Plectin-mediated mitochondrial fusion is necessary for hepatocellular carcinoma cell migration promoted by higher matrix stiffness

Plectin-mediated mitochondrial fusion is necessary for hepatocellular carcinoma cell migration promoted by higher matrix stiffness | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Plectin-mediated mitochondrial fusion is necessary for hepatocellular carcinoma cell migration promoted by higher matrix stiffness Zhihui Wang, Wenbin Wang, Xu Zhang, Qing Luo, Guanbin Song This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6831299/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Increased matrix stiffness is a key physical signal affecting the migration of hepatocellular carcinoma (HCC) cells, and mitochondrial dynamics and function also play important roles in cell migration. Plectin may influence mitochondrial dynamics and function through its cytoskeletal cross-linking function. However, the relationship between these two factors remains unclear. HCC cells were seeded on hydrogels with stiffness of 7 kPa and 53 kPa, respectively, to investigate the effects of matrix stiffness on plectin expression, mitochondrial dynamics and function, and cell migration. Moreover, plectin was knocked down to further assess its specific impacts on mitochondrial dynamics and function, as well as cell migration under different matrix stiffness. Compared with 7 kPa, high matrix stiffness (53 kPa) promotes HCC cell migration by upregulating plectin expression, promoting mitochondrial fusion, and enhancing mitochondrial function. Under high matrix stiffness, plectin knockdown weakens mitochondrial fusion capacity and function, reducing cell migration. Subsequently, we treated cells with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to inhibit mitochondrial function. This treatment significantly suppressed cell migration on high- matrix stiffness. Then, when mitochondrial dynamics were disrupted by the mitochondrial fusion inhibitor 8 (MFI8), mitochondrial function was compromised, and cell migration decreased. High matrix stiffness enhances mitochondrial function by driving mitochondrial fusion through increasing plectin expression, thereby promoting the migration of HCC cells. It provides new insights into the mechanobiological mechanisms underlying matrix stiffness affected HCC cell migration. hepatocellular carcinoma plectin mitochondria migration matrix stiffness Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction HCC is the predominant form of liver cancer, accounting for over 90% of all cases [ 1 ]. Among various malignant tumors, HCC has garnered significant attention due to its high incidence and mortality rates, which are closely associated with its highly aggressive nature and metastatic potential. These characteristics not only complicate treatment but also lead to a poor prognosis [ 2 , 3 ]. The increase in matrix stiffness is a significant driver in the progression of various solid tumors [ 4 , 5 ]. In various cancers, including breast cancer, prostate cancer, and HCC, elevated matrix stiffness has been shown to promote biological behaviors such as tumor cell migration and invasion [ 6 – 8 ]. In HCC, this increase is particularly pronounced. In the liver tissues of patients with hepatic fibrosis and cirrhosis, excessive collagen deposition leads to a significant increase in matrix stiffness [ 9 ]. Elevated stiffness both provides a favorable microenvironment for the growth of HCC cells and enhances their migratory and invasive capabilities through the activation of relevant signaling pathways [ 10 ]. Tumor cell migration is a key process in the development, progression, and metastasis of cancer [ 11 ]. Mitochondria, often referred to as the powerhouses of the cell, are intricately linked to cell migration. Within the cellular environment, mitochondria exhibit a high degree of dynamism, constantly undergoing processes of fusion and fission. These dynamic behaviors ultimately result in the formation of filamentous networks or fragmented, round structures within the cell [ 12 , 13 ]. Mitochondrial dynamics, encompassing both fission and fusion events, significantly influence mitochondrial distribution, quantity, and function. Emerging evidence highlights the critical role of mitochondrial dynamics in tumor cell migration across various cancers, including glioblastoma, breast cancer, lung cancer, and prostate cancer [ 14 – 18 ]. In migrating cancer cells, mitochondria are strategically transported to regions of high energy demand. Specifically, they accumulate at the leading edge of the cell, where energy intensive processes such as focal adhesion dynamics occur [ 19 ]. These findings underscore the importance of mitochondria as a key regulatory hub for tumor cell migration. During cell migration, dynamic changes in the cytoskeleton are crucial for cell movement and deformation [ 20 ]. Plectin, a multifunctional cytoskeletal linker and scaffold protein, plays a significant role in this process. Its actin-binding domain allows it to reorganize actin filaments, thereby influencing cytoskeletal remodeling and cell migration [ 21 – 23 ]. In our previous study, we revealed the crucial role of plectin in the migration of HCC cells influenced by matrix stiffness, finding that it senses the changes in extracellular matrix (ECM) stiffness via modulating F-actin and affects the migration of HCC cells [ 24 ]. Moreover, studies have shown that interactions between the cytoskeleton and mitochondria in myocytes [ 25 ]. Given its cytoskeletal cross-linking function, plectin may critically influence mitochondrial positioning, dynamics, and function. However, the precise relationship between plectin and mitochondria remains largely unexplored. To thoroughly dissect the molecular mechanisms underlying plectin-regulated HCC cell migration by matrix stiffness, this study focuses on the possible role of mitochondrial dynamics in this process. Specifically, we examine whether plectin, in response to changes in extracellular matrix stiffness, modulates mitochondrial dynamics to impact mitochondrial function and, ultimately, adjust the migratory capacity of HCC cells. A comprehensive elucidation of this complex molecular mechanism may offer novel theoretical insights and potential therapeutic targets for HCC treatment. MATERIALS AND METHODS Preparation of polyacrylamide gels Polyacrylamide gels with stiffness levels of 7 KPa (low stiffness) and 53 KPa (high stiffness) were prepared for cell culture, following the methods established in our previous research [ 24 ]. The procedures for culturing and collecting cells on these different stiffness substrates were also conducted in accordance with the protocols previously reported [ 24 , 26 , 27 ]. Cell culture The human HCC cell lines MHCC-97H and MHCC-97L were obtained from the Liver Cancer Institute at Zhongshan Hospital, Fudan University (Shanghai, China). Both the MHCC-97H cell line and the MHCC-97L cell line have been validated using short tandem repeat (STR) profiling. These cells were maintained in high-glucose Dulbecco’ s Modified Eagle’ s Medium (DMEM; Thermo Fisher Scientific; 12800017), enriched with 10% fetal bovine serum (FBS; HyClone; SH30072.03), 2 mM L-glutamine (Solarbio, G8230), 1% penicillin-streptomycin (Solarbio; P1400) in a humidified at 37°C with 5% CO 2 . All experiments were performed with mycoplasma-free cells. Mycoplasma contamination in cell cultures was detected every 6 weeks using a mycoplasma detection kit (Southern Biotech; 13100-01). Virus production and transfection As in our previous study, a stable strain of HCC cells with plectin knockdown was constructed [ 24 , 28 ]. In short, lentiviral particles were produced in 293T cells using a plasmid system consisting of pLKO.1-EGFP-PURO-PLEC (Unibio, Hunan, China.), psPAX2 (Invitrogen, CA, USA), and pMD2.G (Invitrogen, CA, USA). The collected viral supernatant was used to infect HCC cells with the addition of polybrene (Sigma Aldrich, MO, USA) to enhance infection efficiency. After infection, cells were observed under a fluorescence microscope and selected with puromycin (Solarbio, P8230) to obtain stable transfectants. Target protein expression was confirmed by Western blot. The shRNA sequences used for plectin knockdown were: shPlectin-1: 5'-GCCUCUUCAAUGCCAUCAUTT-3' and 5'-AUGAUGGCAUUGAAGAGGCTT-3'; shPlectin-2: 5'-GCCAGUACAUCAAGUUCAUTT-3' and 5'-AUGAACUUGAUGUACUGGCTT-3'. Mitochondrial morphology analysis HCC cells were cultured for 48 hours on substrates with different stiffness levels. The culture medium was then removed, and cells were incubated with Mito Tracker Red (Beyotime, C1049B) working solution in the incubator for 30 minutes. The Mito Tracker Red solution was aspirated and replaced with cell culture medium pre-warmed to 37℃. Cells were imaged and photographed using a confocal microscope. Mitochondrial morphology was analyzed using ImageJ software. Mitochondrial function analysis According to the manufacturer's instructions, intracellular reactive oxygen species (ROS) levels were measured using a ROS Assay Kit- dihydroethidium (DHE) (Solarbio, 104821-25-2), or mitochondrial ROS (mtROS) were assessed using the MitoSOX Red mitochondrial superoxide indicator (MCE, HY-D1055) after respective treatments. In brief, cells were washed 1–2 times with phosphate buffered saline (PBS) (Thermo Fisher Scientific, 10010023) and stained with 5 µM DHE or 5 µM MitoSOX Red at 37°C in a cell culture incubator for 30 minutes. Cells were then washed three times with serum-free DMEM. Finally, images were captured using a confocal microscope (Leica, TCS SP8 DIVE / DMi8, Germany). HCC cells were lysed using the lysis solution provided in the ATP Assay Kit (Beyotime, S0027). The lysates were centrifuged at 12,000 rpm for 5 minutes at 4℃, and the supernatants were collected. The chemiluminescence intensity and protein concentration of the samples were measured using a microplate luminometer (BioTek, Thorold, ON, Canada). ATP content was normalized to protein concentration, and the results were expressed as fold changes relative to the control group. Mitochondrial DNA (mtDNA) damage was assessed by measuring mtDNA copy number. The relative mtDNA copy number was quantified by determining the ratio of mitochondrial genes MT-CO2, MT-ND1, and MT-CYB to the nuclear gene GAPDH using RT-qPCR. Cellular DNA was extracted using a DNA extraction kit (Accurate, AG21009) according to the manufacturer's instructions. Gene expression was analyzed by RT-qPCR using SYBR Green PCR Master Mix (Takara, RR820A). The primer sequences used were as follows: GAPDH: 5'-GGTATGACAACGAATGGC-3' and 5'-GAGCACAGGGTACTTTATTG-3'; MT-CO2: 5'-CAAACCTACGCCAAAATCCA-3' and 5'-GAAATGAATGAGCCTACAGA-3'; MT-ND1: 5'-CCACCTCTAGCCTAGCCGTTTA-3' and 5'-GGGTCATGATGGCAGGAGTAAT-3'; MT-CYB: 5'-ATCACTCGAGACGTAAATTATGGCT-3' and 5'-TGAACTAGGTCTGTCCCAATGTATG-3'. Protein extraction and Western blotting (WB) analysis Total protein was extracted from MHCC-97H and MHCC-97L cells using RIPA lysis buffer (Beyotime, P0013B) containing 1% protease inhibitor (Beyotime, P1005) and 2% phosphatase inhibitor (Beyotime, P1045). Protein concentrations were measured using a BCA protein assay kit (Meilunbio, MA0082-2). The lysates were mixed with 5× loading buffer (Beyotime, P0015), boiled at 100°C for 10 min, and then separated on 8% SDS-PAGE gels at 30 µg per lane. Proteins were transferred to 0.45 µm PVDF membranes (Bio-Rad; 1620177) by electroblotting. The membranes were blocked with 5% skim milk (Biosharp; BS102) in tris-buffered saline with Tween (TBST) (0.05% Tween-20 in Tris-buffered saline) for 1 h at room temperature, then incubated overnight at 4°C with primary antibodies against plectin (Abcam, ab32528), MFN1 (Proteintech, 13798-1-AP), MFN2 (Huabio, ER1802-23), DRP1 (Zenbio 221099), p-DRP1 (Ser616) (Abcam, ab314755) and GAPDH (Proteintech, HRP-60004). After washing with TBST, the membranes were incubated with secondary antibodies (ZSBIO, ZB-2301, ZB-2305) for 1h at room temperature. Following further washing, protein bands were visualized using an ECL detection system (Bio-OI, Guangzhou, China). GAPDH served as a loading control, and band intensities were quantified using ImageJ. Data were normalized by dividing the target protein/GAPDH