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Plectin, a novel regulator in migration, invasion and adhesion of ovarian cancer

Cell & Bioscience volume 15, Article number: 15 (2025) Cite this article

Abstract

Background

Ovarian cancer (OC) is one of the most prevalent gynecologic malignancies and exhibites the highest fatality rate among all gynecologic malignancies. The absence of an early diagnostic biomarker and therapeutic target contributes to an overall 5-year survival rate ranging from 30 to 50%. Plectin (PLEC), a 500 kDa scaffolding protein, has gained prominence in recent years due to its pivotal role in various cellular biological functions such as cell morphology, migration and adhesion, while the accurate role of PLEC in OC remains elusive.

Results

In this study, our findings demonstrate that PLEC exerts a positive influence on the progression of OC, encompassing cellular proliferation, migration, invasion, and adhesion both in vitro and in vivo.

Conclusions

The results providing new insights for the diagnosis and treatment in OC.

Introduction

Ovarian cancer (OC) is an aggressive and progressive gynecological tumor [1], epithelial ovarian cancer (EOC), one of the most common types of ovarian cancer, which constitutes 90% of the malignant ovarian cancer, result in 19,880 estimate new cases and 12,810 estimated deaths in 2022 in United States [2, 3]. The progression of OC often gives rise to metastasizes, thereby resulting in an unfavorable prognosis and elevated mortality rate among [4]. Although the utilization of a combination of biomarkers and multiple clinical factors has been demonstrated to enhance diagnostic accuracy [5], the prognosis of OC remains poor, with an overall 5-year survival rate ranging from 30–50% [6]. The prognosis of OC remains unfavorable due to the absence of early diagnostic makers.

Plectin (PLEC), a 500 kDa scaffolding protein, abundantly expressed in a variety of mammalian tissues and cells [7], consist of a globular domain at the amino terminus, a globular domain at the carboxy terminus and an α-helical rod-like central domain of approximately 200 nm in length connected therebetween [8] It interlinks intermediate filaments with microtubules and microfilaments and anchors intermediate filaments to desmosomes or hemidesmosomes to affect cell adhesion, migration, proliferation, and other biological functions [9,10,11,12]. It has recently been implicated as a pro-tumorigenic regulator of cancer cell biology functions [13]. However, the majority of previous research on PLEC has predominantly focused on its role in skin and skeletal muscles diseases [14]. Although research on the role of PLEC in cancer is now gradually beginning [15], with limited investigations into its involvement in ovarian cancer.

In this study, we investigated the impact of PLEC expression on the progression of EOC by modulating cellular proliferation, migration, invasion, and adhesion both in vitro and in vivo. Furthermore, we conducted an extensive analysis to elucidate the underlying mechanisms involved. Our findings suggest that PLEC has potential as an early diagnostic biomarker and therapeutic target of EOC, providing novel insights into its diagnosis and treatment.

Materials and methods

Cell culture

Human epithelial ovarian cancer cell lines SKOV3 and A2780 were obtained from Procell (Wuhan, Hubei, China) and FENGHUISHENGWU (Changsha, Hunan, China). The SKOV3 and A2780 cells were cultured in MCCOY’S 5 A medium (Solarbio, Beijing, China) with 15% fetal bovine serum (FBS, TIANHANG, China) or 1640 medium (Hyclone, Cytiva, America) with 10% FBS receptively, which both containing 1% penicillin-streptomycin (Solarbio, Beijing, China). Both cells were routinely grown in a humidified incubator with 5% CO2 at 37℃.

