A method for studying the regulatory mechanism of primary breast cancer on distant metastases

Abstract

A research method for the regulatory mechanism of primary breast cancer on distant metastasis, miRNA-210 is intervened in lentiviral transfection of breast cancer cells, changes in Bcl-2 protein expression are detected, Bcl-2 gene luciferase reporter gene plasmid vectors containing miRNA-210 binding sites are constructed, and luciferase reporter gene detection experiments are performed after transfection of breast cancer cells to determine the regulatory relationship between the two; nude mouse tumor-bearing animal model is constructed, primary lesion tissue exosomes are injected into the center of the tumor, changes in tumor volume are recorded, and tumor blocks are taken to detect tumor cell autophagy and cell proliferation; Luciferase reporter gene detection experiments are used to determine the effect of primary lesion tissue exosome miRNA-210 on breast cancer cell proliferation in the in vivo environment, and whether this effect is related to the regulated target cell autophagy. The present invention provides an objective basis for clinical prevention and treatment of distant metastasis of breast cancer by regulating autophagy.

Description

Technical Field

[0001] The present invention relates to a method for studying a regulatory mechanism, and in particular to a method for studying a regulatory mechanism of a primary breast cancer lesion on a distant metastatic lesion. Background Art

[0002] Breast cancer has become the most common cancer in the world. Comprehensive treatment with surgery, radiotherapy, and systemic drugs can significantly improve its prognosis. Epidemiological data show that the 10-year overall survival rate of patients with stage I and II-III breast cancer is as high as 94% and 75%, respectively, but for stage IV breast cancer with distant metastasis, it is only 9%. Approximately 6% of breast cancer patients have distant organ metastases to bones, liver, lungs, and non-axillary lymph nodes at the time of initial diagnosis. Before drug treatment is effective / before drug treatment, there is still a great deal of disagreement as to whether the primary lesion of breast cancer with distant metastases requires surgical resection.

[0003] Previous studies have reported that surgical resection of primary breast cancer can promote the malignant progression of distant metastases, indicating that there may be a check-and-balance relationship between primary breast cancer and distant metastases. When primary breast cancer exists, the tumor volume and blood perfusion of femoral metastases are significantly reduced, while the density of tumor blood vessels metastasized to the bone after resection of the primary lesion is significantly increased, suggesting that the primary lesion of breast cancer has an inhibitory effect on the angiogenesis of distant metastases. However, it is not clear what mechanism regulates distant metastases of breast cancer, how this mechanism is related to the primary lesion, and how the primary lesion regulates this mechanism. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a research method for the regulatory mechanism of primary breast cancer lesions on distant metastases, so as to explore the important mechanism by which primary breast cancer lesions inhibit the growth of distant metastases, that is, tissue exosomes released by primary breast cancer lesions carry miRNA-210 signaling molecules to induce autophagy of breast cancer cells in metastatic lesions, thereby inhibiting cell proliferation.

[0005] The technical scheme of the present invention to achieve the above-mentioned purpose is as follows: A method for studying the regulatory mechanism of primary breast cancer lesions on distant metastatic lesions, comprising:

[0006] An animal model of distant metastasis of breast cancer was constructed. After the model was successfully constructed, exosomes from primary breast cancer tissue were extracted. The differences in the expression of miRNAs signals carried by exosomes from primary breast cancer tissue were detected and compared, and the target molecule miRNA-210 was screened out.

[0007] The miRNA-210 intervention lentivirus was used to transfect breast cancer cells, and the changes in Bcl-2 protein expression were detected. The Bcl-2 gene luciferase reporter gene plasmid vector containing the miRNA-210 binding site was constructed. After transfection into breast cancer cells, the luciferase reporter gene detection experiment was performed to determine the direct regulatory relationship between miRNA-210 and Bcl-2 gene.

[0008] A nude mouse tumor-bearing animal model was constructed, and exosomes from primary lesion tissue obtained from breast cancer distant metastasis animal models with different pretreatment sources were injected into the tumor center. The changes in tumor volume were recorded, and tumor blocks were taken to detect tumor cell autophagy and cell proliferation.

