BmK rAGAP inhibits breast cancer cell proliferation, migration, and invasion by downregulating SCN5A expression and subsequent AKT and MAPK activation.

doi:10.21203/rs.3.rs-1653492/v1

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BmK rAGAP inhibits breast cancer cell proliferation, migration, and invasion by downregulating SCN5A expression and subsequent AKT and MAPK activation.

https://doi.org/10.21203/rs.3.rs-1653492/v1

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BACKGROUND

BmK rAGAP is voltage-gated sodium ion channel-like scorpion venom peptides, which have anti-tumor activity. Voltage-gated sodium ion channels subunit SCN5A was highly expressed in MDA-MB-231 and it was associated with breast cancer malignancy.

METHODS AND RESULTS

In order to explore the effect of BmK rAGAP on MDA-MB-231 and its possible mechanism, the data were downloaded based on TCGA to analyze gene expression of breast cancer patients. Furthermore, MTT, wound healing assay, transwell assay, western blot, immunocytochemistry, fluorescence dual-wavelength colorimetry also were used. We found that BmK rAGAP could inhibit the proliferation, migration, and invasion of MDA-MB-231. BmK rAGAP could reduce expression of SCN5A, and inhibit intracellular [Na⁺]i and affect the expression of E-cadherin, Vimentin, P53, p-ERK, p-p38 MAPK, p-AKT and MMP-2 in MDA-MB-231.

CONCLUSIONS

In conclusion, BmK rAGAP can inhibit the proliferation, migration, and invasion of MDA-MB-231, which may occur by inhibiting the expression of SCN5A and then affecting AKT, p38 MAPK and EMT. It shows that BmK rAGAP was a potential new drug that could be used in the diagnosis and treatment of breast cancer.

BmK rAGAP

voltage-gated sodium channel

SCN5A

the cancer genome atlas

the breast cancer

The latest 2020 global cancer burdens data released by the International Agency for Research on Cancer of the World Health Organization shows that breast cancer has become the world's largest cancer, with a global incidence of 2.26 million ^[1]. The global survey of female cancer deaths shows that breast cancer ranks first in the number of female cancer deaths in the world ^[2]. According to molecular characteristics as the classification criteria, breast cancer is divided into 4 subtypes based on the differential expression of three molecular markers: estrogen receptor (ER), progesterone receptor, PR) and (human epidermal growth factor receptors-2, Her-2) ^[3]. Triple Negative Breast Cancer (TNBC) refers to breast cancer that is negative for ER, PR, and Her-2. It has the characteristics of high invasion, high metastasis, and high recurrence rate. The patient has a short survival time and the worst prognosis ^[4].

Voltage-gated sodium channels (VGSCs) are a multifunctional transmembrane protein which is comprised of a functional α subunit with one or more auxiliary β subunits ^[5]. There are nine α subunits, including SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A, and SCN11A, respectively encoding Na_V1.1 ~ Na_V1.9^[6]. There are four β subunits, including SCN1B, SCN2B, SCN3B, SCN4B, respectively encoding β1/β1B, β2, β3, β4^{[7, 8]}. VGSC is responsible for the initiation and conduction of excitable cell action potentials^{[9, 10]}. It is the material basis for the electrical activities of neuronal cells and muscle cells, and plays an important role in nerve signal conduction and heart rhythm regulation ^[11]. Our previous studies have shown that the expression of SCN5A in TNBC patients is significantly higher than that in other types of breast cancer. TNBC with high SCN5A expression has poor prognosis within 2–5 years. In addition, many studies have shown that Na_V1.5 encoded by SCN5A is involved in the proliferation, migration, and invasion of cancer cells ^[12]. Na_V1.5 expression is higher in the strongly metastatic breast cancer cell MDA-MB-231, and the mRNA expression of SCN5A accounts for about 80% of the total α subunit mRNA expression^{[13, 14]}.