Lentivirus infection

PLEC-Knock Down (KD) cell lines (SKOV3 and A2780) were generated using lentivirus vectors with PLEC shRNA sequence (5’-GGATCCGGTCTCAGTTCCTGAAGTTTATTCAAGAGATAAACTTCAGGAACTGAGACCTTTTTTGAATTC-3’) according to the manufacturer’s instruction, and NC shRNA lentivirus vectors were used as control. Generally speaking, lentivirus vectors were added at a multiplicity of infection (MOI) of 10 into the cells with polybrene when the cells reached 70% confluency, then the medium was replaced after 24 h of infection and the efficiency of transfection was evaluated by western blotting. The PLEC-KD cells were screened with 0.75 µg/ml Puromycin dihydrochloride.

siRNA transfection

The protocol followed our previous paper [16].The siRNAs targeting COL17A1 and ITGβ4 were designed and synthesized by Sangon Biotech (Shanghai, China), and the knockdown efficiency was assessed using Western blot. A2780 and SKOV3 cells that were transfected with negative siRNA were used as controls, and non-siRNA were used as blank, siRNA1151, siRNA454 inA2780 and siRNA492, siRNA114 in SKOV3 (renamed as KD ITGβ4 and KDCOL17A1) were chosen for further experiment.

Western blotting

Total protein preparation for Western Blot was performed as described previously [16]. Generally, proteins were extracted using Column Tissue & Cell Protein Extraction Kit (Epizyme, Shanghai, China), boiled in 1×SDS PAGE loading buffer (RB005-001, Rui Biotech, China), and then separated on 10% SDS-PAGE gels. Immunoblotting was performed with primary antibodies against plectin (ab32528, Abcam, UK) and β-actin (AB0501, Abways, Shanghai), then incubated with AffiniPure Goat Anti-Rabbit IgG H&L/HRP (bs-40295G-HRP, Bioss, Beijing) and visualized using an Omni-ECL™Femto Light Chemiluminescence Kit (SQ201, Epizyme, Shanghai).

Quantitative RT-PCR

Total RNA kit (BSC52M1, BioFlux, China) was used for RNA extraction, and the ReverTra Ace® qPCR RT Kit (FSQ-101, TOYOBO, Shanghai) was used for cDNA synthesize. PCR was carried out in a 20 µl reaction containing 1×SYBR Green PCR Master Mix (AQ601, TransGen, China) with conditions of 95℃ for 3 min, 95℃ for 30 s, 60℃ for 30s and 72℃ for 30s for 40 cycles. All the procedures were carried out according to each manufacturer’s instructions.

Primer sequences for PLEC are:

5’-CCGCCTCTTCAATGCCATCATCC-3’(plectin-F),

5’-TCCAGGTTCTCCAGGTTGGTCTG-3’ (plectin-R).

Primer sequences for GAPDH are:

5’-CAGGAGGCATTGCTGATGAT-3’ (GAPDH-F),

5’-GAAGGCTGGGGCTCATTT-3’ (GAPDH-R).

Colony formation

Cells were seeded into 6-well plates at 1 × 103/well dispersedly and cultured in medium till the cells of majority individual clones have greater than 50 under microscope. Fixed the cells in 4% paraformaldehyde at 20 min, stained by 0.1% crystal violet, and record with camera. All the experiments were repeated 3 times.

CCK-8 assay

Cells were seeded into 96-well plates at 5 × 103/well and cultured in complete medium for 24/48 h, then 10 µl CCK-8 solution (Proteintech, Wuhan, China) was add into each well according to the manufacturer’s instruction. The OD value of 450 nm was determined 4 h later.

Wound healing assay

Cells were seeded into 6-well plates and cultured in complete medium till the cell density reaches about 90%, then scratched vertically along the edge of ruler using 20 µl tips, wash 2 times with PBS, and add the medium with 2% FBS. Results were observed and recorded at 0, 24 and 48 h after scratching using light microscope (CKX53, Olympus, Japan). All the experiments were repeated 3 times.

Cell migration and invasion assays

Cells were seeded into 24-well transwell upper chambers (3422, Corning, USA) at 5 × 103/chamber with serum-free medium, and the lower chambers were filled with 600 µl of complete medium. 100 µg Matrigel (354234, Corning, USA) was precoated at the end of the chamber in the invasion assay. Then fixed the cells in 4% paraformaldehyde at 24 h, stained by 0.1% crystal violet, and recorded using light microscope (CKX53, Olympus, Japan). All the experiments were repeated 3 times.