[0009] The luciferase reporter gene assay was used to determine the effect of exosomal miRNA-210 from primary lesion tissue on breast cancer cell proliferation in vivo, and whether this effect was related to the regulated target cell autophagy.

[0010] As a preferred method for studying the regulatory mechanism of primary breast cancer on distant metastases, breast cancer cells are transfected with lentiviruses that silence or overexpress miRNA-210, silence or overexpress Bcl-2, and simultaneously silence or overexpress miRNA-210 and Bcl-2; after successful transfection, the breast cancer distant metastasis animal model is constructed.

[0011] As a preferred method for studying the regulatory mechanism of primary breast cancer on distant metastases, qRT-PCR and gene chips were used to detect and compare the expression differences of miRNAs signals carried in exosomes of primary tissues. After chip quality evaluation, the result data were analyzed to screen the miRNAs with the most significant expression differences.

[0012] As a preferred method for studying the regulatory mechanism of primary breast cancer on distant metastases, the extracted tissue exosomes were co-cultured with breast cancer cells and clone formation experiments were performed:

[0013] The cells in the co-culture system were treated and inoculated on well plates. After culturing for 2 weeks, they were stained with 0.1% crystal violet and the number of cell clones formed was calculated to verify the effect of exosomes from primary lesion tissue on the proliferation ability of breast cancer cells.

[0014] As a preferred method for studying the regulatory mechanism of primary breast cancer on distant metastases, a cell scratch test is also included: the width of the cell scratch is detected at 0h, 24h, and 48h to verify the effect of primary tissue exocrine secretion on the migration ability of breast cancer cells.

[0015] As a preferred method for studying the regulatory mechanism of primary breast cancer on distant metastases, Transwell migration and invasion experiments are also included: the number of cells penetrating the chamber is detected at 0h, 24h, and 48h to verify the effect of primary tissue exocrine secretion on the migration and invasion ability of breast cancer cells.

[0016] As a preferred method for studying the regulatory mechanism of primary breast cancer on distant metastases, after adding autophagy agonists or autophagy inhibitors to the culture system, the occurrence of cell autophagy in the culture system and the effect on cell proliferation ability were detected to determine whether the exosomal miRNA-210 signal in the primary tissue inhibits the proliferation of breast cancer cells by regulating breast cancer cell autophagy.

[0017] As a preferred method for studying the regulatory mechanism of primary breast cancer lesions on distant metastases, it also includes electron microscopic observation of autophagosomes: detecting the morphology of cellular autophagic ultrastructure through transmission electron microscopy.

[0018] As a preferred method for studying the regulatory mechanism of primary breast cancer lesions on distant metastases, it also includes dual fluorescence detection of autophagy activity and autophagy flow: using the autophagy dual fluorescence detection system, performing fluorescence detection according to the experimental process, and observing and detecting mRFP-LC3 and GFP-LC3 fluorescence staining under a fluorescence microscope to determine changes in cellular autophagy activity and autophagy flow.

[0019] As a preferred method for studying the regulatory mechanism of primary breast cancer lesions on distant metastases, it also includes Western Blot detection related to autophagy formation: detecting the protein expression of LC3-I, LC3-II, and Bcl-2 in each group of cells to evaluate autophagy formation.

[0020] The beneficial effects of the present invention are as follows: by constructing an animal model of distant metastasis of breast cancer, extracting exosomes from primary lesion tissue of breast cancer after successful construction, detecting and comparing the expression differences of miRNAs signals carried in the exosomes of primary lesion tissue, and screening out the target molecule miRNA-210; interfering with miRNA-210 by lentivirus transfection of breast cancer cells, detecting the changes in the expression of Bcl-2 protein, constructing a Bcl-2 gene luciferase reporter gene plasmid vector containing a miRNA-210 binding site, and performing a luciferase reporter gene detection experiment after transfection of breast cancer cells to determine the direct regulatory relationship between miRNA-210 and Bcl-2 gene; constructing a nude mouse tumor-bearing animal model, injecting primary lesion tissue exosomes obtained from distant metastasis animal models of breast cancer from different pretreatment sources into the center of the tumor, recording the changes in tumor volume, and taking tumor blocks to detect the autophagy of tumor cells and cell proliferation; and using a luciferase reporter gene detection experiment to determine the effect of exosome miRNA-210 of primary lesion tissue on the proliferation of breast cancer cells in the in vivo environment, and whether this effect is related to the autophagy of the regulated target cells. The present invention uses tissue exosomes to study the signal transmission from primary breast cancer lesions to distant metastases, which helps to solve the key and difficult problem of judging and monitoring regulatory molecules between primary lesions and distant metastases, and further provides an objective basis for clinical prevention and treatment of distant metastasis of breast cancer by regulating autophagy, making the comprehensive treatment of breast cancer more precise and individualized, reducing unnecessary trauma to patients and social health and economic burdens, and ultimately benefiting a wider range of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0022] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0023] Figure 1 A schematic diagram of a regulatory mechanism designed for a method for studying the regulatory mechanism of a primary breast cancer lesion on a distant metastasis provided in an embodiment of the present invention;

