SiRNA specifically binding to m2 macrophage cd206 and use thereof

Abstract

This invention provides siRNA that specifically binds to CD206 on M2 macrophages. The siRNA is at least one of siRNA-1, siRNA-2, and siRNA-3, or at least one of siRNA-4, siRNA-5, and siRNA-6. The siRNA provided by this invention specifically binds to CD206 on M2 macrophages, thereby reducing CD206 expression and promoting M2 macrophage proliferation, which in turn inhibits tumor cell proliferation and migration.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an siRNA, specifically an siRNA that specifically binds to CD206 of M2 macrophages and its applications. Background Technology

[0002] Macrophages, discovered over 100 years ago, are immune cells widely distributed throughout the body's tissues and blood, playing a crucial role in resisting external bacteria and viruses. They belong to the phagocytic cell system and are among the most important participants in the autoimmune system within the tumor microenvironment, and the diversity of their roles in this environment is constantly being revealed. Recent research on tumor-associated macrophages has provided theoretical and factual evidence for tumor metastasis, recurrence, and drug resistance. The presence of tumor-associated macrophages prevents chemotherapy and other treatments from completely eradicating all tumor cells. While early research considered macrophages as simple tumor killers, subsequent studies have gradually revealed that they may also promote tumor growth, indicating the role of tumor-associated macrophages. Macrophages not only indirectly influence tumor development through their biological behavior but also directly participate in tumor formation, playing a vital role in tumor metastasis.

[0003] 1. Macrophages and their functions

[0004] 1.1 Macrophage origin and function

[0005] Macrophages participate in innate and adaptive immunity, angiogenesis, proliferation, and even the formation of malignant tumors. Macrophages are a heterogeneous group of cells capable of performing different functions in various organs and tissues. Even within a single tissue, their function varies depending on location (Ref. 8). Mononuclear phagocytes are a major component of the innate immune system, non-specifically engulfing foreign substances, bacteria, senescent and mutated cells, maintaining homeostasis, providing innate anti-infection, and anti-tumor immunity. Macrophages exhibit heterogeneity, including cells at different differentiation stages and in different activation states, each with corresponding morphology, phenotype, metabolic characteristics, and biological functions. Macrophages can secrete pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), playing a crucial role in killing and engulfing tumor cells. Increasing research demonstrates that macrophages, as an important component of the tumor microenvironment, participate in tumor growth, invasion and metastasis, angiogenesis, immune escape, and recurrence. Macrophages can polarize / differentiate into two different subtypes in different microenvironments: M1 macrophages and M2 macrophages.

[0006] Classical macrophages are inactive cells, small in size and with weak cytotoxic activity in their resting state. Polarized (or activated) macrophages are divided into two types: typically activated macrophages (M1) and alternately activated macrophages (M2). M1 macrophages preferentially express pro-inflammatory molecules such as nitric oxide, interleukin IL12, CXCL9, CXCL10, CXCL11, and reactive oxygen species, which contribute to tumor suppression. However, M2 macrophages express anti-inflammatory molecules such as ornithine, IL-10, CCL17, CCL18, CCL22, and scavenger receptors. M2 macrophages are thought to be involved in tumor promotion by inducing angiogenesis, immunosuppression, epithelial-mesenchymal transition, promoting stem cell formation, and activating tumor cells. Similarly, when the body is injured, M1 macrophages are the first pro-inflammatory cells activated at the wound site, while M2 macrophages are also known as anti-inflammatory agents and play an important role in the human body.

[0007] Different types of pathogens, inflammatory stimuli, and cytokines such as IFN-γ can activate macrophages. Activated macrophages release various cytokines and chemokines and act as antigen-presenting cells, participating in the initiation and regulation of immune responses. M1 macrophages are induced by Th1 cytokines, such as interferon-γ (IFN-γ), lipopolysaccharide (LPS), and granulocyte-macrophage colony-stimulating factor. They express surface markers such as CD80 and CD86 and secrete cytokines such as interleukin (IL-6), inducible nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNF-α), playing an important role in antibacterial and anti-infective processes.

[0008] Different types of cytokines and their combinations can induce activated macrophages with varying activities and functional characteristics. Macrophages activated by IL-4, IL-10, etc., exhibit weakened antigen-presenting capacity compared to M1 macrophages, and are thus termed alternative activated M2 macrophages, or M2 macrophages. M2 macrophages are induced by Th2 cytokines, express CD163 and CD206, and secrete IL-4, IL-13, IL-10, transforming growth factor-β (TGF-β), arginine 1 (Arg1), chemokines CCL17, and CCL22, among others. Studies have found that M2 macrophages promote tumor cell proliferation, which is closely related to their role in epithelial-mesenchymal transition (EMT) and the promotion of tumor stem cell formation. Studies have found that exosomes derived from lung and breast cancer cells can deliver activated epidermal growth factor receptor (EGFR) to macrophages, leading to reduced interferon-β (IFN-β) secretion and decreased innate immunity in cancer patients through mitogen-activated protein kinase 2 (MEKK2). These studies suggest that macrophages play different roles in the development, progression, and metastasis of tumors.

[0009] 1.2 Tumor-associated macrophages

[0010] Macrophages constitute the largest proportion of cells in the tumor microenvironment (TME), approximately 50%-80%. The macrophage composition in the TME is complex, primarily including M1 and M2 macrophages and tumor-associated macrophages (TAMs). M1 macrophages mainly kill tumor cells, while M2 macrophages play a role in tumor development and progression by promoting tumor growth, tumor angiogenesis, tumor invasion and distant metastasis, tumor immune escape, and tumor drug resistance. In recent years, increasing research has shown that the functions of tumor-associated macrophages (TAMs) are largely consistent with those of M2 macrophages; they can further promote tumor development, including promoting tumor proliferation, increasing tumor cell activity, and promoting tumorigenesis and genetic instability. Therefore, the interaction between TAMs and the tumor or the entire tumor microenvironment (TME) is highly complex, requiring in-depth research and exploration to gain a clear understanding of this interaction. TAMs secrete and release growth factors such as platelet-derived growth factor, hepatocyte growth factor, and epidermal growth factor, which can directly act on tumor cell receptors, promoting mitosis. Additionally, the cytokine IL-6 secreted by TAMs can indirectly promote tumor cell growth by activating intracellular pathways through the tumor cell transcription activator stat3. Furthermore, TAMs can release enzymes that promote tumor growth. Tumor cells require large amounts of nutrients and oxygen. Numerous clinical data indicate a close correlation between the density of local macrophages in tumor tissue and the area of ​​newly formed microvessels. TAMs express the angiotrophin receptor TIE2, and also secrete large amounts of vasoactive molecules such as TNF-α and IL-8, promoting angiogenesis. In promoting tumor invasion and metastasis, TAM secretes proteases that degrade the extracellular matrix, such as matrix metalloproteinases (MMPs), cathepsins, and urokinase fibrinolytic activators, as well as extracellular matrix remodeling enzymes, such as lysine acyl oxidase. These enzymes can degrade the fibrous connective tissue surrounding the tumor, allowing tumor cells to easily invade the in situ carcinoma and enter the peripheral circulatory system for distant metastasis. The proteases further clear obstacles for successful tumor metastasis. The new blood vessels induced by TAM also have high permeability due to the incomplete basement membrane, making it easier for blood vessels to invade the tumor.As a "traitor" to the body's immune system, TAM, in addition to the functions mentioned above, secretes the cytokine IL-10, leading to a decrease in the release of the pro-inflammatory cytokine IL-12. This weakens the ability of natural T cells to produce the cytotoxic factor interferon-γ. High levels of IL-10 also inhibit enzymes required for dendritic cell maturation. Furthermore, TAM secretes arginase I, which hydrolyzes L-arginine, thereby affecting the expression and proliferation of T cell receptors, causing CD4+ and CD8+ T cells to lose their responsiveness to stimuli. The above studies and further research indicate that only M2-TAM is an "accomplice" in tumor development. Further research on M2 may provide valuable references for clinical treatment and prognosis of tumor metastasis.

[0011] 1.3 Macrophage surface markers

[0012] Macrophages have many important molecular markers on their surface, such as IL-12 molecules, CD206 molecules, TGF-β molecules, and MHC molecules. Among these molecules, CD206 is one of the most important surface markers reflecting the functional status of macrophages.

[0013] CD206 is a 175-190 kDa endocytic receptor, also known as mannose receptor C-type 1 (MRC1), macrophage mannose receptor (MMR), or C-type lectin domain family 13 member D (CLEC13D). CD206 is expressed in most macrophage populations, including TAMs, and also in dendritic cells of specific tissues, as well as in liver, spleen, lymphocytes, and dermal microvascular endothelial cells.

