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High matrix stiffness accelerates migration of hepatocellular carcinoma cells through the integrin β1-Plectin-F-actin axis

BMC Biology volume 23, Article number: 8 (2025) Cite this article

Abstract

Background

Abundant research indicates that increased extracellular matrix (ECM) stiffness significantly enhances the malignant characteristics of hepatocellular carcinoma (HCC) cells. Plectin, an essential cytoskeletal linker protein, has recently emerged as a promoter of cancer progression, particularly in the context of cancer cell invasion and metastasis. However, the responsiveness of plectin to changes in ECM stiffness and its impact on HCC progression remain unclear. In this study, we aimed to investigate whether plectin responds to variations in ECM stiffness and to explore its involved molecular mechanisms in regulating HCC cell migration.

Results

Our results showed that, when compared with control group (7 kPa), high ECM stiffness (53 kPa) boosts HCC cell migration by upregulating plectin and integrin β1 expression and increasing F-actin polymerization. Knockdown of integrin β1 negated the high stiffness-upregulated plectin expression. Furthermore, reducing either plectin or integrin β1 levels, or using latrunculin A, effectively prevented the high ECM stiffness-induced F-actin polymerization and HCC cell migration.

Conclusions

These findings demonstrate that integrin β1-plectin-F-actin axis is necessary for high matrix stiffness-driven migration of HCC cells, and provide evidence for the critical role of plectin in mechanotransduction in HCC cells.

Background

The incidence and mortality rates of hepatocellular carcinoma (HCC) are on an upward trajectory globally [1], with a particularly alarming increase observed among younger populations [2]. The high invasion and metastatic potential of HCC are the key factors contributing to its poor prognosis [3]. Most cases of HCC occur in the context of significant liver fibrosis and cirrhosis, characterized by excessive cross-linking and deposition of extracellular matrix (ECM) components, such as collagen, leading to a marked increase in ECM stiffness [4]. In various solid tumors, an ECM stiffness gradient that increases from the tumor core to the periphery has been demonstrated to promote tumor cell proliferation, invasion, metastasis, drug resistance, and recurrence [5, 6]. The increase in matrix stiffness is not only a clinical indicator of disease progression but also a significant driving factor in tumorigenesis.

The dynamic remodeling of cytoskeleton is crucial for cell migration [7]. Plectin, a critical multi-domain cytoskeletal linker protein, interacts with F-actin to participate in the regulation of cytoskeletal dynamics [8, 9]. Plectin plays an essential role in cell adhesion, movement, and deformation—processes that are indispensable for cell migration [10]. Upregulated expression of plectin has been observed in various cancer cells and tumor tissues, suggesting its potential role in promoting tumor development [11]. Furthermore, the expression levels of plectin are closely associated with tumor invasion and patient prognosis, making it a promising candidate as a prognostic biomarker and therapeutic target [11,12,13].

Although studies have indicated that both matrix stiffness and plectin are related to the migration of HCC cells [14,15,16], whether ECM stiffness modulates tumor cell migration through plectin remains an open question. This study aims to fill this knowledge gap by investigating the role of plectin in HCC cell migration under the influence of ECM stiffness and by exploring the underlying molecular mechanisms. A deeper understanding of the interplay between ECM stiffness, plectin, and HCC cell migration will not only provide new insights into the molecular mechanisms of HCC but also offer potential new targets for the development of novel therapeutic strategies and the improvement of patient outcomes.

Results

Plectin is upregulated in HCC tissues, and high matrix stiffness promotes plectin expression in HCC cells