[0024] Figure 2A schematic diagram of a technical route for a method for studying the regulatory mechanism of a primary breast cancer lesion on a distant metastasis provided in an embodiment of the present invention;

[0025] Figure 3 A diagram of constructing a breast cancer lung metastasis model in the method for studying the regulatory mechanism of primary breast cancer on distant metastases provided in an embodiment of the present invention;

[0026] Figure 4 This is a diagram of the extraction of exosomes from primary breast cancer lesions in the method for studying the regulatory mechanism of primary breast cancer lesions on distant metastases provided in an embodiment of the present invention;

[0027] Figure 5 This is an autophagy diagram of breast cancer cells after co-culturing exosomes from primary lesion tissue with breast cancer cells in the method for studying the regulatory mechanism of primary breast cancer lesions on distant metastases provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0028] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0030] Tissue exosomes are a newly discovered way of signal transduction between tissue cells. Unlike the single cell information contained in previous exosomes, they represent the total secretion of exosomes from each cell in the real in vivo microenvironment of the tumor. They contain a richer source of tumor cell information, not only the information of tumor cells in tumor tissues, but also the information of the tumor microenvironment composed of structural cells and immune cells in tumor tissues. Current studies have found that tissue exosomes play an important role in the process of tumor occurrence and development. Tissue exosome molecular signals in peripheral blood have the characteristics of wide distribution, stable expression, and easy detection, which have unique advantages in disease diagnosis and monitoring. Autophagy has an important influence on the malignant proliferation of tumor cells, but its direction of action is variable. Using tissue exosomes to study the signal transmission from primary breast cancer lesions to distant metastases will help solve the key and difficult problem of judging and monitoring regulatory molecules between primary lesions and distant metastases, and thus provide an objective basis for clinical prevention and treatment of distant metastasis of breast cancer by regulating autophagy.

[0031] Tissue exosomes are small vesicles of about 30-150nm in the gaps between tumor tissues. They fully reflect the secretion of exosomes in the tumor microenvironment composed of tumor cells, structural cells and immune cells. They carry the contents of the vesicles to the target cells to achieve intercellular signaling. When tissue exosomes containing TrpC5 are used as signaling mediators, they can transfer chemical resistance to non-chemically resistant receptor breast cancer cells, promote the drug resistance of breast cancer cells and increase their proliferation ability. Tissue exosomes in gastric cancer tissue induce neutrophil autophagy by transmitting HMGB1 / TLR4 / NF-κB signals, promoting the malignant proliferation of gastric cancer cells. In addition, tumor cells in distant metastatic lesions can also serve as receivers of tissue exosome signals, receiving signals from primary lesions and regulating the malignant growth of tumors. Therefore, the embodiments of the present invention believe that tissue exosomes, as carriers of signal transmission between tissue cells, participate in the regulation of primary breast cancer lesions on distant metastases. However, what are the tissue exosome signals released by primary breast cancer lesions, whether these tissue exosome signals are received by breast cancer cells in distant metastases and inhibit their malignant growth, and what is the specific mechanism, need further study.