ratios in each group by the average ratio from the three control groups. Scratch assay The scratch assay was conducted to evaluate cell migration. Inserts (Ibidi Culture-Insert, Germany) were placed in a 24-well plate (NEST, 702002) with hydrogels of different stiffness. Cells were added to both sides of the insert. After the cells reached confluence, the insert was removed, and the cells were washed twice, and cultured in serum-free medium. Wound healing was monitored by photographing at designated time points. The scratch areas were measured using ImageJ. Statistical analysis GraphPad Prism version 9.5.1 (GraphPad Software Inc., San Diego, CA, USA) was utilized for graphing and statistical analysis. Data are expressed as mean ± standard deviation (SD). Comparisons between two groups were made using Student's t-test, while one-way ANOVA was applied for multiple comparisons. Each experiment was conducted independently and replicated at least three times, with p < 0.05 denoting statistical significance. RESULTS High matrix stiffness promotes the migration of HCC cells by upregulating plectin expression As described in our previous study [ 24 ], to verify the response of plectin in HCC cells to changes in ECM stiffness, we cultured the cells for 48 hours on polyacrylamide hydrogels with two different stiffness levels (7 kPa and 53 kPa), representing the physiological elasticity of normal liver and HCC tissues, respectively [ 27 , 29 – 32 ]. WB results showed that the expression level of plectin protein in HCC cells under high matrix stiffness was significantly higher than that in the low matrix stiffness group (Fig. 1 a). Scratch experiment results showed that compared with 7 kPa, HCC cells cultured on 53 kPa hydrogels exhibited a significantly larger scratch healing area within 12 hours (Fig. 1 b). This indicates that high matrix stiffness significantly promotes the migration of HCC cells. Then, the knockdown efficiency of plectin was detected by WB. The results showed that compared with shNC, the plectin expression in cells carrying the shRNA viral fragment was significantly reduced (Fig. 1 c). As shown in Fig. 1 d, compared with the shNC group, the migration of HCC cells in the shPlectin group under high matrix stiffness did not show significant enhancement. This result strongly confirms that plectin is essential for high matrix stiffness-promoted HCC cell migration. High matrix stiffness enhances mitochondrial function in HCC cells Mitochondrial function is closely linked to cell migration. As the cell's energy factory, mitochondrial dysfunction can cause metabolic disorders, which in turn affect cell migration [ 33 ]. Mitochondrial function indicators typically include ROS, mtROS, ATP production, and mtDNA copy number [ 34 ]. Changes in these indicators can directly reflect the functional state of mitochondria. To determine the effect of matrix stiffness on mitochondrial function, the changes in the above indicators were explored. DHE can penetrate the cell membrane of live cells, enter the cytoplasm, and undergo an oxidative reaction with intracellular ROS to form ethidium, which intercalates into chromosomal DNA and produces red fluorescence upon excitation. MitoSOX Red can penetrate the cell membrane and target mitochondria. Once inside the mitochondria, MitoSOX Red is specifically oxidized by superoxide and does not react with other reactive oxygen or nitrogen species. Oxidized MitoSOX Red can bind to nucleic acids in mitochondria and nuclei, producing intense red fluorescence. The results of DHE and MitoSOX Red staining showed that, compared with low matrix stiffness, the levels of ROS and mtROS in HCC cells were reduced under high matrix stiffness (Figs. 2 a-b). This indicates that the oxidative stress level in cells is lower and the redox homeostasis is more balanced, providing a good basis for the execution of normal physiological functions. The mitochondrial respiration process converts the energy from substrates into ATP. When mitochondrial function is impaired, mitochondrial respiration is weakened, leading to a decrease in ATP production. The copy number of mtDNA is commonly used as an indicator of mitochondrial function. Each mitochondrion contains multiple copies, and the number can reflect the mitochondrial biogenesis capacity and energy metabolism level. MT-CO2 encodes cytochrome c oxidase subunit II, an important component of the mitochondrial respiratory chain. MT-ND1 is the largest protein complex in the mitochondrial respiratory chain, responsible for transferring electrons from NADH to ubiquinone (coenzyme Q). This process is the first step in oxidative phosphorylation and the beginning of energy conversion. MT-CYB encodes cytochrome b, a component of mitochondrial respiratory chain complex III. We selected three key mitochondrial-encoded genes and detected their copy number changes using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Meanwhile, it was observed that under high matrix stiffness, the ATP production and mtDNA copy number of HCC cells increased (Figs. 2 c-d). The sufficient ATP may provide a solid energy guarantee for various physiological activities of the cells, and the increase in mtDNA copy number means that the biosynthetic capacity of mitochondria is enhanced, which is conducive to maintaining the normal function and quantity of mitochondria. These findings suggest that high matrix stiffness significantly enhances the mitochondrial function of HCC cells, providing stable support for their continuous metabolism and activity, and further ensuring the migratory potential of HCC cells in the high-stiffness environment. High matrix stiffness enhances HCC mitochondrial function via plectin Mutations in the human plectin gene are closely related to the occurrence of epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) [ 35 , 36 ]. Studies have shown that fibroblasts from the skin of patients with EBS-MD exhibit abnormal mitochondrial networks, as well as significant barriers in key physiological functions such as wound healing and cell migration [ 37 ]. These abnormal manifestations imply the potential impact of plectin deficiency. So, it drives us to explore whether the enhanced mitochondrial function under high matrix stiffness is associated with the upregulation of plectin expression. The results of DHE and MitoSOX staining indicate that, compared with the control group, when plectin is knocked down, the levels of ROS and mtROS in HCC cells under high matrix stiffness are increased (Figs. 3 a-b). Moreover, it was observed that when plectin was knocked down, the promoting effects of high matrix stiffness on ATP production and mtDNA copy number in HCC cells were significantly weakened (Figs. 3 c-d). This suggests that high matrix stiffness enhances mitochondrial function by upregulating plectin expression. Inhibition of mitochondrial function attenuates high stiffness-promoted HCC cell migration To confirm the relationship between mitochondrial function and cell migration, we used CCCP, a mitochondrial oxidative phosphorylation uncoupler, to inhibit mitochondrial function. The results showed that after treatment with CCCP, the levels of ROS and mtROS in cells under high matrix stiffness increased (Figs. 4 a-b), and ATP production and mtDNA copy number decreased (Figs. 4 c-d). In other words, CCCP can inhibit the enhancement of mitochondrial function in HCC cells under high matrix stiffness. The scratch assay results indicated that CCCP can inhibit the promoting effect of high matrix stiffness on the migration of HCC cells (Fig. 4 e). This suggests that high matrix stiffness promotes the migration of HCC cells by enhancing mitochondrial function. High matrix stiffness promotes mitochondrial fusion Mitochondrial dynamics (including mitochondrial fusion and fission) are closely related to mitochondrial function. Mitochondrial fusion helps to form a more interconnected network within the cell, and fused mitochondria can more efficiently exchange their internal DNA, proteins, and lipids, thereby enhancing overall function [ 38 , 39 ]. Mitochondrial fission helps to identify and separate damaged or dysfunctional parts of mitochondria, allowing them to be cleared by the process of mitophagy [ 40 ]. Studies have shown that HCC cells exhibit stronger mitochondrial function under high matrix stiffness. Therefore, it is necessary to explore the changes in mitochondrial dynamics under different matrix stiffness conditions and whether these changes affect mitochondrial function and the migration of HCC cells. The results of Mitotracker RED staining showed that the mitochondria in HCC cells cultured on high stiffness substrates were mostly elongated and tubular, while those on low stiffness substrates formed numerous small and short structures (Fig. 5 a). This clear morphological difference indicates that high matrix stiffness effectively promotes mitochondrial fusion in HCC cells, while low matrix stiffness tends to promote mitochondrial fission. To further verify this phenomenon at the molecular level, we detected the proteins that play key roles in mitochondrial fusion and fission. mitofusin 1 (MFN1) and mitofusin 2 (MFN2) are key proteins in the fusion process, while Dynamin-related protein 1 (DRP1) and its phosphorylated form p-DRP1 (Ser616) are important factors in regulating fission [ 41 , 42 ]. We used WB to detect the expression of MFN1, MFN2, DRP1, and p-DRP1 (Ser616) under different matrix stiffness conditions. As shown in Figs. 5 b-e, high matrix stiffness promotes the expression of MFN1 and MFN2 in HCC cells while inhibiting the expression of DRP1 and p-DRP1 (Ser616). This result further demonstrates that compared with low matrix stiffness, high matrix stiffness enhances mitochondrial fusion and reduces fission capacity in HCC cells. High matrix stiffness promotes mitochondrial fusion via plectin To investigate whether plectin plays a role in the promotion of mitochondrial fusion by high matrix stiffness, we used Mitotracker RED to stain the mitochondria in cells with plectin knockdown. As shown in Fig. 6 a, compared with the control group, the mitochondrial morphology in HCC cells under high matrix stiffness changed from elongated tubular structures to small and fragmented ones after plectin knockdown. This significant morphological difference clearly indicates that plectin knockdown attenuates the pro-fusion effect of high matrix stiffness on HCC cell mitochondria. We further detected the expression levels of MFN1, MFN2, DRP1, and p-DRP1 (Ser616) in the control and plectin knockdown groups under different matrix stiffness using WB. As shown in Figs. 6 b-e, compared with the control group, the expression of MFN1 and MFN2 decreased, while the expression of DRP1 and p-DRP1 (Ser616) increased in the plectin knockdown group under high matrix stiffness. This indicates that plectin knockdown reduces mitochondrial fusion capacity and enhances fission capacity in HCC cells under high matrix stiffness, confirming that plectin plays a key role in regulating mitochondrial dynamics in response to matrix stiffness. MFI8 inhibits mitochondrial fusion in HCC cells To clarify the impact of mitochondrial fusion on mitochondrial function and cell migration, we employed MFI8 to inhibit mitochondrial fusion. The results of Mitotracker RED staining indicated that treatment with MFI8 led to significant changes in the mitochondrial morphology of HCC cells under high matrix stiffness. The originally elongated, tubular shapes were transformed into small, fragmented structures. This morphological difference intuitively reflects the inhibitory effect of MFI8 on mitochondrial fusion (Fig. 7 a). WB analysis was conducted to detect the effects of matrix stiffness on MFN1 and MFN2 in HCC cells after MFI8 treatment. As shown in Figs. 7 b-c, the upregulation of MFN1 and MFN2 were significantly suppressed following MFI8 treatment. These results fully demonstrate that MFI8 can effectively inhibit the promotion of mitochondrial fusion in HCC cells under high matrix stiffness. MFI8 impairs mitochondrial function and attenuates cell migration by inhibiting mitochondrial fusion To further investigate whether changes in mitochondrial dynamics under different matrix stiffness conditions affect mitochondrial