Application of propionate, a metabolite of Fusobacterium nucleatum, in the treatment of breast cancer

Abstract

The present invention discloses the application of propionate, a metabolite of Fusobacterium nucleatum, in the treatment of breast cancer, specifically, propionate, a metabolite of Fusobacterium nucleatum, in regulating the metabolic pathway of M2 macrophages and the survival of breast cancer cells through the FFAR2 / Gαi / PKC signaling pathway, and its application in the treatment of breast cancer. Fusobacterium nucleatum exists in breast cancer tissue, and its important metabolite propionate assists Fusobacterium nucleatum in promoting the malignant phenotype of breast cancer. Propionate regulates the PKC signaling pathway by binding to the FFAR2 receptor, thereby promoting the accumulation of arginine in M2 macrophages and the metastasis and proliferation of breast cancer cells. The present invention reveals the potential mechanism of breast cancer development by clarifying the function of the Fn / Propionate / FFAR2 / Gαi / PKC axis, and provides a scientific basis for exploring new breast cancer treatment options. At the same time, it also provides new ideas for understanding the role of bacteria in cancer development.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, and in particular to application of propionate, a metabolite of Fusobacterium nucleatum, in the treatment of breast cancer. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women. Although significant progress has been made in treatment, tumor heterogeneity, drug resistance, and the metastatic ability of cancer cells still seriously hinder the efficacy of breast cancer treatment. Therefore, the development mechanism of breast cancer needs to be explored in depth.

[0003] The human body is home to a large number of microorganisms. In the past few decades, the microbiome has been considered to be a necessary condition for the normal functioning of human physiology, mainly due to the metabolites they produce. In a healthy state, the microbiome maintains a balance with our body, which is called symbiosis. Once this symbiotic relationship is disrupted, the equilibrium state will be broken, leading to the occurrence of various malignancies. Multiple evidences show that there is an association between microbiome dysbiosis and the occurrence of breast cancer. Periodontal disease, characterized by dysbiosis of oral microbial flora, is associated with systemic diseases such as breast cancer. Periodontal disease can cause the bacterial species in the oral cavity to shift to pathogenic anaerobes, and bacteria can colonize breast cancer tissue from the oral cavity through the blood. Fusobacterium nucleatum (Fn) is a common Gram-negative anaerobic bacterium in the oral cavity and is closely related to the progression of periodontal disease. Studies have shown that Fusobacterium nucleatum exists in human breast cancer tissue and also promotes the progression of breast cancer.

[0004] Bacterial metabolites play a key role in mediating the crosstalk between the microbiota and the host immune system. Metagenomic studies have found that Fusobacterium nucleatum can produce propionate, and dysregulated propionate metabolism increases the metastatic potential of triple-negative breast cancer cells. In addition, short-chain fatty acids (SCFAs) such as propionate are key signaling molecules in many physiological and pathological processes. SCFAs exert their effects by binding to free short-chain fatty acid receptors such as FFAR2 and FFAR3, both of which are members of the G protein-coupled receptor (GPCR) family. SCFAs activate these two receptors through the G protein Gi / o family, thereby activating the PKC pathway.

[0005] Previous studies have mostly focused on the role of SCFAs produced by the microbiota in regulating host homeostasis, while their immunomodulatory effects in the tumor microenvironment have rarely been explored. Tumor-associated macrophages (TAMs) are mainly derived from monocytes and tissue-resident macrophages in the blood circulation. As an important component of the tumor microenvironment (TME), TAMs play a key role in tumor growth, invasion, and metastasis. In response to different microenvironmental signaling factors, TAMs can be divided into classically activated M1 phenotypes and alternatively activated M2 phenotypes. It is worth noting that under the influence of the TME, TAMs often exhibit an immunosuppressive phenotype similar to M2. It is well known that immune cells meet their own needs and exert anti-tumor or immunosuppressive functions through metabolic reprogramming, and metabolic reprogramming greatly affects the polarization state of macrophages. Studies have confirmed that Fn infection increases the infiltration of tumor-associated macrophages (TAMs) and participates in mediating the polarization of M2 macrophages, indicating that Fn infection-induced tumor-associated macrophages have an important pro-tumor effect. However, whether the regulatory mechanism of Fn in TME and its metabolite propionate will change the M2 macrophages in the microenvironment The metabolic process is still unknown. Summary of the invention