[0032] By focusing on the functional signaling molecules carried by tissue exosomes. As carriers of extracellular functional signals, exosomes produce their biological functions from the vesicle contents they carry. microRNA (miRNA) is one of the important components of the vesicle contents of tissue exosomes, and is widely involved in tumor malignant proliferation and multiple biological processes. Previous studies have found that miR-210 is abnormally expressed in exosomes from primary breast cancer tissues. Since the signaling molecules carried by exosomes are derived from cell synthesis, miRNA molecules that show differential expression in exosomes from primary tissues may be involved in regulating the malignant growth of distant metastases. However, whether miR-210 is involved in the signal transduction of exosomes in primary breast cancer tissues, and the relationship and mechanism between its signal transduction and the malignant growth of distant metastases are issues that need to be explored in depth.

[0033] Autophagy is the process by which eukaryotic cells use lysosomes to digest their own structures, but excessive autophagy can lead to cell death. The entire process of autophagy is regulated by autophagy-related genes (ATG), among which the protein Beclin-1 encoded by ATG6 is one of the key proteins in the classic regulatory pathway of autophagy. Under normal circumstances, Beclin-1 and Bcl-2 proteins are in a state of continuous binding complex, which inhibits the former's autophagy-promoting function. When this complex state is broken, free Beclin-1 will be captured by other core proteins that form autophagic vesicles and promote the autophagy process. Studies have found that tumor autophagy can promote tumor cell death and inhibit tumor cell proliferation and growth, and miR-210 can significantly promote cell autophagy. At the same time, miR-210 has been confirmed to be an upstream negative regulator of Bcl-2, participating in the regulation of cell autophagy, and cell autophagy can inhibit the malignant proliferation of tumor cells. However, the role of miR-210 in the inhibition of distant metastasis by exosomes from primary breast cancer tissue has not been published in the literature.

[0034] Based on the above theories and research foundations, see Figure 1 The present invention believes that in the process of primary breast cancer lesions inhibiting the growth of distant metastatic lesions: the primary breast cancer lesions release tissue exosomes, and the miRNA-210 signaling molecules carried by these exosomes are received by tumor cells in distant metastatic lesions and induce cell autophagy to inhibit their proliferation.

[0035] See also Figure 2 , Figure 3 , Figure 4 and Figure 5In view of this, in order to reveal that tissue exosomes released by primary breast cancer lesions carry miRNA-210 signaling molecules to induce autophagy of breast cancer cells in metastatic lesions, thereby inhibiting cell proliferation is an important mechanism by which primary breast cancer lesions inhibit the growth of distant metastatic lesions; to explore whether tissue exosomes and miRNA-210 released by primary breast cancer lesions can be used as important targets for effectively predicting and intervening in distant metastasis of breast cancer, the embodiment of the present invention provides a method for studying the regulatory mechanism of primary breast cancer lesions on distant metastatic lesions, including the following steps:

[0036] An animal model of distant metastasis of breast cancer was constructed. After the model was successfully constructed, exosomes from primary breast cancer tissue were extracted. The differences in the expression of miRNAs signals carried by exosomes from primary breast cancer tissue were detected and compared, and the target molecule miRNA-210 was screened out.

[0037] The miRNA-210 intervention lentivirus was used to transfect breast cancer cells, and the changes in Bcl-2 protein expression were detected. The Bcl-2 gene luciferase reporter gene plasmid vector containing the miRNA-210 binding site was constructed. After transfection into breast cancer cells, the luciferase reporter gene detection experiment was performed to determine the direct regulatory relationship between miRNA-210 and Bcl-2 gene.

[0038] A nude mouse tumor-bearing animal model was constructed, and exosomes from primary lesion tissue obtained from breast cancer distant metastasis animal models with different pretreatment sources were injected into the tumor center. The changes in tumor volume were recorded, and tumor blocks were taken to detect tumor cell autophagy and cell proliferation.

[0039] The luciferase reporter gene assay was used to determine the effect of exosomal miRNA-210 from primary lesion tissue on breast cancer cell proliferation in vivo, and whether this effect was related to the regulated target cell autophagy.

[0040] In this example, the phenomenon of the inhibition of distant metastatic growth induced by differential expression signals of exosome-miRNA in primary breast cancer tissues was first observed and the mechanism was studied.

[0041] Specifically, refer to the previous experimental basis and previous literature reports (Nat Rev Cancer.