[0014] Human CD206 consists of 1456 amino acids (aa), and its extracellular region contains three types of domains: an N-terminal cysteine-rich (CR) domain, a type II fibronectin repeat (FNII) domain, and eight type C lectin domains (CTLD). The CR domain of human CD206 shares 83%, 84%, 89%, 89%, and 90% amino acid sequence identity with the CR domains of mouse, rat, horse, pig, and dog CD206, respectively. The CR domain can bind to sulfated sugars terminated with SO4-3-Gal or SO4-3 / 4-GalNAc and can recognize some pituitary hormones, such as luteinizing hormone (LH), thyroid stimulating hormone (TSH), chondroitin sulfate, and sulfated N-acetylgalactosamine. The FNII domain mediates Ca2+-independent binding to collagen, especially collagen I, II, III, and IV. CTLD participates in Ca2+-dependent recognition of branched sugars such as terminal mannose, fucose, or N-acetylglucosamine, which are ubiquitous in pathogenic microorganisms.

[0015] 1.4 CD206 Functions

[0016] CD206 is an important pattern recognition receptor (PRR) and endocytic receptor in the innate immune system, and it is abundantly expressed in alveolar macrophages, monocyte-derived macrophages, and dendritic cells. CD206 expression has also been found in certain endothelial cell subsets, tracheal smooth muscle cells, retinal epithelial cells, renal mesangial cells, sperm acrosomal cells, and brain microglia. CD206 can recognize and bind to a wide range of endogenous and exogenous ligands, playing a crucial role in maintaining homeostasis, recognizing and phagocytosing pathogens, inducing cytokines, and antigen presentation.

[0017] CD206 is involved in pathogen recognition and phagocytosis. CD206 can recognize polysaccharide components of the cell wall, such as yeast mannan, bacterial capsules, and LPS. Macrophages can use CD206 to phagocytose many non-opsonized microorganisms, including bacteria, fungi, and protozoa. Mannose receptors have been shown to be involved in the recognition and clearance of Candida albicans, Mycobacterium tuberculosis, Staphylococcus aureus, and Lactobacillus acidophilus. After pathogen recognition, macrophages release increased superoxide anions and induced the synthesis of related cytokines. The macrophage membrane, mediated by the actin cytoskeleton, can undergo amoeboid movements to surround pathogens or pathogen-infected target cells, forming phagosomes, which then digest and kill the pathogens.

[0018] CD206 participates in intracellular signal transduction, influencing the expression of numerous genes. The interaction between the mannose receptor (MR) and its ligands triggers a series of amplified intracellular signal cascades, activating transcription and promoting or inhibiting the expression of NO and various cytokines such as TNF-α, IL-12, IL-10, IL-1ra, and IL-RII. The cytoplasmic domain of the MR is short and lacks signal transduction motifs; therefore, its participation in signal transduction requires the involvement of other receptors. Previous studies have confirmed that the induction of IL-8 requires the participation of TLR-2. The MR regulates the body's immune response by mediating the production of different cytokines.

[0019] CD206 participates in antigen processing and presentation. CD206 primarily takes up and concentrates non-self antigens to facilitate antigen processing and presentation. CD206 mediates antigen cross-presentation, where exogenous proteins are internalized and presented to CD8+ T cells via MHC class I molecules. This function is crucial for immune responses to non-directly infected APC viruses and non-endogenously expressed tumor antigens. The translocation of exogenous antigens from the endosomal layer to the cytoplasm for protein degradation is key to antigen cross-presentation.

[0020] 1.5CD206 and tumor-associated macrophages

[0021] In recent years, the tumor microenvironment (TME) has become a key focus in cancer research. The TME includes stromal cells, fibroblasts, immune cells, and the extracellular matrix. Numerous studies have demonstrated a close relationship between the occurrence and development of breast cancer and the TME, with each component playing a crucial, synergistic role in different stages of breast cancer development.

[0022] Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment (TME). In the breast cancer tumor microenvironment, TAMs account for more than 50% of the number of cells within the tumor. Breast cancer cells secrete chemokine (CC motif) ligand 2 (CCL2), CCL5, and CXCL12 to recruit monocytes. Monocytes are stimulated by different signaling molecules in the TME and polarize into TAMs. Most TAMs exhibit the M2 phenotype and are called M2 macrophages.

[0023] Tumor-associated macrophages (TAMs) can promote tumor cell proliferation, invasion, and metastasis, promote tumor angiogenesis, inhibit T cell-mediated anti-tumor immune responses, and regulate tumor drug resistance. TAMs can directly promote tumor cell proliferation by secreting epidermal growth factor (EGF), transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF). TAMs also mediate tumor immune escape by expressing molecules such as programmed death-1 (PD-1) and signal regulatory protein alpha (SIRPα), which inhibit macrophage phagocytosis. Recently, researchers discovered a class of C1q macrophages... + A subset of macrophages, whose m6A methyltransferase METTL14 is dysregulated, leading to increased EBI3 expression, promotes CD8 cell infiltration in tumors. + T cell dysfunction can suppress anti-tumor immune responses. Furthermore, tumor-associated tumor cells (TAMs) can inhibit the anti-tumor effects of chemotherapy drugs, including etoposide, gemcitabine, and the CMF regimen (cyclophosphamide, methotrexate, and 5-fluorouracil). Multiple studies have confirmed that high TAM infiltration density in tumor tissue is closely related to poor prognosis in cancer patients.

[0024] CD206 is highly expressed in M2 macrophages. CD206 not only serves as a marker of M2 macrophage activation but also contributes to their immunosuppressive activity. A novel intronic microRNA (miRNA), named miR-511-3p, was identified in the human and mouse Mrc1 gene. miR-511-3p is a negative regulator of the pro-tumorigenic activity of tumor-associated tumor cells (TAMs), and its forced expression in hematopoietic stem cells has a significant impact on the tumor angiogenesis system and can further inhibit tumor growth.

[0025] A recent study found that activating the tumor-associated macrophage (TAM) surface receptor CD206 can enhance anti-tumor immune responses. Jaynes et al. constructed a decameric amphiphilic host defense peptide analog RP-182, which can selectively induce a conformational shift in CD206, leading to a phenotype change in TAMs. The researchers first discovered that RP-182 mediates CD206 activation in M2 macrophages, subsequently triggering endocytosis, phagosome-lysosome formation, and autophagy. Flow cytometry analysis of RP-182-treated mouse bone marrow-derived macrophage phenotypes showed that RP-182 activation of CD206 reprogrammed M2 macrophages into M1 macrophages. In RP-182-treated tumor tissues, M1 macrophages increased while M2 macrophages decreased. The anti-tumor activity of RP-182 was observed in both a mouse pancreatic cancer model and a patient-derived xenograft model with high CD206 expression. RP-182 also showed good efficacy when used in combination with chemotherapy drugs or immune checkpoint inhibitors in pancreatic cancer. Furthermore, researchers tested RP-182 in animal models of different cancers, finding it effective not only against pancreatic cancer but also against other cancers such as colon cancer, breast cancer, prostate cancer, and melanoma. This study suggests that CD206 can serve as a target for cancer therapy.

[0026] 2.1 The structure of siRNA: A short (typically 20 to 24 bp) double-stranded RNA (dsRNA) with a phosphorylated 5' end and a hydroxylated 3' end with two protruding nucleotides. The siRNA produced by this enzyme consists of a long dsRNA and a small hairpin RNA. siRNA can also be introduced into cells via transfection. Since, in principle, any gene can be knocked down by synthetic siRNA with a complementary sequence, siRNA is an important tool for validating gene function and drug targeting in the post-genomic era. siRNA is typically a 21-nucleotide long double-stranded RNA (dsRNA), with each strand extending two nucleotides beyond the other end of the RNA, such as... Figure 1As shown, each strand has a 5' phosphate terminus and a 3' hydroxyl terminus. This structure is obtained by treating with an enzyme called dicer, which can cleave long double-stranded RNA or small hairpin RNA into siRNA. Furthermore, siRNA can be introduced into cells via various transfection techniques and produce a specific knockdown effect on specific genes. Therefore, the complementarity of appropriately cleaved siRNA can be used to label genes with known sequences, making siRNA an important tool for studying gene function and drug targets. siRNA is similar to miRNA; however, miRNAs are derived from shorter stem-loop RNA products and typically silence genes by inhibiting translation, exhibiting broader specificity. siRNA, on the other hand, usually works by cleaving mRNA before translation and has 100% complementarity, thus exhibiting very strict target specificity.

