siRNA that specifically binds to CD206 on M2-type macrophages and its application

Abstract

The present invention provides an siRNA that specifically binds to CD206 of M2 macrophages, and 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 that specifically binds to CD206 of M2 macrophages provided by the present invention specifically binds to CD206 of macrophages, thereby reducing the expression of CD206, thereby promoting the proliferation of M2 macrophages and suppressing the proliferation and migration of tumor cells.

Description

[Technical field]

[0001] The present invention belongs to the field of biomedicine and relates to siRNA, and more particularly to siRNA that specifically binds to CD206 of M2 macrophages and applications thereof. [Background technology]

[0002] It has been more than 100 years since macrophages were discovered. Macrophages are immune cells that are widely distributed throughout the body, including tissues and blood, and play an important role against bacteria and viruses outside the body. Macrophages are a type of phagocyte and belong to the monocyte system. Macrophages are one of the most important participants in the autoimmune system in the tumor microenvironment in vivo, and the diversity of their roles in the tumor microenvironment is constantly being revealed. In recent years, research on tumor-associated macrophages has found theoretical and factual basis for tumor metastasis, recurrence, and drug resistance. The presence of tumor-associated macrophages makes it impossible to completely kill all tumor cells with chemotherapy or other treatments for tumors. Previous studies considered macrophages to be pure tumor killers, but subsequent studies have gradually revealed that macrophages can also promote tumor growth, that is, tumor-associated macrophages are involved. Macrophages not only indirectly affect tumor development through their own biological behavior, but also use their functions to directly participate in tumors and play a very important role in tumor metastasis.

[0003] 1 Macrophages and their functions 1.1 Origin and function of macrophages Macrophages are involved in innate and adaptive immunity, angiogenesis, proliferation, and even the formation of malignant tumors. Macrophages are a heterogeneous group of cells that can perform various functions in various organs and tissues. Even in a single tissue, their functions vary depending on the location (Reference 8). Mononuclear phagocytes are a major component of the innate immune system, and can nonspecifically phagocytose foreign bodies, bacteria, senescent cells, and mutant cells, maintaining the stability of the internal environment, natural anti-infection immunity, and anti-tumor immunity. Macrophages are heterogeneous cells, including cells in different activation states at different differentiation stages, each with corresponding morphology, phenotype, metabolic characteristics, and biological functions. Macrophages have the ability to secrete inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which are helpful in killing and phagocytosis of tumor cells. More and more studies have demonstrated that macrophages, as an important part of the tumor microenvironment, are involved in the processes of tumor proliferation, invasion, metastasis, neovascularization, immune evasion, and recurrence. Macrophages can polarize / differentiate into two distinct subtypes, M1 and M2 macrophages, in different microenvironments.

[0004] Classically macrophages are non-activated cells that are small in size and have weak killing activity in a resting state. M1 macrophages preferentially express inflammatory molecules such as nitric oxide, interleukin IL12, CXCL9, CXCL10, CXCL11, and reactive oxygen species, which contribute to tumor inhibition. 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 promoting stem cell formation and tumor cell activation through induction of angiogenesis, immune inhibition, and epithelial-mesenchymal stromal transformation. Similarly, when the body is traumatized, M1 macrophages are the first inflammatory cells activated at the wound site. On the other hand, M2 macrophages are also known as anti-inflammatory agents and play an important role in the body.

[0005] Different types of pathogens, inflammatory stimuli, and cytokines such as IFN-γ activate macrophages, which then release a variety of cytokines and chemokines, present them to cells as antigens, and participate in the initiation and regulation of immune responses. M1-type macrophages are induced by Th1 cytokines such as interferon-γ (IFN-γ), lipopolysaccharide (LPS), and granulocyte-macrophage colony-stimulating factor, express surface markers such as CD80 and CD86, and secrete cytokines such as interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNF-α), which play an important role in antibacterial and anti-infective functions.

[0006] Different types of cytokines and their combinations induce activated macrophages with different activity and functional characteristics. Macrophages activated with IL4, IL10, etc. have weaker antigen-presenting ability than M1 macrophages, and are called alternative activated M2 macrophages, i.e., 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), arginase 1 (Arg1), and chemokines CCL17 and CCL22. M2 macrophages have the ability to promote tumor cell proliferation, which has been found to be closely related to the promotion of epithelial-mesenchymal transition (EMT) and the formation of tumor stem cells. Exosomes from lung and breast cancer cells have been found to deliver activated epidermal growth factor receptor (EGFR) to macrophages, reduce interferon-β (IFN-β) secretion, and reduce the innate immunity of tumor patients via mitogen-activated protein kinase 2 (MEKK2). All these studies suggest that macrophages play various roles in the process of tumor development and metastasis.

[0007] 1.2 Tumor-associated macrophages Macrophages are the most prominent cell in the tumor microenvironment (TME), accounting for about 50% to 80%. Macrophage cells in the TME have a complex cell composition, mainly including M1M2 type macrophages and tumor-associated macrophages. M1 type macrophages are mainly involved in killing tumor cells, while M2 type macrophages are mainly involved in promoting tumor growth, neovascularization, invasion and distant metastasis, immune escape and drug resistance, etc. In recent years, more studies have shown that the function of tumor-associated macrophages (TAMs) is more consistent with M2 type macrophages, which can promote the further progression of tumors, such as promoting tumor growth, increasing tumor cell activity, promoting tumor formation, and genetic instability. Thus, in the tumor microenvironment (TME), the interaction between TAMs and tumors or the entire microenvironment is very complex, and further research and exploration are needed to more clearly understand the interaction between TAMs and tumors. TAMs can secrete and release growth factors such as platelet-derived growth factor, hepatocyte growth factor, and epidermal growth factor, and directly act on tumor cell receptors to promote division. In addition, the cytokine IL-6 secreted by TAMs can act on the transcriptional activator stat3 of tumor cells to activate intracellular pathways and indirectly promote tumor cell proliferation. In addition, TAMs can release enzymes that promote tumor proliferation, and because tumor cells require large amounts of nutrients and oxygen, many clinical data have shown that the density of local macrophages in tumor tissues is closely related to the area of ​​neovascular microvessels, and TAMs express the vascular growth factor receptor TIE2, and furthermore, TAMs secrete large amounts of angiogenic molecules such as TNF-α and IL-8 to promote angiogenesis. In promoting tumor invasion and metastasis, TAMs secrete proteases that degrade the extracellular matrix, such as matrix metalloproteinase MMP, histones, and urokinase fibrinogen activator, and also secrete extracellular matrix remodeling enzymes such as lysyl oxidase. These enzymes can degrade the fibrous connective tissue around the tumor, allowing tumor cells to easily invade from the original cancer tissue, enter the surrounding circulatory system, and metastasize to distant sites. In addition, proteases can remove obstacles for smooth metastasis of tumors.The neovessels induced by TAMs are highly permeable due to an incomplete basement membrane, making it easier for blood vessels to invade. In addition to the above functions, TAMs, as "traitors" of the body's immune system, secrete the cytokine IL-10, which leads to a decrease in the release of the inflammatory factor IL-12 from their own bodies, thereby reducing the ability of natural T cells to produce the cytotoxic factor interferon gamma. In addition, high levels of IL-10 inhibit the enzymes required for dendritic cell maturation, and the secretion of arginase I by TAMs has the ability to hydrolyze L-arginine, which affects the expression of T cell receptors and the proliferation of T cells, resulting in the loss of responsiveness of CD4+ and CD8+ T cells to stimuli. The above and further studies have shown that only M2-TAMs are "accomplices" of tumors. More detailed studies on M2 may provide valuable reference for the clinical treatment and prognosis of tumor metastasis.