Expanding on our prior findings regarding the significant upregulation of plectin in both HCC tissues and cells [14], we delved deeper into the interplay between matrix stiffness and plectin expression in HCC tissues. Initially, through comparative analysis of plectin expression levels in HCC tissues versus normal liver tissues, we observed a notable elevation in plectin mRNA expression within HCC tissues (n = 371 vs. n = 276, Fig. 1A). Considering lysyl oxidase (LOX) and collagen 1 (COL1) expression levels as proxies for matrix stiffness [17, 18], we utilized the median expression values of LOX and COL1 to categorize HCC patients into high and low matrix stiffness groups (COL1A1High-LOXHigh, 116 cases; COL1A1Low-LOXLow group, 117 cases). Comparative analysis revealed a significant upregulation of plectin expression in HCC tissues from the high matrix stiffness group compared to the low matrix stiffness group (Fig. 1B). To validate the responsiveness of plectin in HCC cells to changes in ECM stiffness, we employed polyacrylamide hydrogels with two distinct stiffness levels (7 kPa and 53 kPa, representing the physiological elasticity of normal liver and HCC tissues, respectively) for cell culture over a 48-h period [19,20,21,22,23]. Using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot techniques, we assessed plectin expression in MHCC-97H and MHCC-97L HCC cell lines. Results showed significant elevated plectin expression in HCC cells cultured on high matrix stiffness substrates compared to that of cultured on low stiffness substrates (Fig. 1C and D). Our findings underscore plectin's capacity to respond to alterations in ECM stiffness, highlighting its upregulation in HCC cells within high stiffness environments.

Fig. 1
figure 1

Plectin upregulation in HCC. A Plectin mRNA expression in samples from the TCGA database in HCC and normal tissues. B The expression levels of plectin were markedly increased in COL1A1High-LOXHigh TCGA-HCC tissues (n = 119) compared to those in COL1A1Low-LOX.Low TCGA-HCC tissues (n = 120). C Detection of plectin mRNA levels in MHCC-97H and MHCC-97L cells under different ECM stiffness. D Western blot detection of plectin protein levels in MHCC-97H and MHCC-97L cells under different ECM stiffness. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase. Data are presented as mean ± SD; n = 3, * p < 0.05; *** p < 0.001

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High matrix stiffness promotes the migration of HCC cells, and knockdown of plectin inhibits this effect

To explore the influence of matrix stiffness on the migration potential of HCC cells, we conducted scratch assays to assess the migration capabilities under various matrix stiffness conditions. The findings revealed a notable increase in HCC cell migration under high matrix stiffness conditions (Fig. 2A). Given previous studies indicating that elevated matrix stiffness can upregulate plectin expression, we further investigated the potential role of plectin in HCC cell migration. Using shRNA-mediated gene silencing in MHCC-97H and MHCC-97L cells, we successfully reduced plectin expression levels. Western blot analysis confirmed a significant decrease in plectin levels in cells transfected with plectin-specific shRNA (shPlectin) compared to those transfected with non-targeting control shRNA (shNC) (Fig. 2B). For the forthcoming experiments, MHCC-97H cells will be subjected to shPlectin-2 with higher knockdown efficiency, while MHCC-97L cells will be subjected to shPlectin-1. Subsequent scratch assay demonstrated that, even under conditions of high matrix stiffness, the migration of HCC cells in the shPlectin group did not exhibit significant enhancement, contrasting sharply with the shNC group (Fig. 2C). These findings underscore the essential function of plectin in mediating the migration of HCC cells induced by high matrix stiffness.

Fig. 2
figure 2

The ECM stiffness regulates HCC cell migration through plectin. A Wound healing assays of MHCC-97H and MHCC-97L cells cultured on hydrogels of different stiffness for 48 h. B Western blot analysis was performed to assess the efficiency of plectin knockdown in cells, followed by quantitative analysis. C Wound healing assays of MHCC-97H and MHCC-97L cells after plectin knockdown. Data are presented as mean ± SD; n = 3 *p < 0.05, **p < 0.01, *** p < 0.001, Scale bar: 200 μm

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Expression of plectin and integrin β1 are positively correlated In HCC tissues, and high matrix stiffness promotes the expression of integrin β1

The integrin family, as crucial receptors for the ECM, regulates the interaction between cells and the external environment [24]. Integrin β1, an important member of this family, plays a crucial role in the pathogenesis of various cancers. In pancreatic cancer, integrin β1 is closely associated with tumor vascular invasion, distant metastasis, and patient survival rates [25]. In breast cancer, integrin β1 participates in the TGFβ signaling pathway, promoting tumor metastasis to the bone [26]. Additionally, in small cell lung cancer, integrin β1 is instrumental in regulating tumor metastasis [27]. Studies have shown that upregulation of integrin β1 expression and activation of related signaling pathways can enhance cell invasion ability [28]. In gastric cancer patients, integrin β1 also promotes cell proliferation, migration, and invasion [29]. Therefore, integrin β1 not only plays a role in sensing changes in the ECM environment but also participates in cell–cell interactions and activation of signaling pathways, significantly impacting the growth, spread, and invasion of tumor cells.