function and subsequently influence HCC cell migration, we used MFI8 to intervene in mitochondrial fusion and measured mitochondrial function indicators and migration capacity. After treatment with MFI8, the levels of ROS and mtROS in HCC cells under high matrix stiffness were significantly higher than those in the untreated group (Figs. 8 a-b), while ATP production and mtDNA copy number under high stiffness were significantly reduced (Figs. 8 c-d). This indicates that MFI8, by interfering with mitochondrial fusion in HCC cells under high matrix stiffness, further leads to impaired mitochondrial function. Finally, to further validate whether changes in mitochondrial dynamics affect HCC cell migration, we performed scratch assays to measure migration capacity under different matrix stiffness conditions after MFI8 treatment. As shown in Fig. 8 e, high matrix stiffness significantly promoted HCC cell migration, but this effect was markedly inhibited by MFI8 treatment. DISCUSSION In the tumor microenvironment, matrix stiffness has garnered significant attention due to its important impact on tumor progression and treatment efficacy [ 43 , 44 ]. Matrix stiffness not only affects the proliferation and survival of tumor cells but also significantly regulates their migration and invasion capabilities. In recent years, an increasing number of studies have shown that mitochondrial function is closely related to tumor progression driven by matrix stiffness [ 45 , 46 ]. However, the specific molecular mechanisms by which matrix stiffness regulates the migration of HCC cells through its effects on mitochondria have not yet been fully elucidated. Matrix stiffness significantly affects the morphology and subcellular localization of mitochondria. Under low matrix stiffness, mitochondria typically exhibit a fragmented morphology with more branches, whereas under high matrix stiffness, they present with fewer branches and longer tubules [ 47 ]. This morphological change reflects the differences in mitochondrial dynamics. In breast cancer, low matrix stiffness leads to mitochondrial fragmentation, which in turn causes mitochondrial dysfunction and cell apoptosis [ 45 , 46 ]. This indicates that changes in mitochondrial morphology directly affect their function, thereby regulating the metabolism and physiological functions of cells. In our study, we found that high matrix stiffness significantly promotes mitochondrial fusion in HCC cells, resulting in elongated tubular mitochondria, as evidenced by significant increases in mitochondrial perimeter, area, and branch length. This morphological change is closely related to enhanced mitochondrial function, characterized by decreased levels of ROS and mtROS, and increased ATP production and mtDNA copy number. These findings suggest that high matrix stiffness enhances mitochondrial function by promoting mitochondrial fusion, thereby providing favorable conditions for cell migration. Plectin is a multifunctional cytoskeletal linker protein that is widely involved in cytoskeletal remodeling and cell migration. The absence of plectin leads to the separation of mitochondria from myofibrils, decreased activity of mitochondrial respiratory chain complexes, and mitochondrial respiratory dysfunction, which in turn results in insufficient muscle energy supply and functional impairment [ 48 ]. This indicates that plectin plays an important role in maintaining the normal distribution and function of mitochondria. In our study, we found that high matrix stiffness promotes mitochondrial fusion and enhanced function in HCC cells by upregulating plectin expression, which further promotes cell migration. This finding reveals the key regulatory role of plectin in mitochondrial dynamics and function, providing a new perspective for understanding how matrix stiffness affects HCC cell migration by regulating plectin and thereby influencing mitochondria. Mitochondrial metabolism plays a crucial role in the tumorigenesis of various human cancers [ 49 ]. Recent studies have shown that SLC25A35 significantly reprograms mitochondrial metabolism by enhancing fatty acid oxidation, characterized by increased oxygen consumption rate and ATP production, as well as reduced ROS levels. Additionally, SLC25A35 also enhances mitochondrial biogenesis, as evidenced by increased mitochondrial mass and DNA content [ 50 ]. Our study results are similar, with high matrix stiffness upregulating plectin expression to induce mitochondrial fusion and enhance mitochondrial function, thereby providing strong support for the migration of HCC cells. Future research needs to further explore the specific molecular mechanisms between plectin and mitochondria, as well as how plectin affects the migration of HCC cells by regulating mitochondrial dynamics. In conclusion, this study has revealed the key mechanism by which high matrix stiffness promotes the migration of HCC cells: high matrix stiffness upregulates plectin expression, induces mitochondrial fusion, and enhances mitochondrial function, thereby driving the migration of HCC cells (Fig. 9 ). These findings clarify the relationship between high matrix stiffness, plectin, mitochondria, and cell migration, providing an important theoretical basis for the development of novel therapeutic strategies for HCC. Abbreviations BCA Bicinchoninic acid assay CCCP Carbonyl cyanide 3-chlorophenylhydrazone DHE Dihydroethidium DRP1 Dynamin-related protein 1 ECM Extracellular matrix EMT Epithelial-mesenchymal transition HCC Hepatocellular carcinoma mtDNA Mitochondrial DNA mtROS Mitochondrial reactive oxygen species MFI8 Mitochondrial fusion inhibitor 8 MFN1 Mitofusin 1 MFN2 Mitofusin 2 MT-CO2 Mitochondrially encoded cytochrome C oxidase II MT-CYB Mitochondrially encoded cytochrome B MT-ND1 Mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 p-DRP1 Phospho dynamin-related protein 1 RT-qPCR Real-time quantitative reverse transcription polymerase chain reaction PVDF Polyvinylidene fluoride WB Western blotting Declarations Author contributions Zhihui Wang: performed experiments, wrote the manuscript and prepared figures. Wenbin Wang: performed experiments. Xu Zhang: performed experiments. Qing Luo: analyzed and interpreted the data. Guanbin Song: developed the concept, designed the study and edited the manuscript. All authors read and approved the final manuscript. The authors declare that all data were generated in-house and that no paper mill was used. Funding This research work was supported by grant from the National Natural Science Foundation of China (No. 11832008). Data availability No datasets were generated or analyzed during the current study. Ethics approval and consent to participate This article does not contain any studies with human or animal subjects. Consent for publication Not applicable. Conflict of interest The authors declare no competing interests. References A.D. Ladd, S. Duarte, I. Sahin, et al. Mechanisms of drug resistance in HCC. Hepatology. 2024, 79(4): 926-940. A. Sufianov, M. Agaverdiev, A. Mashkin, et al. Targeting microRNA methylation: Innovative approaches to diagnosing and treating hepatocellular carcinoma. Noncoding RNA Res. 2025, 11: 150-157. G. Wu, N. Bajestani, N. Pracha, et al. Hepatocellular Carcinoma Surveillance Strategies: Major Guidelines and Screening Advances. Cancers (Basel). 2024, 16(23). C.C. Lee, J.C. Lin, W.L. Hwang, et al. Macrophage-secreted interleukin-35 regulates cancer cell plasticity to facilitate metastatic colonization. Nat Commun. 2018, 9(1): 3763. R. Liang, G. Song Matrix stiffness-driven cancer progression and the targeted therapeutic strategy. Mechanobiology in Medicine. 2023, 1(2): 100013. M. Quintela-Fandino, B. Bermejo, E. Zamora, et al. High Mechanical Conditioning by Tumor Extracellular Matrix Stiffness Is a Predictive Biomarker for Antifibrotic Therapy in HER2-Negative Breast Cancer. Clin Cancer Res. 2024, 30(22): 5094-5104. I. Samaržija The Potential of Extracellular Matrix- and Integrin Adhesion Complex-Related Molecules for Prostate Cancer Biomarker Discovery. Biomedicines. 2023, 12(1). Y. Zhao, H. Yu, J. Li, et al. A glucose-enriched lung pre-metastatic niche triggered by matrix stiffness-tuned exosomal miRNAs in hepatocellular carcinoma. Nat Commun. 2025, 16(1): 1736. J. Hong, J. Yu, D. Buratto, et al. Unveiling the role of mechanical microenvironment in hepatocellular carcinoma: Molecular mechanisms and implications for therapeutic strategies. Int J Biol Sci. 2024, 20(13): 5239-5253. Z. Shen, L. Tao, Y. Wang, et al. Synergistic anticancer strategy targeting ECM stiffness: Integration of matrix softening and mechanical signal transduction blockade in primary liver cancers. Adv Sci (Weinh). 2025, 12(6): e2403040. S. Wu, Y. Pan, Y. Mao, et al. Current progress and mechanisms of bone metastasis in lung cancer: a narrative review. Transl Lung Cancer Res. 2021, 10(1): 439-451. S. Von Stockum, A. Nardin, E. Schrepfer, et al. Mitochondrial dynamics and mitophagy in Parkinson's disease: A fly point of view. Neurobiol Dis. 2016, 90: 58-67. R.J. Youle, A.M. van der Bliek Mitochondrial fission, fusion, and stress. Science. 2012, 337(6098): 1062-5. T.F. Che, C.W. Lin, Y.Y. Wu, et al. Mitochondrial translocation of EGFR regulates mitochondria dynamics and promotes metastasis in NSCLC. Oncotarget. 2015, 6(35): 37349-66. S.P. Desai, S.N. Bhatia, M. Toner, et al. Mitochondrial localization and the persistent migration of epithelial cancer cells. Biophys J. 2013, 104(9): 2077-88. A. Ferreira-da-Silva, C. Valacca, E. Rios, et al. Mitochondrial dynamics protein Drp1 is overexpressed in oncocytic thyroid tumors and regulates cancer cell migration. PLoS One. 2015, 10(3): e0122308. B. Zhang, J. Wang, Z. Huang, et al. Aberrantly upregulated TRAP1 is required for tumorigenesis of breast cancer. Oncotarget. 2015, 6(42): 44495-508. J. Zhao, J. Zhang, M. Yu, et al. Mitochondrial dynamics regulates migration and invasion of breast cancer cells. Oncogene. 2013, 32(40): 4814-24. D. Senft, Z.A. Ronai Regulators of mitochondrial dynamics in cancer. Curr Opin Cell Biol. 2016, 39: 43-52. Y. Sun, Y. Qiao, Y. Niu, et al. ARP2/3 complex affects myofibroblast differentiation and migration in pancreatic ductal adenocarcinoma. Int J Cancer. 2025, 156(6): 1272-1281. L. Hu, Z. Huang, Z. Wu, et al. Mammalian plakins, giant cytolinkers: Versatile biological functions and roles in cancer. Int J Mol Sci. 2018, 19(4). D.A. Starr, H.N. Fridolfsson Interactions between nuclei and the cytoskeleton are mediated by SUN-KASH nuclear-envelope bridges. Annu Rev Cell Dev Biol. 2010, 26: 421-44. M. Sutoh Yoneyama, S. Hatakeyama, T. Habuchi, et al. Vimentin intermediate filament and plectin provide a scaffold for invadopodia, facilitating cancer cell invasion and extravasation for metastasis. Eur J Cell Biol. 2014, 93(4): 157-69. Z. Wang, W. Wang, Q. Luo, et al. High matrix stiffness accelerates migration of hepatocellular carcinoma cells through the integrin β1-Plectin-F-actin axis. BMC Biol. 2025, 23(1): 8. K. Mado, V. Chekulayev, I. Shevchuk, et al. On the role of tubulin, plectin, desmin, and vimentin in the regulation of mitochondrial energy fluxes in muscle cells. Am J Physiol Cell Physiol. 2019, 316(5): C657-c667. B. Tian, Q. Luo, Y. Ju, et al. A soft matrix enhances the cancer stem cell phenotype of HCC cells. Int J Mol Sci. 2019, 20(11). Q.P. Liu, Q. Luo, B. Deng, et al. Stiffer matrix accelerates migration of hepatocellular carcinoma cells through enhanced aerobic glycolysis via the MAPK-YAP signaling. Cancers (Basel). 2020, 12(2). R. Xu, S. He, D. Ma, et al. Plectin downregulation inhibits migration and suppresses epithelial mesenchymal transformation of hepatocellular carcinoma cells via ERK1/2 signaling. Int J Mol Sci. 2022, 24(1). V.W. Wong, J. Vergniol, G.L. Wong, et al. Diagnosis of fibrosis and cirrhosis using liver stiffness measurement in nonalcoholic fatty liver disease. Hepatology. 2010, 51(2): 454-62. J. Swift, I.L. Ivanovska, A. Buxboim, et al. Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation. Science. 2013, 341(6149): 1240104. P.J. Eddowes, M. Sasso, M. Allison, et al. Accuracy of fibroScan controlled attenuation parameter and liver stiffness measurement in assessing steatosis and fibrosis in patients with nonalcoholic fatty liver disease. Gastroenterology. 