Introduction

Endometrial cancer (EC) is one of the most common malignant tumors of the female reproductive system, accounting for 20–30% of all malignant tumors of the female reproductive tract, and is the sixth leading cause of cancer-related deaths in women worldwide (Crosbie et al. 2022; Makker et al. 2021). In recent years, the incidence of EC has been slowly increasing due to the application of menopausal hormone replacement therapy, but the onset is getting younger and younger, which is a problem and a challenge to be faced by women worldwide (Uccella et al. 2022). Currently, the five-year survival rate of patients with early-stage EC is 90%, decreasing to 20% in late-stage, and specific preventive strategies are still lacking (van den Heerik and Horeweg 2021). Therefore, revealing the key pathways of EC onset and progression will be of significance for early diagnosis and treatment.

Myosin 3B (MYO3B) belongs to the myosin III class and possesses motor and kinase domains, along with actin-activated ATPase and actin translocating activity (Komaba et al. 2003). Current research indicates that MYO3B helps maintain precise stereocilia lengths crucial for normal hearing (Cirilo et al. 2021) and the correlation between MYO3B and tumors remains unestablished. However, numerous studies indicate a significant relationship between the myosin family and tumor progression. For example, MYO3A may be closely associated with cancer metastasis (Baghel et al. 2016). Myosin II promotes tumor progression by restructuring the innate immune microenvironment (Georgouli et al. 2019). However, the molecular mechanism of MYO3B affecting the proliferation of EC remains unclear.

It has been pointed out that MYO3B binds to and regulates the activity of calmodulin (Li et al. 2020), and that ion channels are a key bridge in the interaction between tumor cells and their microenvironment. Changes in intracellular calcium ion (Ca2+) signaling pathways may not be necessary to induce tumor formation, but fluctuations in Ca2+ transport in tumor cells affect tumor progression (Patergnani et al. 2020). And intracellular Ca2+ signaling can induce epithelial mesenchymal transition in tumor cells (Adiga et al. 2022). In addition, studies have reported that intracellular Ca2+ homeostasis regulates the activity of RhoA, a member of the Rho family of small-molecule GTPases, which has a key role in the control of cellular morphology as well as invasive metastasis (Zhu et al. 2017). Ca2+ channel-mediated elevation of intracellular Ca2+ concentration regulates vascular smooth muscle contraction through activation of the RhoA/ROCK1 pathway (Chang et al. 2022). Polyamine-dependent cell migration is also partially dependent on Ca2+-regulated RhoA activity, which promotes myosin II stress fiber formation (Zhang et al. 2022).

Therefore, in this study, we proposed to analyze whether MYO3B regulates endometrial cancer cell proliferation, migration, and invasion by modulating Ca2+-dependent RhoA/ ROCK1 signaling through in vitro and in vivo experiments to provide ideas for the prevention and diagnosis of EC.

Materials and methods

Gathering patient data

Prognostic analysis of immune senescence-associated genes in EC was screened using TCGA database (https://cancergenome.nih.gov/), providing clinical data on 388 patients with EC and identifying genes differentially expressed in normal endometrial and cancerous tissues.

EC tissue specimens

Thirty patients with confirmed EC were collected through the medical records of the inpatient and outpatient departments of the First Hospital of Shanxi Medical University, and normal adjacent tissues of the same patients were used as the control group. Paraformaldehyde (4%) was used to fix the sample; sections were prepared and routinely dewaxed for immunohistochemical staining. The experimental protocol involving humans was approved by the Medical Ethics Committee of the First Hospital of Shanxi Medical University (KYLL-2024-118). Inclusion criteria for the study population: pathologically confirmed diagnosis of EC; no neoadjuvant radiotherapy or endocrine therapy prior to surgery; informed consent of the patients or their families. Exclusion criteria: previous or current comorbidity with other tumors; comorbidity with severe medical or surgical diseases, autoimmune diseases or other contraindications to surgery. Patient survival time: number of days of survival after surgery until 11 June 2024. Patients with postoperative recurrence confirmed by imaging and pathological examination.