Dissociation assay

The protocol was described clearly in previous paper [17]. Briefly, Cells were seeded into 6-well plates at proper density, cultured in complete medium for 24 h. Cells were then separated from the plates with Trypsin-EDTA Solution (Solarbio, Beijing, China). Transfer the dispase solution into a new 6-well plate, next, the cell was mechanically stressed by pipetting up and down 7 times carefully.

Adhesion assay

The adhesion capacity of the cells was measured by cell adhesion detection kit (BestBio Company, Shanghai, China) according to the manufacture’s instruction. Briefly, the coating liquid precoated at the 96-well plates overnight at 4℃, seeded cells into 96-well plates at 5 × 103/well, 37 °C incubator for 30 min. Then added 20 µl staining solution B to cells, and value the OD at 450 nm wavelength at 2 h later.

Confocal microscopy assay

Cells were seeded into confocal dishes at 5 × 103/well, cultured in complete medium, then fixed the cells in 4% paraformaldehyde at 24 h. Target proteins were visualized using primary antibodies against plectin (ab32528, Abcam, UK), collagen XVII (COL17A1, ab184996, Abcam, UK), integrin beta4 (ITGβ4, ab182120, Abcam, UK), and then secondary antibodies including Cy3-labeled Goat Anti-Rabbit IgG (H + L) (A0516, Beyotime, China), FITC-labeled Goat Anti-Rabbit IgG (H + L) (A0562, Beyotime, China). Nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI). The images were recorded under a confocal laser scanning microscope (TCS SP8, Leica, Germany).

Immunohistochemical analysis

Epithelial ovarian cancer tissue microarrays (Ova-809, Alenabio, China) were purchased for PLEC detecting. Paraffin sections were dewaxed and hydrated. Antigen were retrieved by citrate buffer and blocked H2O2 (PV-9003, Goat two-step test kit, ZSGB-BIO) at 37 °C for 20 min, and blocked at room temperature with 1% BSA for 1 h. Slides were incubated with diluted primary antibodies (1:500) including plectin (ab32528, Abcam, UK) and COL17A1 (ab184996, Abcam, UK) and ITGβ4 (ab182120, Abcam, UK) at 4 °C overnight followed by secondary antibody of the Goat two-step test kit for 20 min at room temperature. The slides were stained by diaminobenzidine (DAB) of the Goat two-step test kit and staining in hematoxylin solution for 2 min, mounted by 1% hydrochloric acid alcohol differentiation solution. Pictures were taken by 20× magnification light microscope (U-HGLGPS, Olympus, Japan). The H-Score of each spot on each chip was quantified using the Densito quantitative module of Quant Center 2.1 analysis software, and the H-Score score represents the strength of positive expression. And the details of ovarian tissues are shown in Table 1.

Table 1 Details of ovarian tissues
Full size table

In vivo assays

Female BALB/c nude mice (4-week-old) were purchased (Charles River) and randomized into treatment and control groups (n = 5 mice/group) for further in vivo assays. The A2780 cells were chosen to be used in the in vivo assays.

For the analysis of subcutaneous tumor models, a mixture of A2780 cells (1 × 108/ml) and matrigel matrix (354248, Corning, America) was subcutaneously injected into the BALB/c nude mice (100 µl/mouse). Tumor volume was measured every five days with vernier calipers, excised the tumors from the mice once the tumor volume reached tissues approximately 1000–2000 mm3. Subsequently, the excised tissues were cut into small pieces measuring approximately 2–3 mm3 and implanted in the flank of female mice aged 4-week-old. Size of tumor tissues were measured every 3 days using a caliper.

For the analysis of tumor in situ, 15 µl cells, which carrying a dual GFP/luciferase expression system (KDPLEC) and shEV (Control), were injected into the left ovary of the 4-week-old female BALB/c nude mice at 1 × 108/ml (serum-free medium). The location and numbers of these transduced cells were visualized using IVIS (PE IVIS Spectrum, PerkinElmer) at 5 weeks after the injection.

The resected tumors were fixed in paraffin-embedded blocks for subsequent IHC analysis.