[0027] 2.2 Induction of editing using siRNA or its biosynthetic precursor RNAi

[0028] Gene knockdown via transfection with exogenous siRNA is often unsatisfactory because the effect is only transient, especially in rapidly dividing cells. This can be overcome by generating an expression vector for the siRNA. The siRNA sequence is modified to introduce a short loop between the two strands. The resulting transcript is a short hairpin RNA (shRNA), which can be processed into a functional siRNA by Dicer in its usual manner. A typical transcription cassette uses an RNA polymerase III promoter (e.g., U6 or H1) to direct the transcription of a small RNA (snRNA) (U6 is involved in gene splicing; H1 is a component of RNase P). Theoretically, the resulting siRNA transcript is then processed by Dicer.

[0029] Gene knockdown efficiency can also be improved by using cell squeezing. The activity of siRNA in RNAi largely depends on its binding ability to the RNA-induced silencing complex (RISC). After binding to the RISC, the double-stranded siRNA is unwound and cleaved by an endonuclease. The remaining antisense strand-RISC complex can then bind to the target mRNA to initiate transcriptional silencing.

[0030] 2.3 Posttranscriptional gene silencing editing

[0031] siRNA-induced posttranscriptional gene silencing begins with the assembly of the RNA-induced silencing complex (RISC). This complex silences the expression of certain genes by cleaving the mRNA molecule encoding the target gene. To initiate this process, one of the two siRNA strands (the guide strand) is loaded into the RISC, while the other strand, the guest strand, is degraded. Certain Dicer enzymes may be responsible for loading the guide strand into the RISC. The siRNA then scans and guides the RISC to a sequence that is perfectly complementary to the mRNA molecule. The cleavage of the mRNA molecule is thought to be catalyzed by the Piwi domain of the Argonaute protein of the RISC. The mRNA molecule is then precisely cleaved by cutting the phosphodiester bonds between the target nucleotides paired with siRNA residues 10 and 11, counting from the 5' end. This cleavage leads to further degradation of the mRNA fragment by cellular exonucleases. The 5' fragment is degraded from its 3' end by a foreign body, while the 3' fragment is degraded from its 5' end by 5'-3' exonuclease 1 (XRN1). The dissociation of the target mRNA strand from the RISC after cleavage allows more mRNA to be silenced. This dissociation process is likely promoted by extrinsic factors driven by ATP hydrolysis. Sometimes, cleavage of the target mRNA molecule does not occur. In some cases, endonuclease cleavage of the phosphodiester backbone can be inhibited by mismatches between siRNA and target mRNA near the cleavage site. Other times, even if the target mRNA and siRNA are perfectly paired, the RISC Argonaute protein lacks endonuclease activity. In these cases, gene expression will be silenced by miRNA-induced mechanisms. A simplified version of the Ping-Pong method involves the proteins Aubergine (Aub) and Argonaute-3 (Ago3) cleaving the 3' and 5' ends of piRNA. The Piwi-interacting RNA is responsible for transposon silencing, not the siRNAs.

[0032] Whether siRNA can be used to inhibit TAMs and thus suppress tumor cell migration and proliferation remains unknown. Summary of the Invention

[0033] To address the aforementioned problems, the present invention aims to provide siRNA that specifically binds to CD206 in M2 macrophages. This siRNA inhibits CD206 expression by binding to CD206-specific mRNA in M2 macrophages, thereby inhibiting the proliferation of M2 macrophages and subsequently suppressing the proliferation and migration of tumor cells.

[0034] To achieve the above objectives, the present invention provides a siRNA targeting CD206 in M2 macrophages, wherein the siRNA is:

[0035] 1) One or more of siRNA-1, siRNA-2, siRNA-3, siRNA-4, siRNA-5, and siRNA-6; or,

[0036] 2) One or more of siRNA-7, siRNA-8, siRNA-9, siRNA-10, siRNA-11, and siRNA-12;

[0037] The nucleotide sequence of the sense strand of siRNA-1 is shown in Seq ID No. 1, and the nucleotide sequence of the antisense strand is shown in Seq ID No. 2.

[0038] The nucleotide sequence of the sense strand of siRNA-2 is shown in Seq ID No. 3, and the nucleotide sequence of the antisense strand is shown in Seq ID No. 4.

[0039] The nucleotide sequence of the sense strand of siRNA-3 is shown in Seq ID No. 5, and the nucleotide sequence of the antisense strand is shown in Seq ID No. 6.

[0040] The nucleotide sequence of the sense strand of siRNA-4 is shown in Seq ID No. 7, and the nucleotide sequence of the antisense strand is shown in Seq ID No. 8.

[0041] The nucleotide sequence of the sense strand of siRNA-5 is shown in Seq ID No. 9, and the nucleotide sequence of the antisense strand is shown in Seq ID No. 10.

[0042] The nucleotide sequence of the sense strand of siRNA-6 is shown in Seq ID No. 11, and the nucleotide sequence of the antisense strand is shown in Seq ID No. 12.

[0043] The nucleotide sequence of the sense strand of siRNA-7 is shown in Seq ID No. 13, and the nucleotide sequence of the antisense strand is shown in Seq ID No. 14.

[0044] The nucleotide sequence of the sense strand of siRNA-8 is shown in Seq ID No. 15, and the nucleotide sequence of the antisense strand is shown in Seq ID No. 16.

[0045] The nucleotide sequence of the sense strand of siRNA-9 is shown in Seq ID No. 17, and the nucleotide sequence of the antisense strand is shown in Seq ID No. 18.

[0046] The nucleotide sequence of the sense strand of siRNA-10 is shown in Seq ID No. 19, and the nucleotide sequence of the antisense strand is shown in Seq ID No. 20.

[0047] The nucleotide sequence of the sense strand of siRNA-11 is shown in Seq ID No. 21, and the nucleotide sequence of the antisense strand is shown in Seq ID No. 22.

[0048] The nucleotide sequence of the sense strand of siRNA-12 is shown in Seq ID No. 23, and the nucleotide sequence of the antisense strand is shown in Seq ID No. 24.

[0049] The present invention also provides the use of the above-mentioned siRNA in the preparation of drugs that inhibit the growth or proliferation of tumor cells.

[0050] Preferably, the tumor cells are glioma cells, breast cancer cells, cervical cancer cells, lung cancer cells, gastric cancer cells, colorectal cancer cells, duodenal cancer cells, leukemia cells, prostate cancer cells, endometrial cancer cells, thyroid cancer cells, lymphoma cells, pancreatic cancer cells, liver cancer cells, melanoma cells, skin cancer cells, pituitary tumor cells, germ cell tumor cells, meningoma cells, meningeal cancer cells, glioblastoma cells, astrocytoma cells, oligoglioma cells, oligodendroglioma cells, ependymoma cells, choroid plexus papilloma cells, choroid plexus cancer cells, chordoma cells, ganglioblastoma cells, olfactory neuroblastoma cells, sympathetic nervous system neuroblastoma cells, pineal gland cell tumor cells, pineal blastoma cells, medulloblastoma cells, trigeminal schwannoma cells, facial and auditory nerve tumor cells, glomus jugulare tumor cells, hemangioblastoma cells, craniopharyngioma cells, or granular cell tumor cells.

[0051] The beneficial effects of this invention are as follows:

[0052] This invention provides an siRNA that specifically binds to CD206 in M2 macrophages. This siRNA reduces the expression of CD206 by specifically binding to the mRNA of CD206 in M2 macrophages, thereby reducing the expression of M2 macrophage-related molecules and proteins, and thus inhibiting the proliferation and migration of tumor cells. Attached Figure Description

[0053] Figure 1 This is a schematic diagram of the basic structure of siRNA.

[0054] Figure 2 The qPCR statistical diagram of human siRNA-1 to siRNA-3 transfection on THP1-induced CD206 expression in M2 cells provided by the present invention.

[0055] Figure 3 The qPCR statistical diagram of human siRNA-4 to siRNA-6 transfection on THP1-induced CD206 expression in M2 cells provided by the present invention.

[0056] Figure 4 The image shows a fluorescence photograph of human siRNA-1 transfection on THP1-induced CD206 expression in M2 cells, provided by this invention.

[0057] Figure 5 The qPCR statistical diagram of human siRNA-7 to siRNA-9 transfection on CD206 expression in mouse-derived M2 cells provided by the present invention.

[0058] Figure 6 The qPCR statistical diagram of human siRNA-10 to siRNA-12 transfection on CD206 expression in mouse-derived M2 cells provided by the present invention.