And using siRNA to reduce the expression of SCN5A in MDA-MB-231 showed that the invasion ability of cells was significantly reduced, further indicating that SCN5A can regulate the proliferation, migration, and invasion of breast cancer cells^[12]. Frédéric Gradek ^[12] found that reducing the expression of Na_V1.5 can significantly reverse the epithelial to mesenchymal transition (EMT) and inhibit migration and invasion in MDA-MB-231. However, in the weakly metastatic breast cancer cell MCF-7, the heterologous expression of Na_V1.5 can induce the expression of SNAI1 and ZEB1, thereby enhancing the invasive phenotype of this cell ^[12]. In addition, the continuous Na⁺ current carried by Na_V1.5 can mediate the depolarization of the plasma membrane potential in breast cancer cells, increase the co-localization of Rac1 and phosphatidylserine to activate Rac1 and then affect migration of cells ^[15].

Recombinant analgesic-antitumor peptide (rAGAP) is a α-type long-chain scorpion toxin which was separated and purified by BmK-AGAP (Buthus martensii Karsch-AGAP) by researchers in our laboratory ^[16]. It is a voltage-gated sodium channel-like scorpion venom peptide, which has been shown to have anti-tumor activity ^{[17, 18]}. The latest study found that BmK-AGAP can reduce the phosphorylation of AKT in liver cancer cell Hep G2, reduce the secretion of VEGF and IL-8 and thereby inhibit angiogenesis ^[19]. After adding BmK-AGAP to the mouse breast cancer xenograft model, it was found that the expression of Ki-67, p65/NF-κB and VEGF in the tumor was reduced, and the tumor volume was reduced ^[20]. In summary, BmK-AGAP is a potential new drug that can be used to treat cancer, but the effect and mechanism of BmK-AGAP on breast cancer cells is still unclear. Therefore, this article aims to explore the effect of BmK-AGAP on the proliferation, migration and invasion and its possible mechanism of breast cancer cells. And it to lay a theoretical foundation for the study of the mechanism of BmK-AGAP inhibiting the proliferation, migration, and invasion of breast cancer cells.

Cell Culture

The human breast cancer cells MDA-MB-231 and SK-BR-3 was purchased from Shanghai Cell Bank of Chinese Academy of Sciences. And MCF-7 was purchased from Bina Biological. The short tandem repeat (STR) analysis of MDA-MB-231 and SK-BR-3 was provided by Shanghai Cell Bank of Chinese Academy of Sciences. The short tandem repeat (STR) analysis of MCF-7 was provided by Bina Biological. The MDA-MB-231 cell was routinely maintained in L-15 (Kaiji Biological, China), supplemented with 10% fetal bovine serum (FBS) (Gibco, USA). The cells were maintained in an incubator at 37°C humidified air with 100% air atmospheric condition. The MCF-7 cell was routinely maintained in DMEM (Gibco, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco, USA). The SK-BR-3 cell was routinely maintained in DMEM (Gibco, USA), supplemented with 20% fetal bovine serum (FBS) (Gibco, USA). The MCF-7 and SK-BR-3 cells were maintained in an incubator at 37°C humidified air with 5% CO2 atmospheric condition. The MDA-MB-231, MCF-7, SK-BR-3 cells were routinely subculture every 2–5 days.

Preparation of Recombinant BmK rAGAP

Recombinant BmK AGAP (BmK rAGAP) was isolated and purified by our laboratory. And the Bmk rAGAP was filtered with a 0.22 mm sterile membrane before used.

Cell Proliferation Assay

Inhibition of cell proliferation of BmK rAGAP was evaluated using 3-(4-5- dimethylhiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.MDA-MB-231 cell was seeded in 96-well plates at density of 1×10⁴ cells per well and cultured at 37°C. The cells were then treated with different concentrations of BmK rAGAP (0, 2.5, 5, 10, 20, 40 µM) and incubated in a humidified atmosphere of 100% air at 37°C for 24,48,72,96 hours. Then 200 µL of MTT (0.5 mg/mL) was added to each well. And the cells were cultured for 4–6 hours at 37°C humidified air with 100% air atmospheric condition. Then the MTT solution was discarded, and 150 µL of dimethyl sulfoxide (DMSO) solution was added into each well. The cells were placed on a shaker at 37°C and incubated for 30 minutes in the dark to dissolve the insoluble formazan crystals. Optical density (OD) was measured at a wavelength of 590nm used Infinite 200 Pro Multimode reader (TECAN, Switzerland).