Statistical analysis

Data were statistically analyzed using GraphPad Prism 9 (GraphPad Software), two-tailed unpaired Student’s t-test. Pictures was analyzed using ImageJ. At least 3 times repeat each experiment, and values represent the mean ± standard deviation of the mean. P values less than 0.05 is considered as a standard of significant difference.

Results

The expression of PLEC is highly related with epithelial ovarian cancer

Our previous paper has indicated that the mRNA expression of PLEC was upregulated in ovarian cancers compared with that in normal ovarian tissues based on bioinformatics analysis [16]. Further in this paper, our results demonstrated that the protein expression level of PLEC in pathological tissues is higher than that in normal tissue according to the immunohistochemical results (Fig. 1C), and the typical images were shown as in Fig. 1A and B. But as the TNM stages increased, the expression of PLEC did not show a consistent rise according to the H-Score results (Fig. 1D, E). Taken together, our results in Fig. 1 suggested that the protein expression of PLEC was actually higher in epithelial ovarian cancer tissues at the overall level.

Fig. 1
figure 1

The expression of PLEC is significantly upregulated in EOC. A Expression of PLEC in normal and EOC tissues. B Expression of PLEC in normal tissues and different types of EOC (LGSOC and HGSOC) tissues. C The result of statistical analysis of H-Score expressed by PLEC in A. D The result of statistical analysis of the H-Score of PLEC expression in E. E The expression of PLEC in normal tissues and EOC tissues of different stages (I, II and III). P-value: *<0.05; ***<0.001; ns: no significance, determined by two-tailed Student’s t-test

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PLEC promoted ovarian cancer cell proliferation in vitro and in vivo

To demonstrated the accurate role of PLEC in epithelial ovarian cancer, we constructed two PLEC knock-down and Control epithelial ovarian cancer cell lines (A2780, SKOV3) for further research. The results of Western Blot confirmed that PLEC was successfully knock down (Fig. 2A, B), similar with the result of qRT-PCR (Fig. 2C). While confocal assay also revealed pronounced disparities in PLEC fluorescence intensity (Fig. 2D, E).

Fig. 2
figure 2

Constructed A2780 and SKOV3 PLEC knock-down and control EOC cell lines. A PLEC was knock-down in A2780 and SKOV3. Expression of PLEC was determined by western blotting analysis. B The result of statistical analysis of the grayscale value expressed by PLEC in A, quantified by imageJ, error bars represent SD. C The result of statistical analysis of expression of PLEC determined by qRT-PCR, error bars represent SD. D Confocal assay: A2780 and SKOV3 PLEC knock-down and control EOC cells were seeded into confocal dishes at 5 × 103/well, fixed after 24 h. Target proteins were visualized using antibodies, nuclei were stained by DAPI (green: PLEC, blue: DAPI). E The result of statistical analysis of the fluorescence intensity value expressed by PLEC in D, quantified by imageJ, error bars represent SD. P-value: *<0.05; **<0.01; ***<0.001, **** <0.0001, determined by two-tailed Student’s t-test

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Using the KDPLEC and Control cell lines, we then verified that PLEC played a positive role in the proliferation of EOC cells in vitro. As the results shown in Fig. 3A-D, both two KDPLEC cell lines exhibited significantly decreased colony formation (Fig. 3A, B), cell viability (Fig. 3C) and cell count (Fig. 3D). Similar result was further confirmed in vivo using subcutaneous tumor model, the tumor volume of KDPLEC group is significantly smaller than Control both in vivo (Fig. 3E) and ex vivo (Fig. 3F-G).

