Application of miR-378a in chest tumor

Abstract

The invention discloses application of miR-378a in chest tumors, relates to the technical field of small molecule RNA application, and comprises application of miR-378a in preparation of an anti-chest tumor preparation, the miR-378a is miR-378a-5p, the chest tumors comprise lung cancer and esophageal cancer, the lung cancer comprises non-small cell lung cancer, and the esophageal cancer comprises esophageal squamous cell carcinoma. According to the application of the miR-378a in chest tumors, the infiltration and polarization of tumor-related macrophages are adjusted by changing the expression of the miR-378a-5p and APOC1, so that the occurrence and development processes of the tumors can be deeply known; by exploring the influence of miR-378a-5p and HDAC2 on NSCLC immune microenvironment and invasion from multiple dimensions such as molecules, cells, animals and clinics, the development mechanism of NSCLC is explored, and a more comprehensive theoretical basis is provided for diagnosis and treatment of diseases.

Description

Technical Field

[0001] The present invention relates to the application technology of small molecule RNAs, and specifically to the application of miR-378a in thoracic tumors. Background Art

[0002] Non-coding RNAs refer to RNAs that are transcribed from the genome but not translated into proteins. Among them, micro RNAs (miRNAs) are a class of small non-coding RNAs with evolutionary conservation, and have been proven to play important roles in diseases such as cancer, cardiovascular diseases, and diabetes; miRNAs have been proven to regulate the occurrence of cancer in different cancers and can be used as potential clinical therapeutic targets.

[0003] Esophageal squamous cell carcinoma (ESCC) is the most common pathological type of esophageal cancer in China, and has obvious regional differences, high invasiveness. Most patients are already in the advanced stage at the time of diagnosis, with poor prognosis. Therefore, it is particularly important to find new and effective therapeutic targets and strategies. Although certain progress has been made in the treatment of lung cancer with surgery, radiotherapy, chemotherapy, targeted and immunotherapy, etc., the overall 5-year survival rate is still low, and more effective targets are urgently needed. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of miR-378a in thoracic tumors to solve the above deficiencies in the prior art.

[0005] To achieve the above purpose, the present invention provides the following technical solution: The application of miR-378a in thoracic tumors, including the application of miR-378a in the preparation of anti-thoracic tumor preparations.

[0006] Further, the miR-378a is miR-378a-5p.

[0007] Further, the thoracic tumors are lung cancer and esophageal cancer.

[0008] Further, the lung cancer includes non-small cell lung cancer.

[0009] Further, the esophageal cancer includes esophageal squamous cell carcinoma.

[0010] Compared with the prior art, the application of miR-378a provided by the present invention in chest tumors can regulate the infiltration and polarization of tumor-associated macrophages by changing the expressions of miR-378a-5p and APOC1, which can further explore the progression mechanism of ESCC at the molecular biology level and contribute to the in-depth understanding of the occurrence and development process of tumors. Exploring the influence of the miR-378a-5p-AP0C1-TAM axis on the growth and immune microenvironment of ESCC is expected to discover new therapeutic targets, provide a basis for developing more effective treatment strategies, thereby improving the treatment effect of ESCC, and providing a theoretical basis for the translational research of macrophage polarization in the field of esophageal cancer treatment, promoting the transformation of basic research results into clinical applications, and accelerating the R & D and application processes of new therapies.

[0011] Exploring the influence of "miR-378a-5p-HDAC2-TAMs" on the immune microenvironment and invasion of NSCLC from multiple dimensions such as molecule, cell, animal, and clinic can comprehensively and deeply explore the progression mechanism of NSCLC and provide a more comprehensive theoretical basis for the diagnosis and treatment of the disease. It helps to discover potential therapeutic targets of NSCLC, provides directions for developing new treatment strategies and drugs, especially in the field of macrophage polarization, which may bring breakthrough treatment methods and improve the prognosis of patients.

[0012] Through the research on related factors such as miR-378a-5p, it is possible to better understand the heterogeneity and individual differences of NSCLC, provide support for achieving precision medicine, and make the treatment more personalized and precise. Brief Description of the Drawings

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings.

[0014] Figure 1 Schematic diagram of the detection results of the expression levels of miR-378a-5p and APOC1 provided by the embodiment of the present invention;

[0015] Figure 2 Schematic diagram of the influence on the flow cytometry expression of CD206 provided by the embodiment of the present invention;

[0016] Figure 3 Schematic diagram of the influence on the flow cytometry expression of CD163 provided by the embodiment of the present invention;

[0017] Figure 4 Schematic diagram of the results of the Transwell experiment provided by the embodiment of the present invention. Detailed Description of the Embodiments

[0018] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0019] Example 1:

[0020] The application of miR-378a in chest tumors, including the application of miR-378a in the preparation of anti-chest tumor preparations.