The grouping of MYO3B low and high expression groups was based on the following: the staining intensity of cells was classified as none (0 points), low (1 point), medium (2 points), and high (3 points) by semi-quantitative method. The staining intensity and the proportion of tissue expression were 0–25% (1 point), 26–50% (2 points), 51–75% (3 points) and 76-100% (4 points), and the result of multiplying the two indexes was the final score. The expression of MYO3B was graded according to the scoring results: grade 0 (0–3 points), grade 1 (4–6 points), grade 2 (6–9 points), and grade 3 (9–12 points), with grades 0–1 classified as the low-expression group, and grades 2–3 classified as the high-expression group.

Cell experiments

Human endometrial epithelial cells (EECs, iCell, Shanghai), EC cells (KLE, AN3 CA, HEC-1-B, RL95-2, Ishikawa (IK), iCell, Shanghai) were routinely resuscitated and incubated in DMEM medium containing 10% fetal bovine serum and 100 mg/mL penicillin-streptomycin (Invitrogen, USA) at 37℃ in a 5% CO2 incubator, and then passaged until the cells reached 80% growth.

Set up cell grouping: (i) Control, sh-NC, sh-MYO3B-1, sh-MYO3B-2, sh-MYO3B-3, sh-MYO3B-4, sh-MYO3B-5, and sh-MYO3B-6; (ii) Control, pcDNA-NC, pcDNA-MYO3B; (iii) IK sh-NC, IK sh-MYO3B, KLE pcDNA-NC, KLE pcDNA-MYO3B; (iv) IK sh-NC, IK sh-MYO3B, IK sh-NC + Calmodulin agonist (CALP-2), IK sh-MYO3B + Calmodulin agonist (CALP-2), KLE pcDNA-NC, KLE pcDNA-MYO3B, KLE pcDNA-NC + Calmodulin antagonist (W-7), KLE pcDNA-MYO3B + antagonist (W-7); (v) IK sh-NC, IK sh-MYO3B, IK sh-NC + RhoA agonist (U-46619), IK sh-MYO3B + RhoA agonist (U-46619), KLE pcDNA-NC, KLE pcDNA-MYO3B, KLE pcDNA-NC + RhoA inhibitor (Y27632), KLE pcDNA-MYO3B + RhoA inhibitor (Y27632).

MYO3B knockdown (sh-MYO3B), overexpression (pcDNA-MYO3B), and corresponding negative controls (sh-NC, pcDNA-NC) were produced by GenePharma (Shanghai, China). According to the above grouping, the cells were transfected using Lipofectamine®2000 Transfection Reagent (Invitrogen, Shanghai, China). CALP-2 (57.9 µM, APExBIO, Shanghai, China), W-7 (28 µM, MCE, Shanghai, China), U-46,619 (0.1 µM, MCE, Shanghai, China), and Y27632 (10 µM, MCE, Shanghai, China) were added after cell apposition for 1 h after pretreatment. The cells in each group were cultured for 24 h.

Mouse EC transplantation tumor model

Male BALB/C-NUD mice (4–5 weeks old) were purchased from GemPharmatech Co., Ltd (SCXK (Chuan) 2020-0034) and housed in an SPF-grade environment. After 1 week of acclimatization, the mice were randomly divided into the shRNA-control group (sh-NC, n = 6), shRNA-MYO3B group (sh-MYO3B, n = 6). Ishikawa (IK) cells were transfected with a MYO3B silencing virus (sh-MYO3B; GenePharma) or a negative control (sh-NC; GenePharma). A disposable sterile insulin syringe was used to inject 100 µL of cell suspension into the right axilla of each mouse (1 × 107 cells/mouse); if the maximum diameter of the mass was found to be > 15 mm or the skin on the surface of the tumor was broken in the process of tumor growth, the mice had to be disarticulated and executed immediately (the diameter of the tumor mass could not exceed 15 mm and the volume could not exceed 1500 mm³), and the animals of all the groups on the 28th day of tumor formation, mice were executed by cervical dislocation, and the mice were photographed by peeling out the transplanted tumors. The study complied with the regulations of the Animal Control Committee of First Hospital of Shanxi Medical University (KYLL-2024-118).