Fig. 3
figure 3

PLEC promoted ovarian cancer cell proliferation both in vitro and in vivo.A Colony formation assays of A2780 and SKOV3 PLEC knock-down and control EOC cells. Cells were fixed 2 weeks later, stained with crystal violet. B The result of statistical analysis of the area covered by colonies across average expressed by PLEC in A, quantified by imageJ, error bars represent SD. C Cells were seeded into 96-well plates at 5 × 103/well, The OD value of 450 nm was determined at 24/48 h. Error bars represent SD. D Cells were seeded into 96-well plates at 4 × 103/well, count the cell number at 24/48 h. E Injected the mixture of A2780 PLEC knock-down and control cells (1 × 108/ml) and matrigel matrix subcutaneously into the BALB/c nude mice (n = 5) till the tumor volume reached tissues approximately 1000–2000 mm3. F Excised the tumors from the mice. G Tumor volume was measured every five days with vernier calipers, error bars represent SD. P-value: *<0.05; **<0.01; ***<0.001; ns: no significance, determined by two-tailed Student’s t-test

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PLEC promoted ovarian cancer cell invasion and metastasis in vitro and in vivo

The invasion capacity of two PLEC knockdown cells was observed to decrease when 100 µg Matrigel was precoated at the end of the chamber (Fig. 4A, B), suggesting that PLEC plays a crucial role in promoting cell invasion capacity in vitro. Meanwhile, the IVIS imaging results demonstrated a significant reduction in the number of metastases in KDPLEC group compared to the Control group in vivo (Fig. 4E), and using visual inspection, a widely accepted method when animals were dissected [18,19,20], we found that the number of metastatic nodules was significantly lower in intestine, kidney, and spleen of the KDPLEC group than Control group (Fig. 4C), and the typical images were shown as Fig. 4D, which was consistent with our previous findings on cell invasion capacity in vitro.

Fig. 4
figure 4

PLEC promoted ovarian cancer cell invasion in vitro and in vivo. A Cells were seeded into 24-well transwell upper chambers at 5 × 103/chamber with serum-free medium, and the lower chambers with complete medium. Matrigel was precoated at the end of the chamber, fixed the cells at 24 h, stained by crystal violet. B Cell count quantified by imageJ, error bars represent. C The statistical analysis of the number of tumor metastasis. D Representative intestine, kidney, and spleen tissues of KDPLEC and control. Arrowheads, metastatic tumors. E 15 µl KDPLEC and control cells were injected into the left ovary of the BALB/c nude mice at 1 × 108/ml, visualized using the IVIS in vivo imaging system after 5 weeks. Error bars represent SD. P-value: **<0.01, determined by two-tailed Student’s t-test

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PLEC promoted ovarian cancer cell migration and adhesion in vitro

The impact of PLEC on cell migration was subsequently validated through Wound Healing Assay and Cell Migration Assay. The typical images from the Wound Healing Assay demonstrated a significant increase in the area between two edges of KDPLEC cells compared to the Control cells in both two cell lines, indicating that knocking down PLEC effectively suppressed cell migration at 48 h post-scratching (Fig. 5A), and the results were further confirmed according to the statistical analysis (Fig. 5B and C). Furthermore, the results obtained from the Cell Migration Assay indicated that the migratory cell count of the KDPLEC cells was substantially diminished compared to the Control cells, subsequent to an equivalent duration of cultivation. (Fig. 5D).

Fig. 5
figure 5

PLEC promoted ovarian cancer cell migration in vitro. A Wound Healing Assay of A2780 and SKOV3 PLEC knock-down and control EOC cells, recorded at 0 and 48 h. B The area of blank was quantified by imageJ, error bars represent. C The result of statistical analysis of the cell counts in D, quantified by imageJ, error bars represent. D Cells were seeded into 24-well transwell upper chambers at 5 × 103/chamber with serum-free medium, and the lower chambers with complete medium, fixed the cells at 24 h, stained by crystal violet. P-value: *<0.05; **<0.01; ****<0.0001; ns: no significance, determined by two-tailed Student’s t-test

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Previous studies have demonstrated a strong association between PLEC and focal adhesion [21, 22], suggesting that PLEC plays a regulatory role in cell adhesion. To validate this hypothesis, we performed a dissociation assay which revealed that knockdown PLEC promoted cell dissociation (Fig. 6A, B). Subsequently, we assessed the adhesion capacity of cells using a cell adhesion detection kit and observed a significant suppression in adhesion capacity following PLEC knockdown (Fig. 6C).