[0042] 2007;7(9):659-72.), GFP-MDA-MB-231 breast cancer cells (2×107) were subcutaneously injected into BALB / c nude mice to establish a spontaneous breast cancer lung metastasis model, see Figure 3 The results of in vivo imaging of the animal model showed that the distant metastasis model of breast cancer (lung metastasis) was successfully established. After the successful establishment of the model, the following operations were performed:

[0043] (11) Isolation, culture and identification of exosomes from primary breast cancer lesions:

[0044] Tissue exosomes were isolated and extracted from primary breast cancer lesions. The extracted exosome precipitate was diluted with an equal amount of PBS solution and then subjected to the following exosome identification experiments:

[0045] Electron microscopy: Observe the size and structure of vesicles in the extract to see if they meet the characteristics of exosomes.

[0046] Western Blot method: Detect the expression of exosome marker proteins CD63 and CD81 in the extract.

[0047] See also Figure 4 , for the extraction of exosomes from primary breast cancer tissues, electron microscopic observation and Western Blot detection of the expression of tissue exosome markers CD81 and CD63; breast cancer cell lysate was taken as the control group. The results showed that the extracts were indeed tissue exosomes.

[0048] After the above experiments confirmed that the extracts were indeed tissue exosomes, equal volumes of diluted exosome suspensions were taken to count exosomes for subsequent experiments. (This part of the experiment was completed by Beijing Enze Kangtai Biotechnology Co., Ltd.).

[0049] (12) Detection of differential miRNA signals carried by exosomes in primary breast cancer tissues:

[0050] The exosomes extracted from the primary breast cancer tissue were used to detect the expression of miRNAs in the molecular signals carried by the gene chip. After the chip quality assessment, the result data was analyzed to screen the miRNAs with the most significant expression differences, among which the differential expression of miR-210 was verified. (This part of the experiment was completed by Beijing Enze Kangtai Biotechnology Co., Ltd.)

[0051] (13) Detection of the growth of lung metastases after resection of the primary breast cancer lesion:

[0052] The primary breast cancer lesion was surgically removed, and the growth of lung metastases was observed. Tissue exosomes were labeled with PKH-67 fluorescent dye and then injected into the animal model via tail vein after the primary lesion was removed. The growth of metastases was observed using animal in vivo imaging technology.

[0053] (14) The extracted exosomes from primary breast cancer tissue were added to the culture medium of human breast cancer cell line to conduct the following experimental tests:

[0054] Clone formation experiment: Breast cancer cells were treated and seeded on 6-well plates. After culturing for 2 weeks, they were stained with 0.1% crystal violet and the number of breast cancer cell clones was counted to verify the effect of exosomes from primary lesion tissue on the proliferation ability of breast cancer cells.

[0055] Cell scratch test: The width of cell scratches was detected at 0h, 24h, and 48h to verify the effect of primary lesion tissue exocrine secretion on the migration ability of breast cancer cells.

[0056] Transwell migration and invasion assay: The number of cells penetrating the chamber was detected at 0h, 24h, and 48h to verify the effect of primary tissue exocrine secretion on the migration and invasion ability of breast cancer cells.

[0057] The above experiments were used to detect the effects of exosomes from primary lesion tissue on the proliferation, apoptosis, migration and invasion of breast cancer cells.

[0058] In this example, we investigated whether tissue exosomes carrying miRNA-210 signals affect breast cancer cell proliferation by regulating cell autophagy and explored the specific mechanism.

[0059] Specifically, a luciferase reporter gene plasmid vector of the Bcl-2 gene (wild type and mutant) containing the miR-210 binding site was constructed and identified (the technology of this part of the experiment is mature and can be completed by the company on a commercial basis), the wild-type or mutant luciferase reporter plasmid and the miR-210 mimic and mimic NC predicted to have binding sites were co-transfected into breast cancer cells, and the firefly and Renilla luciferase activities were detected. The final activity values ​​of different co-transfection groups were used to evaluate the regulatory relationship between the miR-210 and Bcl-2 genes.

