP/YPet ratio images of Lyn-FAK in HeLa cells transfected with miRNA mimic and mimic with inhibitor along with the Lyn-FAK sensor. All error bars represent SEM (gray, green, and orange; n = 54, 42, and 65, respectively; ^∗∗∗:p < 0.001, ^∗∗: p < 0.01, Student's t-test).

In addition, FAK activity, assessed via the Lyn-FAK biosensor, was significantly diminished in miR-548t-3p-treated cells, further supporting the mechanistic link between nuclear mechanics and focal adhesion stability (Fig. 2 E and F). Notably, FAK activity was restored upon co-transfection with the miRNA inhibitor, reinforcing the role of lamin A/C in maintaining the structural and functional integrity of focal adhesions. These findings establish lamin A/C as a central regulator of nuclear-cytoskeletal interactions essential for the maintenance of nuclear tension and focal adhesion signaling, highlighting the broader implications of nuclear mechanotransduction in cellular responses to mechanical stimuli.

miR-548t-3p impairs nuclear envelope mechanosensitivity by targeting lamin A/C

Cells dynamically sense and respond to changes in the mechanical properties of the ECM through mechanotransduction pathways mediated by the LINC complex (37,39). This mechanosensitivity is essential for regulating nuclear morphology, cytoskeletal organization, and adhesion dynamics. The LINC complex, which includes nesprin, actin, talin1, and tensin1, facilitates the transmission of mechanical tension from the ECM to the nuclear envelope (11). To examine the impact of miR-548t-3p on nuclear mechanosensitivity, we utilized FRET-based nesprin biosensors to quantify nuclear tension across substrates of varying stiffness (1, 5, and 40 kPa).

Under control conditions, nesprin-mediated nuclear tension increased significantly with substrate stiffness, demonstrating enhanced force transmission at 40 kPa compared with softer matrices (Fig. 3 A, B, and E). However, miR-548t-3p-treated cells exhibited a loss of this stiffness-dependent nesprin tension response, as the FRET ratio remained unchanged across all ECM stiffness conditions (Fig. 3 C and F). This finding indicates a disruption in nuclear force transmission due to miR-548t-3p-mediated depletion of lamin A/C. Notably, co-transfection with a miR-548t-3p inhibitor restored the normal tension gradient, as nuclear tension levels once again increased proportionally to ECM stiffness (Fig. 3 D and G). Quantitative analysis of the FRET-based nesprin biosensor further confirmed that miR-548t-3p significantly reduced nesprin tension across all tested substrate stiffness conditions, particularly in cells cultured on 40 kPa gels (Fig. 3 H–J). These findings establish miR-548t-3p as a critical regulator of nuclear mechanotransduction by impairing lamin A/C expression, thereby compromising nuclear envelope mechanosensitivity and stiffness-dependent force transmission.

Figure 3.

LMNA degraded by miRNA exhibits unaltered nuclear membrane tension in response to increased matrix stiffness. (A) Schematic representation of cell shapes in ECM with varying stiffness. Representative images show the Nespin-TS biosensor in HeLa cells seeded at 1, 5, and 40 kPa. (B) Vehicle, (C) miR-548t-3p, and (D) mir-548t-3p transfected with Inh. Scale bar, 10 μm. FRET of the nuclear envelope was extracted using ImageJ. The FRET ratio of the Nesprin-TS biosensor is significantly increased when the substrate stiffness is 40 kPa compared with 1 kPa. Normalized CFP/FRET ratios of the Nesprin-TS biosensor after treatment with (E) vehicle, (F) miR-548t-3p, and (G) miR-548t-3p plus inhibitor under mechanical stresses of 1 kPa, 5 kPa, and 40 kPa. All error bars represent SEM (black, gray, and charcoal; (E) n = 80, 110, and 120, (F) n = 70, 160, and 130, (G) n = 30, 10, and 70, respectively; ^∗∗: p < 0.01, ^∗∗∗∗: p < 0.0001, Student's t-test). (H) Normalized CFP/FRET ratio of Nesprin-TS biosensor in HeLa cells treated with miRNA mimic and miRNA inhibitor seeded on 1 kPa. Under identical extracellular matrix stiffness conditions, miRNA-mediated inhibition of LMNA results in a reduction of the FRET ratio of the Nesprin-TS biosensor. All error bars represent SEM (gray, blue, and green; n = 80, 70, and 30, respectively; ^∗: p < 0.05, ^∗∗: p < 0.01, Student's t-test). (I) Normalized CFP/FRET ratio of Nesprin-TS biosensor in HeLa cells treated with miRNA mimic and miRNA inhibitor seeded on 5 kPa. Under identical extracellular matrix stiffness conditions, miRNA-mediated inhibition of LMNA results in a reduction of the FRET ratio of the Nesprin-TS biosensor. All error bars represent SEM (gray, blue, and green; n = 110, 160, and 10, respectively; ^∗∗: p < 0.01, ^∗∗∗∗: p < 0.0001, Student's t-test). (J) Normalized CFP/FRET ratio of Nesprin-TS biosensor in HeLa cells treated with miRNA mimic and miRNA inhibitor seeded on 40 kPa. Under identical extracellular matrix stiffness conditions, miRNA-mediated inhibition of LMNA results in a reduction of the FRET ratio of the Nesprin-TS biosensor. All error bars represent SEM (gray, blue, and green; n = 120, 130, and 70, respectively; ^∗∗∗∗: p < 0.0001, Student's t-test).