Fig. 6
figure 6

PLEC promoted ovarian cancer cell adhesion in vitro with COL17A1 and ITGβ4. A The image fragmented cell monolayers which were detached with dispase and mechanically fragmented by pipetting. B The area of those cell fragments quantification by imageJ, error bars represent. C pre-coating the 96-well plate with the coating solution from the adhesion detection kit, seeding the cells in the 96-well plate at a density of 5 × 104 cells per well, adding 10 µl of cell staining solution to each well for 2 h after incubation, and subsequently measuring the OD value of the sample wells after 4 h, followed by statistical analysis of the data. D COL17A1 and ITGβ4 are highly related with PLEC (https://cn.string-db.org/). E Confocal assays (green: PLEC, red: ITGβ4/COL17A1, blue: DAPI). F IHC of tumor slides. G The result of statistical analysis of the fluorescence intensity value expressed by PLEC, ITGβ4 and COL17A1 in B, quantified by imageJ, error bars represent SD. P-value: *<0.05; **<0.01; ***<0.001; ****<0.0001, determined by two-tailed Student’s t-test

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PLEC modulates cell adhesion through its interaction with COL17A1 and ITGβ4

In an endeavor to delineate the underlying mechanism by which PLEC regulates the adhesive properties of ovarian cancer cells, an investigation was conducted into various proteins that are reported to associated with PLEC on STRING (https://cn.string-db.org/), The findings revealed a multitude of proteins that interact with PLEC, including RPS15, COL17A1, ITGβ4, SYNE3 and CASP8 etc. Notably, the strongest correlations at the protein level were established between PLEC and COL17A1, ITGβ4, CASP8 at protein level (Fig. 6D). Given that COL17A1 and ITGβ4 play crucial roles in the adhesion process in cancer [23, 24], they were selected for further detailed inquiry in OC.

Subsequently, the target proteins were subjected to immunofluorescence staining and visualized imaging using a confocal laser microscope. The results revealed predominant colocalization of PLEC with COL17A1 and ITGβ4 mostly in both two cell lines (Fig. 6E), corroborating our previous findings. The IHC results obtained from resected tumors in vivo strongly demonstrated a significant decrease in the expression levels of COL17A1 and ITGβ4 upon knockdown of PLEC (Fig. 6F, G).

To verify the specific roles of COL17A1 and ITGβ4 in OC adhesion, we constructed the COL17A1 and ITGβ4 knockdown A2780 and SKOV3 cell lines via siRNA transfection. The results of Western blot confirmed the successful reduction in the expression levels of COL17A1 and ITGβ4 (Fig. 7A-D). Subsequent dissociation assay revealed that the adhesion capacity of A2780 and SKOV3 cells was significantly inhibited following the knockdown of COL17A1 and ITGβ4 (Fig. 7E, F). Besides, cell adhesion assays using a cell adhesion detection kit further affirmed that the adhesion ability of A2780 and SKOV3 cells was significantly reduced after knockdown of COL17A1 and ITGβ4 compared to the Control group (Fig. 7G).

Fig. 7
figure 7

PLEC modulates cell adhesion through its interaction with COL17A1 and ITGβ4. A, B ITGβ4 and COL17A1 was knock-down in A2780 and SKOV3. Expression of ITGβ4 and COL17A1 was determined by western blotting analysis. C, D The result of statistical analysis of the grayscale value expressed by ITGβ4 and COL17A1 in A, quantified by imageJ, error bars represent SD. E The image fragmented cell monolayers which were detached with dispase and mechanically fragmented by pipetting. F The area of those cell fragments in E, quantification by imageJ, error bars represent. G Adhesion assay. P-value: *<0.05; **<0.01; ***<0.001; ****<0.0001; ns: no significance, determined by two-tailed Student’s t-test

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Notably, knockdown of PLEC precipitated a significant decline in the expression levels of COL17A1 and ITGβ4 in both two cell lines, indicating that PLEC may interact with these proteins to modulate their expression. In conclusion, the findings in Figs. 6 and 7supports that PLEC plays a regulatory role in cell adhesion through its interaction COL17A1 and ITGβ4.