[0008] 1.3 Macrophage surface markers There are many important molecular markers on the surface of macrophages, including IL series molecules, CD series molecules, TGF series molecules, and MHC series molecules. Among these molecules, CD206 is one of the most important surface markers that respond to the functional status of macrophages.

[0009] CD206 is a 175-190 kDa endocytic receptor also known as mannose receptor C type 1 (MRC1), macrophage mannose receptor (MMR), and C-type lectin domain family 13, member D (CLEC13D). CD206 is expressed on most macrophage populations, including TAMs, on dendritic cells in certain tissues, and on liver, spleen, lymphatic system, and dermal microvascular endothelial cells.

[0010] Human CD206 consists of 1456 amino acids (aa) and contains three structural domains in its extracellular region, including one N-terminal cysteine-rich (CR) structural domain, one fibronectin type II repeat (FNII), and eight C-type lectin domains (CTLD). The CR domain of human CD206 shares 83%, 84%, 89%, and 90% amino acid sequence identity with mouse, rat, horse, pig, and dog CD206, respectively. The CR domain binds sulfated glycans terminated with SO4-3-Gal or SO4-3 / 4-GalNAc and recognizes pituitary hormones such as lutein-stimulating hormone (LH) and thyroid-stimulating hormone (TSH). The FNII structural domain mediates Ca2+-independent binding to collagens, particularly collagens I, II, III, and IV. CTLDs are involved in the Ca2+-dependent recognition of branched-chain sugars of terminal mannose, fucose, and N-acetylaminoglucose, sugar molecules commonly found in pathogenic microorganisms.

[0011] 1.4 CD206 Features CD206 is an important pattern recognition receptor (PRR) and endocytosis receptor in the innate immune system, and is highly expressed on alveolar macrophages, monocyte-derived macrophages, and dendritic cells. It has also been found on several endothelial cell subpopulations, tracheal smooth muscle cells, retinal epithelial cells, renal vascular media cells, sperm acrosomal cells, and cerebral microglia. CD206 can recognize and bind a wide range of endogenous and exogenous ligands and plays an important role in processes such as endothelial homeostasis, pathogen recognition and phagocytosis, cytokine induction, and antigen presentation.

[0012] CD206 is involved in the recognition and phagocytosis of pathogens. CD206 recognizes polysaccharide components of cell walls, such as yeast mannan, bacterial podosomes, and LPS. Macrophages can phagocytose many non-regulated microorganisms, including bacteria, fungi, and protozoa, via CD206. Mannose receptors have been shown to be involved in the recognition and clearance of Candida, Mycobacterium tuberculosis, Staphylococcus aureus, and acidophilic Lactobacillus. Upon pathogen recognition, the release of superoxide anion from macrophages increases and the synthesis of related cytokines is induced. Macrophage membranes undergo deformation movements via the actin cytoskeleton to surround pathogens and pathogen-infected target cells, form phagocytic vesicles, and digest and kill pathogens.

[0013] CD206 is involved in intracellular signal transduction and affects the expression of many genes. Interaction of MR (mannose receptor) with ligands triggers a series of intracellular signal transduction cascades that amplify, transactivate, promote or inhibit the expression of NO and various cytokines, such as TNF-α, IL-12, IL-10, IL-1ra, and IL-RII. The cytoplasmic structural domain of MR is very short and does not contain signal transduction motifs, so the involvement of other receptors is also required for MR to participate in signal transduction. Previous studies have shown that TLR-2 is required for the induction of IL-8, and MR controls immune responses by mediating the production of various cytokines.

[0014] CD206 is involved in antigen processing and presentation. CD206 mainly ingests and concentrates non-self antigens, and then processes and presents them. CD206 mediates antigen cross-presentation, i.e., exogenous proteins are internalized and presented to CD8+ T cells via MHC class I molecules. This function is important for immune responses to indirect viral infection of APCs and non-endogenous expression of tumor antigens. Translocation of exogenous antigens from endosomes to the cytoplasm for proteolysis is important for antigen cross-presentation.

[0015] 1.5 CD206 and tumor-associated macrophages In recent years, the tumor microenvironment (TME) has become a key component of tumor research. The tumor microenvironment includes stromal cells, fibroblasts, immune cells, extracellular matrix, etc. Many studies have demonstrated that the occurrence and development of breast cancer is closely related to the TME, and that the components of the TME play an important role in the development of breast cancer.

[0016] Tumor-associated macrophages (TAMs) are the most abundant immune cells in the TME. In the tumor microenvironment of breast cancer, TAMs account for more than 50% of cells in the tumor. Breast cancer cells secrete chemokine (CC motif) ligand 2 (CCL2), CCL5, and CXCL12 to recruit monocytes. Monocytes are stimulated by various signaling molecules in the TME and polarize into TAMs. Most TAMs display an M2 phenotype and are called M2-type macrophages.

[0017] TAMs promote tumor cell proliferation, invasion and metastasis, promote tumor angiogenesis, inhibit T cell-mediated antitumor immune responses, and modulate tumor chemo-resistance. TAMs can directly promote tumor cell proliferation through secretion of epidermal growth factor (EGF), transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), etc. TAMs mediate tumor immune escape by inhibiting phagocytosis by macrophages through expression of molecules such as programmed death receptor-1 (PD-1) and signal-regulatory protein α (SIRPα). Recently, researchers have reported that tumor-associated macrophages express C1q + We identified a subpopulation of macrophages in which dysregulation of the m6A methylase METTL14 increases the expression of EBI3, contributing to the dysfunction of tumor-infiltrating CD8 T cells, thus inhibiting antitumor immune responses. In addition, TAMs can inhibit the antitumor effects of chemotherapeutic agents such as etoposide, gemcitabine, and CMF regimens (cyclophosphamide, methotrexate, and 5-fluorouracil). Several studies have confirmed that high infiltration density of TAMs in tumor tissues is closely associated with poor prognosis of cancer patients.

[0018] CD206 is highly expressed in M2 macrophages. CD206 not only serves as a marker for M2 macrophage activation but also contributes to immune inhibitory activity. A novel intronic microRNA (miRNA) called miR-511-3p was identified in the human and mouse Mrc1 gene. miR-511-3p is a negative regulator of the tumorigenic activity of TAMs, and enhanced expression in hematopoietic stem cells can have a significant effect on the tumor angiogenesis system and further inhibit tumor growth.

[0019] Recent studies have shown that activating the tumor-associated surface receptor CD206 enhances antitumor immune responses. Jaynes et al. constructed a 10-mer amphiphilic host defense peptide analogue, RP-182, to selectively induce conformational transformation of CD206, resulting in phenotypic transformation of TAMs. The research team first found that RP-182 mediated CD206 activation in M2 macrophages, triggering the programs of endocytosis, phagolysosome-lysosome formation, and autophagy. Flow cytometric analysis of the phenotype of mouse bone marrow-derived macrophages treated with RP-182 showed that CD206 activation by RP-182 reedited M2 macrophages into M1 macrophages. In tumor tissues treated with RP-182, M1 macrophages were increased, while M2 macrophages were decreased. The antitumor activity of RP-182 was observed in mouse pancreatic cancer models and patient-derived xenograft models with high CD206 expression. RP-182 also showed promising effects in pancreatic cancer when combined with chemotherapy agents and immune checkpoint inhibitors. Furthermore, the researchers tested RP-182 in various animal models of cancer and found that it was effective not only against pancreatic cancer, but also against other cancers such as colon cancer, breast cancer, prostate cancer, and melanoma. This study indicates that CD206 can be used as a target for cancer therapy.