Cellular traction force is dependent on nuclear tension mediated by miRNA-548t-3p

Cellular traction force plays a crucial role in mechanotransduction by regulating cell adhesion, migration, and mechanical interactions with the extracellular matrix (ECM) (40,41,42). Given the observed effects of miR-548t-3p on nuclear tension, we hypothesized that this microRNA would also influence traction force generation. To test this, we measured cellular traction forces using the fluorescent bead displacement method during trypsin-mediated detachment (41).

Traction force maps revealed that cells treated with miR-548t-3p exhibited a significant reduction in traction force compared with control cells, particularly at sites of focal adhesion (Fig. 4 A). Despite this reduction in traction force, the projected cell area remained largely unaffected by miR-548t-3p treatment (Fig. 4 B). However, quantitative analysis of root mean-square (RMS) traction forces demonstrated a marked decrease in miR-548t-3p-treated cells on both 5 kPa and 40 kPa substrates (Fig. 4 C). Notably, cells treated with miR-548t-3p on 40 kPa gels generated traction forces comparable to control cells on 5 kPa gels, suggesting that lamin A/C depletion impairs stiffness-dependent force generation. These results demonstrate that miR-548t-3p-mediated depletion of lamin A/C compromises cellular traction force by disrupting nuclear tension and cytoskeletal organization.

Figure 4.

Cell adhesion and actomyosin-induced traction are regulated by lamin A/C expression. (A) The traction force images and graph in HeLa cells transfected with miRNA mimic (red; n = 7). Hot and cold colors of RMS traction indicate high and low traction force. Scale bar, 10 μm. Quantification of (B) Projected cell area and (C) RMS traction of HeLa cells. The box and whisker plots display the median, interquartile range, and the positions of the minimum and maximum values. All error bars represent SEM (gray, blue, charcoal, and navy; n = 7, 4, 8, and 6, respectively, ^∗: p < 0.05, Student's t-test, gray, pink, charcoal, and red; n = 8, 6, 8, and 5, respectively; ^∗: p < 0.05,^∗∗∗: p < 0.001, Student's t-test).

Discussion

This study demonstrates that miR-548t-3p directly targets LMNA, reducing lamin A/C expression and altering nuclear mechanical properties. Lamin A/C depletion compromises nuclear-cytoskeletal connectivity, resulting in decreased nuclear tension and impaired focal adhesion dynamics (Figs. 1 and 2). Furthermore, miR-548t-3p attenuates nuclear mechanosensitivity across substrates of varying stiffness, as evidenced by reduced traction forces (Figs. 3 and 4). These findings establish miR-548t-3p as a key regulator of cellular mechanotransduction through modulation of lamin A/C.