2019, 156(6): 1717-1730. N. Li, X. Zhang, J. Zhou, et al. Multiscale biomechanics and mechanotransduction from liver fibrosis to cancer. Adv Drug Deliv Rev. 2022, 188: 114448. S. Madan, B. Uttekar, S. Chowdhary, et al. Mitochondria lead the way: Mitochondrial dynamics and function in cellular movements in development and disease. Front Cell Dev Biol. 2021, 9: 781933. C. Xu, L. Qiao, L. Ma, et al. Biogenic selenium nanoparticles synthesized by Lactobacillus casei ATCC 393 alleviate intestinal epithelial barrier dysfunction caused by oxidative stress via Nrf2 signaling-mediated mitochondrial pathway. Int J Nanomedicine. 2019, 14: 4491-4502. D. Kiritsi, L. Tsakiris, F. Schauer plectin in skin fragility disorders. Cells. 2021, 10(10). M.M. Zrelski, S. Hösele, M. Kustermann, et al. Plectin deficiency in fibroblasts deranges intermediate filament and organelle morphology, migration, and adhesion. J Invest Dermatol. 2024, 144(3): 547-562.e9. M.J. Castañón, G. Wiche identifying plectin isoform functions through animal models. Cells. 2021, 10(9). J. Nunnari, W.F. Marshall, A. Straight, et al. Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA. Mol Biol Cell. 1997, 8(7): 1233-42. K. Okamoto, P.S. Perlman, R.A. Butow The sorting of mitochondrial DNA and mitochondrial proteins in zygotes: preferential transmission of mitochondrial DNA to the medial bud. J Cell Biol. 1998, 142(3): 613-23. D.F. Kashatus, K.H. Lim, D.C. Brady, et al. RALA and RALBP1 regulate mitochondrial fission at mitosis. Nat Cell Biol. 2011, 13(9): 1108-15. Y. Li, Y. Gao, G. Yu, et al. G6PD protects against cerebral ischemia-reperfusion injury by inhibiting excessive mitophagy. Life Sci. 2025, 362: 123367. A. Santel, M.T. Fuller Control of mitochondrial morphology by a human mitofusin. J Cell Sci. 2001, 114(Pt 5): 867-74. R.L. Barrett, E. Puré Cancer-associated fibroblasts and their influence on tumor immunity and immunotherapy. Elife. 2020, 9. M. Chirivì, F. Maiullari, M. Milan, et al. Tumor extracellular matrix stiffness promptly modulates the phenotype and gene expression of infiltrating T lymphocytes. Int J Mol Sci. 2021, 22(11). Y. Chen, P. Li, X. Chen, et al. Endoplasmic reticulum-mitochondrial calcium transport contributes to soft extracellular matrix-triggered mitochondrial dynamics and mitophagy in breast carcinoma cells. Acta Biomater. 2023, 169: 192-208. Y. Chen, P. Li, Y. Peng, et al. Protective autophagy attenuates soft substrate-induced apoptosis through ROS/JNK signaling pathway in breast cancer cells. Free Radic Biol Med. 2021, 172: 590-603. P. Daga, B. Thurakkal, S. Rawal, et al. Matrix stiffening promotes perinuclear clustering of mitochondria. Mol Biol Cell. 2024, 35(7): ar91. L. Winter, A.V. Kuznetsov, M. Grimm, et al. Plectin isoform P1b and P1d deficiencies differentially affect mitochondrial morphology and function in skeletal muscle. Hum Mol Genet. 2015, 24(16): 4530-44. P.E. Porporato, N. Filigheddu, J.M.B. Pedro, et al. Mitochondrial metabolism and cancer. Cell Res. 2018, 28(3): 265-280. H.C. Yu, L. Bai, L. Jin, et al. SLC25A35 enhances fatty acid oxidation and mitochondrial biogenesis to promote the carcinogenesis and progression of hepatocellular carcinoma by upregulating PGC-1α. Cell Commun Signal. 2025, 23(1): 130. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6831299","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":475163419,"identity":"68c121fc-5314-4b57-9234-26c187462b96","order_by":0,"name":"Zhihui Wang","email":"","orcid":"","institution":"Chongqing University","correspondingAuthor":false,"prefix":"","firstName":"Zhihui","middleName":"","lastName":"Wang","suffix":""},{"id":475163420,"identity":"038e11ab-7e50-4767-b686-9bf3e493b185","order_by":1,"name":"Wenbin Wang","email":"","orcid":"","institution":"Chongqing University","correspondingAuthor":false,"prefix":"","firstName":"Wenbin","middleName":"","lastName":"Wang","suffix":""},{"id":475163421,"identity":"f77948d3-f167-4186-a87c-96eeedb8d4c1","order_by":2,"name":"Xu Zhang","email":"","orcid":"","institution":"Chongqing University","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Zhang","suffix":""},{"id":475163422,"identity":"6fecb863-c66b-427e-ad32-e7b363fc6ffd","order_by":3,"name":"Qing Luo","email":"","orcid":"","institution":"Chongqing University","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Luo","suffix":""},{"id":475163423,"identity":"22e2b2e4-a368-41bc-8eda-a6bf1d3d0ac1","order_by":4,"name":"Guanbin Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYLCCDxDKgHgdjDNI1sLMQ5IW+fbew69taqwTG9ibt0kw1NwhrMXgzLk065xj6YkNPMfKJBiOPSNCi0SOmXFuw+HEBiBDgrHhMBEOmwHUYgnSIv+GSC0MN3KMHzOCbeEhUovBmTNmjD3H0o3beNKKLRKOEeOw9h7jDz9qrGX72Q9vvPGhhhiHMTCwSQCjhoENxEwgSgNQ+QeQllEwCkbBKBgFOAEASF02JCMQ/z8AAAAASUVORK5CYII=","orcid":"","institution":"Chongqing University","correspondingAuthor":true,"prefix":"","firstName":"Guanbin","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2025-06-05 17:38:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6831299/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6831299/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85377316,"identity":"27f1f1d0-2ecb-4478-bede-ec943bf62c2f","added_by":"auto","created_at":"2025-06-25 08:39:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":524530,"visible":true,"origin":"","legend":"\u003cp\u003eHigh matrix stiffness promotes the migration of HCC cells by upregulating plectin expression\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. WB detection of plectin protein levels in MHCC-97H and MHCC-97L cells under different ECM stiffness.\u003cstrong\u003e b\u003c/strong\u003e. Wound healing assays of MHCC-97H and MHCC-97L cells cultured on hydrogels of different stiffness for 12 hours. \u003cstrong\u003ec\u003c/strong\u003e. WB analysis was performed to assess the efficiency of plectin knockdown in cells, followed by quantitative analysis. \u003cstrong\u003ed\u003c/strong\u003e. Wound healing assays of MHCC-97H and MHCC-97L cells after plectin knockdown. Data are presented as mean ± SD; n = 3 *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, Scale bar: 200 μm.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6831299/v1/ceec2ea170dd388a780d6a6d.png"},{"id":85377302,"identity":"90a28f56-ac4c-4955-b2e3-d9ce17c46aea","added_by":"auto","created_at":"2025-06-25 08:39:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":281399,"visible":true,"origin":"","legend":"\u003cp\u003eHigh matrix stiffness enhances mitochondrial function in HCC cells\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eMatrix stiffness modulates ROS levels in HCC cells. \u003cstrong\u003eb.\u003c/strong\u003e Matrix stiffness alters mtROS production in HCC cells. \u003cstrong\u003ec.\u003c/strong\u003e Matrix stiffness regulates ATP generation in HCC cells. \u003cstrong\u003ed.\u003c/strong\u003e Matrix stiffness affects mtDNA copy number in HCC cells Data are presented as mean ± SD; n = 3, * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, Scale bar: 100 μm.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6831299/v1/78be3ba5c2754c9a1122c4b2.png"},{"id":85377309,"identity":"40b74cf8-1b30-4b0d-b0dc-935443249d13","added_by":"auto","created_at":"2025-06-25 08:39:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":401091,"visible":true,"origin":"","legend":"\u003cp\u003eHigh matrix stiffness enhances mitochondrial function in HCC cells through plectin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eModulation of ROS levels in plectin-knockdown HCC cells by matrix stiffness. \u003cstrong\u003eb.\u003c/strong\u003eModulation of mtROS levels in plectin-knockdown HCC cells by matrix stiffness. \u003cstrong\u003ec.\u003c/strong\u003eInfluence of matrix stiffness on ATP production in plectin-knockdown HCC cells. \u003cstrong\u003ed.\u003c/strong\u003e Influence of matrix stiffness on mtDNA copy number in plectin-knockdown HCC cells. Data are presented as mean ± SD; n = 3 *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Scale bar: 100 μm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6831299/v1/10054d73f2b087ce5479d11c.png"},{"id":85377315,"identity":"56384622-852d-4cde-90c3-56611117574c","added_by":"auto","created_at":"2025-06-25 08:39:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":620091,"visible":true,"origin":"","legend":"\u003cp\u003eImpairment of mitochondrial function inhibits the migration of HCC cells promoted by high stiffness\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eEffect of matrix stiffness on ROS in HCC cells after CCCP treatment, Scale bar: 100 μm. \u003cstrong\u003eb.\u003c/strong\u003e Effect of matrix stiffness on mtROS in HCC cells after CCCP treatment. Scale bar: 100 μm. \u003cstrong\u003ec.\u003c/strong\u003e Effect of matrix stiffness on ATP production in HCC cells after CCCP treatment. \u003cstrong\u003ed.\u003c/strong\u003e Effect of matrix stiffness on mtDNA copy number of HCC cells after CCCP treatment. \u003cstrong\u003ee.\u003c/strong\u003eEffect of matrix stiffness on HCC cell migration after CCCP treatment, Scale bar: 200 μm. Data are presented as mean ± SD; n=3, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6831299/v1/0e9545b0992829fe750590db.png"},{"id":85377301,"identity":"624398b0-7788-4408-a938-583d28931d6a","added_by":"auto","created_at":"2025-06-25 08:39:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":557191,"visible":true,"origin":"","legend":"\u003cp\u003eHigh matrix stiffness promotes mitochondrial fusion in HCC cells\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eInfluence of matrix stiffness on mitochondrial morphology. \u003cstrong\u003eb-e.\u003c/strong\u003e WB detected the protein expression of MFN1, MFN2, DRP1, p-DRP1 (Ser616). Data are presented as mean ± SD; n=3, * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, Scale bar: 10 μm.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6831299/v1/78756f8353962d3d9dd28ba8.png"},{"id":85377788,"identity":"216ecd7b-625c-4807-8ed9-b3f3a47bfca4","added_by":"auto","created_at":"2025-06-25 08:47:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":439506,"visible":true,"origin":"","legend":"\u003cp\u003eHigh matrix stiffness promotes mitochondrial fusion in HCC cells through plectin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eInfluence of matrix stiffness on mitochondrial morphology after plectin knockdown. \u003cstrong\u003eb-e.\u003c/strong\u003e WB detected the protein expression of MFN1, MFN2, DRP1, p-DRP1 (Ser616) after plectin knockdown. Data are presented as mean ± SD; n=3, * \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, Scale bar: 10 μm.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6831299/v1/f3f27a0910a5c9d164255bfc.png"},{"id":85377324,"identity":"91b6310f-a434-4af8-b781-e602bbbc5ae7","added_by":"auto","created_at":"2025-06-25 08:39:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":315388,"visible":true,"origin":"","legend":"\u003cp\u003eImpaired mitochondrial fusion inhibits mitochondrial function and migration in HCC cells\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eInfluence of matrix stiffness on mitochondrial morphology after MFI8 treatment. \u003cstrong\u003eb-c.\u003c/strong\u003e WB detected the protein expression of MFN1, MFN2 after MFI8 treatment. Data are presented as mean ± SD; n=3, * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, Scale bar: 10 μm.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6831299/v1/3953628997428dbcb2ae7e24.png"},{"id":85377825,"identity":"af0571c3-6339-4c4e-97e7-a7bf42f72111","added_by":"auto","created_at":"2025-06-25 08:47:28","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":622630,"visible":true,"origin":"","legend":"\u003cp\u003eMFI8 impairs mitochondrial function and slows cell migration by inhibiting mitochondrial fusion\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003eThe impact of matrix stiffness on ROS in HCC cells after MFI8 treatment, Scale bar: 100 μm. \u003cstrong\u003eb.\u003c/strong\u003e The impact of matrix stiffness on mtROS in HCC cells after MFI8 treatment, Scale bar: 100 μm.\u003cstrong\u003e c.\u003c/strong\u003e The impact of matrix stiffness on ATP production in HCC cells after MFI8 treatment. \u003cstrong\u003ed.\u003c/strong\u003e The impact of matrix stiffness on mtDNA copy number of HCC cells after MFI8 treatment. \u003cstrong\u003ee.