[0020] 2.1 Defined siRNA structure: It has a short (usually 20-24 bp) double-stranded RNA (dsRNA) with a phosphorylated 5' end and a hydroxylated 3' end with two prominent nucleotides. The siRNAs generated by this Dicer enzyme include long dsRNAs and small hairpin RNAs. siRNAs can also be introduced into cells by transfection. In principle, any gene can be knocked down by synthetic siRNAs with complementary sequences, making siRNAs an important tool for validating gene function and drug targets in the post-genomic era. As shown in Figure 1, siRNAs are typically 21-nucleotide long double-stranded RNAs (dsRNAs) with two strands each extending two nucleotides beyond the other end from the end of the RNA. Each strand has a 5' phosphate end and a 3' hydroxyl end. This structure is created using an enzyme called Dicer, which cleaves longer double-stranded RNA or small hairpin RNA into siRNAs. In addition, siRNAs can also be introduced into cells through various transfection techniques to produce specific knockdown effects for specific genes. Thus, the complementarity of appropriately tailored siRNAs can be used to identify genes with known sequences. This phenomenon makes siRNA an important tool for studying gene function and drug targeting. siRNAs are similar to miRNAs, but miRNAs are derived from short stem-loop RNA products and usually silence genes by inhibiting translation, with broader specificity of action. siRNAs, on the other hand, usually act by cleaving mRNA before translation and are 100% complementary, making their target specificity very strict.

[0021] 2.2 Editing induction by siRNA or its biosynthetic precursor RNAi Knockdown by transfection of exogenous siRNA is usually unsatisfactory, especially in rapidly dividing cells, since the effect is transient. This can be overcome by creating an expression vector for siRNA. The sequence of the siRNA is modified to introduce a short loop between the two strands. The resulting transcripts are short hairpin RNAs (shRNAs) that can be processed into functional siRNAs by Dicer in the usual way. A typical transcript set uses an RNA polymerase III promoter (e.g. U6 or H1) to direct the transcription of small RNAs (snRNAs) (U6 is involved in gene splicing and H1 is a component of RNase). In theory, the resulting siRNA transcripts are then processed by Dicer.

[0022] Gene knockdown efficiency can also be improved by using cell extrusion. The activity of siRNA in RNAi is highly dependent on its ability to bind to the RNA-induced silencing complex (RISC). When double-stranded siRNA binds to RISC, it is degraded by endonucleases, resulting in cleavage of the sense strand. The remaining antisense strand-RISC complex can bind to target mRNA to initiate transcriptional silencing.

[0023] 2.3 Post-transcriptional gene silencing editing siRNA-induced post-transcriptional gene silencing begins with the assembly of an RNA-induced silencing complex (RISC). This complex silences specific gene expression by cleaving the mRNA molecule that codes for 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 passenger strand, is degraded. A specific Dicer enzyme may be involved in loading the guide strand into the RISC. The siRNA then scans and guides the RISC to the perfectly complementary sequence on the mRNA molecule. The cleavage of the mRNA molecule is thought to be catalyzed by the Piwi domain of the Argonaute protein in the RISC. The mRNA molecule is then precisely cleaved by cleaving the phosphodiester bond between the target nucleotides paired with the 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' fragments are degraded from the 3' end by the exosome, whereas the 3' fragments are degraded from the 5' end by 5'-3' exoribonuclease 1 (XRN1). Dissociation of the target mRNA strand from RISC after cleavage results in silencing of more mRNAs. This dissociation process is most likely promoted by an external factor triggered by ATP hydrolysis. In some cases, cleavage of the target mRNA molecule may not occur. In some cases, endonucleolytic cleavage of the phosphodiester backbone may be inhibited by a mismatch between the siRNA and the target mRNA near the cleavage site. In other cases, the Argonaute protein of RISC may lack endonuclease activity even when the target mRNA and siRNA are perfectly paired. In this case, gene expression is inhibited by a miRNA-guided mechanism. A simplified version of the ping-pong method involves the proteins Aubergine (Aub) and Argonaute-3 (Ago3), which cleave the 3' and 5' ends of piRNAs. Piwi-interacting RNAs are involved in silencing transposons, but not siRNAs.

[0024] Moreover, it remains to be seen whether inhibition of TAMs can be achieved using siRNA to block tumor cell migration and proliferation. Summary of the Invention [Problem to be solved by the invention]

[0025] In order to solve the above problems, an object of the present invention is to provide an siRNA that specifically binds to CD206 in M2 macrophages, thereby inhibiting the expression of CD206 by specifically binding to CD206 mRNA in M2 macrophages, thereby inhibiting the proliferation of M2 macrophages, and further inhibiting the proliferation and migration of tumor cells. [Means for solving the problem]

[0026] In order to achieve the above object, the present invention provides an siRNA targeting CD206 of M2 macrophages, the siRNA comprising: 1) one or more of siRNA-1, siRNA-2, siRNA-3, siRNA-4, siRNA-5, and siRNA-6; or 2) one or more of siRNA-7, siRNA-8, siRNA-9, siRNA-10, siRNA-11, and siRNA-12; Among them, The nucleotide sequence of the sense strand of the 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 the siRNA-2 is shown in SEQ ID NO:3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:4; The nucleotide sequence of the sense strand of the siRNA-3 is shown in SEQ ID NO:5, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:6; The nucleotide sequence of the sense strand of the siRNA-4 is shown in SEQ ID NO:7, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:8; The nucleotide sequence of the sense strand of the siRNA-5 is shown in SEQ ID NO:9, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:10; The nucleotide sequence of the sense strand of the siRNA-6 is shown in SEQ ID NO:11, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:12; The nucleotide sequence of the sense strand of the siRNA-7 is shown in SEQ ID NO: 13, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO: 14; The nucleotide sequence of the sense strand of the siRNA-8 is shown in SEQ ID NO:15, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:16; The nucleotide sequence of the sense strand of the siRNA-9 is shown in SEQ ID NO:17, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:18; The nucleotide sequence of the sense strand of the siRNA-10 is shown in SEQ ID NO:19, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:20; The nucleotide sequence of the sense strand of the siRNA-11 is shown in SEQ ID NO:21, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:22; The nucleotide sequence of the sense strand of the siRNA-12 is shown in SEQ ID NO: 23, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO: 24. siRNA is provided.

[0027] The present invention also provides use of the above-described siRNA in the manufacture of a medicament for inhibiting the growth or proliferation of tumor cells.