Lamin A/C is a critical structural component that mediates nuclear mechanosensing and adaptation to mechanical stimuli (4). It maintains nuclear architecture, facilitates coupling between the cytoskeleton and the nucleus, and preserves mechanical homeostasis (43,44). Loss of lamin A/C leads to increased nuclear deformability, impaired mechanotransduction, and disruption of force-sensitive signaling pathways (45). Notably, LMNA expression itself is regulated by substrate stiffness, increasing under high-rigidity conditions to sustain nuclear integrity and mechanotransduction capacity (46,47).

miRNAs have emerged as potent mechanotransducers that orchestrate cellular responses to mechanical cues (22,23,48). For instance, miR-21 is upregulated by shear stress and promotes endothelial cell survival (49), whereas miR-20a regulates osteoblast differentiation under mechanical loading (17). Expanding this concept, we identify miR-548t-3p as a novel regulator of nuclear mechanosensitivity and focal adhesion dynamics via its modulation of lamin A/C. These findings implicate miR-548t-3p in the regulation of nuclear-cytoskeletal coupling and mechanical homeostasis. Importantly, although our study focuses on mechanical and molecular phenotypes such as nuclear tension and traction force, it does not directly examine functional consequences such as cell migration, spreading, or mechanical adaptation. In addition, because lamin A/C plays a central role in maintaining nuclear structure and gene regulatory networks, we cannot rule out the possibility that miR-548t-3p-induced lamin A/C depletion may have indirectly altered the expression of other genes before the assays. Therefore, some of the observed phenotypes may reflect broader transcriptional effects rather than direct mechanotransduction mechanisms. Future studies should clarify these indirect effects and examine how mechanical and gene expression changes are interrelated following LMNA suppression.

Mechanical homeostasis governs fundamental physiological and pathological processes such as stem cell fate decisions, metastasis, and tissue remodeling (50,51). miRNAs are well positioned to influence these processes by rapidly coordinating signaling cascades in response to mechanical inputs (22,52). A deeper understanding of miR-548t-3p-mediated mechanotransduction may thus provide insights into novel diagnostic or therapeutic strategies for diseases involving aberrant nuclear mechanics.

Further investigation should explore whether miR-548t-3p regulates additional nuclear envelope or cytoskeletal elements beyond lamin A/C and delineate its downstream signaling pathways. Mapping its broader gene regulatory network will be crucial for understanding how this miRNA orchestrates cellular mechanics. Notably, reduced lamin A/C expression has been associated with enhanced nuclear deformability and invasiveness in breast cancer cells (53,54), whereas elevated levels of miR-548 correlate with poor prognosis and metastasis in gastric cancer (55). These observations suggest that miR-548t-3p and lamin A/C expression profiles may serve as prognostic indicators in mechanically responsive tumors.

Beyond cancer, lamin A/C dysregulation is implicated in disorders involving aberrant tissue mechanics, including cardiomyopathies and muscular dystrophies (56). Mechanosensitive nuclear signaling is also critical in stem cell differentiation and tissue regeneration, highlighting the therapeutic potential of targeting miRNAs to modulate nuclear mechanics (57). By identifying miR-548t-3p as a regulator of lamin A/C-dependent mechanotransduction, this study provides mechanistic insights into miRNA-mediated mechanical homeostasis and suggests its relevance as a therapeutic target in regenerative medicine and diseases associated with disrupted mechanobiology (58).

Conclusions

In this study, we identified miR-548t-3p as a key regulator of nuclear mechanics through its direct targeting and downregulation of lamin A/C. Using a combination of FRET-based biosensors, immunofluorescence imaging, and traction force microscopy, we demonstrated that miR-548t-3p significantly reduces lamin A/C expression, resulting in decreased nuclear tension, altered nuclear morphology, impaired focal adhesion dynamics, and diminished cellular traction force generation. Importantly, these effects culminate in the loss of stiffness-dependent mechanosensitivity, underscoring the critical role of lamin A/C in nuclear mechanotransduction. Our findings reveal a previously unrecognized role for miR-548t-3p in regulating mechanical homeostasis at the single-cell level and highlight its potential involvement in pathological conditions where mechanical signaling is disrupted. These insights advance our understanding of the molecular mechanisms underlying cell–matrix interactions and suggest that targeting the miR-548t-3p-lamin A/C axis could offer new therapeutic avenues for diseases associated with aberrant mechanotransduction.

Acknowledgments

This work was supported by National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIT) (RS-2023-00279771 and RS-2024-00400827).

Author contributions

D. L., J.S., and T.-J.K. conceptualized the project, designed the experiments, and wrote the manuscript. D.L performed the experiments. D.L., J.S., Y.J., and M.S. analyzed and discussed the data. All the authors have read and approved the final version of this manuscript.

Declaration of interests

The authors declare no competing interests.

Editor: Guy Genin.