Immunohistochemical staining

Endometrial cancer tissue and mouse tumor tissue Sect. (5 μm) were deparaffinized, antigenically repaired, endogenous peroxidase blocked with 3% hydrogen peroxide, serum blocked, and incubated overnight at 4℃ with the addition of primary antibody Ki-67 (1:400, HuaBio, Hangzhou, China), MYO3B (1:600, Affinity, Suzhou, China), ROCK1 (1:200, Abcam, UK), RhoA (1:100, ABclonal, Wuhan, China), and F-actin (1:200, GeneTex, Shanghai, China). Add secondary antibody (HRP labeled goat anti-rabbit, 1:100, Servicebio, Wuhan, China) and incubate at 37℃ for 30 min. DAB color development, hematoxylin re-staining, sealing, digital trinocular camera microcamera system (BA400Digital, Motic, Xiamen, China) for image acquisition, and Halo data analysis system (Indica labs, USA) was used to calculate the percentage of positive area (% DAB Positive Tissue) in each image.

CCK-8 assay

After the cells were incubated for 24 h, the medium was replaced with fresh medium, and 10 µL of CCK-8 reagent (Biosharp, Guangzhou, China) was added to each well. The plates were incubated in the dark, and cell survival was calculated based on the absorbance at 450 nm detected via a microplate reader (ELx800, BioTek, USA).

Flow cytometry assay

Cells were collected, centrifuged at 1000 r/min for 5 min, resuspended with 500 µL Binding Buffer, add 5 µL of Annexi V (KeyGEN, Nanjing, China), and add 5 µL of PI (KeyGEN, Nanjing, China), mix gently, and incubate for 15 min at room temperature under the condition of avoiding light, and then apoptosis was detected by Cytoflex flow cytometer (Beckman Coulter, USA) within 1 h. Ca2+ content analysis: Cell precipitates were obtained, cells were resuspended by adding 500 µL Fluo-4 AM (Beyotime, Beijing, China) dilution, incubated for 40 min at 37℃ away from light, and the supernatant was discarded by centrifugation at 1000 r/min. Subsequently, 500 µL PBS was added and washed twice, incubated at 37℃ for 20 min, centrifuged at 1000 r/min for 5 min, resuspended in 300 µL PBS, and analyzed by flow cytometry.

Scratch assay

When the cells were full grown to monolayer, the supernatant was aspirated, the pipette gun was scratched, the cells were washed twice with PBS, the scratched cells were removed, and the cells were cultured in 37℃ and 5% CO2 incubator according to the grouping. Samples were taken at the time points of 0 h and 24 h, and the scratched state of the cells was photographed with a microscope (DMI1, LEICA, Germany).

Transwell assay

Pre-chilled at 4℃ with 1:8 dilution of Matrigel (Corning, Suzhou, China) was added to the Transwell upper chamber, spread well, and dried at 37℃ for 70 min. Groups were prepared with cell suspensions, and the cell concentration was adjusted to 5 × 104 cells/mL, 200 µL of cell suspension was added to the upper chamber, and 600 µL of medium containing 20% FBS was added to the lower chamber as a chemotactic factor. The small chambers were incubated at 5% CO2 and 37℃ for 24 h. The cells in the upper chamber were wiped off with cotton swabs, rinsed with PBS, fixed with methanol, and stained with 0.1% crystal violet (Bomei, Hefei, China). Three fields of view of each well were selected and photographed under a light microscope, and the number of migrated cells in each group was counted.

Immunofluorescence staining

Cell crawls were washed 3 times with PBS, membrane-breaking solution (Servicebio, Wuhan, China) was added to cover the cells and incubated at room temperature for 10 min, bovine serum (Servicebio, Wuhan, China) was closed at room temperature for 20 min, and antibodies to F-actin (1:200, Abcam, UK) and Paxinllin (1:50, Abcam, UK) was incubated overnight at 4℃, and the antibody was added dropwise for 30 min at 37℃. Subsequently, DAPI (Servicebio, Wuhan, China) was added dropwise and incubated for 10 min at room temperature, and then sealed with anti- fluorescence quenching sealer. Images of the sections were captured using scanning and browsing software (OlyVIA, OLYMPUS, Japan), and the fluorescence intensity of all the captured images was measured using Image-J (National Institutes of Health, USA).