[0059] Figure 7 A fluorescent photograph of mouse siRNA-9 transfection on CD206 expression in mouse-derived M2 cells, provided by this invention.

[0060] Figure 8 The CCK8 assay results show the effect of different culture conditions on the proliferation of 4T1-luc cells.

[0061] Figure 9A Photographs of tumors generated from a mouse breast cancer tumor model constructed using siRNA-9 provided by this invention.

[0062] Figure 9B A statistical graph of tumor weight was generated to construct a mouse breast cancer tumor model using siRNA-9 provided in this invention.

[0063] Figure 9C A statistical graph of tumor volume was generated to construct a mouse breast cancer tumor model using siRNA-9 provided in this invention.

[0064] Figure 10A Imaging images of animals that have been injected with 4T1-luc via the tail vein to construct a mouse breast cancer tumor model using siRNA-9 provided in this invention.

[0065] Figure 10B A bar graph showing the fluorescence intensity of 4T1-luc injected into the tail vein of a mouse breast cancer tumor model constructed using siRNA-9 provided by this invention.

[0066] Figure 11AAnimal imaging observation images of a mouse breast cancer tumor model constructed using siRNA-9 provided by this invention, after tail vein inoculation with M-M2 macrophages and 4T1-luc.

[0067] Figure 11B A bar graph showing the fluorescence intensity of M-M2 macrophages and 4T1-luc injected into the tail vein of a mouse breast cancer tumor model constructed using siRNA-9 provided in this invention. Detailed Implementation

[0068] To facilitate understanding of the present invention, a more complete description of the invention will be provided below with reference to embodiments, of which preferred embodiments are given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are provided so that a thorough and complete understanding of the disclosure of the present invention may be achieved. It should be understood that experimental methods not specifically described in the following embodiments are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. All commonly used reagents used in the embodiments are commercially available products.

[0069] Material

[0070] 1. Human breast cancer cell line MCF-7, human mononuclear cell line THP-1, mouse breast cancer cell line 4T1, and mouse breast cancer cell line 4T1-luc (4T1-luc is a mouse breast cancer cell line expressing fluorescent labeling) were all purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee.

[0071] 2. RPMI 1640 complete culture medium was purchased from Thermo Fisher Scientific, USA, product number: 31800022

[0072] 3. PMA was purchased from Sigma-Aldrich LLC in the United States, product number: P1585.

[0073] 4. LPS was purchased from Sigma-Aldrich LLC in the United States, product number: L2630.

[0074] 5. IFNγ was purchased from PeproTech in the United States, product number: 300-02

[0075] 6. IL-4 was purchased from PeproTech in the United States, product number: 200-04

[0076] 7. IL-13 was purchased from PeproTech in the United States, product number: 200-13

[0077] 8.riboFECT TM The CP transfection kit was purchased from Guangzhou Ribo Biotechnology Co., Ltd., product number: C10511-05.

[0078] 9. The primary antibody was purchased from Abcam in the United States, product number: ab64693.

[0079] 10. The secondary antibody was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., product number: ZF-0316

[0080] 11. The fluorescent secondary antibody was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., product number: ZF-0311.

[0081] 12. Mouse ICRs were purchased from the Department of Animal Science, Peking University School of Medicine.

[0082] 13. Trizol reagent was purchased from ThermoFisher Scientific, USA, product number: 15596018.

[0083] 14. The RevertAid RT reverse transcription kit was purchased from ThermoFisher Scientific, USA, product number: K1691.

[0084] 15. PowerUp TM SYBR TM Green premix was purchased from ThermoFisher Scientific, USA, product number: A25742.

[0085] Example 1

[0086] Based on the human CD206 gene, a suitable siRNA sequence was searched in the GenBank gene bank.

[0087] Based on the GenBank search sequence NM_002438.4, six DNA target sequences were obtained, as shown in Table 1:

[0088] Table 1 DNA target sequences

[0089] Serial Number Sequence (5'→3') 1 Seq ID No. 25 GTGTGACCATGTATTCAAA 2 Seq ID No. 26 CAACCAGGATGCCGAATCA 3 Seq ID No. 27 GGATCGCCCTGAACAGTAA 4 Seq ID No. 28 GTAACTTGACTGATAATCA 5 Seq ID No. 29 GATTGTTCAGAAATGTTGA 6 Seq ID No. 30 GGCTTAAATGACATTAAGA

[0090] Using the above 6 DNA target sequences as templates, the corresponding siRNAs were chemically synthesized, and TT was added to the 3' end of the sense and antisense strands of each siRNA to increase the stability of the siRNA.

[0091] The siRNA sequences with a 3' TT terminus are shown in Table 2:

[0092] Table 2 siRNA sequences

[0093]

[0094]

[0095] The aforementioned siRNA-1 to siRNA-6 were chemically synthesized by Beijing Qingke Biotechnology Co., Ltd. For ease of subsequent detection, FAM fluorescent labeling was applied to the 5' ends of both the sense and antisense strands of siRNA-1 to siRNA-6 using a standard labeling method. Simultaneously, the company provided a non-homologous negative control siRNA-NC based on the target sequence.

[0096] Example 2

[0097] Human siRNA-1 to siRNA-6 and siRNA-NC prepared in Example 1 were used with riboFECT. TM The CP transfection kit was used to transfect THP1-induced M2 macrophages, and the expression levels of CD206 and downstream related molecules in M2 macrophages were detected.

[0098] 1. Induction of M2 macrophages

[0099] 1) Human mononuclear cell line THP-1 cells were seeded in culture flasks containing RPMI 1640 complete medium and cultured stably for 2-4 generations;

[0100] 2) Collect THP-1 cells into 15mL centrifuge tubes, centrifuge at 800rpm for 3min, and adjust the cell density to 5×10⁻⁶. 5 / mL, seeded into a new culture dish containing RPMI 1640 complete medium, add PMA to a final concentration of 100ng / mL and culture for 24h, then observe under a microscope whether THP-1 cells have completely adhered to the culture dish. Complete adhesion proves that they have differentiated into M0 macrophages.

[0101] 3) Discard the culture medium, add RPMI1640 complete medium containing 20 ng / mL LPS and 20 ng / mL IFNγ and culture for 48 h to induce M1 macrophages; then add RPMI1640 complete medium containing 20 ng / mL IL-4 and IL-13 and culture for 48 h to obtain M2 macrophages.

[0102] 2. qPCR detection of CD206 expression and downstream related molecules in M2 macrophage cells

[0103] siRNA-1, siRNA-2, siRNA-3, siRNA-4, siRNA-5, siRNA-6, and siRNA-NC were administered according to riboFECT. TM Following the instructions in the CP transfection kit, transfect the cells into M2 macrophage cells. After transfection, place the cell culture plate (6-well plate) in a 37°C, 5% CO2 and saturated humidity incubator for 24-72 h.

[0104] 1) RNA extraction

[0105] Each cell sample transfected with siRNA-1, siRNA-2, siRNA-3, siRNA-4, siRNA-5, siRNA-6, and siRNA-NC was tested in triplicate.

[0106] a. Discard the original culture medium in the cell culture plate, add PBS, gently shake the culture dish, and wash 2-3 times.

[0107] b. Add 1 mL of Trizol reagent to each well of the cell culture plate, let stand at room temperature for 5 min, and collect the cell suspension into 1.5 mL centrifuge tubes in (1) after several blows and aspirations, 1 mL per tube.

[0108] c. Add 200 μL of chloroform to each tube, shake vigorously for 15 seconds, and let stand at room temperature for 2-3 minutes.

[0109] d. Place the above centrifuge tubes into a high-speed low-temperature centrifuge and centrifuge at 4°C and 10000g for 15 minutes. Carefully aspirate the supernatant and transfer it to another clean 1.5mL centrifuge tube. Add an equal volume (about 600μL) of isopropanol and let it stand at room temperature for 10 minutes.

[0110] f. Place the above centrifuge tubes into a high-speed low-temperature centrifuge and centrifuge at 4°C and 10000g for 10 minutes. Discard the supernatant and add 1 mL of pre-cooled 80% ethanol to wash the precipitate.

[0111] g. Place the above centrifuge tubes into a high-speed low-temperature centrifuge and centrifuge at 4°C and 7500g for 5 minutes, then discard the supernatant.

[0112] h. Repeat step (7).

[0113] i. After centrifugation, discard the supernatant and air dry for 5-10 minutes to allow the ethanol to evaporate completely.