Wound Healing Assay

Wound Healing Assay was used to analyze the effect of BmK rAGAP on cell migration ability. The breast cancer cells which in the logarithmic growth phases were seeded in 24-well plates, and when then the cells were cultured until 90% confluence reaches, used a 10 µL pipette tip to streak vertically. The cells were then treated with different concentrations of BmK rAGAP (0, 2.5, 5, 10 µM) and incubated in a humidified atmosphere of 100% air at 37°C for 24 hours. The wound width was photographed by Inverted microscope and measured by Image J at 0 h and 24 h.

Transwell Migration and Invasion Assays

The migration ability of breast cancer cells was determined by transwell migration assay. The breast cancer cells which in the logarithmic growth phases were seeded into the upper chamber of the transwell without Matrigel (Corning, USA). L-15 supplemented with 15% FBS was added as an attractant to the bottom chamber. After 24 and 48 hours, the cells in the upper chamber were swabbed of by cotton swabs. The migration cells were fixed with methanol, stained with Trypan blue. The migration cells were photographed with Inverted microscope and counted in ten random fields at 10× magnification. The breast cancer cells were seeded into the upper chamber of the transwell with Matrigel (Corning, USA) to determine the invasion ability. The rest of steps were similar to the migration assay, and both the migration and invasion assays were determined simultaneously.

Western Blot Assay

According to the manufacturer’s protocol of the whole protein extraction kit (KAJI Bio, China), the total protein were extracted, and the protein was measured by BCA assay. Protein extraction from the cells was separated by 10% SDS-PAGE with an equal amount and then transferred onto PVDF (Millipore, USA). The membrane was blocked with 5% goat serum or skimmed milk powder for 1 hour of 28°C, and probed into specific antibodies against Na_V1.5(1:2000, CST, USA), AKT(1:1000, CST, USA), p-AKT(1:2000, CST, USA),Vimentin(1:200, CST, USA), p-p38 MAPK(1:1000, CST, USA), p-ERK(1:1000, CST, USA), P53(1:2000, proteintech, USA), MMP-2(1:500, CST, USA), E-cadherin(1:10000, Abcam, USA), overnight at 4°C. Incubate with specific secondary antibodies(goat anti-rabbit IgG, 1:10000, Abcam, US and goat anti-mouse IgG, 1:10000, Abcam, UK) for 1 hour of 28°C. The antibodies binding were detected with enhanced chemiluminescence kit (ECL, Thermo, USA) and the relative amount of protein were analyzed with Image J.

Real-Time PCR

Total RNAs from breast cancer cells was extracted using Trizol (Takara, Japan) according to the manufacturer’s instruction. RNA was reversing transcribed for cDNA according to the manufacturer’s instruction of PrimeScriptTM RT reagent Kit (Takara, Japan). R-T PCR was performed according to the manufacturer’s protocol by using SYBR Premix Ex TaqTM (Takara, Japan).

Fluorescence Dual-Wavelength Colorimetry

The cells were digested and centrifuged, when the cells were cultured until 80%~90% confluence reaches. And then the cells were resuspended in Lock’s buffer stained with Dye loading buffer and incubated for 1-1.5 hours of a shaker at 37°C under dark conditions. The cells were washed and resuspended with Lock’s buffer, then Na⁺ concentration was measured used Infinite 200 Pro Multimode reader (TECAN, Switzerland). The cells were excited at 505nm, detected at 340nm and 380nm.

Immunocytochemistry

The cells which in the logarithmic growth phase was inoculated into 24-well plates containing the cells climbing film, continued to culture the cells until reached confluency 80 ~ 90%, the medium was removed. The cells were fixed with 4% paraformaldehyde solution, and then incubated for 10 minutes in PBS containing 0.25% Triton-X100 (polyethylene glycol octyl phenyl ether). The cells were blocked with blocking solution to 30 minutes, incubated at 4°C for 12 to 16 hours with specific antibody, and incubated for 1 hour of room temperature in the dark with fluorescent secondary antibody. Added 0.1 µg/mL DAPI to the cells and stained the nucleus of 10 minutes in a dark room, then added anti-fluorescence quencher to the cells. The cells were observed and photographed under a laser confocal microscope with filters with wavelengths of 408 nm and 488 nm.