[0028] 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, germinoma cells, meningioma cells, meningeal cancer cells, glioblastoma cells, astrocytic glial cells, oligodendroglioma cells, astrocytic oligodendroglioma cells, ependymoma cells, choroid plexus papilloma cells, choroid plexus carcinoma cells, chordoma cells, ganglioneuroma cells, olfactory neuroblastoma cells, sympathetic nervous system neuroblastoma cells, pinealocytoma cells, pineoblastoma cells, meloblastoma cells, trigeminal neurinoma cells, facial acoustic neuroma cells, jugular spheroma cells, hemangioreticuloma cells, craniopharyngioma cells or granulosa cell tumor cells. Effect of the Invention

[0029] The beneficial effects of the present invention are as follows: The present invention provides siRNA that specifically binds to CD206 in M2 macrophages, thereby reducing the expression of CD206 by specifically binding to CD206 mRNA in M2 macrophages, thereby reducing the expression of M2 macrophage-associated molecules and proteins, and further inhibiting the proliferation and migration of tumor cells. [Brief description of the drawings]

[0030] [Figure 1] Schematic diagram of the basic structure of siRNA. [Diagram 2] FIG. 13 shows qPCR statistics of human siRNA-1 to siRNA-3 transfection provided by the present invention on THP1-induced M2 cell CD206 expression. [Diagram 3] FIG. 13 shows qPCR statistics of human siRNA-4 to siRNA-6 transfection provided by the present invention on THP1-induced M2 cell CD206 expression. [Figure 4] FIG. 13 is a fluorescent photograph of human siRNA-1 transfection provided by the present invention on THP1-induced M2 cell CD206 expression. [Diagram 5]FIG. 13 shows qPCR statistics of human siRNA-7 to siRNA-9 transfections provided by the present invention on mouse-derived M2 cell CD206 expression. [Figure 6] FIG. 13 shows qPCR statistics for mouse-derived M2 cell CD206 expression after transfection with human siRNA-10 to siRNA-12 provided by the present invention. [Figure 7] FIG. 13 is a fluorescent photograph showing the expression of CD206 in mouse-derived M2 cells after siRNA-9 transfection of mice provided by the present invention. [Figure 8] FIG. 13 shows the results of a CCK8 assay for the effect of 4T1-luc cells on growth viability in different conditioned media. [Figure 9A] FIG. 1 is a photograph of a tumor generated from a mouse breast cancer tumor model constructed using siRNA-9 provided by the present invention. [Figure 9B] FIG. 13 is a statistical diagram of tumor weights generated from a mouse breast cancer tumor model constructed using siRNA-9 provided by the present invention. [Figure 9C] FIG. 13 is a statistical diagram of tumor volumes generated from a mouse breast cancer tumor model constructed using siRNA-9 provided by the present invention. [Figure 10A] 1 shows animal images of a mouse breast cancer tumor model constructed using siRNA-9 provided by the present invention, in which 4T1-luc was inoculated into the tail vein. [Figure 10B] 13 is a histogram of fluorescence intensity when 4T1-luc was inoculated into the tail vein of a mouse breast cancer tumor model constructed using siRNA-9 provided by the present invention. [Figure 11A] 1 shows animal images of a mouse breast cancer tumor model constructed using siRNA-9 provided by the present invention, in which M-M2 macrophages and 4T1-luc were inoculated into the tail vein. [Figure 11B] 13 is a histogram of fluorescence intensity when M-M2 macrophages and 4T1-luc were inoculated into the tail vein of a mouse breast cancer tumor model constructed using siRNA-9 provided by the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] In order to facilitate the understanding of the present invention, the present invention will be described in more detail with reference to the following embodiments, and the preferred embodiments of the present invention are listed below. However, the present invention can be embodied in many different forms and is not limited to the embodiments described herein. These examples are provided to provide a thorough and comprehensive understanding of the present disclosure. It should be understood that the experimental methods without specific conditions in the following examples are generally in accordance with conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or conditions recommended by the manufacturer. All of the various commonly used reagents used in the examples are commercially available. EXAMPLES

[0032] material 1. Human breast cancer cell line MCF-7, human monocytic 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 a fluorescent marker), all purchased from the Representative Culture Collection Committee Cell Bank of the Chinese Academy of Sciences. 2. RPMI1640 complete medium was purchased from ThermoFisher Scientific, USA, product number: 31800022 3. PMA was purchased from Sigma-Aldrich LLC, USA, product number: P1585. 4. LPS was purchased from Sigma-Aldrich LLC, USA, product number: L2630. 5. IFNγ was purchased from PeproTech, USA, product number: 300-02 6.IL-4 purchased from PeproTech, USA, Product No.: 200-04 7.IL-13 purchased from PeproTech, USA, product number: 200-13 8.riboFECT TMCP transfection kit was purchased from Guangzhou Ruibo Biotechnology Co., Ltd., product number: C10511-05. 9. Primary antibody was purchased from Abcam, USA, product number: ab64693. 10.Secondary antibody was purchased from Beijing Nakasugi Jinqiao Biotechnology Co., Ltd., product number: ZF-0316 11. Fluorescent secondary antibody was purchased from Beijing Nakasugi Jinqiao Biotechnology Co., Ltd., product number: ZF-0311. 12. Mice ICR purchased from the Department of Animals, School of Medicine, Peking University. 13. Trizol reagent purchased from ThermoFisher Scientific, USA, product number: 15596018. 14.RevertAid RT Reverse Transcription Kit was purchased from ThermoFisher Scientific, USA, product number: K1691. 15. PowerUp TM SYBR TM Green premix was purchased from ThermoFisher Scientific, USA, product number: A25742.

[0033] Example 1 Based on the human CD206 gene, Genbank was searched to find suitable siRNA sequences. According to the Genbank search sequence NM_002438.4, six DNA target sequences were analyzed and shown in Table 1. [Table 1]

[0034] Using the above six 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 enhance the stability of the siRNA.

[0035] The siRNA sequences with 3' terminal TT are shown in Table 2. [Table 2]

[0036] The chemical synthesis of the above siRNA-1 to siRNA-6 was commissioned to Beijing Qingke Biotechnology Co., Ltd. For the convenience of subsequent assays, the 5' ends of the sense and antisense strands of siRNA-1 to siRNA-6 were both FAM fluorescently labeled, using a conventional labeling method. Meanwhile, the company provided the non-homologous negative control siRNA-NC according to the target sequence.

[0037] Example 2 The human siRNA-1 to siRNA-6 and siRNA-NC prepared in Example 1 were subjected to riboFECT TM The CP transfection kit was used to transfect THP1-induced M2 macrophages, and changes in the expression of CD206 and its downstream related molecules in M2 macrophage cells were measured.

[0038] 1. Induction of M2-type macrophages 1) Human monocytic cell line THP-1 cells were inoculated into culture bottles containing RPMI1640 complete medium and stably cultured for 2 to 4 generations. 2) Collect THP-1 cells in a 15 mL centrifuge tube and centrifuge at 800 rpm for 3 min to reduce the cell density to 5 × 10 5 The THP-1 cells were then adjusted to 100 ng / mL and inoculated into a new culture dish containing complete RPMI 1640 medium, treated with 100 ng / mL PMA for 24 hours, and observed under a microscope to see whether the THP-1 cells had completely adhered to the wall. Complete adhesion indicated that they had differentiated into M0-type macrophages. 3) The medium was discarded, and RPMI1640 complete medium containing 20 ng / mL LPS and 20 ng / mL IFNγ was added and cultured for 48 hours to induce M1-type macrophages. After that, RPMI1640 complete medium containing 20 ng / mL IL-4 and IL-13 was added and cultured for 48 hours to induce M2-type macrophages.