\u003c/strong\u003e The impact of matrix stiffness on HCC cell migration after MFI8 treatment, Scale bar: 200 μm. Data are presented as mean ± SD; n=3, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6831299/v1/ff510ef452a5d82e955e2f24.png"},{"id":85377292,"identity":"c57f8654-2904-44dd-91f0-6337396ced1a","added_by":"auto","created_at":"2025-06-25 08:39:22","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":311995,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of matrix stiffness regulating HCC cell migration through plectin-mediated mitochondrial dynamics/function\u003c/p\u003e\n\u003cp\u003eHigh matrix stiffness upregulates the expression of plectin, which induces mitochondrial fusion (characterized by increased expression of MFN1 and MFN2, and decreased expression of DRP1 and p-DRP1), enhances mitochondrial function (marked by decreased levels of ROS and mtROS, and increased ATP production and mtDNA copy number), and thereby further promotes the migration of HCC cells.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6831299/v1/da59cae18b4216c504d92f2f.png"},{"id":101729398,"identity":"296a0011-e51d-4aa7-b2d3-ccc6c6dccb33","added_by":"auto","created_at":"2026-02-03 05:41:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5036706,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6831299/v1/67e4292b-f204-47a1-89ad-aeb1919793ee.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Plectin-mediated mitochondrial fusion is necessary for hepatocellular carcinoma cell migration promoted by higher matrix stiffness","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHCC is the predominant form of liver cancer, accounting for over 90% of all cases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among various malignant tumors, HCC has garnered significant attention due to its high incidence and mortality rates, which are closely associated with its highly aggressive nature and metastatic potential. These characteristics not only complicate treatment but also lead to a poor prognosis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The increase in matrix stiffness is a significant driver in the progression of various solid tumors [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In various cancers, including breast cancer, prostate cancer, and HCC, elevated matrix stiffness has been shown to promote biological behaviors such as tumor cell migration and invasion [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In HCC, this increase is particularly pronounced. In the liver tissues of patients with hepatic fibrosis and cirrhosis, excessive collagen deposition leads to a significant increase in matrix stiffness [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Elevated stiffness both provides a favorable microenvironment for the growth of HCC cells and enhances their migratory and invasive capabilities through the activation of relevant signaling pathways [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTumor cell migration is a key process in the development, progression, and metastasis of cancer [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Mitochondria, often referred to as the powerhouses of the cell, are intricately linked to cell migration. Within the cellular environment, mitochondria exhibit a high degree of dynamism, constantly undergoing processes of fusion and fission. These dynamic behaviors ultimately result in the formation of filamentous networks or fragmented, round structures within the cell [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Mitochondrial dynamics, encompassing both fission and fusion events, significantly influence mitochondrial distribution, quantity, and function. Emerging evidence highlights the critical role of mitochondrial dynamics in tumor cell migration across various cancers, including glioblastoma, breast cancer, lung cancer, and prostate cancer [\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In migrating cancer cells, mitochondria are strategically transported to regions of high energy demand. Specifically, they accumulate at the leading edge of the cell, where energy intensive processes such as focal adhesion dynamics occur [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These findings underscore the importance of mitochondria as a key regulatory hub for tumor cell migration.\u003c/p\u003e \u003cp\u003eDuring cell migration, dynamic changes in the cytoskeleton are crucial for cell movement and deformation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Plectin, a multifunctional cytoskeletal linker and scaffold protein, plays a significant role in this process. Its actin-binding domain allows it to reorganize actin filaments, thereby influencing cytoskeletal remodeling and cell migration [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In our previous study, we revealed the crucial role of plectin in the migration of HCC cells influenced by matrix stiffness, finding that it senses the changes in extracellular matrix (ECM) stiffness via modulating F-actin and affects the migration of HCC cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Moreover, studies have shown that interactions between the cytoskeleton and mitochondria in myocytes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Given its cytoskeletal cross-linking function, plectin may critically influence mitochondrial positioning, dynamics, and function. However, the precise relationship between plectin and mitochondria remains largely unexplored.\u003c/p\u003e \u003cp\u003eTo thoroughly dissect the molecular mechanisms underlying plectin-regulated HCC cell migration by matrix stiffness, this study focuses on the possible role of mitochondrial dynamics in this process. Specifically, we examine whether plectin, in response to changes in extracellular matrix stiffness, modulates mitochondrial dynamics to impact mitochondrial function and, ultimately, adjust the migratory capacity of HCC cells. A comprehensive elucidation of this complex molecular mechanism may offer novel theoretical insights and potential therapeutic targets for HCC treatment.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of polyacrylamide gels\u003c/h2\u003e \u003cp\u003ePolyacrylamide gels with stiffness levels of 7 KPa (low stiffness) and 53 KPa (high stiffness) were prepared for cell culture, following the methods established in our previous research [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The procedures for culturing and collecting cells on these different stiffness substrates were also conducted in accordance with the protocols previously reported [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eThe human HCC cell lines MHCC-97H and MHCC-97L were obtained from the Liver Cancer Institute at Zhongshan Hospital, Fudan University (Shanghai, China). Both the MHCC-97H cell line and the MHCC-97L cell line have been validated using short tandem repeat (STR) profiling. These cells were maintained in high-glucose Dulbecco\u0026rsquo; s Modified Eagle\u0026rsquo; s Medium (DMEM; Thermo Fisher Scientific; 12800017), enriched with 10% fetal bovine serum (FBS; HyClone; SH30072.03), 2 mM L-glutamine (Solarbio, G8230), 1% penicillin-streptomycin (Solarbio; P1400) in a humidified at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. All experiments were performed with mycoplasma-free cells. Mycoplasma contamination in cell cultures was detected every 6 weeks using a mycoplasma detection kit (Southern Biotech; 13100-01).\u003c/p\u003e\n\u003ch3\u003eVirus production and transfection\u003c/h3\u003e\n\u003cp\u003eAs in our previous study, a stable strain of HCC cells with plectin knockdown was constructed [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In short, lentiviral particles were produced in 293T cells using a plasmid system consisting of pLKO.1-EGFP-PURO-PLEC (Unibio, Hunan, China.), psPAX2 (Invitrogen, CA, USA), and pMD2.G (Invitrogen, CA, USA). The collected viral supernatant was used to infect HCC cells with the addition of polybrene (Sigma Aldrich, MO, USA) to enhance infection efficiency. After infection, cells were observed under a fluorescence microscope and selected with puromycin (Solarbio, P8230) to obtain stable transfectants. Target protein expression was confirmed by Western blot. The shRNA sequences used for plectin knockdown were:\u003c/p\u003e \u003cp\u003eshPlectin-1: 5'-GCCUCUUCAAUGCCAUCAUTT-3' and 5'-AUGAUGGCAUUGAAGAGGCTT-3'; shPlectin-2: 5'-GCCAGUACAUCAAGUUCAUTT-3' and 5'-AUGAACUUGAUGUACUGGCTT-3'.\u003c/p\u003e\n\u003ch3\u003eMitochondrial morphology analysis\u003c/h3\u003e\n\u003cp\u003eHCC cells were cultured for 48 hours on substrates with different stiffness levels. The culture medium was then removed, and cells were incubated with Mito Tracker Red (Beyotime, C1049B) working solution in the incubator for 30 minutes. The Mito Tracker Red solution was aspirated and replaced with cell culture medium pre-warmed to 37℃. Cells were imaged and photographed using a confocal microscope. Mitochondrial morphology was analyzed using ImageJ software.\u003c/p\u003e\n\u003ch3\u003eMitochondrial function analysis\u003c/h3\u003e\n\u003cp\u003eAccording to the manufacturer's instructions, intracellular reactive oxygen species (ROS) levels were measured using a ROS Assay Kit- dihydroethidium (DHE) (Solarbio, 104821-25-2), or mitochondrial ROS (mtROS) were assessed using the MitoSOX Red mitochondrial superoxide indicator (MCE, HY-D1055) after respective treatments. In brief, cells were washed 1\u0026ndash;2 times with phosphate buffered saline (PBS) (Thermo Fisher Scientific, 10010023) and stained with 5 \u0026micro;M DHE or 5 \u0026micro;M MitoSOX Red at 37\u0026deg;C in a cell culture incubator for 30 minutes. Cells were then washed three times with serum-free DMEM. Finally, images were captured using a confocal microscope (Leica, TCS SP8 DIVE / DMi8, Germany).\u003c/p\u003e \u003cp\u003eHCC cells were lysed using the lysis solution provided in the ATP Assay Kit (Beyotime, S0027). The lysates were centrifuged at 12,000 rpm for 5 minutes at 4℃, and the supernatants were collected. The chemiluminescence intensity and protein concentration of the samples were measured using a microplate luminometer (BioTek, Thorold, ON, Canada). ATP content was normalized to protein concentration, and the results were expressed as fold changes relative to the control group.\u003c/p\u003e \u003cp\u003eMitochondrial DNA (mtDNA) damage was assessed by measuring mtDNA copy number. The relative mtDNA copy number was quantified by determining the ratio of mitochondrial genes MT-CO2, MT-ND1, and MT-CYB to the nuclear gene GAPDH using RT-qPCR. Cellular DNA was extracted using a DNA extraction kit (Accurate, AG21009) according to the manufacturer's instructions. Gene expression was analyzed by RT-qPCR using SYBR Green PCR Master Mix (Takara, RR820A). The primer sequences used were as follows:\u003c/p\u003e \u003cp\u003eGAPDH: 5'-GGTATGACAACGAATGGC-3' and 5'-GAGCACAGGGTACTTTATTG-3'; MT-CO2: 5'-CAAACCTACGCCAAAATCCA-3' and 5'-GAAATGAATGAGCCTACAGA-3'; MT-ND1: 5'-CCACCTCTAGCCTAGCCGTTTA-3' and 5'-GGGTCATGATGGCAGGAGTAAT-3'; MT-CYB: 5'-ATCACTCGAGACGTAAATTATGGCT-3' and 5'-TGAACTAGGTCTGTCCCAATGTATG-3'.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and Western blotting (WB) analysis\u003c/h2\u003e \u003cp\u003eTotal protein was extracted from MHCC-97H and MHCC-97L cells using RIPA lysis buffer (Beyotime, P0013B) containing 1% protease inhibitor (Beyotime, P1005) and 2% phosphatase inhibitor (Beyotime, P1045). Protein concentrations were measured using a BCA protein assay kit (Meilunbio, MA0082-2). The lysates were mixed with 5\u0026times; loading buffer (Beyotime, P0015), boiled at 100\u0026deg;C for 10 min, and then separated on 8% SDS-PAGE gels at 30 \u0026micro;g per lane. Proteins were transferred to 0.45 \u0026micro;m PVDF membranes (Bio-Rad; 1620177) by electroblotting. The membranes were blocked with 5% skim milk (Biosharp; BS102) in tris-buffered saline with Tween (TBST) (0.05% Tween-20 in Tris-buffered saline) for 1 h at room temperature, then incubated overnight at 4\u0026deg;C with primary antibodies against plectin (Abcam, ab32528), MFN1 (Proteintech, 13798-1-AP), MFN2 (Huabio, ER1802-23), DRP1 (Zenbio 221099), p-DRP1 (Ser616) (Abcam, ab314755) and GAPDH (Proteintech, HRP-60004). After washing with TBST, the membranes were incubated with secondary antibodies (ZSBIO, ZB-2301, ZB-2305) for 1h at room temperature. Following further washing, protein bands were visualized using an ECL detection system (Bio-OI, Guangzhou, China). GAPDH served as a loading control, and band intensities were quantified using ImageJ. Data were normalized by dividing the target protein/GAPDH ratios in each group by the average ratio from the three control groups.