Statistical Analysis

Each assay was performed at least three times. All experimental data were presented as the mean ± SD. GraphPad Prism 7.0 was used for experimental data. The Student´s t-test and one-way ANOVA were used for data analysis. The former were used for comparison between groups, the latter was used for comparison of three or more groups.

TCGA Database Analysis

Open the TCGA database website (https://portal.gdc.cancer.gov/), download and preprocess the transcriptome RNA expression and clinical data of the breast cancer patient data set. R studio was used for TCGA database data. The specific process of data analysis is shown in the Fig. 1.

CCLE Database Analysis

The date was obtained from the Cancer Cell Line Encyclopedia (CCLE) online website (https://portals.broadinstitute.org/ccle). GraphPad Prism 7.0 was used for CCLE data.

1 SCN5A correlation analysis and GO, KEGG analysis

Our previous studies have shown that the expression of SCN5A in TNBC patients is significantly higher than that in other types of breast cancer. And the literature shows that SCN5A is related to the proliferation, migration, and invasion of breast cancer cells. Therefore, in order to find the diagnosis and treatment targets of breast cancer, explore the differentially expressed genes related to SCN5A, analyze the RNA expression of the transcriptome of breast cancer patients in the TCGA database, and screen for differences expressed genes in breast cancer tissues and normal breast tissues.

The breast cancer related data were downloaded from the TCGA database, and the genes differentially expressed in breast cancer tissue and normal breast tissue and related to SCN5A were screened for GO and KEGG analysis (Fig. 2, Table 1). 8 related signal pathways were enriched through KEGG analysis, including the signal pathways related to cell proliferation, Cell Cycle pathway, G1 to S cell cycle pathway, FAK/PI3K/AKT/mTOR pathway, and MAPK pathway, TGF-β, Ras and pathways related to cell migration and invasion include EMT, Integrin, TGF-β pathway, FAK/PI3K/AKT/mTOR pathway, and MAPK pathway.

Table 1

**The signaling pathway related genes**
	Signaling Pathway(n = the number of genes)
Proliferation related	G1 to S cell cycle control (n = 21)	Cell Cycle (n = 32)	FAK/PI3K/AKT/mTOR (n = 44)
Proliferation related	MAPK (n = 27)	TGF-β (n = 26)	Ras (n = 24)
Migration and invasion related	EMT (n = 24)	Integrin pathway (n = 19)	TGF-β (n = 26)
Migration and invasion related	FAK/PI3K/AKT/mTOR (n = 44)	MAPK (n = 27)

2 SCN5A differentially expressed in breast cancer cells.

In order to analyze the expression of SCN5A in breast cancer cells, the RNA expression data ofbreast cancer cells was downloaded based on the CCLE database. The results showed that there was no significant difference in the mRNA expression of SCN5A in Luminal A-type, Luminal B-type, and Her-2-type cells. The mRNA expression of SCN5A in TNBC-type cells was higher than that in Her-2-type cells, but there was no statistical difference between TNBC type cells and the other two types of cells (Fig. 3A). From the downloaded data, the data of three breast cancer cells used in our laboratory were screened, and the mRNA expression of SCN5A in these three cells was analyzed (Fig. 3B). The mRNA expression of SCN5A in MDA-MB-231 cells was significantly higher than that in MCF-7 and SK-BR-3 cells.

Further analyzed the expression of Na_V1.5 encoded by SCN5A in MDA-MB-231, MCF-7, SK-BR-3 cells. The whole cell proteins of these three cells were extracted and Western Blot was performed to analyze the expression of Na_V1.5 in these three cells. The results showed that the protein expression of Na_V1.5 in MDA-MB-231 cells was significantly higher than that of the other two cells (Fig. 3C). The results of immunocytochemistry showed that the expression of Na_V1.5 in MDA-MB-231 was significantly higher than that in MCF-7 (Fig. 3D). In conclusion, mRNA and protein expressions of SCN5A were higher in MDA-MB-231.