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from the cells using an ultra-pure RNA extraction kit (YEASEN, Shanghai, China), and 5 µL of RNA was taken to detect the integrity of RNA. The residual genomic DNA in the RNA was digested with a DNase I kit (Takara, Japan) and reverse transcription was performed using a reverse transcription kit (Takara, Japan). Amplification was performed using TB Green TM Premix Ex Taq™ II (Takara, Japan). The primer sequence: MYO3B, Forward sequences (5’-3’): TGAATCACTTCCAGATCCCACAG AC, Reverse sequences (5’-3’): CCAGGCTCCCATCTCTCTTGTTAG; GAPDH, Forward sequences (5’-3’): TGACTTCAACAGCGACACCCA, Reverse sequences (5’-3’): CACCCT GTTGCTGTAGCCAAA. GAPDH was used as an internal control. The relative expression of each target gene was quantified by the 2−ΔΔCt method; where ΔCt = Ct target gene – Ct internal reference and ΔΔCt = ΔCt experiment -ΔCt control.

Western blot analysis

RIPA cell lysis buffer was added to lyse the cells and tissue, which were subsequently centrifuged to collect the protein supernatant. SDS loading buffer was added, and the samples were boiled in boiling water. SDS-PAGE was used to separate proteins, which were transferred to PVDF membranes. Membranes were then blocked with 5% skim milk powder at room temperature. After incubation with primary antibodies at 4℃ overnight, including anti-MYO3B (1:2000), anti-RhoA (1:1000), anti-RhoB (1:2000), anti-RhoC (1:1000), anti-ROCK1 (1:1000), anti-LIMK (1:5000), anti-p-LIML (1:2000), anti-cofilin (1:1000), anti-p-cofilin (1:2000), and anti-β-actin (1:50000); Antibody from ABclonal (Wuhan, China), the membranes were washed with PBS three times, and incubated with goat anti-rabbit IgG (H + L) secondary antibody (1:5000; Affbiotech, Wuhan, China) for 1 h at room temperature. Developed by enhanced chemiluminescence (ECL, Zenbio, Shanghai, China), and the bands were exposed with Fluorescence Image Analysis System Software V2.0 (Tanon, Shanghai), and the results were scanned by Gel-Pro analyzer4 software and expressed as the integrated optical density (IOD) of the target protein.

Statistical analysis

Factors affecting recurrence in patients with EC (age, survival time, tumor size, metastasis, muscle layer, infiltration, histologic grade, and MYO3B expression) were analyzed using a logistic regression model with SPSS 20.0 software (IBM, USA). And the independent influencing factors affecting EC recurrence and MYO3B expression were also analyzed using binary logistic regression equations. Data were expressed as mean ± standard deviation (SD). Comparisons of data among groups were carried out by one-way ANOVA; the LSD test was used if the variance was homogeneous, and Tamhane’s T2 test was used if the variance was not homogeneous. A P-value < 0.05 was considered significant.

Results

Expression of MYO3B in clinical tissue samples of endometrial cancer and its clinical significance

Firstly, we preliminarily succeeded in identifying six important model genes associated with prognosis (Fig. 1A), including NOG, MYO3B, ASPM, FBN3, MMP1 and GRB7. Subsequently, we found that the expression of MYO3B in EC tissues showed high expression (Fig. 1B and C, P < 0.01), suggesting that MYO3B may be associated with EC progression. In addition, we found that MYO3B expression was an influential factor in the recurrence of EC (Table 1). Metastasis was included in the binary logistic regression equation, and the analysis showed that metastasis was an independent risk factor for recurrence in patients (Table 2). Finally, factors with significant differences were included in the binary logistic regression equation, and the results of the analyses showed that Age, Survival time, and Tumor size were not independent influences on MYO3B expression (Table 3).

Fig. 1
figure 1

Expression of MYO3B in clinical tissue samples of endometrial cancer and its clinical significance. (A) Important model genes from databases associated with prognosis. (B) Expression of MYO3B in tissues. (C) Immunohistochemical staining of MYO3B in tissues (200×, Scale bar, 50 μm). The data are expressed as the mean ± SD. **P < 0.01

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Table 1 Analysis of factors influencing EC recurrence
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Table 2 Analysis of independent factors affecting EC recurrence
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Table 3 Analysis of factors influencing the expression of MYO3B
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Effect of MYO3B expression on proliferation, apoptosis, invasion, and migration of endometrial cancer