[0039] 2. qPCR detection of CD206 expression and downstream related molecules in M2-type macrophages siRNA-1, siRNA-2, siRNA-3, siRNA-4, siRNA-5, siRNA-6 and siRNA-NC were TM Following the steps in the CP transfection kit instructions, M2 macrophage cells were transfected with each of the vectors. After transfection, the cell culture plate (6-well plate) was left in a culture box at 37°C, 5% CO2 and saturated humidity for 24-72 hours. 1) RNA extraction Each sample of cell samples transfected with siRNA-1, siRNA-2, siRNA-3, siRNA-4, siRNA-5, siRNA-6 and siRNA-NC was run in triplicate in parallel. a. Discard the original medium in the cell culture plate, add PBS, and gently shake the culture dish to wash it 2 to 3 times. b. 1 mL of Trizol reagent was added to each well of the cell culture plate, and the mixture was left at room temperature for 5 minutes. After pipetting several times, the cell suspension was collected in 1.5 mL centrifuge tubes in (1), 1 mL per tube. c. 200 μL chloroform was added to each tube, shaken vigorously for 15 seconds, and allowed to stand at room temperature for 2-3 minutes. d. Place the above centrifuge tube in a high-speed low-temperature centrifuge and centrifuge at 4°C and 10,000 g for 15 minutes. Carefully aspirate and transfer the supernatant into another clean 1.5 mL centrifuge tube, add an equal volume (approximately 600 μL) of isopropanol, and leave at room temperature for 10 minutes. f. The centrifuge tube was placed in a high-speed low-temperature centrifuge and centrifuged at 4° C. and 10,000 g for 10 minutes. The supernatant was discarded and 1 mL of pre-chilled 80% ethanol was added to wash the precipitate. g. The above centrifuge tube was placed in a high-speed low-temperature centrifuge and centrifuged at 4°C and 7,500 g for 5 minutes, and the supernatant was discarded. h. Step (7) was repeated. i. After centrifugation was completed, the supernatant was discarded and the mixture was dried in air for 5 to 10 minutes to thoroughly evaporate the ethanol. j. Add 30 to 50 μL of RNAase-free ddH2O to the centrifuge tube, aspirate and mix repeatedly to obtain total RNA. k. Collect 1-2 μL samples from each group and measure the concentration using an ultra-microspectrophotometer, and the appropriate concentration was used for subsequent experiments or stored in a -80℃ ultra-low temperature refrigerator.

[0040] 2) RNA reverse transcription RNA reverse transcription was performed using the RevertAid RT kit, and the reagents used were included in the RevertAid RT kit. a. RNA reverse transcription reaction system 1 was prepared as shown in Table 3. [Table 3]

[0041] b. Mix by gentle aspiration, briefly centrifuge, incubate in a metal bath at 65°C for 5 minutes, and place on ice for 2 minutes. c. RNA reverse transcription reaction system 2 was prepared as shown in Table 4. [Table 4]

[0042] d. RNA reverse transcription reaction system 2 was added to RNA reverse transcription reaction system 1, mixed gently by suction, centrifuged immediately, and then placed in a PCR device. The reaction conditions were 25°C for 5 minutes; 42°C for 60 minutes; 70°C for 5 minutes; cooled to 4°C to obtain a cDNA template.

[0043] After completing the reverse transcription, the cDNA samples were either used for subsequent qPCR experiments or stored in a -80°C ultra-low temperature refrigerator.

[0044] 3) qPCR The qPCR reactions were prepared as shown in Table 5. [Table 5]

[0045] Wherein, 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.

[0046] The above components were mixed uniformly by gentle suction, centrifuged instantly, and then the StepOne TM The reaction conditions were as shown in Table 6. [Table 6]

[0047] qPCR was used to detect the interference of CD206 expression in THP1-induced M-M2 macrophages by siRNA, and the results are shown in Figure 2 and Figure 3. Among them, HS1 to HS6 correspond to the PCR products after transfection with siRNA-1 to siRNA-6, respectively, and NC is the PCR product after transfection with siRNA-NC, which represents the negative control, and showed the interference of CD206 expression at 24 hours, 48 ​​hours, and 72 hours. The experimental data were shown as mean ± standard deviation (mean ± SD) (n = 3). *: P < 0.05 compared with NC group; **: P < 0.01 compared with NC group; ***: P < 0.001 compared with NC group; ****: P < 0.0001 compared with NC group.

[0048] From Figure 2 and Figure 3, it can be seen that siRNA-1 to siRNA-6 all have the effect of inhibiting the expression of CD206. Here, NC was used as a negative control, and HS1-HS6 were shown. The large variation in the difference between different time points is due to the fact that different siRNAs bind to different parts of the gene, which may result in different inhibitory effects, appropriate action time, and strength of action. siRNA-1 was used as a representative in the subsequent experiments.

[0049] 3. Immunofluorescence assay to detect the expression of related proteins: 1) M2 macrophages were harvested after transfection with siRNA-1 for 24, 48, and 72 hours, and the cell density was adjusted to 1×10 5 The resulting solution was adjusted to 1 / mL and inoculated into a confocal dish. Non-transfected M2-type macrophages served as a negative control (NC). 2) After the cells had adhered to the wall, the medium was discarded, PBS was added, and the cells were shaken on a horizontal decolorizing shaker for 5 minutes and washed three times. 3) Fixation: 4% paraformaldehyde was added, and the sections were left at room temperature for 10 minutes, and then washed three times with PBS. 4) Blocking: 5% BSA in PBS was added and blocking was carried out for 30 minutes in an incubator at 37°C. 5) Primary antibody reaction: After discarding the blocking solution, 200 μL (1:500 dilution) of the corresponding primary antibody was added to each group; an equal volume of 5% BSA was added to the negative control group and incubated at 4°C overnight. 6) Secondary antibody reaction: The primary antibody was aspirated and discarded, PBS was added, and the cells were shaken on a horizontal destaining shaker for 5 minutes and washed three times. Corresponding fluorescent secondary antibodies (1:500 dilution) were added to each group, and an equal volume of secondary antibody was added to the negative control group, and the cells were incubated at 37°C for 1 hour in a dark incubator. 7) DAPI: Aspirate and discard the secondary antibody, add PBS, and wash 3 times. Add 200 μL of DAPI and incubate at room temperature for 20 minutes; aspirate and discard DAPI, add PBS, and wash 3 times.

[0050] The cell luminescence of each group was observed and photographed using a Leica laser confocal microscope. Taking siRNA-1 as an example, the results are shown in Figure 4, in which DAPI indicates the blue fluorescence of DAPI labeling the cell nucleus, CD206-TRITC indicates the red fluorescence of TRITC labeling CD206, Merge indicates the coating overlay result of DAPI and CD206-TRITC results, and T-M2 NC was the negative control group, and T-M2 HS1 24h indicates the result of 24-hour treatment with siRNA-1, and T-M2 HS148h indicates the result of 48 hours of treatment with siRNA-1, and T-M2 HS1 72h shows the results of 72 hours of treatment with siRNA-1.

[0051] As can be seen from Figure 4, when fluorescently labeled siRNA-1 was transfected into human M2 macrophage cells, the expression level of CD206 in M2 macrophages was significantly lower than that in the blank control group and the negative control group, and after 72 hours of treatment, CD206 expression was basically not observed, indicating that the siRNA-1 provided by the present invention can effectively inhibit the expression of CD206.

[0052] Example 3 Based on the mouse CD206 gene, appropriate siRNA sequences were searched in Genbank. As shown in Table 7, six DNA target sequences were analyzed according to the Genbank search sequence NM_008625.2. [Table 7]

[0053] Using the above six 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 enhance the stability of the siRNA.