3 BmK rAGAP suppressed proliferation, migration, and invasion of MDA-MB-231 cells

BmK rAGAP is a sodium channel-like scorpion venom polypeptide, which has been shown to have anti-tumor activity. In order to study the effect of BmK rAGAP on breast cancer cells, this study analyzed the effect of BmK rAGAP on cell proliferation, migration, and invasion through MTT colorimetric assay, wound healing assay, and transwell assay. Firstly, BmK rAGAP significantly inhibited the proliferation of MDA-MB-231. Prolonging the action time of BmK rAGAP, the IC₅₀ value gradually decreased (Fig. 4A).

In this study, determined the inhibitory concentration value of BmK rAGAP (Fig. 4B). Under the same action time, the greater the concentration of BmK rAGAP, the greater the inhibition rate of cell proliferation. BmK rAGAP inhibited cell proliferation in a dose-dependent manner. At the same dose, the longer the action time of BmK rAGAP, the greater the degree of inhibition of cell proliferation. The effect of BmK rAGAP on the viability of MDA-MB-231 cells after 48h was determined (Fig. 4C). The results showed that BmK rAGAP had no effect on cell viability at concentrations of 2.5, 5, 10, 20µM, but at a concentration of 40µM, cell viability decreased remarkably.

Secondly, BmK rAGAP significantly inhibited the migration of MDA-MB-231. To determine the effect of BmK rAGAP on the migration of MDA-MB-231 cells, a wound healing assay was used to determine the effect of BmK rAGAP on the migration of MDA-MB-231 after 24 hours (Fig. 4D, E). The results of wound healing assay showed that BmK rAGAP significantly inhibited the migration of MDA-MB-231. The transwell assay was used to determine the effect of BmK rAGAP on migration. The results showed that after BmK rAGAP was treated for 24 hours, the migration rate decreased significantly (Fig. 4F, G). The inhibitory effect on cell migration was enhanced after the treatment time was extended to 48h (Fig. 4H, I).

Finally, BmK rAGAP significantly inhibited the invasion of MDA-MB-231. The transwell assay determined the effect of BmK rAGAP on the invasion of MDA-MB-231. The results showed that BmK rAGAP can significantly inhibit the invasion of MDA-MB-231. After BmK rAGAP was treated for 24 hours, the cell invasion rate decreased in a dose-dependent manner (Fig. 4J, K). And the inhibitory effect on cell invasion was weakened when the treatment time was 48h (Fig. 4L, M). In conclusion, BmK rAGAP significantly suppressed proliferation, migration, and invasion of MDA-MB-231 cells.

4 BmK rAGAP decreased the [Na⁺]i and inhibited the mRNA and protein expression of SCN5A in MDA-MB-231 cells.

BmK rAGAP suppressed proliferation, migration, and invasion of MDA-MB-231 cells. In order to investigate the mechanism of BmK rAGAP, this study treated MDA-MB-231 cells with different concentrations of BmK rAGAP to observe the intracellular [Na⁺]i, as well as the SCN5A mRNA and protein expression. MDA-MB-231 cells which in the logarithmic growth phases were cultured in medium containing different concentrations of BmK rAGAP for 24 and 48 hours, and the intracellular [Na⁺]i was detected by fluorescence dual-wavelength colorimetry. The results showed that the intracellular [Na⁺]i decreased significantly (Fig. 5A, B). RT-PCR was used to detect the mRNA expression of SCN5A in the cells. Inoculate MDA-MB-231 into a culture dish, and different concentrations of BmK rAGAP were added, continue to culture the cells for 24 and 48 hours, and then the total cell RNA was extracted. The results showed that the mRNA expression of SCN5A in MDA-MB-231 was significantly decreased (Fig. 5C, D).

Western Blot was used to analyze the effect of BmK rAGAP on the expression of Na_V1.5. After adding different concentrations of BmK rAGAP for 24 and 48 hours, the whole cell protein was extracted and the expression of Na_V1.5 was determined. The results showed that BmK rAGAP could significantly reduce the Na_V1.5 expression (Fig. 5E). In summary, BmK rAGAP decreased the intracellular [Na⁺]i and inhibited the mRNA and protein expression of SCN5A in MDA-MB-231. The literatures show that SCN5A is related to cell proliferation, migration, and invasion phenotypes. Therefore, it is speculated that BmK rAGAP may affect the proliferation, migration, and invasion of MDA-MB-231 by inhibiting the expression of SCN5A.