Therefore, we first analyzed the gene expression data of plectin and integrin β1 in HCC tissues. The results showed that plectin and integrin β1 are significantly positively correlated in HCC tissues (Fig. 3A), suggesting that they may have synergistic effects in the HCC development. Further analysis revealed that compared to normal liver tissues, integrin β1 is significantly upregulated in HCC tissues (Fig. 3B). This confirms the potential important role of integrin β1 in the development of HCC. In addition, we found that compared to the low matrix stiffness group, the expression of integrin β1 is significantly upregulated in the high matrix stiffness group (HCC tissues, Fig. 3C). This suggests that changes in ECM stiffness may regulate the biological behavior of HCC cells by transmitting mechanical signals through integrin β1. The expression of integrin β1 in HCC cells cultured under different matrix stiffness conditions was detected by RT-qPCR and Western blot. The results showed that high matrix stiffness significantly promotes the expression of integrin β1 in HCC cells (Fig. 3D and E).

Fig. 3
figure 3

Correlation of plectin and integrin β1 expression in HCC tissues. A Gene correlation: Scatter plot and fitted line from Spearman correlation analysis between plectin and integrin β1 expression. Each point represents a sample, with gene expression on the X and Y axes. B Integrin β1 mRNA expression in samples from TCGA database in HCC and normal tissues. C The expression levels of integrin β1 were significantly increased in COL1A1High-LOXHigh TCGA-HCC tissues (n = 119) compared to those in COL1A1Low-LOX.Low TCGA-HCC tissues (n = 120). D Detection of integrin β1 mRNA levels in MHCC-97H and MHCC-97L cells under different ECM stiffness. E Western blot detection of integrin β1 protein levels in MHCC-97H and MHCC-97L cells under different ECM stiffness. Data are presented as mean ± SD; n = 3, *p < 0.05, ***p < 0.001

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Knockdown of integrin β1 inhibits high matrix stiffness-increased plectin expression and HCC cell migration

It has been established that high matrix stiffness can promote the expression of integrin β1. To investigate whether plectin senses changes in ECM stiffness through integrin β1, we transiently knocked down integrin β1 in HCC cells using siRNA. Western blot showed that compared to the control group (siNC), the knockdown of integrin β1 (si-Integrin β1) was successful, accompanied by a decrease in plectin expression (Fig. 4A). This finding suggests that integrin β1 may be involved in regulating the expression of plectin. Further Western blot analysis revealed that under high matrix stiffness conditions, the si-Integrin β1 group did not upregulate plectin expression (Fig. 4B). This result indicates that integrin β1 is crucial for maintaining the upregulation of plectin expression under high matrix stiffness, supporting the hypothesis that plectin responds to changes in ECM stiffness through integrin β1.

Fig. 4
figure 4

Integrin β1 knockdown inhibits high ECM stiffness-increased plectin expression and HCC cell migration. A Western blot analysis was conducted to assess the efficiency of integrin β1 knockdown in cells, along with plectin expression, followed by quantitative analysis. B Following integrin β1 knockdown, Western blot analysis was performed to investigate the effect of ECM stiffness on plectin expression. C Wound healing assays were conducted to analyze migration of MHCC-97H and MHCC-97L cells after integrin β1 knockdown. Data are presented as mean ± SD; n = 3, * p < 0.05, ** p < 0.01, Scale bar: 200 μm

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To assess the impact of integrin β1 knockdown on the migration of HCC cells, we performed scratch assays. The results showed that under high matrix stiffness conditions, knockdown of integrin β1 significantly inhibited the promoting effect of high matrix stiffness on the migration ability of HCC cells (Fig. 4C). This finding highlights the indispensable function of integrin β1 in facilitating the migration of HCC cells triggered by increased ECM stiffness.