[0060] Among them, after constructing lentiviral vectors for silencing or overexpressing miRNA-210, silencing or overexpressing Bcl-2, and silencing or overexpressing both at the same time and transfecting GFP-MDA-MB-231 breast cancer cells, qRT-PCR was used to detect whether the expression of miRNA-210 and Bcl-2 was consistent with expectations. After successful transfection, the above GFP-MDA-MB-231 (2×107) was subcutaneously injected into BALB / c nude mice to establish a spontaneous breast cancer lung metastasis model. After the model was successfully established, the following operations were performed:

[0061] (21) The exosomes extracted from the primary lesion tissues of each group were co-cultured with breast cancer cells to detect the differences in the occurrence of cell autophagy:

[0062] a1 Electron microscopic observation of autophagosomes: The morphology of the ultrastructure of cell autophagy is detected by transmission electron microscopy. Under the electron microscope, the occurrence of autophagy can be judged based on various characteristics of autophagy (such as autophagosomes formed by vacuolar double-layer membrane-like structures, autophagolysosomes whose cytoplasm has been degraded by single-layer membrane structures, etc.).

[0063] b1 Dual fluorescence detection of autophagy activity and autophagy flow: Use the autophagy dual fluorescence detection system to perform fluorescence detection according to the experimental process, and observe and detect mRFP-LC3 and GFP-LC3 fluorescence staining under a fluorescence microscope to determine the changes in cell autophagy activity and autophagy flow.

[0064] c1 Western Blot detection related to autophagy formation: The protein expressions of LC3-I, LC3-II, and Bcl-2 in each group of cells were detected to evaluate the formation of autophagy.

[0065] (22) The exosomes extracted from the primary lesion tissues of each group were co-cultured with breast cancer cells, and the occurrence of cellular autophagy and the differences in cell proliferation ability were detected after adding autophagy agonists (rapamycin) or autophagy inhibitors (3-MA) ​​to the culture system:

[0066] a2 detection method is the same as a1.

[0067] b2 Clone formation experiment: After treatment, cells were inoculated on 6-well plates, and after 2 weeks of culture, they were stained with 0.1% crystal violet and the number of cell clones was counted to verify the effect of exosomes from primary lesion tissue on the proliferation ability of breast cancer cells. Figure 5 After breast cancer cells were co-cultured with exosomes from primary lesion tissue, their autophagy level was significantly increased, while Bcl-2 expression was decreased.

[0068] In this example, spontaneous breast cancer lung metastasis models of different treatment groups were constructed, and exosomes from primary lesion tissues of each group were extracted after successful modeling. A nude mouse tumor-bearing animal model was then constructed, and tissue exosomes extracted from different groups (primary lesion tissue exosomes, primary lesion tissue exosomes after regulating miR-210 expression) were injected into the tumor center of the nude mouse, and then the changes in tumor volume were recorded. After the feeding was completed, the tumor block was taken to detect tumor cell autophagy and cell proliferation:

[0069] (31) Western Blot detection related to autophagy formation: After the cells were collected, the protein expressions of LC3-I, LC3-II, and Bcl-2 in each group of cells were detected by Western Blot to evaluate the formation of autophagy.

[0070] (32) Immunohistochemical detection of the expression of proliferation-related indicators: KI-67, Bcl-2, Bax, etc.

[0071] In this example, a dual luciferase vector construction experiment report was conducted. The PCR method was used to design the amplification primers according to the BCL-2 (human) 3'UTR sequence information. The 3'UTR sequence of the BCL-2 gene was amplified by PCR using 293T genomic DNA as a template and cloned into pmiR-RB-REPORT. TMIn the dual luciferase reporter vector, the reporter fluorescence of the vector used is the Renilla luciferase gene (hRluc), and the firefly luciferase gene (hLuc) is used as the internal reference gene. The relative changes in the activity of Renilla luciferase are detected to identify the regulatory effect of miRNA on the gene. The experiment showed that the exosomal miRNA-210 of the primary lesion tissue has an effect on the proliferation of breast cancer cells in the in vivo environment, and this effect is related to the autophagy of the regulated target cells.

[0072] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0073] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of exosome miR-210 from primary breast cancer tissue in the preparation of drugs for inhibiting breast cancer lung metastasis.

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