[0114] j. Add 30-50 μL of RNAase-free ddH2O to the centrifuge tube, mix thoroughly by pipetting, and obtain total RNA.

[0115] k. Take 1-2 μL of sample from each group and determine the concentration using an ultra-micro spectrophotometer. If the concentration is appropriate, it can be used for subsequent experiments or stored in an ultra-low temperature freezer at -80℃.

[0116] 2) RNA reverse transcription

[0117] RNA reverse transcription was performed using the RevertAid RT kit, and the reagents used were those specified in the RevertAid RT kit.

[0118] a. Prepare RNA reverse transcription reaction system 1, as shown in Table 3.

[0119] Table 3 RNA reverse transcription reaction system 1

[0120]

[0121] b. Gently blow and aspirate to mix, then briefly centrifuge and incubate in a 65°C metal bath for 5 minutes, followed by 2 minutes on ice.

[0122] c. Prepare RNA reverse transcription reaction system 2, as shown in Table 4.

[0123] Table 4 RNA reverse transcription reaction system 2

[0124]

[0125] d. Add RNA reverse transcription reaction system 2 to RNA reverse transcription reaction system 1, gently pipette to mix, briefly centrifuge, and then place in a PCR instrument. The reaction conditions are: 25℃, 5 min; 42℃, 60 min; 70℃, 5 min; cool at 4℃ to obtain cDNA template.

[0126] After the cDNA reversal is completed, the cDNA sample can be used for subsequent qPCR experiments or stored in an ultra-low temperature freezer at -80°C.

[0127] 3) qPCR

[0128] Prepare the qPCR reaction system as shown in Table 5.

[0129] Table 5 qPCR reaction system

[0130]

[0131] The sense strand is the sense strand of siRNA-1 to siRNA-6 or siRNA-NC, and the antisense strand is the antisense strand of siRNA-1 to siRNA-6 or siRNA-NC.

[0132] Gently mix the above ingredients by blowing and aspiration, then briefly centrifuge and place in StepOne. TMReal-Time PCR instrument. Reaction conditions are shown in Table 6.

[0133] Table 6. qPCR reaction conditions (Tm≥60℃)

[0134]

[0135] The expression of CD206 in THP1-induced M-M2 macrophages was detected using qPCR, and the results are as follows: Figure 2 and Figure 3 As shown, HS1 to HS6 correspond to the PCR products after transfection with siRNA-1 to siRNA-6, respectively. NC represents the PCR product after transfection with siRNA-NC, indicating the negative control and representing the interference of CD206 expression at 24h, 48h, and 72h. Experimental data are expressed as mean ± standard deviation (mean ± SD) (n = 3). *: P < 0.05 compared with the NC group; **: P < 0.01 compared with the NC group; ***: P < 0.001 compared with the NC group; ****: P < 0.0001 compared with the NC group.

[0136] from Figure 2 and Figure 3 It can be seen that siRNA-1 to siRNA-6 all have an inhibitory effect on CD206 expression. NC serves as the negative control, showing the variation between HS1-HS6. The significant differences at different time points are due to the fact that different siRNAs bind to different sites on the gene, potentially causing different inhibitory effects, at different appropriate times, and with varying strengths. Therefore, at the same time point, the inhibitory effects of different siRNAs are not entirely the same. Subsequent experiments will use siRNA-1 as a representative.

[0137] 3. Immunofluorescence assay for the expression of related proteins:

[0138] 1) Collect M2 macrophages transfected with siRNA-1 at 24h, 48h, and 72h, and adjust the cell density to 1×10⁻⁶. 5 / mL, seeded in confocal dishes, with untransfected M2 macrophages as the negative control group (NC).

[0139] 2) After the cells have adhered to the wall, discard the culture medium, add PBS, and shake on a horizontal decolorizing shaker for 5 minutes, then wash 3 times.

[0140] 3) Fixation: Add 4% paraformaldehyde, let stand at room temperature for 10 minutes, and then wash 3 times with PBS.

[0141] 4) Blocking: Add 5% BSA prepared with PBS and block in an incubator at 37°C for 30 min.

[0142] 5) Primary antibody reaction: After discarding the blocking solution, add 200 μL of the corresponding primary antibody (1:500 dilution) to each group; add an equal volume of 5% BSA to the negative control group and incubate overnight at 4°C.

[0143] 6) Secondary antibody reaction: Discard the primary antibody, add PBS, and shake horizontally on a shaker for 5 min, then wash 3 times. Add the corresponding fluorescent secondary antibody (1:500 dilution) to each group, and add an equal volume of secondary antibody to the negative control group. Incubate at 37℃ in the dark for 1 h.

[0144] 7) DAPI: Discard the secondary antibody and wash 3 times with PBS. Add 200 μL of DAPI and incubate at room temperature for 20 min; discard the DAPI and wash 3 times with PBS.

[0145] The luminescence of cells in each group was observed and photographed under a Leica laser confocal microscope. Taking siRNA-1 as an example, the results are as follows: Figure 4 As shown, DAPI represents the blue fluorescence of DAPI labeled in the cell nucleus, CD206-TRITC represents the red fluorescence of TRITC labeled with CD206, Merge represents the superposition result of the DAPI and CD206-TRITC coatings, and T-M2 NC As the negative control group, T-M2 HS1 24h indicates siRNA-1 treatment for 24 hours, T-M2 HS1 48h indicates siRNA-1 treatment for 48 hours, T-M2 HS1 72h indicates that the siRNA-1 treatment lasted for 72 hours.

[0146] from Figure 4 As can be seen, when fluorescently labeled siRNA-1 was transfected into human M2 macrophages, the expression level of CD206 in M2 macrophages was significantly reduced compared with the blank control group and the negative control group. After 72 hours of treatment, the expression of CD206 was basically unobservable, which indicates that the siRNA-1 provided by the present invention can effectively inhibit the expression of CD206.

[0147] Example 3

[0148] Based on the mouse CD206 gene, a search was conducted in the GenBank gene bank to find suitable siRNA sequences. Based on the GenBank search sequence NM_008625.2, six DNA target sequences were obtained, as shown in Table 7.

[0149] Table 7 DNA target sequences

[0150] Serial Number Sequence (5'→3') 1 Seq ID No.31 GCAAGCATTTGTTACCTAT 2 Seq ID No. 32 GCATGAAGCAGAGACATAT 3 Seq ID No. 33 GTGGTATGCAGACTGCACC 4 Seq ID No. 34 GGCATTCTTTACCAGATAA 5 Seq ID No. 35 GGCTTACGGTGAACCAAAT 6 Seq ID No. 36 CCACTGACTACGACAAAGA

[0151] Using the above 6 DNA target sequences as templates, the corresponding siRNAs were chemically synthesized, and TT was added to the 3' end of the sense and antisense strands of each siRNA to increase the stability of the siRNA.

[0152] The siRNA sequences with a 3' TT terminus are shown in Table 8:

[0153] Table 8 siRNA sequences

[0154]

[0155]

[0156] Beijing Qingke Biotechnology Co., Ltd. was commissioned to chemically synthesize the aforementioned siRNA-7 to siRNA-12. For ease of subsequent detection, two siRNAs with the same sequence were synthesized for both the sense and antisense strands of siRNA-7 to siRNA-12. One siRNA was labeled with FAM fluorescently at the 5' end using a conventional labeling method. An unlabeled siRNA with the same sequence was also synthesized. Simultaneously, the company provided a non-homological negative control siRNA-NC based on the target sequence.

[0157] Example 4

[0158] The mouse siRNA-7 to siRNA-12 prepared in Example 3 were used for riboFECT. TM The CP transfection kit was used to transfect induced bone marrow M2 macrophages, and the expression levels of CD206 and downstream related molecules in M2 macrophages were detected.

[0159] 1. Preparation of mouse M2 macrophages:

[0160] (1) Select 6-8 week old BALB / c mice, sacrifice them by cervical dislocation and soak them in 75% alcohol for 5 minutes.

[0161] (2) Use sterile ophthalmic scissors to cut open the skin in the groin of the mouse and peel off the hind limb. Cut along the root of the femoral head upwards, being careful not to cut the femur. Carefully remove the muscle and soak the clean bone in PBS.

[0162] (3) Soak the removed bones in 75% alcohol for 2 minutes, and then wash them twice with PBS.

[0163] (4) Cut off both ends of the bone with ophthalmic scissors, draw up PBS with a 5mL syringe, insert it into the bone marrow cavity to rinse the cells into a 15mL centrifuge tube, and gently blow and aspirate repeatedly to disperse the cells.