Knockdown of plectin and integrin β1 attenuates the promoting effect of high matrix stiffness on F-actin polymerization

Cell migration heavily relies on the pivotal involvement of the cytoskeletal protein F-actin [30], and its regulation is closely associated with plectin. Immunofluorescence experiments revealed that HCC cells cultured under high matrix stiffness exhibited higher fluorescence intensity of F-actin, thicker and longer actin stress fibers, increased quantity, and clearer cytoskeletal structure (Fig. 5A). To further investigate the roles of plectin and integrin β1 in high matrix stiffness-induced F-actin polymerization, we cultured HCC cells from control groups (shNC, siNC) and knockdown groups (shPlectin, si-Integrin β1) on substrates with different matrix stiffness. Immunofluorescence results showed that knockdown of plectin and integrin β1 both weakened the promoting effect of high matrix stiffness on F-actin polymerization (Fig. 5B and C).

Fig. 5
figure 5

Plectin or integrin β1 knockdown attenuates high ECM stiffness-promoted F-actin polymerization. A Confocal immunofluorescence images of F-actin in MHCC-97H and MHCC-97L cells cultured on different stiffness hydrogels for 48 h. B Representative immunofluorescence images of the effect of matrix stiffness on F-actin after plectin knockdown. C Representative immunofluorescence images of the effect of matrix stiffness on F-actin after integrin β1 knockdown. D Representative immunofluorescence images of the effect of matrix stiffness on vimentin after plectin knockdown. (Scale bar: 25 μm)

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In addition to binding with F-actin, plectin is a major intermediate filament (IF) binding protein [31]. Vimentin, a crucial component of the IF family, and its expression level is closely related to the migration, invasion, and metastasis of tumor cells [32, 33]. We also determined the changes of IF and found that under high matrix stiffness conditions, the expression of vimentin significantly increases, and this enhancement can be inhibited by plectin knockdown (Fig. 5D), indicating a complicated interaction between plectin and cytoskeleton proteins.

Latrunculin A reverses high matrix stiffness-induced F-actin aggregation and HCC cell migration

After confirming the role of matrix stiffness in F-actin aggregation, we used Latrunculin A (Lat-A) to depolymerize F-actin in order to investigate the relationship between F-actin and HCC cell migration under matrix stiffness conditions. Immunofluorescence experiments revealed that treatment with Lat-A significantly reversed the promotion of F-actin polymerization induced by high matrix stiffness (Fig. 6A). Subsequently, scratch assays were conducted to assess the effect of Lat-A treatment on HCC cell migration. The results showed that under high matrix stiffness conditions, treatment with Lat-A markedly reduced the migration of HCC cells (Fig. 6B). These findings demonstrate the significant impact of F-actin in HCC cell migration.

Fig. 6
figure 6

Latrunculin A reverses high ECM stiffness-induced F-actin polymerization and HCC cell migration. A Representative immunofluorescence images of high ECM stiffness-induced F-actin polymerization after Latrunculin A treatment. Scale bar: 25 μm. B Wound healing assays of MHCC-97H and MHCC-97L cells in the presence of Latrunculin A. Data are presented as mean ± SD; n = 3, * p＜0.05, ** p＜0.01, Scale bar: 200 μm

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Discussion

Recent studies have emphasized the profound influence of matrix stiffness within the tumor microenvironment on various behaviors of tumor cells, including migration, proliferation, survival, and signal transduction [12, 34, 35]. Thus, delving deeper into the regulatory mechanisms underlying matrix stiffness on cancer cell migration undoubtedly presents an ideal avenue for gaining a better understanding of metastasis and uncovering novel targets for cancer intervention. Moreover, emerging evidence suggests that plectin, a cytoskeletal linker protein, could be a central factor in orchestrating tumor cell migration [12, 13, 36, 37]. However, the molecular mechanism of its regulation under matrix stiffness remains unclear. Hence, elucidating how plectin responds to alterations in matrix stiffness and subsequently modulates cancer cell migration is imperative. This study investigates the interaction between ECM stiffness, plectin, integrin β1, and F-actin, revealing that plectin can regulate F-actin polymerization and affect HCC cell migration by responding to changes in ECM stiffness through integrin β1 (Fig. 7), thereby offering novel insights into the molecular mechanisms governing HCC cell migration.