Here, we analyzed the effects of MYO3B expression on endometrial cancer proliferation, apoptosis, invasion and migration. We first examined the expression of MYO3B in endometrial cancer cell lines, and the results showed that the expression of MYO3B was significantly lower in KLE cells compared with EECs cells, but its expression was significantly increased in IK cells (Fig. 2A and D, P < 0.01), so IK and KLE cells were used for the subsequent experiments. Next, we screened the MYO3B knockdown sequences, as shown in Fig. 2B and D, the knockdown effect of sh-MYO3B-6 was better (P < 0.01). The effect of MYO3B overexpression vector was identified as shown in Fig. 2C and D, the expression of MYO3B was significantly enhanced after overexpressing MYO3B (P < 0.01). Moreover, knockdown of MYO3B inhibited endometrial cancer cell proliferation, promoted apoptosis, and attenuated cell invasion and migration (Fig. 2E-H, P < 0.01), and then, overexpression of MYO3B had the opposite effect to knockdown of MYO3B.

Fig. 2
figure 2

Effect of MYO3B expression on proliferation, apoptosis, invasion, and migration of endometrial cancer. (A) MYO3B mRNA and protein expression in cells. (B) MYO3B mRNA and protein expression in the knockdown group. (C) MYO3B mRNA and protein expression in overexpression group. (D) Western blot protein bands, protein band calculated as a ratio relative to β-actin protein levels. (E) Cell proliferation, apoptosis, migration, and invasion analysis. (F) Apoptosis flow cytogram. (G) Representative pictures of cellular invasion (crystal violet stain, 100×, Scale bar, 100 μm). (H) Representative images of cell migration (40×, Scale bar, 250 μm). The data are expressed as the mean ± SD. *P < 0.05, **P < 0.01

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Effect of MYO3B expression on Ca2+ homeostasis and RhoA/ROCK1 signaling in endometrial cancer cells

Next, we examined the effects of MYO3B expression on Ca2+ homeostasis and RhoA/ROCK1 signaling in endometrial cancer cells. The results revealed that knockdown of MYO3B expression significantly reduced intracellular Ca2+ content (Fig. 3A and D, P < 0.05) and inhibited the expression of Paxinllin and F-actin (Fig. 3B and C, P < 0.01), while overexpression of MYO3B had the opposite effect. Addition of the agonist CALP-2 significantly increased intracellular Ca2+ content compared with knockdown of MYO3B (Fig. 3D and E, P < 0.01). In addition, knockdown of MYO3B expression significantly decreased the protein expression of RhoA and ROCK1, in contrast, overexpression of MYO3B promoted the protein expression of RhoA and ROCK1 (Fig. 3F, P < 0.05). Compared with sh-MYO3B, sh-MYO3B + CALP-2 significantly enhanced the protein expression of RhoA, ROCK1, and p-LIMK, and compared with pcDNA-MYO3B, pcDNA-MYO3B + W-7 significantly decreased the protein expression of RhoA, ROCK1, and p-LIMK (Fig. 3G, P < 0.05), indicating that MYO3B may be dependent on Ca2+/calmodulin-dependent protein kinase signaling to activate RhoA/ROCK1 signaling.

Fig. 3
figure 3

Effect of MYO3B expression on Ca2+ homeostasis and RhoA/ROCK1 signaling in endometrial cancer cells. (A) Flow cytometric detection of intracellular Ca2+ content. (B) Fluorescence intensity of Paxinllin and F-actin. (C) Immunofluorescence co-staining of Paxinllin and F-actin (60×, Scale bar, 10 μm). (D) Flow cytogram of Ca2+ content. (E) Ca2+ content. (F) Western blot results of RhoA, RhoB, RhoC, and ROCK1 expression in cells, protein band calculated as a ratio relative to β-actin protein levels. (G) Western blot results of RhoA, ROCK1, LIMK, and p-LIMK expression in cells, protein band calculated as a ratio relative to β-actin protein levels. The data are expressed as the mean ± SD. *P < 0.05, **P < 0.01

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MYO3B promotes endometrial cancer cell proliferation, migration, and invasion through activation of RhoA/ROCK1 signaling