Discussion

Plakins is a family of cytoskeletal binding proteins expressed in a variety of tissues and cells, which anchor the cytoskeletal network to each other [25]. Plectin, a member of plakins family, is a substantial cytoskeletal binding protein with a dumbbell-shaped weighing no less than 500 kDa [9]. It has gained significant attention in recent years due to its pivotal role in various cancer progression [26, 27]. In our previous study, we identified a strong correlation between PLEC and OC, suggesting its potential as a diagnostic biomarker for OC [16, 28]. However, further research in this area remains vacant. Therefore, we conducted additional investigations to elucidate the precise role of PLEC in in the progression of OC.

In this study, our analysis of tissue microarrays revealed a significantly higher expression level of PLEC in OC tumor tissues compared to normal tissues, providing further evidence for the involvement of PLEC in OC progression (Fig. 1). However, the result in Fig. 1D indicated that with the increasing TNM stages, the expression of PLEC did not show a consistent rise according to the H-Score results. This may be caused by two reasons: firstly, the microarrays of ovarian cancer using for IHC was consist of different subtypes of ovarian cancer tissues, including mucinous, serous papillary, endometrioid, clear cell, mucinous papillary and serous carcinomas, the expression score and percentage of PLEC was differential in these subtypes (Table 1), and this was also confirmed in other research results [29]. Secondly, this may be caused by the limitation of tissues number including normal and malignant. Consistent with our result, the expression level of PLEC is significantly altered in various other cancer types, including prostate cancer, colon cancer, and pancreatic cancer [30,31,32]. Therefore, we generated two PLEC knock-down and Control EOC cell lines for the further investigation (Fig. 2). It has been established that PLEC influences cell proliferation in multiple cell lines due to its impact on mitosis [33]. Our results confirmed a significant decrease in proliferation of PLEC knockdown cell lines both in vitro and in vivo (Fig. 3), providing compelling evidence for the promotion of OC cell proliferation by PLEC.

During tissue cell morphogenesis, post-injury repair and cancer progression, cells usually migrate collectively in groups, chains and sheets [34]. The cytoskeleton which composed of F-actin, MTs and IFs plays a pivotal role in facilitating cell migration, while PLEC exerts its influence on cell migration by interacting with IFs through its C-terminal high-affinity IFs structural domain [35,36,37]. Previous studies have verified that PLEC promotes cell migration in various diseases and cancers [38, 39], but limited in OC. Therefore, we conducted a series of migration-related experiments, which confirmed the acceleration of OC cell migration and invasion by PLEC both in vitro and in vivo (Figs. 4 and 5), similar to observations made in other cancers [40].

Knockdown of PLEC disrupts adhesion junctions by abolishing wave protein-actin crosslinks [41], indicating of PLEC in cell adhesion at adhesion junctions. Our study further confirmed that that knockdown PLEC in OC cells inhibits cell adhesion (Fig. 6). Osmanagic-Myers S et al. proved that keratinocytes lacking PLEC exhibit enhanced migration compared to normal cells, suggesting a stabilizing function of PLEC in matrix attachment to underlying substrates [42]. Additionally, previous studies have reported the promotion of cancer cell adhesion by PLEC in other types of cancer as well [39, 43, 44], implying its diverse roles across different diseases.

OC metastasis is the main cause of poor clinical outcomes, while colonization is an essential part in OC metastasis, the successful colonization at the target location of the transfer directly determines the success of the metastasis [45]. The enhanced ability of cancer cells to adhere facilitates successful colonization, which is necessary for metastasis to occur, allowing cancer cells to colonize distant sites [46].To elucidate the underlying mechanisms by which PLEC facilitates cell adhesion, our investigation focuses on two adhesion-related molecules: COL17A1 and ITGβ4, based on bioinformatics predictions (Fig. 6D). COL17A1 is a protein involved in the formation of the hemidesmosomes, which anchored to the underlying basement membrane [47]. ITGβ4, a transmembrane adhesion molecule composed of hemidesmosomes, plays a vital role in various cancer cell biology functions [48].Plectin and ITGβ4 have been shown to interact to link with other cytoskeletal networks, thereby contributing to maintain cellular structural organization [27], and previous studies have demonstrated that ITGβ4 and PLEC in promoting prostate cancer progression [30, 49], however, their involvement in OC remains elusive.