Fig. 7
figure 7

Proposed schematic diagram of matrix stiffness regulation on HCC cell migration through the integrin β1-plectin-F-actin Axis. Under the high ECM stiffness, plectin sense the change of mechanical environment through integrin β1 and upregulates. The upregulation of plectin, thereby enhances F-actin polymerization and accelerates the migration of HCC cells

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The relationship between the cell cytoskeleton and the external milieu is both complex and intimate [38, 39]. The tension generated by cytoskeletal contraction serves as a means to sense and respond to the mechanical properties of the ECM [40,41,42]. Furthermore, plectin has been shown to influence the organization of IF cytoskeleton, thereby exerting an influence on cell morphology and behavior [31, 43, 44], hinting at its potential role as a crucial mechanical sensor within cells. Our study validates this notion by demonstrating the upregulation of plectin in HCC tissues, which correlates positively with matrix stiffness. Moreover, at the cellular level, we confirm that plectin indeed responds to changes in matrix stiffness, with elevated matrix stiffness robustly inducing its expression.

It is well-established that liver disease progression is accompanied by an increase in matrix stiffness, with mounting evidence suggesting that this heightened stiffness significantly enhances the malignant properties of HCC cells [45,46,47]. Intriguingly, our findings reveal that elevated matrix stiffness promotes HCC cell migration, while plectin knockdown attenuates this effect, further confirming the key role of plectin in regulating HCC cell migration under matrix stiffness.

As a critical ECM receptor [48], integrin β1 exhibits a positive correlation with plectin expression in HCC tissues, with elevated matrix stiffness further augmenting integrin β1 expression. Integrin β1 is crucial for sustaining the upregulation of plectin expression under conditions of high matrix stiffness, and it plays a pivotal role in matrix stiffness-induced HCC cell migration. Therefore, targeting integrin β1 and its associated signaling pathways may represent an effective therapeutic strategy. However, the mechanisms of action of integrin β1 in HCC are likely to be quite complex, involving a variety of signaling pathways and cellular biological processes. Consequently, future research is required to further explore the specific mechanisms of action of integrin β1 in HCC.

Plectin, through its interactions with actin and microtubules, exerts an influence on the mechanical properties and dynamic characteristics of the cell cytoskeleton system [8, 49, 50]. F-actin, in particular, mediates the formation of cellular structures such as lamellipodia, filopodia, stress fibers, and focal adhesions, thereby directly participating in various mechanobiological processes, including cell migration, cytokinesis, intracellular substance transport, and endocytosis [51,52,53]. Immunofluorescence experiments demonstrate that high matrix stiffness promotes F-actin polymerization, while plectin and integrin β1 knockdown attenuate this phenomenon. Lastly, we observe that Latrunculin-A can reverse high matrix stiffness-induced F-actin polymerization and HCC cell migration, further validating the critical importance of F-actin in HCC cell migration. However, our results in this study are acquired from in vitro experiments, consequently, further in vivo studies are needed to substantiate these findings down the line.

Beyond its interaction with F-actin, plectin serves as a primary binding protein for IFs in a variety of tissues and cells [54,55,56]. IFs are essential for preserving cellular integrity and resisting mechanical stress, and their structural reorganization during tumor development is closely related to cellular migration [57, 58]. Studies have shown that the rearrangement of IFs, including vimentin, nestin, and keratins, is associated with the malignancy of cancer [59,60,61]. We have observed that under conditions of high matrix stiffness, the fluorescence intensity, coverage area, and expression levels of vimentin are all increased, and the knockdown of plectin significantly weakens this promotional effect. Plectin plays a significant role in modulating the effects of matrix stiffness on vimentin expression, but it is currently unclear whether other IFs are also subject to similar regulation. Moreover, plectin is capable of linking IF with microtubules, actin, and membrane components, forming a complex cytoskeletal network that is crucial for the mechanical stability and function of cells [31, 62]. Therefore, future research could focus on exploring the interactions between plectin and other IF proteins, as well as their roles within the cytoskeletal network.