5. The effect of BmK rAGAP on the expression of pathway proteins in MDA-MB-231 cells.

Based on the SCN5A-related genes and related signal pathways obtained from the above TCGA database analysis, Western Blot analysis of pathway proteins was performed. MDA-MB-231 cells were treated with BmK rAGAP (0, 2.5, 5, 10 µmol/L) for 48h, the whole cell protein was extracted and then Western Blot was performed to determine the expression of pathway proteins in the cells (Fig. 6).

The results showed that BmK rAGAP can increase the expression of E-cadherin and Vimentin, reduce the expression of MMP-2 protein but BmK rAGAP promotes the expression of p-ERK. BmK rAGAP has no effect on the expression of AKT, but significantly reduces the expression of p-AKT. In addition, BmK rAGAP promoted the expression of P53 protein and p-p38 MAPK. It is speculated that BmK rAGAP may affect the proliferation of MDA-MB-231 through AKT and p38 MAPK pathways. And it may affect the migration and invasion of MDA-MB-231 through EMT.

Several literatures shown that Na_V1.5 is encoded by SCN5A, which is highly expressed in strongly metastatic breast cancer cells MDA-MB-231, and is involved in cell proliferation, migration, and invasion ^{[12, 13, 14]}. In previous, we analysis of patients with different types of breast cancer found that the mRNA expression level of SCN5A in patients with triple-negative breast cancer was significantly higher than that in patients with other types of breast cancer.

BmK rAGAP affects the expression of SCN5A, which may inhibit the proliferation, migration, and invasion of breast cancer cells MDA-MB-231. BmK rAGAP is a sodium channel scorpion toxin protein, which can significantly to inhibit the mRNA and protein expression of SCN5A, and SCN5A is significantly related to the proliferation, migration, and invasion of breast cancer. Therefore, in order to explore how BmK rAGAP affects breast cancer cell proliferation, migration, and invasion through SCN5A, we used the TCGA database to analyze the differentially expressed genes in breast cancer patients and perform SCN5A correlation analysis to obtain SCN5A-related genes.

On this basis, KEGG analysis of SCN5A-related genes was performed to obtain the signaling pathways that may be related to SCN5A, and the protein expression of the above signaling pathways was analyzed. We found that BmK rAGAP could increase the expression of E-cadherin, Vimentin, P53, p-ERK and p-p38 MAPK, and reduce p-AKT, MMP-2 expression in cells MDA-MB-231. Therefore, we speculated that BmK rAGAP may affect the proliferation of breast cancer cells MDA-MB-231 through AKT and P38 MAPK pathways, and affect the migration and invasion of breast cancer cells MDA-MB-231 through EMT. The abnormal expression of AKT pathway in tumor cells, which can promote cells proliferation, angiogenesis, and inhibit cells apoptosis. In addition, the clinical use of AKT inhibitors to treat breast cancer has a significant effect ^[21]. p38 MAPK is a mitogen-activated protein kinase (MAPK), which participates in gene expression regulation, cell growth, development, cycle regulation, apoptosis, and other processes. And it is the hub of many signal pathways ^[22]. P53 is a tumor suppressor gene and a cell cycle regulator, which is related to cell apoptosis, differentiation, DNA repair and other functions. About 50% of cancers are related to P53 mutations ^{[23, 24]}. On the one hand, BmK rAGAP could inhibit AKT phosphorylation and promote p-p38 MAPK and P53 protein expression, suggested that BmK rAGAP may promote cell apoptosis through the AKT/ p38 MAPK-P53 pathway, thereby affected cell proliferation.

On the other hand, BmK rAGAP inhibited the phosphorylation of AKT, that was, promoted the inactivation of the AKT pathway, leading to the weakening of the inhibitory effect of AKT on P53, which leaded to increase in P53 expression, inhibited the expression of anti-apoptotic proteins and promoted cell apoptosis. BmK rAGAP activated the P38 MAPK pathway to promote the expression ofP53 protein, and the increased expression of P53 protein could affect gene transcription, which could promote cell apoptosis and inhibit cell proliferation.