[0164] (5) Filter the cell suspension through a 70μm (200 mesh) cell filter into a new 15mL centrifuge tube and centrifuge at 1000rpm for 10min.

[0165] (6) Discard the supernatant, add 2-3 mL of red blood cell lysis buffer to resuspend, and lyse at 4°C for 10 min.

[0166] (7) Add DMEM complete culture medium to 10 mL to terminate the red cleavage, and centrifuge at 1500 rpm for 5 min.

[0167] (8) Resuspend the cells in DMEM complete medium containing a final concentration of 50 ng / mL mMCSF, count the cells, adjust the cell concentration, and seed them in 6-well or 24-well plates.

[0168] (9) Change the medium on the 3rd day after plating, and mature macrophages can be harvested on the 7th day, which are M-M0 macrophages.

[0169] (10) Polarize mature macrophages, discard the original culture medium, add DMEM complete culture medium containing 100 ng / mL LPS and IFNγ and culture for 24 h to obtain M-M1 macrophages; add DMEM complete culture medium containing 20 ng / mL IL-4 and culture for 24 h to obtain M-M2 macrophages.

[0170] 2. qPCR was used to detect the expression level of CD206 in mouse-derived M2 macrophages.

[0171] siRNA-7, siRNA-8, siRNA-9, siRNA-10, siRNA-11, siRNA-12, and siRNA-NC were administered according to riboFECT. TM The CP transfection kit was followed according to the instructions to transfect the cells into M2 macrophages. After transfection, the culture plates were incubated at 37°C, 5% CO2, and saturated humidity for 24-72 hours. CD206 expression in mouse M2 macrophages was detected by qPCR.

[0172] The specific method is the same as the qPCR detection section in Example 2.

[0173] The expression of CD206 in M2 macrophages derived from mice was detected using qPCR, and the results are as follows: Figure 5 and Figure 6As shown, MS1 to MS6 correspond to the PCR products after transfection with siRNA-7 to siRNA-12, respectively. NC represents the PCR product after transfection with siRNA-NC, indicating the negative control and representing the interference of CD206 expression at 24h, 48h, and 72h. Experimental data are expressed as mean ± standard deviation (mean ± SD) (n = 3). *: P < 0.05 compared with the NC group; **: P < 0.01 compared with the NC group; ***: P < 0.001 compared with the NC group; ****: P < 0.0001 compared with the NC group.

[0174] from Figure 5 and Figure 6 It can be seen that, from Figure 5 and Figure 6 It can be seen that siRNA-7 to siRNA-12 all have an inhibitory effect on CD206 expression. Subsequent experiments will be conducted using siRNA-9 as a representative.

[0175] 3. Immunofluorescence assay for the expression of related proteins:

[0176] Cells transfected with siRNA-9 were treated using the same method as in Example 2.

[0177] The luminescence of cells in each group was observed and photographed under a Leica laser confocal microscope. Taking siRNA-9 as an example, the results are as follows: Figure 7 As shown, DAPI represents the blue fluorescence of DAPI labeled in the cell nucleus, CD206-TRITC represents the red fluorescence of TRITC labeled with CD206, Merge represents the superposition result of the DAPI and CD206-TRITC coatings, and T-M2 NC As the negative control group, T-M2 MS3 24h indicates siRNA-9 treatment for 24 hours, T-M2 MS3 48h indicates siRNA-9 treatment for 48 hours, T-M2 MS3 72h indicates that the siRNA-9 treatment lasted for 72 hours.

[0178] from Figure 7 As can be seen, when fluorescently labeled siRNA-9 was transfected into mouse-derived M2 macrophages, the expression level of CD206 in the macrophages was significantly reduced compared with the blank control group and the negative control group. After 72 hours of treatment, the expression of CD206 was basically unobservable, which indicates that the siRNA-9 provided by the present invention can effectively silence the expression of CD206.

[0179] Example 5: Overall experimental study on the effects of CD206 downregulation on M2 macrophages on breast cancer tumor development and metastasis.

[0180] 1. Effects of CD206-downregulated M-M2 macrophage conditioned medium on mouse breast cancer tumor growth.

[0181] To further verify the effect of CD206 downregulation on the growth of breast cancer tumors, 4T1-luc cells treated with different culture conditions were seeded into the left forelimb axilla of Balb / c mice to construct a mouse breast cancer tumor model for the study.

[0182] Conditioned culture media M-M2 CM and M-M2 were prepared separately. NC CM, M-M2 MS3 CM, the specific method is as follows:

[0183] Preparation of M-M2 CM conditioned medium: Collect the culture supernatant of mouse M2 macrophages (M-M2 macrophages) prepared in Example 4, centrifuge at 1000 rpm for 10 min, add fresh complete culture medium at a ratio of 1:1 and mix well to prepare M-M2 macrophage conditioned medium, denoted as M-M2 CM.

[0184] Conditioned culture medium M-M2 NC Preparation of CM: The culture supernatant of M2 macrophages transfected with siRNA-NC 48 h after Example 4 was collected, centrifuged at 1000 rpm for 10 min, and then mixed with fresh complete culture medium at a 1:1 ratio to prepare M-M2. NC Macrophage conditioned medium, denoted as M-M2 NC CM.

[0185] Conditioned culture medium M-M2 MS3 Preparation of CM: The culture supernatant of M2 macrophages transfected with siRNA-9 48 h after Example 4 was collected, centrifuged at 1000 rpm for 10 min, and then mixed with fresh complete culture medium at a 1:1 ratio to prepare M-M2. MS3 Macrophage conditioned medium, denoted as M-M2 MS3 CM.

[0186] Conditioned medium 4T1 CM is the supernatant of the culture medium for culturing 4T1-luc cells. It is prepared by centrifuging at 1000 rpm for 10 min and then adding fresh complete culture medium at a 1:1 ratio.

[0187] Using the above-mentioned conditioned medium 4T1 CM, M-M2 CM, M-M2 NC CM, M-M2 MS34T1-luc cells were cultured in CM for 12 h, 24 h, 36 h, and 48 h, respectively, and their proliferation capacity was detected using CCK8 assay. The results are shown in Table 9. Figure 8 As shown.

[0188] Table 9. Effect of CD206 downregulation on 4T1 cell proliferation activity as detected by CCK-8 assay.

[0189]

[0190] Table 8 shows the ratios of OD values ​​measured at different time points to 0h, with data presented as mean ± standard deviation (mean ± SD) (n = 3). *: p < 0.05 compared to the 4T1 CM group; **: p < 0.01 compared to the 4T1 CM group; ***: p < 0.001 compared to the 4T1 CM group; #: p < 0.05 compared to the M-M2 CM group; ##: p < 0.01 compared to the M-M2 CM group; &: p < 0.01 compared to the M-M2 CM group. NC Compared with the CM group, p<0.05; &&&: compared with M-M2 NC Compared with the CM group, p<0.001.

[0191] From Table 9 and Figure 8 It can be seen that, compared with the 4T1 CM group, M-M2 MS3 There was no significant difference in cell proliferation activity between the CM group and the M-M2 group (p>0.05). NC Cell proliferation activity was enhanced in the CM group, and the differences were statistically significant (p<0.05); compared with the M-M2CM group, the M-M2 group showed increased cell proliferation activity. MS3 Cell proliferation activity was reduced in the CM group, and the differences were statistically significant (p<0.05); compared with M-M2 NC Compared to the CM group, M-M2 MS3 Cell proliferation activity was reduced in the CM group, and the differences were statistically significant (p<0.05). The results indicate that downregulation of CD206 weakens the ability of M-M2 macrophages to promote the proliferation of 4T1 breast cancer cells.

[0192] Collect 4T1-luc cells and adjust the cell density to 2×10⁻⁶. 5 / mL, 100μl of cell suspension was inoculated into the left forelimb axilla of mice and labeled as group 4T1.

[0193] Collect conditioned media M-M2 CM and M-M2 respectively NC CM, M-M2 MS3 4T1-luc cells cultured in CM for 24 hours were then adjusted to a cell density of 2×10⁻⁶. 5 / mL, 100μl of cell suspension was inoculated into the left forelimb axilla of mice and labeled as 4T1 / M-M2CM and 4T1 / M-M2. NC CM, 4T1 / M-M2 MS3 CM group. Mice were euthanized by cervical dislocation 14 days later, dissected, and tissue samples were taken. The tumor mass was weighed and its volume was calculated. The statistics are shown in Table 10.

[0194] Table 10 Effects of CD206-downregulated M-M2 macrophage conditioned medium on mouse breast cancer tumor growth.