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eScratch assay\u003c/h3\u003e\n\u003cp\u003eThe scratch assay was conducted to evaluate cell migration. Inserts (Ibidi Culture-Insert, Germany) were placed in a 24-well plate (NEST, 702002) with hydrogels of different stiffness. Cells were added to both sides of the insert. After the cells reached confluence, the insert was removed, and the cells were washed twice, and cultured in serum-free medium. Wound healing was monitored by photographing at designated time points. The scratch areas were measured using ImageJ.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism version 9.5.1 (GraphPad Software Inc., San Diego, CA, USA) was utilized for graphing and statistical analysis. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Comparisons between two groups were made using Student's t-test, while one-way ANOVA was applied for multiple comparisons. Each experiment was conducted independently and replicated at least three times, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 denoting statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHigh matrix stiffness promotes the migration of HCC cells by upregulating plectin expression\u003c/h2\u003e \u003cp\u003eAs described in our previous study [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], to verify the response of plectin in HCC cells to changes in ECM stiffness, we cultured the cells for 48 hours on polyacrylamide hydrogels with two different stiffness levels (7 kPa and 53 kPa), representing the physiological elasticity of normal liver and HCC tissues, respectively [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. WB results showed that the expression level of plectin protein in HCC cells under high matrix stiffness was significantly higher than that in the low matrix stiffness group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Scratch experiment results showed that compared with 7 kPa, HCC cells cultured on 53 kPa hydrogels exhibited a significantly larger scratch healing area within 12 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). This indicates that high matrix stiffness significantly promotes the migration of HCC cells. Then, the knockdown efficiency of plectin was detected by WB. The results showed that compared with shNC, the plectin expression in cells carrying the shRNA viral fragment was significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, compared with the shNC group, the migration of HCC cells in the shPlectin group under high matrix stiffness did not show significant enhancement. This result strongly confirms that plectin is essential for high matrix stiffness-promoted HCC cell migration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHigh matrix stiffness enhances mitochondrial function in HCC cells\u003c/h2\u003e \u003cp\u003eMitochondrial function is closely linked to cell migration. As the cell's energy factory, mitochondrial dysfunction can cause metabolic disorders, which in turn affect cell migration [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Mitochondrial function indicators typically include ROS, mtROS, ATP production, and mtDNA copy number [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Changes in these indicators can directly reflect the functional state of mitochondria.\u003c/p\u003e \u003cp\u003eTo determine the effect of matrix stiffness on mitochondrial function, the changes in the above indicators were explored. DHE can penetrate the cell membrane of live cells, enter the cytoplasm, and undergo an oxidative reaction with intracellular ROS to form ethidium, which intercalates into chromosomal DNA and produces red fluorescence upon excitation. MitoSOX Red can penetrate the cell membrane and target mitochondria. Once inside the mitochondria, MitoSOX Red is specifically oxidized by superoxide and does not react with other reactive oxygen or nitrogen species. Oxidized MitoSOX Red can bind to nucleic acids in mitochondria and nuclei, producing intense red fluorescence. The results of DHE and MitoSOX Red staining showed that, compared with low matrix stiffness, the levels of ROS and mtROS in HCC cells were reduced under high matrix stiffness (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b). This indicates that the oxidative stress level in cells is lower and the redox homeostasis is more balanced, providing a good basis for the execution of normal physiological functions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mitochondrial respiration process converts the energy from substrates into ATP. When mitochondrial function is impaired, mitochondrial respiration is weakened, leading to a decrease in ATP production. The copy number of mtDNA is commonly used as an indicator of mitochondrial function. Each mitochondrion contains multiple copies, and the number can reflect the mitochondrial biogenesis capacity and energy metabolism level. MT-CO2 encodes cytochrome c oxidase subunit II, an important component of the mitochondrial respiratory chain. MT-ND1 is the largest protein complex in the mitochondrial respiratory chain, responsible for transferring electrons from NADH to ubiquinone (coenzyme Q). This process is the first step in oxidative phosphorylation and the beginning of energy conversion. MT-CYB encodes cytochrome b, a component of mitochondrial respiratory chain complex III. We selected three key mitochondrial-encoded genes and detected their copy number changes using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Meanwhile, it was observed that under high matrix stiffness, the ATP production and mtDNA copy number of HCC cells increased (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-d). The sufficient ATP may provide a solid energy guarantee for various physiological activities of the cells, and the increase in mtDNA copy number means that the biosynthetic capacity of mitochondria is enhanced, which is conducive to maintaining the normal function and quantity of mitochondria.\u003c/p\u003e \u003cp\u003eThese findings suggest that high matrix stiffness significantly enhances the mitochondrial function of HCC cells, providing stable support for their continuous metabolism and activity, and further ensuring the migratory potential of HCC cells in the high-stiffness environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHigh matrix stiffness enhances HCC mitochondrial function via plectin\u003c/h2\u003e \u003cp\u003eMutations in the human plectin gene are closely related to the occurrence of epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Studies have shown that fibroblasts from the skin of patients with EBS-MD exhibit abnormal mitochondrial networks, as well as significant barriers in key physiological functions such as wound healing and cell migration [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These abnormal manifestations imply the potential impact of plectin deficiency. So, it drives us to explore whether the enhanced mitochondrial function under high matrix stiffness is associated with the upregulation of plectin expression. The results of DHE and MitoSOX staining indicate that, compared with the control group, when plectin is knocked down, the levels of ROS and mtROS in HCC cells under high matrix stiffness are increased (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b). Moreover, it was observed that when plectin was knocked down, the promoting effects of high matrix stiffness on ATP production and mtDNA copy number in HCC cells were significantly weakened (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-d). This suggests that high matrix stiffness enhances mitochondrial function by upregulating plectin expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of mitochondrial function attenuates high stiffness-promoted HCC cell migration\u003c/h2\u003e \u003cp\u003eTo confirm the relationship between mitochondrial function and cell migration, we used CCCP, a mitochondrial oxidative phosphorylation uncoupler, to inhibit mitochondrial function. The results showed that after treatment with CCCP, the levels of ROS and mtROS in cells under high matrix stiffness increased (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-b), and ATP production and mtDNA copy number decreased (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-d). In other words, CCCP can inhibit the enhancement of mitochondrial function in HCC cells under high matrix stiffness. The scratch assay results indicated that CCCP can inhibit the promoting effect of high matrix stiffness on the migration of HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). This suggests that high matrix stiffness promotes the migration of HCC cells by enhancing mitochondrial function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHigh matrix stiffness promotes mitochondrial fusion\u003c/h2\u003e \u003cp\u003eMitochondrial dynamics (including mitochondrial fusion and fission) are closely related to mitochondrial function. Mitochondrial fusion helps to form a more interconnected network within the cell, and fused mitochondria can more efficiently exchange their internal DNA, proteins, and lipids, thereby enhancing overall function [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Mitochondrial fission helps to identify and separate damaged or dysfunctional parts of mitochondria, allowing them to be cleared by the process of mitophagy [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Studies have shown that HCC cells exhibit stronger mitochondrial function under high matrix stiffness. Therefore, it is necessary to explore the changes in mitochondrial dynamics under different matrix stiffness conditions and whether these changes affect mitochondrial function and the migration of HCC cells.\u003c/p\u003e \u003cp\u003eThe results of Mitotracker RED staining showed that the mitochondria in HCC cells cultured on high stiffness substrates were mostly elongated and tubular, while those on low stiffness substrates formed numerous small and short structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). This clear morphological difference indicates that high matrix stiffness effectively promotes mitochondrial fusion in HCC cells, while low matrix stiffness tends to promote mitochondrial fission. To further verify this phenomenon at the molecular level, we detected the proteins that play key roles in mitochondrial fusion and fission. mitofusin 1 (MFN1) and mitofusin 2 (MFN2) are key proteins in the fusion process, while Dynamin-related protein 1 (DRP1) and its phosphorylated form p-DRP1 (Ser616) are important factors in regulating fission [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. We used WB to detect the expression of MFN1, MFN2, DRP1, and p-DRP1 (Ser616) under different matrix stiffness conditions. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-e, high matrix stiffness promotes the expression of MFN1 and MFN2 in HCC cells while inhibiting the expression of DRP1 and p-DRP1 (Ser616). This result further demonstrates that compared with low matrix stiffness, high matrix stiffness enhances mitochondrial fusion and reduces fission capacity in HCC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHigh matrix stiffness promotes mitochondrial fusion via plectin\u003c/h2\u003e \u003cp\u003eTo investigate whether plectin plays a role in the promotion of mitochondrial fusion by high matrix stiffness, we used Mitotracker RED to stain the mitochondria in cells with plectin knockdown. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, compared with the control group, the mitochondrial morphology in HCC cells under high matrix stiffness changed from elongated tubular structures to small and fragmented ones after plectin knockdown. This significant morphological difference clearly indicates that plectin knockdown attenuates the pro-fusion effect of high matrix stiffness on HCC cell mitochondria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further detected the expression levels of MFN1, MFN2, DRP1, and p-DRP1 (Ser616) in the control and plectin knockdown groups under different matrix stiffness using WB. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-e, compared with the control group, the expression of MFN1 and MFN2 decreased, while the expression of DRP1 and p-DRP1 (Ser616) increased in the plectin knockdown group under high matrix stiffness. This indicates that plectin knockdown reduces mitochondrial fusion capacity and enhances fission capacity in HCC cells under high matrix stiffness, confirming that plectin plays a key role in regulating mitochondrial dynamics in response to matrix stiffness.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMFI8 inhibits mitochondrial fusion in HCC cells\u003c/h2\u003e \u003cp\u003eTo clarify the impact of mitochondrial fusion on mitochondrial function and cell migration, we employed MFI8 to inhibit mitochondrial fusion. The results of Mitotracker RED staining indicated that treatment with MFI8 led to significant changes in the mitochondrial morphology of HCC cells under high matrix stiffness. The originally elongated, tubular shapes were transformed into small, fragmented structures. This morphological difference intuitively reflects the inhibitory effect of MFI8 on mitochondrial fusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). WB analysis was conducted to detect the effects of matrix stiffness on MFN1 and MFN2 in HCC cells after MFI8 treatment. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-c, the upregulation of MFN1 and MFN2 were significantly suppressed following MFI8 treatment. These results fully demonstrate that MFI8 can effectively inhibit the promotion of mitochondrial fusion in HCC cells under high matrix stiffness.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMFI8 impairs mitochondrial function and attenuates cell migration by inhibiting mitochondrial fusion\u003c/h2\u003e \u003cp\u003eTo further investigate whether changes in mitochondrial dynamics under different matrix stiffness conditions affect mitochondrial function and subsequently influence HCC cell migration, we used MFI8 to intervene in mitochondrial fusion and measured mitochondrial function indicators and migration capacity. After treatment with MFI8, the levels of ROS and mtROS in HCC cells under high matrix stiffness were significantly higher than those in the untreated group (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea-b), while ATP production and mtDNA copy number under high stiffness were significantly reduced (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec-d). This indicates that MFI8, by interfering with mitochondrial fusion in HCC cells under high matrix stiffness, further leads to impaired mitochondrial function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, to further validate whether changes in mitochondrial dynamics affect HCC cell migration, we performed scratch assays to measure migration capacity under different matrix stiffness conditions after MFI8 treatment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee, high matrix stiffness significantly promoted HCC cell migration, but this effect was markedly inhibited by MFI8 treatment.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn the tumor microenvironment, matrix stiffness has garnered significant attention due to its important impact on tumor progression and treatment efficacy [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Matrix stiffness not only affects the proliferation and survival of tumor cells but also significantly regulates their migration and invasion capabilities. In recent years, an increasing number of studies have shown that mitochondrial function is closely related to tumor progression driven by matrix stiffness [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, the specific molecular mechanisms by which matrix stiffness regulates the migration of HCC cells through its effects on mitochondria have not yet been fully elucidated.\u003c/p\u003e \u003cp\u003eMatrix stiffness significantly affects the morphology and subcellular localization of mitochondria. Under low matrix stiffness, mitochondria typically exhibit a fragmented morphology with more branches, whereas under high matrix stiffness, they present with fewer branches and longer tubules [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This morphological change reflects the differences in mitochondrial dynamics. In breast cancer, low matrix stiffness leads to mitochondrial fragmentation, which in turn causes mitochondrial dysfunction and cell apoptosis [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. This indicates that changes in mitochondrial morphology directly affect their function, thereby regulating the metabolism and physiological functions of cells. In our study, we found that high matrix stiffness significantly promotes mitochondrial fusion in HCC cells, resulting in elongated tubular mitochondria, as evidenced by significant increases in mitochondrial perimeter, area, and branch length. This morphological change is closely related to enhanced mitochondrial function, characterized by decreased levels of ROS and mtROS, and increased ATP production and mtDNA copy number. These findings suggest that high matrix stiffness enhances mitochondrial function by promoting mitochondrial fusion, thereby providing favorable conditions for cell migration.\u003c/p\u003e \u003cp\u003ePlectin is a multifunctional cytoskeletal linker protein that is widely involved in cytoskeletal remodeling and cell migration. The absence of plectin leads to the separation of mitochondria from myofibrils, decreased activity of mitochondrial respiratory chain complexes, and mitochondrial respiratory dysfunction, which in turn results in insufficient muscle energy supply and functional impairment [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. This indicates that plectin plays an important role in maintaining the normal distribution and function of mitochondria. In our study, we found that high matrix stiffness promotes mitochondrial fusion and enhanced function in HCC cells by upregulating plectin expression, which further promotes cell migration. This finding reveals the key regulatory role of plectin in mitochondrial dynamics and function, providing a new perspective for understanding how matrix stiffness affects HCC cell migration by regulating plectin and thereby influencing mitochondria.\u003c/p\u003e \u003cp\u003eMitochondrial metabolism plays a crucial role in the tumorigenesis of various human cancers [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Recent studies have shown that SLC25A35 significantly reprograms mitochondrial metabolism by enhancing fatty acid oxidation, characterized by increased oxygen consumption rate and ATP production, as well as reduced ROS levels. Additionally, SLC25A35 also enhances mitochondrial biogenesis, as evidenced by increased mitochondrial mass and DNA content [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Our study results are similar, with high matrix stiffness upregulating plectin expression to induce mitochondrial fusion and enhance mitochondrial function, thereby providing strong support for the migration of HCC cells. Future research needs to further explore the specific molecular mechanisms between plectin and mitochondria, as well as how plectin affects the migration of HCC cells by regulating mitochondrial dynamics.\u003c/p\u003e \u003cp\u003eIn conclusion, this study has revealed the key mechanism by which high matrix stiffness promotes the migration of HCC cells: high matrix stiffness upregulates plectin expression, induces mitochondrial fusion, and enhances mitochondrial function, thereby driving the migration of HCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). These findings clarify the relationship between high matrix stiffness, plectin, mitochondria, and cell migration, providing an important theoretical basis for the development of novel therapeutic strategies for HCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eBCA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBicinchoninic acid assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCCCP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCarbonyl cyanide 3-chlorophenylhydrazone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDHE\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDihydroethidium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDRP1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDynamin-related protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eECM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eEMT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpithelial-mesenchymal transition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHCC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHepatocellular carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003emtDNA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial DNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003emtROS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial reactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMFI8\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrial fusion inhibitor 8\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMFN1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitofusin 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMFN2\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitofusin 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMT-CO2\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrially encoded cytochrome C oxidase II\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMT-CYB\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrially encoded cytochrome B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMT-ND1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ep-DRP1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhospho dynamin-related protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRT-qPCR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReal-time quantitative reverse transcription polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePVDF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolyvinylidene fluoride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eWB\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWestern blotting\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZhihui Wang:\u003c/strong\u003e performed experiments, wrote the manuscript and prepared figures. \u003cstrong\u003eWenbin Wang:\u003c/strong\u003e performed experiments. \u003cstrong\u003eXu Zhang:\u003c/strong\u003eperformed experiments. \u003cstrong\u003eQing Luo:\u003c/strong\u003e analyzed and interpreted the data. \u003cstrong\u003eGuanbin Song:\u003c/strong\u003e developed the concept, designed the study and edited the manuscript. All authors read and approved the final manuscript. The authors declare that all data were generated in-house and that no paper mill was used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research work was supported by grant from the National Natural Science Foundation of China (No. 11832008).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analyzed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human or animal subjects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eA.D. Ladd, S. Duarte, I. Sahin, et al. Mechanisms of drug resistance in HCC. Hepatology. 2024, 79(4): 926-940.\u003c/li\u003e\n\u003cli\u003eA. Sufianov, M. Agaverdiev, A. Mashkin, et al. Targeting microRNA methylation: Innovative approaches to diagnosing and treating hepatocellular carcinoma. Noncoding RNA Res. 2025, 11: 150-157.\u003c/li\u003e\n\u003cli\u003eG. Wu, N. Bajestani, N. Pracha, et al. Hepatocellular Carcinoma Surveillance Strategies: Major Guidelines and Screening Advances. Cancers (Basel). 2024, 16(23).\u003c/li\u003e\n\u003cli\u003eC.C. Lee, J.C. Lin, W.L. Hwang, et al. Macrophage-secreted interleukin-35 regulates cancer cell plasticity to facilitate metastatic colonization. Nat Commun. 