Conclusions

In this study, we demonstrate that elevated ECM stiffness induces the upregulation of plectin via integrin β1, subsequently fostering F-actin polymerization, which in turn markedly boosts the migration of HCC cells. Our study elucidates the intricate interactions among matrix stiffness, plectin, integrin β1 and F-actin, highlighting their vital modulatory functions in HCC cell migration. These findings not only provide a better understanding of HCC cell migration from a mechanical microenvironment-dependent plectin regulation mechanism, but also suggest a promising therapeutic strategy targeting plectin in HCC treatment.

Materials and methods

Preparation of polyacrylamide (PAA) gels

Polyacrylamide (PA) hydrogels coated with collagen type I (COL1) and possessing adjustable mechanical properties were prepared by varying the concentrations of acrylamide (Sigma-Aldrich, MO, USA) and bis-acrylamide (Sigma-Aldrich, MO, USA). The preparation of PA hydrogels and the measurement of their Young’s modulus followed our previously reported protocol [63]. In brief, acrylamide and bis-acrylamide were mixed in desired proportions (e.g., 40% acrylamide and 2% bis-acrylamide) along with 10% ammonium persulfate (APS; 1/200 volume, Beyotime, Shanghai, China) and N,N,N′,N′-Tetramethylethylenediamine (TEMED; 1/2000 volume, Macklin, Shanghai, China). Upon polymerization, the gels were thoroughly washed with distilled water to remove residual reagents. Subsequently, the gels were further cross-linked using sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino) hexanoate (sulfo-SANPAH; Thermo Fisher Scientific, MA, USA) under 365 nm ultraviolet exposure and subsequently conjugated with 0.1 mg/mL rat-tail COL1 (Hangzhou Shengyou Biotechnology, Zhejiang, China).

Bioinformatics analysis

Transcriptional expression data of the plectin gene from both human liver tissue and HCC tissue, along with relevant clinical information, were acquired from The Cancer Genome Atlas (TCGA) website (https://portal.gdc.cancer.gov/). The RNA-seq gene expression data were presented in transcripts per million (TPM) format per thousand bases of mapped reads and were subjected to log2 transformation for subsequent analysis. Studies have indicated that the expression levels of LOX and COL1A1 can be used to assess the grade of matrix stiffness [17, 18, 46]. HCC patients were stratified based on the expression of LOX and COL1A1, with the median expression value serving as the boundary. Patients with expression values above the 50th percentile were defined as High, while those below were defined as Low. Among 363 patients, 116 cases with high expression of both COL1A1 and LOX were categorized into the high matrix stiffness group, and 117 cases with low expression of both COL1A1 and LOX were categorized into the low matrix stiffness group. The ggpubr package was utilized to plot the expression of the target genes between patients with different stiffness levels. All data were downloaded from the TCGA database, and readers can obtain the data from the official website or request all codes and cleaned data from the authors via email, thus circumventing the need for ethical approval.

Cell culture

The human HCC cell lines MHCC-97H, MHCC-97L were procured from the Liver Cancer Institute, Zhongshan Hospital, Fudan University (Shanghai, China). Cells were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Gbico, Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, UT, USA), 2 mM glutamine (Solarbio, G8230, Beijing, China), 100 μg/mL streptomycin (Solarbio Life Sciences, Beijing, China), and 100 U/mL penicillin (Solarbio Life Sciences, Beijing, China) at 37 °C in a humidified atmosphere containing 5% CO2. Upon reaching 80–90% confluence, cells were detached using a solution of 0.25% trypsin-0.02% EDTA (Procell, Wuhan, China) and sub-cultured at a density of 2.5 × 103 cells/cm2.