In this part, we analyzed whether MYO3B promotes EC cell proliferation, migration, and invasion through activation of RhoA/ROCK1 signaling. Compared with sh-MYO3B group, sh-MYO3B + U-46,619 significantly promoted cell proliferation (Fig. 4A, P < 0.05), remarkably attenuated apoptosis (Fig. 4B and C, P < 0.01), and significantly increased cell migration and invasion (Fig. 4D-G, P < 0.01). In contrast, pcDNA-MYO3B + Y27632 observably inhibited cell proliferation (Fig. 4A, P < 0.05), significantly increased apoptosis (Fig. 4B and C, P < 0.01), and memorably attenuated cell migration and invasion (Fig. 4D-G, P < 0.01), as compared to the pcDNA-MYO3B group. Furthermore, the expression of Paxinllin, F-actin, RhoA, ROCK1, p-LIMK, and p-cofilin was significantly elevated in the sh-MYO3B + U-46,619 group compared to the sh-MYO3B group (Fig. 5, P < 0.05), however, the opposite effect was associated with pcDNA-MYO3B + Y27632, demonstrating that MYO3B promotes endometrial cancer cell proliferation, migration, and invasion through activation of RhoA/ROCK1 signaling.

Fig. 4
figure 4

MYO3B promotes endometrial cancer cell proliferation, migration, and invasion through activation of RhoA/ROCK1 signaling. (A) Cell proliferation analysis. (B) Cell apoptosis analysis. (C) Apoptosis flow cytogram. (D) Representative images of cell migration (40×, Scale bar, 250 μm). (E) Representative pictures of cellular invasion (crystal violet stain, 100×, Scale bar, 100 μm). (F) Cell migration analysis. (G) Cell invasion analysis. The data are expressed as the mean ± SD. *P < 0.05, **P < 0.01

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Fig. 5
figure 5

MYO3B promotes endometrial cancer cell proliferation, migration, and invasion through activation of RhoA/ROCK1 signaling. (A) Fluorescence intensity of Paxinllin and F-actin. (B) Immunofluorescence co-staining of Paxinllin and F-actin (60×, Scale bar, 10 μm). (C) Western blot results of RhoA, ROCK1, LIMK, p-LIMK, cofilin and p-cofilin expression in cells, protein band calculated as a ratio relative to β-actin protein levels. The data are expressed as the mean ± SD. *P < 0.05, **P < 0.01

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Effect of knockdown of MYO3B on endometrial cancer cell growth in vivo

Additionally, the effect of knockdown of MYO3B on the growth of EC cells in vivo was observed. In vivo experiments showed that knockdown of MYO3B significantly suppressed tumor size and tumor volume in endometrial cancer compared with the sh-NC group (Fig. 6A, P < 0.05). In addition, knockdown of MYO3B significantly attenuated the expression of Ki-67, MYO3B, ROCK1, RhoA, and F-actin in the tumor tissues (Fig. 6B and C, P < 0.05), showing that knockdown of MYO3B inhibited the growth of endometrial cancer in mice.

Fig. 6
figure 6

Effect of knockdown of MYO3B on endometrial cancer cell growth in vivo. (A) Tumor size and Tumor volume. (B) Expression of Ki-67, MYO3B, ROCK1, RhoA, and F-actin in tumor tissues. (C) Immunohistochemical staining of Ki-67, MYO3B, ROCK1, RhoA, and F-actin in tumor tissues (40×, Scale bar, 50 μm). The data are expressed as the mean ± SD. *P < 0.05, **P < 0.01

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Discussions

Distant metastasis is a key determinant of EC prognosis (Nees et al. 2022), and MYO3B may be associated with EC progression. However, the molecular mechanisms by which MYO3B affects EC remain unclear. The present study provides insights into this issue. We demonstrated that the expression of MYO3B was an influential factor in EC recurrence, and the expression of MYO3B was significantly up-regulated in EC tissues and cells, but down-regulated in KLE cells, and MYO3B knockdown inhibited the proliferation, migration, and invasion ability of EC cells and promoted apoptosis, suggesting that MYO3B plays a tumor-promoting role in EC. Additionally, we provide evidence that MYO3B knockdown decreased Ca2+ concentration in EC cells and the RhoA/ROCK1 signaling pathway was inhibited, and the effect of MYO3B knockdown on RhoA/ROCK1 signaling was reversed by treatment with the Calmodulin agonist CALP-2, and the effects of MYO3B knockdown on cell proliferation, migration, and invasion were reversed after treatment with the RhoA agonist U-46,619.