[0006] The present invention found that Fusobacterium nucleatum exists in breast cancer tissues, and its important metabolite propionate regulates the Gαi / PKC axis by binding to the FFAR2 receptor, thereby inhibiting the Arg-1 expression of M2 macrophages, promoting arginine accumulation, and the metastasis and proliferation of breast cancer cells. Based on this, the present invention provides the following technical solutions:

[0007] Fusobacterium nucleatum metabolite propionate regulates the metabolic pathway of M2 macrophages and the survival of breast cancer cells through the FFAR2 / Gαi / PKC signaling pathway, and its application in breast cancer treatment. As an important metabolite of Fusobacterium nucleatum, propionate assists Fusobacterium nucleatum in promoting the malignant phenotype of breast cancer. Propionate regulates the / Gαi / PKC axis by binding to the FFAR2 receptor, thereby inhibiting the expression of Arg-1 in M2 macrophages, promoting arginine accumulation, and the metastasis and proliferation of breast cancer cells.

[0008] In the above application technology solution, the pathogenic bacteria is Fusobacterium nucleatum, which is a Gram-negative anaerobic bacterium.

[0009] The present invention also provides the use of an antibacterial agent for inhibiting Fusobacterium nucleatum in the treatment of breast cancer.

[0010] In the above application technology solution, the presence of Fusobacterium nucleatum indicates a poor prognosis for breast cancer patients.

[0011] In the above application technology scheme, the propionate treatment concentration is 10uM in both in vivo and in vitro experiments.

[0012] The present invention also provides the use of the drug for inhibiting the generation of propionate in the treatment of breast cancer.

[0013] In the above-mentioned application technology scheme, propionate promotes the accumulation of arginine in M2 macrophages through the FFAR2 / Gαi / PKC signaling pathway.

[0014] The present invention also provides the use of an agent for inhibiting the binding of propionate to FFAR2 in a drug for inhibiting the occurrence and development of breast cancer.

[0015] In the above application technology scheme, the reagent inhibits the FFAR2 / Gαi / PKC signaling pathway and the arginine content of M2 macrophages.

[0016] The drug for preventing breast cancer is in the form of any one of powder, suppository, gel, oral solution, hard capsule and soft capsule.

[0017] The beneficial results of the present invention are: revealing the immunomodulatory effects and related mechanisms of Fusobacterium nucleatum metabolites on the tumor microenvironment. The present invention has confirmed that Fusobacterium nucleatum exists in breast cancer tissues, and its important metabolite propionate regulates the Gαi / PKC axis by binding to the FFAR2 receptor, thereby inhibiting the Arg-1 expression of M2 macrophages, promoting arginine accumulation, and the metastasis and proliferation of breast cancer cells. The present invention reveals the potential molecular mechanism of breast cancer development by clarifying the function of the Fn / Propionate / FFAR2 / Gαi / PKC axis, providing a scientific basis for exploring new breast cancer treatment options. At the same time, it also provides new ideas for understanding the role of bacteria in cancer development. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 These are the research results on the distribution of Fusobacterium nucleatum in breast cancer tissues and macrophages and its promotion of macrophage M2 polarization.

[0017] Figure 2 These are the results of a research experiment on how Fusobacterium nucleatum promotes the proliferation and metastasis of breast cancer cells.

[0018] Figure 3 This is the result of a research experiment that propionate, a metabolite of Fusobacterium nucleatum, inhibits the expression of Arg-1 in M2 macrophages and promotes their arginine accumulation.

[0019] Figure 4 This is the result of a research experiment that propionate promotes the proliferation and metastasis of breast cancer cells.

[0020] Figure 5These are the results of a research experiment on how Fusobacterium nucleatum promotes macrophage M2 polarization and arginine accumulation through FFAR2.

[0021] Figure 6 These are the experimental results of a study on how propionate promotes arginine accumulation in M2 macrophages through the FFAR2 / Gαi / PKC axis.