Virus production and transfection

The plasmid system employed the following plasmids: pLKO.1-EGFP-PURO-PLEC (Unibio, Hunan, China.), psPAX2 (Invitrogen, CA, USA), and pMD2.G (Invitrogen, CA, USA), for lentiviral infection. Initially, the mixed plasmids were transfected into 293 T cells, gently agitated, and then incubated in a cell culture incubator for 48 h. The unpurified viral supernatant was collected and stored at -80 °C. Subsequently, approximately 1 × 104 liver cancer cells per well (MHCC-97H/MHCC-97L) were seeded in a 12-well plate and infected with the collected viral supernatant. Additionally, 8 μg/mL polybrene (Sigma Aldrich, MO, USA) was added to enhance the infection rate. After 48 h of incubation, the fluorescence intensity of the cells was observed using an inverted fluorescence microscope. Then, 2 μg/mL puromycin was added, and stable clones were selected for one week. Finally, Western blotting was performed to assess the expression of the target protein. The target sequences of shRNA gene silencing plasmids were as follows:

shPlectin-1: 5'-GCCUCUUCAAUGCCAUCAUTT-3' and 5'-AUGAUGGCAUUGAAGAGGCTT-3'; shPlectin-2: 5'-GCCAGUACAUCAAGUUCAUTT -3' and 5'- AUGAACUUGAUGUACUGGCTT-3'.

siRNA transfection

The specific siRNA and negative control siRNA were purchased from Tsingke (Beijing, China). The siRNA sequences are as follows: siNC: 5'-UUCUCCGAACGUGUCACGUTT-3' and 5'-ACGUGACACGUUCGGAGAATT-3'. si-Integrin β1: 5'-CCACAGCAGUUGGUUUUGCTT-3' and 5'-GCAAAACCAACUGCUGUGGTT-3'. For transfection experiments, Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, MA, USA) was used to transiently transfect siRNA into HCC cells for 48 h, after which the cells were collected for further experiments.

Real-time quantitative reverse transcription PCR

According to the manufacturer's instructions, cellular total RNA was obtained using the RNA extraction kit (Takara, Kyoto, Japan). Subsequently, RNA was converted into cDNA using the ReverTra Ace RT-qPCR kit (RR047Q, Takara, Japan) following the manufacturer's protocol. Gene expression was analyzed by RT-qPCR using SYBR Green PCR Master Mix (RR820A, Takara, Japan), with GAPDH serving as an internal control. The primer information is as follows: GAPDH: 5’-GGTATGACAACGAATTTGGC-3’ and 5’-GAGCACAGGGTACTTTATTG-3’, integrin β1: 5’-GCATCCCTGAAAGTCCCAA-3’ and 5’-CACTGTCCGCAGACGCAC-3’, plectin: 5’-CTCGGAGCTGGAGCTGAC-3’ and 5’-ACCAGGCTGATGGTCTTGA-3’. The relative expression of RNAs was calculated using the 2−ΔΔCt method and normalized to GAPDH transcripts.

Protein extraction and Western blot

Total protein from MHCC-97H and MHCC-97L cells was extracted on ice using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with 1% protease inhibitor (Beyotime, Shanghai, China) and 2% phosphatase inhibitor. The protein concentration of the lysates was determined using the BCA protein assay kit (Beyotime, Shanghai, China). The lysates were mixed with 5 × loading buffer, boiled at 100 °C for 10 min to denature the proteins, and then separated on 8% SDS-PAGE gels with each protein sample loaded at 30 μg. Subsequently, the proteins were transferred onto 0.45 μm PVDF membranes (Millipore, Billerica, MA, USA) by electroblotting. The membranes were blocked with 5% skim milk in TBST buffer (0.05% Tween-20 in Tris-buffered saline) for 1 h at room temperature. Then, the membranes were incubated overnight at 4 °C with primary antibodies diluted in antibody dilution buffer (Beyotime, Shanghai, China), including plectin (ab32528, Abcam, UK), integrin β1 (ab179471, Abcam, UK), and GAPDH (bsm-33033 M, Bioss, China). After washing the membranes with TBST four times for 5 min each, they were incubated with appropriate secondary antibodies (ZSBIO, Beijing, China) diluted in 5% skim milk at room temperature for 1 h. The membranes were then washed with TBST four times for 5 min each and visualized using the ECL detection system (Bio-OI, Guangzhou, China). GAPDH on the same membrane was used as a loading control, and it was quantified by ImageJ 1.8.0 (National Institutes of Health). The average ratio of target protein to GAPDH from the three control groups is used as the reference value for normalization, where the ratios of target protein to GAPDH in the control groups are each divided by this average to normalize the data.