Currently, the correlation between MYO3B and tumor is still undetermined, in order to explore the significance of MYO3B in EC, we firstly analyzed the expression of MYO3B in EC tissues, MYO3B was presented as a high expression in EC tissues, and the results of fresh tissues were consistent with the database data. Moreover, the results of EC cell lines were similar to the tissue results, the expression of MYO3B was elevated in Ishikawa, RL95-2 cells, but the expression of MYO3B in KLE cells presented a low expression, which may be due to the different local environments and differentiation states within the tumor (Pei et al. 2022). Meanwhile, the analysis of MYO3B expression and patients’ clinical characteristics showed that MYO3B expression was an influencing factor for EC recurrence, and patients’ age, survival time, tumor size, infiltration, and histologic grade were not independent influencing factors for MYO3B expression. This suggests that MYO3B can be used as an independent factor to predict poor prognosis of EC.

Studies have shown that sustained proliferation, evasion of apoptosis, and genomic instability are three of the most prevalent characteristics in human cancers that can drive cancer progression (Wang et al. 2023). Therefore, inhibiting the proliferation, migration, and invasion of cancer cells is of significance in attenuating cancer progression (Garrett et al. 2024). Studies have reported that silencing of S100A4 significantly attenuated the migration and invasion of EC cells (Hua et al. 2016). The results of the present study were consistent with the report, and we found that knockdown of MYO3B inhibited the proliferation, migration, and invasion ability of EC cells and promoted apoptosis. Similarly, knockdown of MYO3B in EC model mice significantly inhibited tumor size, volume and proliferation, suggesting that knockdown of MYO3B inhibited EC progression. Ca2+ plays an important role in endoplasmic reticulum cellular responses, signal transduction pathways, and transcriptional regulation, and its balance is a prerequisite and basis for maintaining normal cellular structure and function (Prevarskaya et al. 2014). It has been reported that in the resting state, intracellular Ca2+ is maintained at a low level, and when the cells respond to stimuli, the intracellular Ca2+ concentration rises rapidly (Zheng et al. 2023). The Ca2+ concentration can influence tumor progression, e.g., in prostate cancer, inhibition of TRPV2 reduces the Ca2+ concentration, and invasive ability is attenuated (Liberati et al. 2014). In this experiment, MYO3B knockdown down-regulated Ca2+ concentration in EC cells, Calmodulin is one of the major Ca2+-binding proteins in cells and plays multiple roles in a variety of Ca2+-signaling pathways, regulating the activity of other proteins (Tokumitsu and Sakagami 2022). The attenuating effect of knockdown of MYO3B on Ca2+ in EC cells was reversed by the addition of the Calmodulin agonist CALP-2, suggesting that MYO3B regulates Ca2+ signaling in EC.

It has been noted that RhoA/ROCK1 signaling is an important intracellular signaling pathway involved in the regulation of a wide range of cellular functions (Dong et al. 2023). Rho GTPases are frequently activated in human cancers and play a key role in cancer cell invasion and metastasis through the regulation of cell motility and the dynamic organization of the actin cytoskeleton (Yin et al. 2016). Previous studies have shown that overexpression of RhoA together with ROCK1 and ROCK2 supports spreading and migration of HeLa cells (Tang et al. 2021). In addition, the study reported that intracellular Ca2+ homeostasis regulates RhoA activity (Zhu et al. 2017). In EC, TRPV4 and calcium regulate cytoskeletal-induced metastasis via the RhoA/ROCK/LIMK/cofilin pathway (Li et al. 2020), and our results are similar to it. Our findings suggest that knockdown of MYO3B inhibited RhoA/ROCK1 signaling pathway activation in EC cells, and the effect of MYO3B knockdown on the RhoA/ROCK1 signaling pathway was reversed with the Calmodulin agonist CALP-2, suggesting that MYO3B may mediate the activation of the RhoA/ROCK1 signaling pathway by affecting Ca2+ release and that RhoA/ROCK1 occurs in a Ca2+-dependent manner. Furthermore, our results showed that activation of RhoA/ROCK reduced the inhibitory effect of MYO3B knockdown on EC cell migration and invasion, which demonstrated that MYO3B may be involved in the EC process through Ca2+-mediated RhoA/ROCK1 pathway.

Conclusion

In conclusion, MYO3B mediated the RhoA/ROCK1 signaling pathway by regulating Ca2+ levels, promoted EC cell proliferation, enhanced cell migration and invasion, and inhibited apoptosis, suggesting that MYO3B promotes the progression of EC and can be used as a target for further study of EC therapy.