[0022] Figure 7 The results are from a study using a 4T1 xenograft tumor model constructed in BALB / C mice to explore the effects of Fusobacterium nucleatum and propionate on breast cancer growth. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0024] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0025] Example 1

[0026] 1 Materials and methods

[0027] 1.1 Cell lines and strain culture

[0028] Breast cancer cells MCF-7, MDA-MB-231 cells and THP-1 cells were purchased from the ATCC cell bank in the United States. MCF-7 and MDA-MB-231 were cultured in DMEM medium (BasalMedia, Shanghai, China) supplemented with 10% fetal bovine serum (vivacell, Germany) and 1% penicillin / streptomycin. THP-1 was cultured in RPMI 1640 medium (BasalMedia, Shanghai, China) supplemented with 10% fetal bovine serum (vivacell, Germany) and 1% penicillin / streptomycin. All cells were cultured at 5% CO2 and 37°C. The Fn strain (ATCC 25586) was cultured in Brucella blood agar culture dishes, cultured overnight under anaerobic conditions at 37°C, and the bacterial suspension was prepared after washing three times with sterile PBS. The turbidity of the bacterial suspension was determined by turbidimetry, and its concentration was adjusted to 1×10 8 CFU mL-1. The following experiments were all conducted at a multiplicity of infection (MOI) of 100:1, where MOI represents the ratio of bacteria to cells.

[0029] 1.2 Preparation of relevant conditioned medium (CM)

[0030] (1) The supernatant culture medium of MDA-MB-231 cells was collected and mixed with fresh RPMI1640 supplemented with 10% fetal bovine serum at a ratio of 1:1 to prepare a conditioned culture medium containing 5% fetal bovine serum. Conditioned medium (CM). After 24 hours, the cells were infected with bacteria for another 24 hours. The groups were: (2) In serum-free medium, nonspecific siRNA (siNC) or FFAR2-siRNA (siFFAR2) was transfected into the cells using lipofectamine2000 reagent at a final concentration of 50 nM. In the cells, 6 hours after transfection, the culture medium was replaced with fresh culture medium containing 10% fetal bovine serum, and Fn or propionate was added to each group. CM was collected and prepared 48 hours later, and the method was the same as above. The groups were: (3) Treatment with CM containing MDA-MB-231 cells After 24 h, the cells were replaced with serum-free medium and then transfected with nonspecific siNC or siArg-1 at a final concentration of 50 nM using lipofectamine2000 reagent. In cells, the supernatant was collected 6 hours after transfection and added to fresh culture medium supplemented with 10% fetal bovine serum, mixed at a ratio of 1:1 to prepare conditioned medium (CM) containing 5% fetal bovine serum. MDA-MB-231 and MCF-7 cells were treated with the conditioned medium.

[0031] 1.3 Cell transfection

[0032] Cells were transfected with siRNA (Qingke Biotechnology, Beijing, China) or scrambled according to the manufacturer's protocol. The siRNA sequences are shown in Table 1.

[0033] 1.4 Immunoblotting

[0034] The whole protein of the cells was collected by centrifugation and lysed in RIPA supplemented with PMSF (phenylmethylsulfonyl fluoride), a protease inhibitor. The protein concentration was determined using a BCA protein detection kit (Beyotime, China), and the extracted protein samples were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, Germany). The membrane was blocked with 5% skim milk powder for 1-2 hours at room temperature, incubated with the primary antibody at 4°C overnight, and then the membrane was incubated with the secondary antibody at room temperature for 1 hour. Finally, the target band on the PVDF membrane was developed in a chemiluminometer using ECL luminescent solution, and the image was collected and saved.

[0035] 1.5 RNA extraction and fluorescence quantitative PCR detection

[0036] Total RNA of cells was extracted with Trizol reagent (Takara, Japan) and reverse transcribed into cDNA, and then fluorescent quantitative PCR was performed using SYBR Premix Ex Taq II (Aibotek, China). The reaction was performed in a volume of 10 mL using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad). All experiments were performed according to the manufacturer's instructions. The primer sequences are shown in Table 1:

[0037] Table 1 Sequence

[0038] The reaction system was placed in a fluorescent quantitative PCR instrument and the program was set as follows: 95°C pre-denaturation for 2 min, 95°C denaturation for 10 s, 60°C annealing and extension for 30 s, for a total of 40 cycles.

[0039] 1.6 Arginine content detection

[0040] After centrifugation of the cell suspension at 3000 rpm for 5 minutes, the supernatant was discarded and the cell pellet was resuspended in PBS. The arginine content was measured using a specific kit according to the manufacturer's instructions.

[0041] 1.7 Fluorescence in situ hybridization

[0042] The localization of Fn in THP-1 cells and the distribution in breast cancer tissues were detected using a FISH kit (GenePharma, China). The FISH probe was added to the paraffin sections and cell slides and placed in a 37°C incubator for 12-16 hours. The next day, 10ul of a fluorescence quencher containing DAPI was added and the cells were stained in the dark for 20 minutes. Photos were taken using a confocal microscope (Leica TCSSP8).