Epithelial-mesenchymal transition (EMT) is the first step for tumors to have an aggressive and metastatic phenotype. The adhesion junctions between epithelial cells are degraded, leading to loss of integrity of epithelial cells, which leads to migration and invasion. One of the driving factors of degradation is matrix metalloproteinase (MMP) ^[25]. E-cadherin, a marker of epithelial cells, is negatively correlated with tumor invasion and metastasis, and Vimentin, a marker of mesenchymal cells, is positively correlated with tumor invasion and metastasis ^[26].

Studies report that as the degree of tumor malignancy increases, E-cadherin shows a decreasing expression, while Vimentin expression increases. In TNBC patients, E-cadherin and Vimentin are related to distant tumor metastasis and clinical staging ^[27]. Western Blot results showed that BmK rAGAP could reduce the expression of MMP-2 and increase the expression of E-cadherin, indicating that BmK rAGAP could reduce the epithelial-mesenchymal transition by inhibiting the degradation of extracellular matrix, thereby inhibiting cell migration and invasion.

In addition, we also found that BmK rAGAP has a feedback activation effect on the ERK pathway. BmK rAGAP can promote ERK phosphorylation and activate the ERK pathway in MDA-MB-231 cells. In breast cancer endocrine therapy, excessive activation of the ERK pathway is associated with drug resistance in breast cancer patients ^[28], which suggests that BmK rAGAP may interact with drug resistance genes to activate the ERK pathway. In this paper, KEGG analysis of SCN5A-related genes was performed to obtain AKT, MAPK, TGF-β, EMT and other pathways. The experiment showed that BmK rAGAP could inhibit the activation of AKT, P-P38 MAPK, and EMT pathways, which laid a theoretical foundation for establishing the connection between SCN5A and the above pathways. However, the mechanism of how BmK rAGAP affects AKT, p-p38 MAPK, and EMT through SCN5A remains unclear. Based on the existing results, we will further analyze the mechanism of BmK rAGAP and the relationship between SCN5A and related pathways. It provides support for exploring the mechanism of BmK rAGAP and discovering new targets for diagnosis and treatment of breast cancer.

SCN5A was highly expressed in breast cancer cells MDA-MB-231 and TNBC patients. BmK rAGAP could suppress the proliferation, migration, and invasion of breast cancer cells MDA-MB-231, it could also reduce the mRNA and protein expression of SCN5A and inhibit intracellular [Na+]i. It indicates that BmK rAGAP may affect proliferation, migration, and invasion by inhibiting the expression of SCN5A. KEGG analysis of SCN5A-related genes was performed to obtain the relevant signal pathways. Western Blot, which was based on the pathway obtained by KEGG, found that BmK rAGAP could increase the expression of E-cadherin, Vimentin, P53, p-ERK and p-p38 MAPK, and reduce p-AKT, MMP-2 expression in cells MDA-MB-231. BmK rAGAP may affect the proliferation of breast cancer cells MDA-MB-231 through AKT and p38 MAPK signaling pathways, and affect the migration and invasion of MDA-MB-231 through EMT. BmK rAGAP has significant potential in the diagnosis and treatment of breast cancer.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

All authors contributed to the study conception and design. Conceptualization were performed by Jinghai ZHANG, Yanfeng LIU and Weiping GE. Material preparation, data collection and analysis were performed by Weiping GE, Xueji OU, Mei GENG, Tingting ZHANG, Jinghai ZHANG and Yanfeng LIU. Formal analysis, investigation and data curation were performed by Weiping GE, Xueji OU, Mei GENG, Xuanwen DING and Xing LI. Software was performed by Zili XU. The first draft of the manuscript was written by Weiping GE and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no competing financial or nonfinancial conflict of interest.

Ethical approval

The present research did not involve Human Participants and/or Animals.

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BmK rAGAP inhibits breast cancer cell proliferation, migration, and invasion by downregulating SCN5A expression and subsequent AKT and MAPK activation.

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Abstract

Figures

Introduction

Materials And Methods

Cell Culture

Cell Proliferation Assay

Wound Healing Assay

Transwell Migration and Invasion Assays

Western Blot Assay

Real-Time PCR

Fluorescence Dual-Wavelength Colorimetry

Immunocytochemistry

Statistical Analysis

TCGA Database Analysis

CCLE Database Analysis

Results

Discussion

Conclusion

Declarations

References

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