[0054] The siRNA sequences with a 3' terminal TT are shown in Table 8. [Table 8]

[0055] The chemical synthesis of the above siRNA-7 to siRNA-12 was entrusted to Beijing Qingke Biotechnology Co., Ltd. For the convenience of subsequent assays, when synthesizing the sense and antisense strands of siRNA-7 to siRNA-12, two siRNAs with the same sequence were synthesized, one of which was FAM fluorescently labeled at the 5' end of the siRNA, and the labeling method was the general method, and the other was an siRNA with the same sequence but without FAM labeling. Meanwhile, the company provided a negative control siRNA-NC with no homology according to the target sequence.

[0056] Example 4 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 changes in the expression of CD206 and its downstream related molecules in M2 macrophage cells were measured. 1. Preparation of mouse-derived M2-type macrophages: (1) 6- to 8-week-old BALB / c mice were selected, killed by cervical dislocation, and immersed in 75% alcohol for 5 minutes. (2) Cut the skin at the crotch with sterile ophthalmic scissors, peel off the mouse's hind limbs, cut upward along the base of the femoral head, being careful not to cut the femur, carefully remove the muscle, and immerse the clean bone in PBS. (3) The extracted bones were immersed in 75% alcohol for 2 minutes and washed twice with PBS. (4) The bone end was cut with ophthalmic scissors, PBS was aspirated with a 5 mL syringe, and the syringe was inserted into the bone marrow cavity to rinse the cells. The syringe was then placed in a 15 mL centrifuge tube and gently blown repeatedly to disperse the cells. (5) The cell suspension was filtered through a 70 μm (200 mesh) cell filter, placed in a new 15 mL centrifuge tube, and centrifuged at 1,000 rpm for 10 minutes. (6) The supernatant was discarded, and 2 to 3 mL of red blood cell lysis solution was added to resuspend the cells, followed by lysis at 4°C for 10 minutes. (7) 10 mL of DMEM complete medium was added to complete erythrocyte lysis, and the mixture was centrifuged at 1,500 rpm for 5 minutes. (8) The cells were resuspended in DMEM complete medium containing a final concentration of 50 ng / mL mM CSF, counted, and then the cell concentration was adjusted and seeded into 6-well or 24-well plates. (9) The fluid was replaced on the third day after plate inoculation, and mature macrophages were harvested on the seventh day as M-M0 macrophages. (10) Mature macrophages were polarized, the original medium was discarded, and DMEM complete medium containing 100 ng / mL LPS and IFNγ was added and cultured for 24 hours to obtain M-M1 type macrophages. DMEM complete medium containing 20 ng / mL IL-4 was added and cultured for 24 hours to obtain M-M2 type macrophages.

[0057] 2. qPCR assay to detect the expression level of CD206 in mouse-derived M2-type macrophage cells. siRNA-7, siRNA-8, siRNA-9, siRNA-10, siRNA-11, siRNA-12, siRNA-NC, riboFECT TM Following the instructions in the CP transfection kit, M2 macrophage cells were transfected with each of the CP transfection kits. After transfection, the cell culture plate was placed in a culture box at 37°C, 5% CO2 and saturated humidity for 24-72 hours. qPCR was performed to detect the expression of CD206 in mouse M2 macrophages. The specific method was performed with reference to the qPCR assay section in Example 2. qPCR was used to detect the interference of CD206 expression in mouse-derived M2 macrophages by siRNA, and the results are shown in Figure 5 and Figure 6. Among them, MS1 to MS6 correspond to the PCR products after transfection with siRNA-7 to siRNA-12, respectively, and NC is the PCR product after transfection with siRNA-NC, representing the negative control, and showed the interference of CD206 expression at 24 hours, 48 ​​hours, and 72 hours. The experimental data were shown 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. As can be seen from Figures 5 and 6, all of siRNA-7 to siRNA-12 had the effect of inhibiting the expression of CD206, and siRNA-9 was used as a representative for the subsequent experiments.

[0058] 3. Immunofluorescence assay to detect the expression of related proteins: After siRNA-9 transfection, the cells were treated in the same manner as in Example 2. The cell luminescence of each group was observed and photographed using a Leica laser confocal microscope. Taking siRNA-9 as an example, the results are shown in Figure 7, in which DAPI indicates the blue fluorescence of DAPI labeling the cell nucleus, CD206-TRITC indicates the red fluorescence of TRITC labeling CD206, Merge indicates the coating overlay result of DAPI and CD206-TRITC results, and T-M2 NC was the negative control group, and T-M2 MS3 24h indicates the result of 24-hour treatment with siRNA-9, T-M2 MS3 48h shows the results of 48-hour treatment with siRNA-9, and T-M2 MS3 72h shows the results of 72 hours of treatment with siRNA-9. As can be seen from Figure 7, fluorescently labeled siRNA-9 was transfected into mouse-derived M2 macrophage cells, and the expression level of CD206 in the macrophages was significantly lower than that of the blank control group and the negative control group. After 72 hours of treatment, CD206 expression was basically not observed, indicating that the siRNA-9 provided by the present invention can effectively inhibit the expression of CD206.

[0059] Example 5: A complete experiment on the effect of M2-type macrophages with downregulated CD206 expression on the development and metastasis of breast cancer tumors 1.Effect of CD206 down-regulated M-M2 macrophage conditioned medium on mouse breast cancer tumor growth To further verify the effect of CD206-downregulated M2-type macrophages on breast cancer tumor growth, 4T1-luc cells treated with different conditioned media were inoculated into the left forelimb axilla of Balb / c mice to establish a mouse breast cancer tumor model.

[0060] Conditioned medium M-M2 CM, M-M2 NC CM, M-M2 MS3 CM was prepared respectively, and the specific method was as follows.

[0061] Preparation of conditioned medium M-M2 CM: The culture supernatant of mouse-derived M2 macrophages (M-M2 macrophages) prepared in Example 4 was collected and centrifuged at 1000 rpm for 10 minutes. Fresh complete medium was added in a 1:1 ratio and mixed uniformly to prepare M-M2 macrophage conditioned medium, which was named M-M2 CM.

[0062] Conditioned medium M-M2 NC Preparation of CM: The culture supernatant of the siRNA-NC-M2 macrophages transfected for 48 hours in Example 4 was collected, centrifuged at 1000 rpm for 10 minutes, and fresh complete medium was added in a 1:1 ratio and mixed uniformly to prepare M-M2 NC Prepare macrophage conditioned medium, M-M2 NCIt was made into a commercial.

[0063] Conditioned medium M-M2 MS3 Preparation of CM: The culture supernatant of the siRNA-9-M2 macrophages transfected for 48 hours in Example 4 was collected and centrifuged at 1000 rpm for 10 minutes, and fresh complete medium was added in a 1:1 ratio and mixed uniformly to prepare M-M2 MS3 Prepare macrophage conditioned medium, M-M2 MS3 It was made into a commercial.

[0064] Conditioned medium 4T1 CM is the culture supernatant of 4T1-luc cells cultured at 1000 rpm for 10 minutes, and was prepared by adding fresh complete medium in a 1:1 ratio and mixing uniformly.

[0065] Above conditioned medium 4T1 CM, M-M2 CM, M-M2 NC CM, M-M2 MS3 4T1-luc cells were cultured in CM for 12, 24, 36, and 48 hours, respectively, and then the 4T1 proliferation ability was evaluated using CCK8 assay. The results are shown in Table 9 and Figure 8. [Table 9]

[0066] The data in Table 9 are the ratios of OD values ​​measured at different time points to 0 hours, and the data are the mean ± SD (n=3). *: p<0.05 compared with 4T1 CM group; **: p<0.01 compared with 4T1 CM group; ***: p<0.001 compared with 4T1 CM group; #: p<0.05 compared with M-M2 CM group; ##: p<0.01 compared with M-M2 CM group; &: p<0.001 compared with M-M2 CM group. NC Compared with the CM group, p<0.05;&&&:M-M2 NC p<0.001 compared with the CM group.