[0043] 1.8 Immunofluorescence

[0044] Tumor sections were dewaxed in xylene and immersed in graded alcohol for hydration. The sections were placed in boiling citrate buffer for antigen retrieval. Hydrogen peroxide was used to block endogenous peroxidase activity, and goat serum was added to the sections to block nonspecific binding. The slides were stained with a multiple fluorescent staining kit (AIFang Bio Kit, AFIHC024) according to the manufacturer's instructions, and finally anti-fluorescence quenching sealing agent was added to seal the slides and photographed under a microscope.

[0045] 1.9 Immunohistochemistry

[0046] Tumor sections were cut into 4 μm thick sections, baked in a 70°C oven for 2-4 hours, then dewaxed in xylene and soaked in graded alcohol for hydration. The sections were placed in boiling citrate buffer for antigen retrieval, soaked in 3% hydrogen peroxide for 20 minutes to block endogenous peroxidase, and then blocked with goat serum blocking solution for 30 minutes. The sections were incubated with the primary antibody at 4°C overnight, and then incubated with the HRP-conjugated secondary antibody at room temperature for 30 minutes. DAB color development, counterstaining and sealing were then performed according to the procedures of the kit.

[0047] 1.10 Cell proliferation assay

[0048] A cell counting kit (Biosharp, China) was used to detect the viability of cell growth. The density is 2x10 3 MCF-7 and MDA-MB-231 cells were seeded in 96-well plates. After 24, 48, and 72 hours of culture, 10% CCK-8 solution was added and incubated for 2 hours. Finally, the optical density (OD) value at 450 nm was measured using a 680 microplate reader (Bio-Rad).

[0048] 1.11 Wound healing assay

[0049] The density is 5x10 5 MCF-7 and MDA-MB-231 cells were inoculated into 6-well plates. After forming a monolayer of cells, a wound was cut at the confluence using a 200uL pipette tip. The culture dish was then gently washed three times with PBS to remove detached cells and continued to be incubated in serum-free basal medium for 24-48 hours. At 0h, 24h, and 48h after scratching, scratch images of the same position were taken using a phase contrast microscope (Olympus, Japan).

[0050] 1.12 Clone formation experiment

[0051] The density is 8x10 2 -1x10 3 MDA-MB-231 and MCF-7 cells were seeded in culture dishes, and 5 mL of DMEM medium and 500 uL of fetal bovine serum were added to each dish and incubated for 10-14 days. Then, the cells were washed 3 times with PBS, fixed with paraformaldehyde for 15 min, and finally stained with 0.1% crystal violet.

[0052] 1.13 Flow cytometry

[0053] Collect the cell pellet by centrifugation, resuspend the cells with 500uL PBS, and add CD16 / 32 antibody to block for 30 minutes. Wash the cell pellet with 100uL PBS, add CD86 and CD206 antibodies, and incubate at 4℃ for 30 minutes in the dark. Wash with PBS three times, add 100uL PBS to resuspend the cell pellet, and detect on the machine.

[0054] 1.14 Xenograft Mouse Model

[0055] Animal experiments were carried out in strict accordance with the Chongqing Animal Protection Standard Guidelines and the Chongqing Medical University Experimental Animal Management Committee. 4T1 cells (5×10 5 ) was injected subcutaneously into 4-week-old BALB / c The mice were randomly divided into three groups, with 6 mice in each group. The same volume of PBS (100uL), Fn (1×10 6 / 100uL), propionate (10uM / 100uL), and injected twice a week. Before each injection, the tumor volume was measured with a caliper and calculated using the following formula: tumor volume (mm 3) =1 / 2L×W 2 On the 17th day after injection, the mice were killed and the tumor tissues were collected. The tumors were weighed, photographed, and used for further immunohistochemistry (IHC) staining, flow cytometry, and immunofluorescence to detect the expression of related proteins.

[0056] 1.14 Statistical analysis

[0057] All experimental data were analyzed using SPSS 20.0 software and GraphPad Prism 8.2, and Student-t test was used to analyze the significance of the differences between the two groups. The results are expressed as mean ± SD, and P values ​​< 0.05 were considered statistically significant.