Immunofluorescence staining

Cells cultured on PA hydrogels with different substrate stiffness in 24-well plates were subjected to respective treatments. Subsequently, the cells were fixed in 4% paraformaldehyde for 15 min and washed three times with PBS (Biosharp, Hefei, China). Next, cells were permeabilized and blocked with 0.25% Triton X-100 (Solarbio, Beijing, China) and 0.5% BSA (Biosharp, Hefei, China) in PBS for 1 h, followed by three washes with PBS. Then, cells were incubated with primary antibody overnight at 4 °C, after five washes in PBS, and then incubated with the secondary antibody at 37'C for 1 h. After three washes with PBS, cells were stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Solarbio, Beijing, China) for 10 min. Following five washes with PBS, PA hydrogels were removed from the 24-well plate and placed on glass slides. Images were acquired using confocal microscopy (Leica, TCS SP8 DIVE / DMi8, Germany). The antibodies employed in this study included Actin-Tracker Red-Rhodamine (dilution, 1:200; C2207S, Beyotime, Shanghai, China), vimentin rabbit monoclonal antibody (dilution, 1:200, ab92547, Abcam, UK), and Alexa Fluor 647-labeled Goat Anti-Rabbit IgG (H + L) secondary antibodies (dilution, 1:500; A0468, Beyotime, Shanghai, China).

Scratch assay

The scratch assay is a method used to assess cell migration. Inserts (Ibidi Culture-Insert, Germany) are placed in a 24-well plate coated with polyacrylamide (PA) hydrogels of varying matrix stiffness. Cell suspension (approximately 1 × 106 cells in 100 μL) is added on both sides of the insert. Once the cells reach confluence, the insert is removed with sterilized tweezers, and the cells are washed twice with culture medium to remove any cellular debris. Subsequently, cells were cultured in serum-free culture medium. Wound healing was front photographed at indicated time point. Scratch areas were quantified using ImageJ software.

Statistical analysis

GraphPad Prism version 9.5.1 (GraphPad Software Inc., San Diego, CA, USA) was used to prepare graphs and statistics. Data are presented as mean ± standard deviation (SD). The Student's t-test was employed for comparing statistics between two groups and one-way analysis of variance (ANOVA) was used for multiple comparations. Each experiment was performed independently and repeated at least three times, with p < 0.05 indicating a statistically significant difference.

The STAR-counts data and corresponding clinical information for HCC were downloaded from the TCGA database (https://portal.gdc.cancer.gov). Subsequently, we extracted the data in TPM format along with the clinical data and performed normalization using the log2(TPM + 1) method. After retaining samples that had both RNA sequencing data and complete clinical information, we proceeded with 371 samples for subsequent analysis. Using R software and the ggplot2 package, we analyzed the relationship between two genes and visualized the results. We employed Spearman's correlation analysis to describe the correlation between quantitative variables that were not normally distributed, considering a p-value less than 0.05 as statistically significant.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

We express our deep gratitude to the National Natural Science Foundation of China for their substantial support and generous sponsorship of our research project. At the same time, we would like to extend our sincere thanks to Rui Liang for the valuable support and assistance he provided in the bioinformatics analysis of this study.

Funding

This research work was supported by grant from the National Natural Science Foundation of China (No. 11832008).

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Authors and Affiliations

  1. College of Bioengineering, Chongqing University, Chongqing, 400030, China

    Zhihui Wang, Wenbin Wang, Qing Luo & Guanbin Song

  2. Key Laboratory of Biorheological Science & Technology, Ministry of Education, Chongqing University, Chongqing, 400030, China

    Zhihui Wang, Wenbin Wang, Qing Luo & Guanbin Song

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Z.W., performed experiments, wrote the manuscript and prepared figures. W.W., performed experiments. Q.L., analyzed and interpreted the data. G.S., developed the concept, designed the study and edited the manuscript. All authors reviewed the manuscript.

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Correspondence to Guanbin Song.

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Wang, Z., Wang, W., Luo, Q. et al. High matrix stiffness accelerates migration of hepatocellular carcinoma cells through the integrin β1-Plectin-F-actin axis. BMC Biol 23, 8 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12915-025-02113-1

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