[0067] As can be seen from Table 9 and Figure 8, compared with the 4T1 CM group, M-M2 MS3There was no significant difference in cell proliferation and viability between the CM group (p>0.05), and M-M2 CM and M-M2 NC The cell proliferation and viability of the CM group increased, and both were significantly different (p<0.05); compared with the M-M2 CM group, the M-M2 MS3 The cell proliferation and viability rate of the CM group decreased, and there was a significant difference in both groups (p<0.05); NC Compared with the CM group, M-M2 MS3 The cell proliferation and viability of the CM group was decreased, and both were significantly different (p<0.05). As a result, M-M2 type macrophages with CD206 downregulation promoted the decrease in the proliferation ability of breast cancer cell 4T1.

[0068] 4T1-luc cells were harvested and the cell density was adjusted to 2 × 10 5 / mL, and 100 μl of the cell suspension was inoculated into the left forelimb axilla of each mouse, which was labeled as the 4T1 group.

[0069] Conditioned medium M-M2 CM, M-M2 NC CM, M-M2 MS3 After culturing in CM for 24 hours, 4T1-luc cells were harvested and the cell density was adjusted to 2 × 10 5 The cell suspension was adjusted to 100 μl / mL, and 100 μl of the cell suspension was inoculated into the left forelimb axilla of each mouse. NC CM, 4T1 / M-M2 MS3 The mice were labeled as group CM. After 14 days, the mice were killed by cervical dislocation, dissected, and the tumors were excised, weighed, and the tumor volumes were calculated. The statistical values ​​are shown in Table 10. [Table 10]

[0070] As can be seen from Table 10, compared with the 4T1 control group, 4T1 / M-M2 CM, 4T1 / M-M2 NC There was no obvious difference in tumor weight in the CM group, but the tumor volume tended to increase. MS3 The tumor weight in the CM group showed a tendency to decrease, and the tumor volume was significantly decreased; compared with the 4T1 / M-M2 CM group, the tumor weight in the CM group showed a tendency to decrease, and the tumor volume was significantly decreased;MS3 The tumor weight in the CM group showed a tendency to decrease, and the tumor volume was significantly decreased; 4T1 / M-M2 NC Compared with the CM group, 4T1 / M-M2 MS3 The tumor weight in the CM group showed a tendency to decrease, and the tumor volume was significantly reduced.M-M2 macrophage conditioned medium with downregulated CD206 expression could inhibit the ability of M2 macrophages to promote mouse breast cancer tumor growth, and further inhibit mouse breast cancer tumor growth.

[0071] 2. Effect of M-M2 macrophages with downregulated CD206 expression on mouse breast cancer tumor growth To further examine the effect of CD206 downregulated M2 macrophages on breast cancer tumor growth, we performed studies using mouse breast cancer tumor models established by co-inoculating M-M2 macrophages and 4T1-luc cells (4:1) into the left forelimb axilla of Balb / c mice. NC and M-M2 MS3 Harvest the cells and reduce the cell density to 2 x 10 5 100 μl of 4T1-luc cell suspension and 25 μl of M-M2, adjusted to 10 μl / mL, M-M2 NC , M-M2 MS3 The cell suspensions were mixed and co-inoculated into the left forelimb axilla of mice in a 4:1 ratio, as 4T1+M-M2, 4T1+M-M2, respectively. NC , 4T1+M-M2 MS3 After 14 days, the mice were killed by cervical dislocation, dissected, and the tumors were excised, weighed, and calculated. The statistics are shown in Table 11 and Figures 9A to 9C.

[0072] FIG. 9A shows mouse breast cancer tumor models in which 4T1-luc cells and M-M2 macrophages (4:1) were co-inoculated into the left forelimb axilla. NC , 4T1+M-M2 MS3 Figure 9B shows tumor photographs of the 4T1, 4T1+M-M2, and 4T1+M-M2 groups. NC , 4T1+M-M2 MS3Histograms of tumor weights of groups are shown. Figure 9C shows the histograms of tumor weights of 4T1, 4T1+M-M2, 4T1+M-M2 NC , 4T1+M-M2 MS3 The histograms of tumor volume of each group are shown. Experimental data are shown as mean ± SD (n = 4). *: P < 0.05 compared with 4T1 group; **: P < 0.01 compared with 4T1 group; ##: P < 0.01 compared with 4T1 + M-M2 group; &: P < 0.01 compared with 4T1 + M-M2 group. NC P < 0.05 compared with the group. [Table 11]

[0073] As can be seen from Figures 9A to 9C and Table 10, compared with the 4T1 control group, 4T1+M-M2, 4T1+M-M2 NC The tumor weights of the 4T1+M-M2 groups increased significantly, and the tumor volumes showed a tendency to increase. MS3 The tumor volume and weight of the 4T1+M-M2 group showed a tendency to decrease compared with the 4T1+M-M2 group. MS3 The tumor volume and weight of the 4T1+M-M2 group were significantly reduced. NC Compared with the 4T1+M-M2 group, MS3 The tumor volume and weight of the mice were significantly reduced in both groups. These results suggest that M-M2 macrophages with downregulated CD206 expression could inhibit the ability of M2 macrophages to promote mouse breast cancer tumor growth and further inhibit mouse breast cancer tumor growth.

[0074] 3. Effect of CD206 down-regulated M-M2 macrophage conditioned medium on mouse breast cancer metastasis To further verify the effect of CD206 downregulated M2 macrophages on breast cancer metastasis, we used conditioned medium M-M2 CM, M-M2 CM, and M-M2 CM, respectively. NC CM, M-M2 MS3 The mouse breast cancer tumor model was established by inoculating 4T1-luc cells treated with α-lactam 1 (Ala) into Balb / c mice via the tail vein. 4T1-luc cells were harvested and the cell density was adjusted to 2 × 105 100 μl of the 4T1-luc cell suspension was injected via the tail vein and labeled as the 4T1 group. Conditioned medium M-M2 CM, M-M2 NC CM, M-M2 MS3 After culturing in CM for 24 hours, 4T1-luc cells were harvested and the cell density was adjusted to 2 × 10 5 The cell suspension was adjusted to 100 μl / mL and 100 μl of the cell suspension was injected via the tail vein. NC CM, 4T1 / M-M2 MS3 The animals were subjected to imaging analysis after 7 days, and the results are shown in FIG.

[0075] FIG. 10A shows mouse breast cancer tumor metastasis models, 4T1, 4T1 / M-M2 CM, and 4T1 / M-M2 CM, inoculated with conditioned medium-treated 4T1-luc via the tail vein. NC CM, 4T1 / M-M2 MS3 Figure 10B shows the observation images of the animal images of the CM group. NC CM, 4T1 / M-M2 MS3 The histograms of fluorescence intensity of animal images from the CM group are shown. Experimental data are shown as mean ± standard deviation (mean ± SD) (n = 4). *: P < 0.05 compared with the 4T1 group; &&: 4T1 / M-M2 NC Compared with the CM group, P < 0.01. [Table 12]

[0076] As can be seen from Figures 10A to 10B and Table 11, compared with the 4T1 control group, 4T1 / M-M2 CM, 4T1 / M-M2 NC The lung fluorescence intensity in the CM group showed a tendency to increase, and the 4T1 / M-M2 MS3 The lung fluorescence intensity in the CM group was significantly decreased compared with the 4T1 / M-M2 CM group; MS3 The lung fluorescence intensity in the CM group showed a tendency to decrease; 4T1 / M-M2 NC Compared with the CM group, 4T1 / M-M2MS3 The lung fluorescence intensity of the CM group was significantly decreased. As a result, M-M2 macrophage conditioned medium with downregulated CD206 expression could inhibit the ability of M2 macrophages to promote mouse breast cancer tumor metastasis and further inhibit mouse breast cancer tumor metastasis.