[0058] 2 Experimental results

[0059] 2.1 Fusobacterium nucleatum exists in breast cancer tissue and promotes M2 polarization of macrophages in the tumor microenvironment

[0060] Numerous studies have shown that there are a large number of Fusobacterium nucleatum (Fn) in breast cancer tissue. To verify this conclusion, we performed FISH staining on tissue sections of breast cancer patients. As expected, we detected Fn ( Figure 1A). As we all know, macrophages are very important immune cells in the tumor microenvironment (TME), which greatly affect the progression of the tumor. At the same time, Fn is an intracellular bacterium, so we speculate that Fn may invade macrophages. The FISH results show that Fn is clearly distributed around the nucleus of macrophages ( Figure 1 B). In the TME, M2 macrophages can promote tumor growth, angiogenesis, invasion and metastasis. Therefore, in order to simulate the tumor microenvironment, we treated THP-1 cells with CM, a conditioned medium of MDA-MB-231 cells, and then treated them with Fn ( Figure 1 C). We found that Fn reduced the expression of M1 marker genes. The mRNA and protein levels of CXCR4, CD80, and INOS were decreased, while the mRNA and protein levels of M2 marker genes CD206 and TGF-β were increased ( Figure 1 DF). The same results were observed in flow cytometry ( Figure 1 G). The above results indicate that Fn can promote the M2 polarization of macrophages in the tumor microenvironment.

[0061] 2.2 Fusobacterium nucleatum promotes the proliferation and migration of breast cancer cells

[0062] To further investigate the Fn-stimulated After the effects of Fn on the proliferation and migration of MDA-MB-231 and MCF-7 cells, we treated breast cancer cells in different ways and conducted wound healing assays, CCK8 and clone formation assays. In the wound healing assay, Fn enhanced the migration and repair ability of breast cancer cells ( Figure 2 AB). CCK8 and clone formation assays were used to test the proliferation potential of each group of cells. The average cell proliferation rate of the treated group of breast cancer cells was significantly higher than that of the control group ( Figure 2 CF). In short, Fn can promote the proliferation and migration of breast cancer cells.

[0063] 2.3 Propionate, a metabolite of Fusobacterium nucleatum, inhibits Arg-1 expression and promotes arginine accumulation in M2 macrophages

[0064] A large number of studies have shown that metabolic reprogramming can regulate the polarization state of macrophages. We found that there are several key enzymes in the urea cycle: ODC1, Arg-1, and ASL. Among them, Arg-1 is an important marker gene for M2 macrophages. Therefore, we have a hypothesis: whether there are differences in metabolic processes between M2 macrophages induced by Fn and tumor-associated macrophages. We found that ODC1 and ASL are highly expressed in M2 macrophages, while only Arg-1 is reduced in M2 macrophages infected with Fn ( Figure 3 AB). It is known that Arg-1 can decompose arginine. We found that the arginine level of M2 macrophages after Fn infection was significantly increased by using an arginine content detection kit ( Figure 3 C), this result is logical. Increasingly, studies have shown that many metabolites of the gut microbiota play a key role in mediating the crosstalk between the microbiota and the host immune system. To further investigate the potential mechanisms, we focused on the metabolites of Fn, including formate, acetate, propionate, and butyrate. The mRNA levels of Arg-1 in the 247 samples of the rats were increased without significant differences. However, propionate significantly decreased the The mRNA level of Arg-1 in Figure 3 D) Propionate-treated mice showed a significant difference in The protein level of Arg-1 was also significantly decreased in Figure 3 E) Arg-1 is a key enzyme that catalyzes the production of ornithine and urea from arginine. Therefore, we used a kit to test arginine levels and found that propionate did increase Arginine levels ( Figure 3 F). The above results indicate that propionate has an important effect on the expression of Arg-1. Therefore, we speculate that propionate assists Fn in regulating Arg-1 in M2 macrophages. We treated the conditioned medium of MDA-MB-231 cells with After treatment with propionate (NaP), the mRNA and protein expression levels of M1 marker genes CXCR4 and INOS decreased, while the mRNA and protein expression levels of M2 marker genes CD206 and TGF-β increased ( Figure 3 GH). This suggests that propionate can also promote the M2 polarization of macrophages in the tumor microenvironment. Both the mRNA and protein levels of Arg-1 were significantly decreased ( Figure 3 IJ). The arginine content test also obtained consistent results ( Figure 3 K). We have previously demonstrated that Fn can downregulate M2 However, it has not been confirmed that the M2 type of Arg-1 knockout Whether it can also promote the proliferation and migration of breast cancer cells. Through wound healing experiments, CCK8 and clone formation experiments, we found that after knocking down Arg-1 in M2 macrophages, the proliferation and migration abilities of MDA-MB-231 and MCF-7 cells were significantly improved ( Figure 3 LO). In summary, propionate plays a key role in the regulation of Arg-1 expression and arginine content in M2 macrophages by Fusobacterium nucleatum.