[0195]

[0196] As shown in Table 10, compared with the 4T1 control group, the CM values ​​of 4T1 / M-M2 and 4T1 / M-M2 were significantly different. NC There was no significant difference in tumor weight among the CM group, but tumor volume showed an increasing trend, 4T1 / M-M2 MS3 The tumor weight in the CM group showed a decreasing trend, and the tumor volume was significantly reduced; compared with the 4T1 / M-M2 CM group, the 4T1 / M-M2 group showed a decreasing trend. MS3 The tumor weight in the CM group showed a decreasing trend, and the tumor volume was significantly reduced; compared with 4T1 / M-M2 NC Compared to the CM group, 4T1 / M-M2 MS3 Tumor weight in the CM group showed a decreasing trend, and tumor volume was significantly reduced. Conditioned culture medium for M-M2 macrophages with downregulated CD206 expression inhibited the ability of M2 macrophages to promote mouse breast cancer tumor growth and further suppressed mouse breast cancer tumor growth.

[0197] 2. Effects of CD206-downregulated M-M2 macrophages on breast cancer tumor growth in mice

[0198] To further verify the effect of CD206 downregulation on M2 macrophages on breast cancer tumor growth, M-M2 macrophages and 4T1-luc cells (4:1) were co-inoculated into the left forelimb axilla of Balb / c mice to establish a mouse breast cancer tumor model for the study. 4T1-luc, M-M2, and M-M2 cells were collected. NC and M-M2 MS3 Cells, adjust cell density to 2×10 5 / mL, take 100μl of 4T1-luc cell suspension and mix it with 25μl of M-M2 and M-M2 respectively. NC M-M2 MS3 The cell suspensions were mixed at a 4:1 ratio and co-inoculated into the left forelimb axilla of mice, labeled as 4T1+M-M2 and 4T1+M-M2, respectively. NC 4T1+M-M2 MS3Group. Mice were euthanized by cervical dislocation 14 days later, dissected, and tissue samples were taken. The tumor mass was weighed and its volume calculated. The results are shown in Table 11. Figures 9A to 9C As shown.

[0199] Figure 9A This indicates that a mouse breast cancer tumor model was co-inoculated with 4T1-luc cells and M-M2 macrophages (4:1) in the left forelimb axilla, including 4T1, 4T1+M-M2, and 4T1+M-M2 macrophages. NC 4T1+M-M2 MS3 Photos of a group of tumors. Figure 9B Represents 4T1, 4T1+M-M2, and 4T1+M-M2 NC 4T1+M-M2 MS3 A bar chart showing the weight of tumors in each group. Figure 9C Represents 4T1, 4T1+M-M2, and 4T1+M-M2 NC 4T1+M-M2 MS3 A bar chart of tumor volume for each group. Experimental data are expressed as mean ± standard deviation (mean ± SD) (n = 4). *: P < 0.05 compared with the 4T1 group; **: P < 0.01 compared with the 4T1 group; ##: P < 0.01 compared with the 4T1+M-M2 group; &: P < 0.01 compared with the 4T1+M-M2 group. NC For group comparisons, P < 0.05.

[0200] Table 11 Effects of CD206-downregulated M-M2 macrophages on breast cancer tumor growth in mice.

[0201]

[0202] from Figures 9A to 9C As shown in Table 10, compared with the 4T1 control group, 4T1+M-M2 and 4T1+M-M2... NC The tumor weight of the group increased significantly, and the tumor volume showed an increasing trend, 4T1+M-M2 MS3 The tumor volume and weight of the group showed a decreasing trend; compared with the 4T1+M-M2 group, the 4T1+M-M2 group showed a decreasing trend. MS3 The tumor volume and weight of the group were significantly reduced; compared with 4T1+M-M2 NC Compared to the previous group, 4T1+M-M2 MS3 The tumor volume and weight were significantly reduced in the group. This indicates that M-M2 macrophages with downregulated CD206 expression inhibit the ability of M2 macrophages to promote the growth of mouse breast cancer tumors, and can further inhibit the growth of mouse breast cancer tumors.

[0203] 3. Effects of CD206-downregulated M-M2 macrophage conditioned medium on mouse breast cancer tumor metastasis.

[0204] To further verify the effect of CD206 downregulation on M2 macrophages on breast cancer tumor metastasis, M-M2 CM and M-M2 macrophages were cultured in conditioned medium, respectively. NC CM, M-M2 MS3 The treated 4T1-luc cells were injected into Balb / c mice via the tail vein to establish a mouse breast cancer tumor model for further study.

[0205] Collect 4T1-luc cells and adjust the cell density to 2×10⁻⁶. 5 / mL, 100μl of cell suspension was injected via tail vein and labeled as group 4T1. Conditioned culture media M-M2 CM and M-M2 were collected. NC CM, M-M2 MS3 4T1-luc cells cultured in CM for 24 hours were then adjusted to a cell density of 2×10⁻⁶. 5 / mL, 100μl of cell suspension was injected into Balb / c mice via the tail vein and labeled as 4T1 / M-M2 CM and 4T1 / M-M2, respectively. NC CM, 4T1 / M-M2 MS3 CM group. Seven days later, animal luminescence imaging analysis was performed, and the results are as follows: Figure 9A See Figure 9B and Table 12.

[0206] Figure 10A This indicates a mouse model of breast cancer tumor metastasis induced by tail vein inoculation with conditioned medium treated with 4T1-luc, including 4T1, 4T1 / M-M2 CM, and 4T1 / M-M2. NC CM, 4T1 / M-M2 MS3 Imaging observation images of animals in the CM group. Figure 10B Indicates 4T1, 4T1 / M-M2 CM, 4T1 / M-M2 NC CM, 4T1 / M-M2 MS3 A bar chart of fluorescence intensity in CM group animals. Experimental data are expressed as mean ± standard deviation (mean ± SD) (n = 4). *: P < 0.05 compared with 4T1 group; &&: P < 0.05 compared with 4T1 / M-M2 group. NC For the CM group comparison, P<0.01.

[0207] Table 12 Effects of CD206-downregulated M-M2 macrophage conditioned medium on mouse breast cancer tumor metastasis.

[0208]

[0209] from Figures 10A to 10B As shown in Table 11, compared with the 4T1 control group, the CM of 4T1 / M-M2 and 4T1 / M-M2... NCThe lung fluorescence intensity in the CM group showed an increasing trend, 4T1 / M-M2 MS3 The fluorescence intensity in the lungs of the CM group was significantly reduced; compared with the 4T1 / M-M2 CM group, the 4T1 / M-M2 group showed a significant decrease in lung fluorescence intensity. MS3 The lung fluorescence intensity in the CM group showed a decreasing trend; compared with 4T1 / M-M2 NC Compared to the CM group, 4T1 / M-M2 MS3 The fluorescence intensity in the lungs of the CM group was significantly reduced. These results indicate that the conditioned medium for M-M2 macrophages with downregulated CD206 expression inhibited the ability of M2 macrophages to promote mouse breast cancer tumor metastasis and further suppressed mouse breast cancer tumor metastasis.

[0210] 4. Effects of CD206-downregulated M-M2 macrophages on breast cancer tumor metastasis in mice

[0211] To further verify the effect of CD206 downregulation on M2 macrophages on breast cancer tumor metastasis, M-M2 macrophages and 4T1-luc cells (4:1) were co-inoculated into Balb / c mice via tail vein to construct a mouse breast cancer tumor model for the study.

[0212] Collect 4T1-luc, M-M2, M-M2 NC and M-M2 MS3 Cells, adjust cell density to 2×10 5 / mL, take 100μl of 4T1-luc cell suspension and mix it with 25μl of M-M2 and M-M2 respectively. NC M-M2 MS3 Cell suspensions were mixed and co-inoculated into Balb / c mice via tail vein at a ratio of 4:1, and labeled as 4T1+M-M2 and 4T1+M-M2, respectively. NC 4T1+M-M2 MS3 Group. Seven days later, animal luminescence imaging analysis was performed, and the results are as follows: Figures 11A to 11B As shown in Table 13.

[0213] Figure 11A A mouse model of breast cancer tumor metastasis was established by co-inoculating M-M2 macrophages and 4T1-luc (4:1) via tail vein. The cell types were 4T1, 4T1+M-M2, and 4T1+M-M2. NC 4T1+M-M2 MS3 Group of animal imaging observation images. Figure 11B For 4T1, 4T1+M-M2, 4T1+M-M2 NC 4T1+M-M2 MS3A bar chart of fluorescence intensity in the imaging of the animals. Experimental data are expressed as mean ± standard deviation (mean ± SD) (n = 4). *: P < 0.05 compared with group 4T1; ##: P < 0.01 compared with group 4T1+M-M2; &&&&: P < 0.0001 compared with group 4T1+M-M2.