[0077] 4. Effect of M-M2 macrophages with downregulated CD206 expression on mouse breast cancer metastasis To further verify the effect of CD206-downregulated M2 macrophages on breast cancer tumor metastasis, we performed a study using a mouse breast cancer tumor model established by co-inoculating M-M2 macrophages and 4T1-luc cells (4:1) via the tail vein into Balb / c mice. 4T1-luc, M-M2, M-M2 NC and M-M2 MS3 Harvest the cells and reduce the cell density to 2 x 10 5 100 μl of 4T1-luc cell suspension was added to each well, and 25 μl of M-M2 and M-M2 were added to each well. NC , M-M2 MS3 The cells were mixed with the cell suspension and inoculated into Balb / c mice via the tail vein in a 4:1 ratio, resulting in 4T1+M-M2, 4T1+M-M2, and 4T1+M-M2, respectively. NC , 4T1+M-M2 MS3 After 7 days, animals were imaged and the results are shown in Figures 11A-11B and Table 13.

[0078] FIG. 11A shows mouse breast cancer tumor metastasis models in which M-M2 macrophages and 4T1-luc (4:1) were co-inoculated via the tail vein: 4T1, 4T1+M-M2, and 4T1+M-M2. NC , 4T1+M-M2 MS3 Figure 11B shows the observation images of animals in the 4T1, 4T1+M-M2, and 4T1+M-M2 groups. NC , 4T1+M-M2 MS3Figure 1 shows histograms of animal image fluorescence intensity in groups. Experimental data are shown as mean ± SD (n=4). *: P<0.05 compared with 4T1 group; ##: P<0.01 compared with 4T1+M-M2 group; &&&&: P<0.0001 compared with 4T1+M-M2 group. [Table 13]

[0079] As can be seen from Table 13 and Figures 11A to 11B, compared with the 4T1 control group, 4T1+M-M2, 4T1+M-M2 NC The lung fluorescence intensity of the 4T1+M-M2 group was significantly increased. MS3 The lung fluorescence intensity in the 4T1+M-M2 group was significantly decreased compared with that in the 4T1+M-M2 group. MS3 The lung fluorescence intensity of the 4T1+M-M2 group was significantly decreased. NC Compared with the 4T1+M-M2 group, MS3 The lung fluorescence intensity of the M-M2 macrophage-conditioned medium with downregulated CD206 expression was found to inhibit the ability of M2 macrophages to promote mouse breast cancer tumor metastasis, and further inhibit mouse breast cancer tumor metastasis.

[0080] As can be seen from the above Examples, the siRNA provided by the present invention targets CD206 in M2 macrophages and inhibits the expression of CD206 in M2 macrophages, thereby inhibiting the ability of M2 macrophages to promote the proliferation of breast cancer tumors and suppressing the metastasis of breast cancer tumors.

[0081] In order to confirm that the siRNA provided by the present invention inhibits M2-type macrophages and thereby suppresses the growth and metastasis of breast cancer tumors, only breast cancer cells were used. However, since the siRNA provided by the present invention acts directly on M2-type macrophages, it is possible to inhibit not only breast cancer cells but also 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, and pancreatic cancer cells. It is also applied to tumor cells, liver cancer cells, melanoma cells, skin cancer cells, pituitary tumor cells, germinoma cells, meningioma cells, meningeal cancer cells, glioblastoma cells, astrocytic glial cells, oligodendroglioma cells, astrocytic oligodendroglioma cells, ependymoma cells, choroid plexus papilloma cells, choroid plexus carcinoma cells, chordoma cells, ganglioneuroma cells, olfactory neuroblastoma cells, sympathetic neuroblastoma cells, pinealocyte tumor cells, pineoblastoma cells, medullary neurinoma cells, trigeminal neurinoma cells, facial acoustic neuroma cells, jugular spheroma cells, hemangioreticuloma cells, craniopharyngioma cells or granulosa cell tumor cells. Therefore, the siRNA provided by the present invention can inhibit the expression of CD206 in M2 macrophages, thereby suppressing the proliferation and metastasis of multiple types of tumor cells, and can achieve the effect of treating multiple tumors with one route.

[0082] The above-mentioned embodiments are merely more specific and detailed representations of some embodiments of the present invention, and are not to be construed as limiting the patent scope of the present invention. It should be noted that a person skilled in the art can make some modifications and improvements without departing from the concept of the present invention, all of which are within the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be subject to the scope of the attached claims.

Claims

1. An siRNA that specifically binds to CD206 of M2 macrophages, the siRNA comprising: 1) one or more of siRNA-1, siRNA-2, siRNA-3, siRNA-4, siRNA-5, and siRNA-6; or 2) one or more of siRNA-7, siRNA-8, siRNA-9, siRNA-10, siRNA-11, and siRNA-12; The nucleotide sequence of the sense strand of the 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 the siRNA-2 is shown in SEQ ID NO:3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:4; The nucleotide sequence of the sense strand of the siRNA-3 is shown in SEQ ID NO:5, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:6; The nucleotide sequence of the sense strand of the siRNA-4 is shown in SEQ ID NO:7, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:8; The nucleotide sequence of the sense strand of the siRNA-5 is shown in SEQ ID NO:9, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:10; The nucleotide sequence of the sense strand of the siRNA-6 is shown in SEQ ID NO:11, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:12; The nucleotide sequence of the sense strand of the siRNA-7 is shown in SEQ ID NO:13, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:14; The nucleotide sequence of the sense strand of the siRNA-8 is shown in SEQ ID NO:15, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:16; The nucleotide sequence of the sense strand of the siRNA-9 is shown in SEQ ID NO:17, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:18; The nucleotide sequence of the sense strand of the siRNA-10 is shown in SEQ ID NO:19, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:20; The nucleotide sequence of the sense strand of the siRNA-11 is shown in SEQ ID NO:21, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:22; The nucleotide sequence of the sense strand of the siRNA-12 is shown in SEQ ID NO:23, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:

24. An siRNA characterized by:

2. 13. Use of the siRNA of claim 1 in the manufacture of a medicament for inhibiting the growth or proliferation of tumor cells.

3. 3. The use according to claim 2, characterized in that 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, germinoma cells, meningioma cells, meningeal cancer cells, glioblastoma cells, astrocytic glial cells, oligodendroglioma cells, astrocytic oligodendroglioma cells, ependymoma cells, choroid plexus papilloma cells, choroid plexus carcinoma cells, chordoma cells, ganglioneuroma cells, olfactory neuroblastoma cells, sympathetic neuroblastoma cells, pinealocytoma cells, pineoblastoma cells, meloblastoma cells, trigeminal neurinoma cells, facial acoustic neuroma cells, jugular spheroma cells, hemangioreticuloma cells, craniopharyngioma cells or granulosa cell tumor cells.

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