[0065] 2.4 Propionate promotes the proliferation and migration of breast cancer cells

[0066] Considering the important role of propionate in this process and the fact that propionate has been previously shown to affect the metastatic potential of breast and lung cancer, we believe that propionate may also affect the proliferation and migration of breast cancer cells. Increased the migration and repair capabilities of breast cancer cells ( Figure 4 AB). Under the same treatment conditions, CCK8 and clone formation experiments also showed that propionate also significantly promoted the proliferation of breast cancer cells ( Figure 4 CF). The above results indicate that propionate can promote the malignant phenotype of breast cancer cells.

[0067] 2.5 Fusobacterium nucleatum promotes arginine accumulation in M2 macrophages via FFAR2

[0068] Previous experiments have demonstrated the important role of the Fn metabolite propionate, and the next step is to further explore its potential molecular mechanisms. We focus on a class of free short-chain fatty acid receptors, including FFAR2 and FFAR3. They are G protein-coupled receptors (GPCRs) that bind to SCFAs. These free fatty acid receptors can be activated by acetate, propionate, and butyrate. SCFAs are the main driving force for FFAR2 and FFAR3. Although FFAR2 and FFAR3 are both receptors for SCFAs, only FFAR2 is negatively correlated with the survival rate of breast cancer patients ( Figure 5 A) In addition, the M2 type after Fn infection Both the mRNA and protein levels of FFAR2 were significantly increased in Figure 5 BC). The above results suggest that FFAR2 plays an important role in the M2 type of Fn infection. However, high expression of FFAR2 is associated with M2 Therefore, we investigated the effect of siFFAR2 knockdown on the M2 type of Fn-infected mice. After successful knockdown of FFAR2 expression ( Figure 5 D, G), the mRNA levels of M2 marker genes CD206 and TGF-β increased, but the mRNA levels of M1 marker genes CXCR4, CD80, and iNOS decreased ( Figure 5 EF) Fn-infected M2 type was detected by immunoblotting Knockdown of FFAR2 reduced the protein levels of M2 marker genes CD206 and TGF-β, while the protein levels of M1 marker genes CD86 and iNOS were significantly increased ( Figure 5 H). The mRNA and protein levels of Arg-1 were also restored ( Figure 5 IJ). In addition, knockdown of FFAR2 also significantly reduced the content of arginine ( Figure 5 K). In summary, FFAR2 is the key reason why Fn induces M2 polarization of macrophages and promotes arginine accumulation in these cells.

[0069] 2.6 Propionate promotes arginine accumulation in M2 macrophages via the FFAR2 / Gαi / PKC axis

[0070] We have demonstrated that FFAR2 is involved in the effects of Fn infection on macrophage polarization and the regulation of Arg-1 expression, but it is unclear whether the same effect exists after knocking down FFAR2 in macrophages treated with the Fusobacterium nucleatum metabolite propionate. We first confirmed that the mRNA and protein levels of FFAR2 in M2 macrophages treated with propionate were significantly higher than those in the control group ( Figure 6 AB). We then analyzed the M2 phenotype in the siFFAR2 knockdown group. As expected, after successful knockdown of the FFAR2 gene ( Figure 6 C, F), the mRNA levels of M2 marker genes CD206 and TGF-β were significantly suppressed, while the mRNA levels of M1 marker genes CXCR4, CD80 and iNOS were restored ( Figure 6 DE) Propionate-treated M2 phenotype detected by immunoblotting After inhibiting the expression of FFAR2, the protein levels of M2 marker genes CD206 and TGF-β decreased, while the protein levels of M1 marker genes CXCR4 and iNOS increased significantly ( Figure 6 G). At the same time, the mRNA and protein levels of Arg-1 were restored ( Figure 6 HI). In addition, downregulation of FFAR2 also significantly reduced the arginine content in M2 macrophages ( Figure 6 J). Previous studies have shown that FFAR2 can bind to pertussis toxin-sensitive Gi / o proteins and Gq proteins. SCFAs activate FFAR2 through the Gi / o family of G proteins, thereby inhibiting the production of cAMP and activating the PKC / IP3 pathway. Therefore, we wanted to explore which pathway plays a key role in the regulation of propionate on the expression of Arg-1 and arginine levels in M2 macrophages. Figure 6As shown in Figure K, Forskolin (cAMP agonist) and MCU-i4 (IP3 inhibitor) had no significant effect on the expression of Arg-1, while STS (PKC inhibitor) significantly restored the protein expression level of Arg-1. At the same time, the arginine level was also reduced after inhibiting PKC with STS. The above results indicate that propionate promotes arginine accumulation in M2 macrophages through the FFAR2 / Gαi / PKC axis.