In this study, the confocal assay results revealed a significant decrease in the colocalization of PLEC with COL17A1 and ITGβ4, along with a reduction in the expression levels of these two proteins following PLEC knockdown in both two cell lines (Fig. 6E). Subsequently, the IHC assay demonstrated a remarkable decrease in the expression of COL17A1 and ITGβ4 upon PLEC knockdown in vivo (Fig. 6F, G). Upon siRNA-mediated knockdown of COL17A1 and ITGβ4 in A2780 and SKOV3 cells, the detachment assay demonstrated a significant inhibition of cell adhesion (Fig. 7E and F). Additionally, the cell adhesion assays further supported these findings, demonstrating a significant reduction in adhesion capability compared to the control group (Fig. 7G). These findings indicated that PLEC may modulate the expression of COL17A1 and ITGβ4 through its functional interaction with them, and supporting the role of PLEC in cell adhesion.

Conclusions

In this study, we demonstrated that PLEC exerts a positive influence on the proliferation, migration, invasion and adhesion of ovarian cancer cells. Furthermore, it regulates cell adhesion through its interactions with COL17A1 and ITGβ4. These findings suggest that PLEC has potential as an early diagnostic biomarker and therapeutic target of OC, providing novel insights into the diagnosis and treatment of PLEC in this disease.

Data availability

Data available on request.

Abbreviations

OC:

Ovarian cancer

PLEC:

Plectin

EOC:

Epithelial ovarian cancer

FBS:

Fetal bovine serum

KD:

Knock Down

MOI:

Multiplicity of infection

DAPI:

4′,6-diamidino-2-phenylindole

COL17A1:

Collagen XVII

ITGβ4:

Integrin beta4

DAB:

Diaminobenzidine

IHC:

Immunohistochemical analysis

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Acknowledgements

We thank the lab members for their in-depth discussions and careful reading of this review.

Funding

The present study was supported by the National Natural Science Foundation of China (grant numbers: 81501683), and the Natural Science Foundation of Shandong Province (grant No. ZR2023MH193, ZR2019MH047, ZR2015HL057), Youth Innovation Team of Shandong Province (2022KJ261).

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Authors and Affiliations

  1. School of Basic Medicine Sciences, Shandong Second Medical University, Weifang, Shandong, 261053, P. R. China

    Lanning Bai & Xiaodong Cui

  2. School of Life Science and Technology, Shandong Second Medical University, Weifang, Shandong, 261053, P. R. China

    Xueqian Qian, Hui Zhang, Yi Yuan & Yangyang Han

  3. Department of Physiology, Shandong Second Medical University, Weifang, Shandong, 261053, P. R. China

    Min Cheng

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Contributions

Bai Lanning: Conceptualization, Methodology and Writing - Original Draft. Qian Xueqian: Investigation, Resources. Zhang Hui and Yuan Yi: Data Curation and Formal analysis. Cui Xiaodong: Writing - Review & Editing. Han Yangyang and Cheng Min organized members to discuss the manuscript. All authors read and approved the final manuscript.

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Correspondence to Xiaodong Cui, Min Cheng or Yangyang Han.

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Ethical approval

The study entitled “Plectin, a novel regulator of ovarian cancer progression” was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals published by the Weifang Medical University. The protocol was approved by the Committee on the Ethics of Animal Experiments of Weifang Medical University (Permit Number: 2021SDL505). All surgery was performed under isoflurane, and every effort was made to minimize suffering.

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Bai, L., Qian, X., Zhang, H. et al. Plectin, a novel regulator in migration, invasion and adhesion of ovarian cancer. Cell Biosci 15, 15 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13578-025-01349-2

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Cell & Bioscience

ISSN: 2045-3701

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