2018, 9(1): 3763.\u003c/li\u003e\n\u003cli\u003eR. Liang, G. Song Matrix stiffness-driven cancer progression and the targeted therapeutic strategy. Mechanobiology in Medicine. 2023, 1(2): 100013.\u003c/li\u003e\n\u003cli\u003eM. Quintela-Fandino, B. Bermejo, E. Zamora, et al. High Mechanical Conditioning by Tumor Extracellular Matrix Stiffness Is a Predictive Biomarker for Antifibrotic Therapy in HER2-Negative Breast Cancer. Clin Cancer Res. 2024, 30(22): 5094-5104.\u003c/li\u003e\n\u003cli\u003eI. Samaržija The Potential of Extracellular Matrix- and Integrin Adhesion Complex-Related Molecules for Prostate Cancer Biomarker Discovery. Biomedicines. 2023, 12(1).\u003c/li\u003e\n\u003cli\u003eY. Zhao, H. Yu, J. Li, et al. A glucose-enriched lung pre-metastatic niche triggered by matrix stiffness-tuned exosomal miRNAs in hepatocellular carcinoma. Nat Commun. 2025, 16(1): 1736.\u003c/li\u003e\n\u003cli\u003eJ. Hong, J. Yu, D. Buratto, et al. Unveiling the role of mechanical microenvironment in hepatocellular carcinoma: Molecular mechanisms and implications for therapeutic strategies. Int J Biol Sci. 2024, 20(13): 5239-5253.\u003c/li\u003e\n\u003cli\u003eZ. Shen, L. Tao, Y. Wang, et al. Synergistic anticancer strategy targeting ECM stiffness: Integration of matrix softening and mechanical signal transduction blockade in primary liver cancers. Adv Sci (Weinh). 2025, 12(6): e2403040.\u003c/li\u003e\n\u003cli\u003eS. Wu, Y. Pan, Y. Mao, et al. Current progress and mechanisms of bone metastasis in lung cancer: a narrative review. Transl Lung Cancer Res. 2021, 10(1): 439-451.\u003c/li\u003e\n\u003cli\u003eS. Von Stockum, A. Nardin, E. Schrepfer, et al. Mitochondrial dynamics and mitophagy in Parkinson\u0026apos;s disease: A fly point of view. Neurobiol Dis. 2016, 90: 58-67.\u003c/li\u003e\n\u003cli\u003eR.J. Youle, A.M. van der Bliek Mitochondrial fission, fusion, and stress. Science. 2012, 337(6098): 1062-5.\u003c/li\u003e\n\u003cli\u003eT.F. Che, C.W. Lin, Y.Y. Wu, et al. Mitochondrial translocation of EGFR regulates mitochondria dynamics and promotes metastasis in NSCLC. Oncotarget. 2015, 6(35): 37349-66.\u003c/li\u003e\n\u003cli\u003eS.P. Desai, S.N. Bhatia, M. Toner, et al. Mitochondrial localization and the persistent migration of epithelial cancer cells. Biophys J. 2013, 104(9): 2077-88.\u003c/li\u003e\n\u003cli\u003eA. Ferreira-da-Silva, C. Valacca, E. Rios, et al. Mitochondrial dynamics protein Drp1 is overexpressed in oncocytic thyroid tumors and regulates cancer cell migration. PLoS One. 2015, 10(3): e0122308.\u003c/li\u003e\n\u003cli\u003eB. Zhang, J. Wang, Z. Huang, et al. Aberrantly upregulated TRAP1 is required for tumorigenesis of breast cancer. Oncotarget. 2015, 6(42): 44495-508.\u003c/li\u003e\n\u003cli\u003eJ. Zhao, J. Zhang, M. Yu, et al. Mitochondrial dynamics regulates migration and invasion of breast cancer cells. Oncogene. 2013, 32(40): 4814-24.\u003c/li\u003e\n\u003cli\u003eD. Senft, Z.A. Ronai Regulators of mitochondrial dynamics in cancer. Curr Opin Cell Biol. 2016, 39: 43-52.\u003c/li\u003e\n\u003cli\u003eY. Sun, Y. Qiao, Y. Niu, et al. ARP2/3 complex affects myofibroblast differentiation and migration in pancreatic ductal adenocarcinoma. Int J Cancer. 2025, 156(6): 1272-1281.\u003c/li\u003e\n\u003cli\u003eL. Hu, Z. Huang, Z. Wu, et al. Mammalian plakins, giant cytolinkers: Versatile biological functions and roles in cancer. Int J Mol Sci. 2018, 19(4).\u003c/li\u003e\n\u003cli\u003eD.A. Starr, H.N. Fridolfsson Interactions between nuclei and the cytoskeleton are mediated by SUN-KASH nuclear-envelope bridges. Annu Rev Cell Dev Biol. 2010, 26: 421-44.\u003c/li\u003e\n\u003cli\u003eM. Sutoh Yoneyama, S. Hatakeyama, T. Habuchi, et al. Vimentin intermediate filament and plectin provide a scaffold for invadopodia, facilitating cancer cell invasion and extravasation for metastasis. Eur J Cell Biol. 2014, 93(4): 157-69.\u003c/li\u003e\n\u003cli\u003eZ. Wang, W. Wang, Q. Luo, et al. High matrix stiffness accelerates migration of hepatocellular carcinoma cells through the integrin \u0026beta;1-Plectin-F-actin axis. BMC Biol. 2025, 23(1): 8.\u003c/li\u003e\n\u003cli\u003eK. Mado, V. Chekulayev, I. Shevchuk, et al. On the role of tubulin, plectin, desmin, and vimentin in the regulation of mitochondrial energy fluxes in muscle cells. Am J Physiol Cell Physiol. 2019, 316(5): C657-c667.\u003c/li\u003e\n\u003cli\u003eB. Tian, Q. Luo, Y. Ju, et al. A soft matrix enhances the cancer stem cell phenotype of HCC cells. Int J Mol Sci. 2019, 20(11).\u003c/li\u003e\n\u003cli\u003eQ.P. Liu, Q. Luo, B. Deng, et al. Stiffer matrix accelerates migration of hepatocellular carcinoma cells through enhanced aerobic glycolysis via the MAPK-YAP signaling. Cancers (Basel). 2020, 12(2).\u003c/li\u003e\n\u003cli\u003eR. Xu, S. He, D. Ma, et al. Plectin downregulation inhibits migration and suppresses epithelial mesenchymal transformation of hepatocellular carcinoma cells via ERK1/2 signaling. Int J Mol Sci. 2022, 24(1).\u003c/li\u003e\n\u003cli\u003eV.W. Wong, J. Vergniol, G.L. Wong, et al. Diagnosis of fibrosis and cirrhosis using liver stiffness measurement in nonalcoholic fatty liver disease. Hepatology. 2010, 51(2): 454-62.\u003c/li\u003e\n\u003cli\u003eJ. Swift, I.L. Ivanovska, A. Buxboim, et al. Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation. Science. 2013, 341(6149): 1240104.\u003c/li\u003e\n\u003cli\u003eP.J. Eddowes, M. Sasso, M. Allison, et al. Accuracy of fibroScan controlled attenuation parameter and liver stiffness measurement in assessing steatosis and fibrosis in patients with nonalcoholic fatty liver disease. Gastroenterology. 2019, 156(6): 1717-1730.\u003c/li\u003e\n\u003cli\u003eN. Li, X. Zhang, J. Zhou, et al. Multiscale biomechanics and mechanotransduction from liver fibrosis to cancer. Adv Drug Deliv Rev. 2022, 188: 114448.\u003c/li\u003e\n\u003cli\u003eS. Madan, B. Uttekar, S. Chowdhary, et al. Mitochondria lead the way: Mitochondrial dynamics and function in cellular movements in development and disease. Front Cell Dev Biol. 2021, 9: 781933.\u003c/li\u003e\n\u003cli\u003eC. Xu, L. Qiao, L. Ma, et al. Biogenic selenium nanoparticles synthesized by Lactobacillus casei ATCC 393 alleviate intestinal epithelial barrier dysfunction caused by oxidative stress via Nrf2 signaling-mediated mitochondrial pathway. Int J Nanomedicine. 2019, 14: 4491-4502.\u003c/li\u003e\n\u003cli\u003eD. Kiritsi, L. Tsakiris, F. Schauer plectin in skin fragility disorders. Cells. 2021, 10(10).\u003c/li\u003e\n\u003cli\u003eM.M. Zrelski, S. H\u0026ouml;sele, M. Kustermann, et al. Plectin deficiency in fibroblasts deranges intermediate filament and organelle morphology, migration, and adhesion. J Invest Dermatol. 2024, 144(3): 547-562.e9.\u003c/li\u003e\n\u003cli\u003eM.J. Casta\u0026ntilde;\u0026oacute;n, G. Wiche identifying plectin isoform functions through animal models. Cells. 2021, 10(9).\u003c/li\u003e\n\u003cli\u003eJ. Nunnari, W.F. Marshall, A. Straight, et al. Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA. Mol Biol Cell. 1997, 8(7): 1233-42.\u003c/li\u003e\n\u003cli\u003eK. Okamoto, P.S. Perlman, R.A. Butow The sorting of mitochondrial DNA and mitochondrial proteins in zygotes: preferential transmission of mitochondrial DNA to the medial bud. J Cell Biol. 1998, 142(3): 613-23.\u003c/li\u003e\n\u003cli\u003eD.F. Kashatus, K.H. Lim, D.C. Brady, et al. RALA and RALBP1 regulate mitochondrial fission at mitosis. Nat Cell Biol. 2011, 13(9): 1108-15.\u003c/li\u003e\n\u003cli\u003eY. Li, Y. Gao, G. Yu, et al. G6PD protects against cerebral ischemia-reperfusion injury by inhibiting excessive mitophagy. Life Sci. 2025, 362: 123367.\u003c/li\u003e\n\u003cli\u003eA. Santel, M.T. Fuller Control of mitochondrial morphology by a human mitofusin. J Cell Sci. 2001, 114(Pt 5): 867-74.\u003c/li\u003e\n\u003cli\u003eR.L. Barrett, E. Pur\u0026eacute; Cancer-associated fibroblasts and their influence on tumor immunity and immunotherapy. Elife. 2020, 9.\u003c/li\u003e\n\u003cli\u003eM. Chiriv\u0026igrave;, F. Maiullari, M. Milan, et al. Tumor extracellular matrix stiffness promptly modulates the phenotype and gene expression of infiltrating T lymphocytes. Int J Mol Sci. 2021, 22(11).\u003c/li\u003e\n\u003cli\u003eY. Chen, P. Li, X. Chen, et al. Endoplasmic reticulum-mitochondrial calcium transport contributes to soft extracellular matrix-triggered mitochondrial dynamics and mitophagy in breast carcinoma cells. Acta Biomater. 2023, 169: 192-208.\u003c/li\u003e\n\u003cli\u003eY. Chen, P. Li, Y. Peng, et al. Protective autophagy attenuates soft substrate-induced apoptosis through ROS/JNK signaling pathway in breast cancer cells. Free Radic Biol Med. 2021, 172: 590-603.\u003c/li\u003e\n\u003cli\u003eP. Daga, B. Thurakkal, S. Rawal, et al. Matrix stiffening promotes perinuclear clustering of mitochondria. Mol Biol Cell. 2024, 35(7): ar91.\u003c/li\u003e\n\u003cli\u003eL. Winter, A.V. Kuznetsov, M. Grimm, et al. Plectin isoform P1b and P1d deficiencies differentially affect mitochondrial morphology and function in skeletal muscle. Hum Mol Genet. 2015, 24(16): 4530-44.\u003c/li\u003e\n\u003cli\u003eP.E. Porporato, N. Filigheddu, J.M.B. Pedro, et al. Mitochondrial metabolism and cancer. Cell Res. 2018, 28(3): 265-280.\u003c/li\u003e\n\u003cli\u003eH.C. Yu, L. Bai, L. Jin, et al. SLC25A35 enhances fatty acid oxidation and mitochondrial biogenesis to promote the carcinogenesis and progression of hepatocellular carcinoma by upregulating PGC-1\u0026alpha;. Cell Commun Signal. 2025, 23(1): 130.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"hepatocellular carcinoma, plectin, mitochondria, migration, matrix stiffness","lastPublishedDoi":"10.21203/rs.3.rs-6831299/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6831299/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIncreased matrix stiffness is a key physical signal affecting the migration of hepatocellular carcinoma (HCC) cells, and mitochondrial dynamics and function also play important roles in cell migration. Plectin may influence mitochondrial dynamics and function through its cytoskeletal cross-linking function. However, the relationship between these two factors remains unclear. HCC cells were seeded on hydrogels with stiffness of 7 kPa and 53 kPa, respectively, to investigate the effects of matrix stiffness on plectin expression, mitochondrial dynamics and function, and cell migration. Moreover, plectin was knocked down to further assess its specific impacts on mitochondrial dynamics and function, as well as cell migration under different matrix stiffness. Compared with 7 kPa, high matrix stiffness (53 kPa) promotes HCC cell migration by upregulating plectin expression, promoting mitochondrial fusion, and enhancing mitochondrial function. Under high matrix stiffness, plectin knockdown weakens mitochondrial fusion capacity and function, reducing cell migration. Subsequently, we treated cells with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to inhibit mitochondrial function. This treatment significantly suppressed cell migration on high- matrix stiffness. Then, when mitochondrial dynamics were disrupted by the mitochondrial fusion inhibitor 8 (MFI8), mitochondrial function was compromised, and cell migration decreased. High matrix stiffness enhances mitochondrial function by driving mitochondrial fusion through increasing plectin expression, thereby promoting the migration of HCC cells. It provides new insights into the mechanobiological mechanisms underlying matrix stiffness affected HCC cell migration.\u003c/p\u003e","manuscriptTitle":"Plectin-mediated mitochondrial fusion is necessary for hepatocellular carcinoma cell migration promoted by higher matrix stiffness","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-25 08:39:09","doi":"10.21203/rs.3.rs-6831299/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c63801f3-62f4-4ab2-8f25-eafa4c363e3c","owner":[],"postedDate":"June 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-13T10:09:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-25 08:39:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6831299","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6831299","identity":"rs-6831299","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}