[0071] 2.7 Fusobacterium nucleatum and its metabolite propionate can promote the growth of subcutaneous tumors

[0072] Finally, we constructed a 4T1 xenograft tumor model using BALB / C mice to support in vitro studies. 4T1 cells (5×10 5 ) was injected subcutaneously into 4-week-old BALB / c female mice. The mice were randomly divided into three groups, with 6 mice in each group. Eight days later, each group was injected with the same volume of PBS (100uL), Fn (1×10 6 / 100uL), propionate (10uM / 100uL), injected twice a week, and the mice were killed after 30 days ( Figure 7 A). Both Fn and propionate significantly promoted tumor growth ( Figure 7 BD). In addition, the results of the Ki67 experiment further demonstrated that both Fn and propionate can promote the proliferation of breast cancer cells ( Figure 7 E). Flow cytometry revealed that the proportion of M2 macrophages in the tissues of mice treated with Fn or propionate was higher than that in the control group, and their Arg-1 expression level was significantly lower than that in the control group. Immunofluorescence triple staining results showed that compared with the control group, the CD206 level in the Fn and propionate groups was significantly increased, while the Arg-1 level was significantly inhibited ( Figure 7 G). In conclusion, the above results are consistent with the in vitro experimental results: Fn or propionate induces M2 polarization of macrophages in the tumor microenvironment by inhibiting Arg-1 expression, thereby promoting the growth of breast cancer tumors.

[0073] 3 Analysis

[0074] In summary, this study showed that propionate, a metabolite of Fusobacterium nucleatum, inhibited Arg-1 expression in M2 macrophages through the FFAR2 / Gαi / PKC signaling pathway, thereby promoting arginine accumulation and the proliferation and migration of breast cancer cells.

Claims

1. Propionate, a metabolite of Fusobacterium nucleatum, regulates the metabolic pathway of M2 macrophages and the survival of breast cancer cells through the FFAR2 / Gαi / PKC signaling pathway, and its application in the treatment of breast cancer, characterized in that: Propionate, as an important metabolite of Fusobacterium nucleatum, assists in promoting the malignant phenotype of breast cancer by Fusobacterium nucleatum; propionate regulates the / Gαi / PKC axis by binding to the FFAR2 receptor, thereby inhibiting the expression of Arg-1 in M2 macrophages, promoting arginine accumulation, and the metastasis and proliferation of breast cancer cells.

2. The use according to claim 1, characterized in that: The pathogenic bacteria is Fusobacterium nucleatum, which is a Gram-negative anaerobic bacterium.

3. The use according to claim 1, characterized in that: The propionate treatment concentration was 10 μM in both in vivo and in vitro experiments.

4. The use according to claim 1, characterized in that: Propionate promotes arginine accumulation in M2 macrophages through the FFAR2 / Gαi / PKC signaling pathway.

5. Use of the antibacterial agent for inhibiting Fusobacterium nucleatum according to claim 2 in the treatment of breast cancer.

6. The use according to claim 3, characterized in that: The presence of Fusobacterium nucleatum predicts a poor prognosis in breast cancer patients.

7. Use of the drug for inhibiting propionate production according to claim 5 in the treatment of breast cancer.

8. Application of agents that inhibit the binding of propionate to FFAR2 in drugs that inhibit the occurrence and development of breast cancer.

9. The use according to claim 8, characterized in that: The reagent inhibits the FFAR2 / Gαi / PKC signaling pathway and the arginine content of M2 macrophages.

10. A drug for preventing breast cancer, characterized in that: The drug comprises the inhibitor according to claim 6.

11. The drug for preventing breast cancer according to claim 10, characterized in that: The dosage form of the drug is any one of powder, suppository, gel, oral solution, hard capsule and soft capsule.

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