[0214] Table 13 Effects of CD206-downregulated M-M2 macrophages on tumor metastasis in mouse breast cancer.

[0215]

[0216] From Table 13 and Figures 11A to 11B It can be seen that, compared with the 4T1 control group, 4T1+M-M2 and 4T1+M-M2... NC The lung fluorescence intensity of the group was significantly increased, 4T1+M-M2 MS3 The fluorescence intensity in the lungs of the group was significantly reduced; compared with the 4T1+M-M2 group, the 4T1+M-M2 group showed a significant decrease. MS3 The fluorescence intensity in the lungs of the group was significantly reduced; compared with 4T1+M-M2 NC Compared to the previous group, 4T1+M-M2 MS3 The fluorescence intensity in the lungs of the group was significantly reduced. The results indicate that M-M2 macrophages with downregulated CD206 expression inhibited the ability of M2 macrophages to promote the growth of mouse breast cancer tumors and further inhibited the metastasis of mouse breast cancer tumors.

[0217] As can be seen from the above embodiments, the siRNA provided by the present invention can target CD206 of M2 macrophages. By inhibiting the expression of CD206 of M2 macrophages, it inhibits the ability of M2 macrophages to promote the growth of breast cancer tumors, thereby inhibiting the metastasis of breast cancer tumors.

[0218] Although the siRNA provided in this invention is only used in breast cancer to demonstrate its ability to inhibit breast cancer tumor growth and metastasis by suppressing M2 macrophages, the siRNA provided in this invention acts directly on M2 macrophages. M2 macrophages can act not only on breast cancer cells but also on other types of tumor cells, such as glioma cells, breast cancer cells, cervical cancer cells, lung cancer cells, gastric cancer cells, colorectal cancer cells, duodenal cancer cells, leukemia cells, prostate cancer cells, endometrial cancer cells, thyroid cancer cells, lymphoma cells, pancreatic cancer cells, liver cancer cells, melanoma cells, skin cancer cells, pituitary adenoma cells, germ cell tumor cells, meningoma cells, meningioma cells, glioblastoma cells, astrocytoma cells, oligodendroglioma cells, and oligodendroglioma cells. Ependymoma cells, choroid plexus papilloma cells, choroid plexus cancer cells, chordoma cells, gangliocytoma cells, olfactory neuroblastoma cells, sympathetic nervous system neuroblastoma cells, pineal cell tumor cells, pinealoblastoma cells, medulloblastoma cells, trigeminal schwannoma cells, facial and auditory nerve tumor cells, glomus jugulare tumor cells, hemangioblastoma cells, craniopharyngioma cells, or granular cell tumor cells. Therefore, the siRNA provided by this invention can inhibit the growth and metastasis of multiple types of tumor cells by inhibiting the expression of CD206 in M2 macrophages, achieving the effect of treating multiple tumors through a single approach.

[0219] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. SEQUENCE LISTING <110> Niu Gang, Tan Huanran <120> siRNA that specifically binds to CD206 in M2 macrophages and its applications <130> DOME <160> 36 <170> PatentIn version 3.3 <210> 1 <211> twenty one <212> DNA <213> Artificial sequence <400> 1 gugugaccau guauucaaat t 21 <210> 2 <211> twenty one <212> DNA <213> Artificial sequence <400> 2 uuugaauaca uggucacact t 21 <210> 3 <211> twenty one <212> DNA <213> Artificial sequence <400> 3 caaccaggau gccgaaucat t 21 <210> 4 <211> twenty one <212> DNA <213> Artificial sequence <400> 4 ugauucggca uccugguugt t 21 <210> 5 <211> twenty one <212> DNA <213> Artificial sequence <400> 5 ggaucgcccu gaacaguaat t 21 <210> 6 <211> twenty one <212> DNA <213> Artificial sequence <400> 6 uuacuguuca gggcgaucct t 21 <210> 7 <211> twenty one <212> DNA <213> Artificial sequence <400> 7 guaacuugac ugauaaucat t 21 <210> 8 <211> twenty one <212> DNA <213> Artificial sequence <400> 8 ugauuaucag ucaaguuact t 21 <210> 9 <211> twenty one <212> DNA <213> Artificial sequence <400> 9 gauuguucag aaauguugat t 21 <210> 10 <211> twenty one <212> DNA <213> Artificial sequence <400> 10 ucaacauuuc ugaacaauct t 21 <210> 11 <211> twenty one <212> DNA <213> Artificial sequence <400> 11 ggcuuaaaug acauuaagat t 21 <210> 12 <211> twenty one <212> DNA <213> Artificial sequence <400> 12 ucuuaauguc auuuaagcct t 21 <210> 13 <211> twenty one <212> DNA <213> Artificial sequence <400> 13 gcaagcattt gttacctatt t 21 <210> 14 <211> twenty one <212> DNA <213> Artificial sequence <400> 14 auagguaucu aaugcuugct t 21 <210> 15 <211> twenty one <212> DNA <213> Artificial sequence <400> 15 gcaugaagca gagacauaut t 21 <210> 16 <211> twenty one <212> DNA <213> Artificial sequence <400> 16 auaugucucu gcuucaugct t 21 <210> 17 <211> twenty one <212> DNA <213> Artificial sequence <400> 17 gugguaugca gacugcacct t 21 <210> 18 <211> twenty one <212> DNA <213> Artificial sequence <400> 18 ggugcagucu gcauaccuct t 21 <210> 19 <211> twenty one <212> DNA <213> Artificial sequence <400> 19 ggcauucuuu accagauaat t 21 <210> 20 <211> twenty one <212> DNA <213> Artificial sequence <400> 20 uuaucuggua aagaaugcct t 21 <210> twenty one <211> twenty one <212> DNA <213> Artificial sequence <400> twenty one ggcuuacggu gaaccaaaut t 21 <210> twenty two <211> twenty one <212> DNA <213> Artificial sequence <400> twenty two auuugguuca ccguaagcct t 21 <210> twenty three <211> twenty one <212> DNA <213> Artificial sequence <400> twenty three ccacugacua cgacaaagat t 21 <210> twenty four <211> twenty one <212> DNA <213> Artificial sequence <400> twenty four ucuuugucgu agucaguggt t 21 <210> 25 <211> 19 <212> DNA <213> Homo sapiens <400> 25 gtgtgaccat gtattcaaa 19 <210> 26 <211> 19 <212> DNA <213> Homo sapiens <400> 26 caaccaggat gccgaatca 19 <210> 27 <211> 19 <212> DNA <213> Artificial sequence <400> 27 ggatcgccct gaacagtaa 19 <210> 28 <211> 19 <212> DNA <213> Homo sapiens <400> 28 gtaacttgac tgataatca 19 <210> 29 <211> 19 <212> DNA <213> Homo sapiens <400> 29 gattgttcag aaatgttga 19 <210> 30 <211> 19 <212> DNA <213> Homo sapiens <400> 30 ggcttaaatg acattaaga 19 <210> 31 <211> 19 <212> DNA <213> Mus musculus <400> 31 gcaagcattt gttacctat 19 <210> 32 <211> 19 <212> DNA <213> Mus musculus <400> 32 gcatgaagca gagacatat 19 <210> 33 <211> 19 <212> DNA <213> Mus musculus <400> 33 gtggtatgca gactgcacc 19 <210> 34 <211> 19 <212> DNA <213> Mus musculus <400> 34 ggcattcttt accagataa 19 <210> 35 <211> 19 <212> DNA <213> Mus musculus <400> 35 ggcttacggt gaaccaaat 19 <210> 36 <211> 19 <212> DNA <213> Mus musculus <400> 36 ccactgacta cgacaaaga 19

Claims

1. A siRNA that specifically binds to CD206 on M2 macrophages, characterized in that, The siRNA is: 1) siRNA-1; or 2) siRNA-9; The nucleotide sequence of the sense strand of siRNA-1 is shown in Seq ID No. 1, and the nucleotide sequence of the antisense strand is shown in Seq ID No.

2. The nucleotide sequence of the sense strand of siRNA-9 is shown in Seq ID No. 17, and the nucleotide sequence of the antisense strand is shown in Seq ID No.

18.

2. The use of siRNA as described in claim 1 in the preparation of drugs that inhibit the growth or proliferation of tumor cells, characterized in that, The tumor cells are breast cancer cells.

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