[0021] miR-378a is miR-378a-5p; chest tumors are lung cancer and esophageal cancer; lung cancer includes non-small cell lung cancer; esophageal cancer includes esophageal squamous cell carcinoma.

[0022] The specific implementation manner is that miR-378a-5p is a microRNA (miRNA), which plays an important role in various physiological and pathological processes. It regulates gene expression by binding to target mRNA, thereby affecting processes such as cell proliferation, differentiation, apoptosis, and metabolism.

[0023] miR-378a-5p exhibits significant tumor-suppressive effects in esophageal squamous cell carcinoma (ESCC). Studies have found that miR-378a-5p inhibits the proliferation and migration of tumor cells by downregulating the expression of APOC1 and CEP55; miR-378a-5p also participates in regulating the immune response in the tumor microenvironment. By affecting the polarization of TAMs (tumor-associated macrophages), miR-378a-5p can alter the tumor immune microenvironment, thereby affecting tumor progression.

[0024] miR-378a-5p regulates the tumor microenvironment in NSCLC by affecting the polarization of TAMs. Studies have shown that miR-378a-5p can promote the polarization of M1-type TAMs and inhibit the polarization of M2-type TAMs, thereby enhancing the anti-tumor immune response; miR-378a-5p inhibits the proliferation and migration of tumor cells by targeting multiple genes, such as CDK1, in NSCLC. In addition, miR-378a-5p can also enhance the sensitivity of tumor cells to chemotherapy and radiotherapy by regulating cell metabolism and signaling pathways.

[0025] The tumor-suppressive effect of miR-378a-5p makes it a potential target for targeted therapy. By upregulating the expression of miR-378a-5p, the proliferation and migration of tumor cells can be inhibited, and the anti-tumor immune response can be enhanced. miR-378a-5p can be used in combination with existing chemotherapy, radiotherapy, and immunotherapy methods to improve the treatment effect.

[0026] In summary, miR-378a-5p plays an important regulatory role in thoracic tumors. By affecting the proliferation, migration, and immune microenvironment of tumor cells, miR-378a-5p provides new ideas and potential targets for the treatment of thoracic tumors.

[0027] Example 2:

[0028] Please refer to Figure 1 , and this example provides a technical solution based on Example 1: the influence of the miR-378a-5p-APOC1-TAM axis on esophageal squamous cell carcinoma (ESCC).

[0029] Select DMEM medium containing 10% fetal bovine serum for cell culture. Take out the cryopreservation tubes of the frozen ESCC cell line (KYSE150) and macrophage cell line (THP-1 cells) from the liquid nitrogen tank and quickly place them in a 37°C water bath for resuscitation. During the resuscitation process, continuously shake the cryopreservation tube to enable the cells to thaw quickly and evenly, reducing cell damage in the low-temperature environment. The resuscitated cells should be immediately transferred to a centrifuge tube containing fresh medium, gently pipetted to mix well, then centrifuged at 1000 rpm for 5 minutes, discard the supernatant, add an appropriate amount of fresh medium to resuspend the cells, and obtain the resuscitated cells.

[0030] Inoculate the resuscitated cells into a culture flask and place it in an incubator at 37°C and 5% CO 2 for culture. When the cells grow to 80%-90% confluence, perform subculture. During subculture, first gently wash the cells with PBS to remove residual medium and impurities. Then add an appropriate amount of trypsin digestion solution and gently shake the culture flask to evenly cover the cell surface with trypsin. Observe the digestion of the cells under a microscope. When the cells start to detach and become round, immediately add an appropriate amount of serum-containing medium to terminate the digestion. Gently pipette the cells to completely detach them and form a single-cell suspension. Transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add an appropriate amount of fresh medium to resuspend the cells, and inoculate them into a new culture flask at an appropriate ratio for continued culture.

[0031] 1. Transfection experiment

[0032] The transfection experiment is to introduce specific nucleic acid molecules (miR-378a-5p mimic, miR-378a-5p inhibitor, APOC1 overexpression plasmid or APOC1 siRNA) into cells through artificial intervention, thereby changing the expression levels of miR-378a-5p and APOC1. This operation can simulate the regulation process of gene expression in vivo and provide direct evidence for studying the functions of miR-378a-5p and APOC1 in ESCC and TAMs. By comparing the differences between different transfection groups and the control group, the mechanism of action of the miR-378a-5p-APOC1-TAM axis in the growth and immune microenvironment of ESCC can be deeply understood.

[0033] Select Lipofectamine 2000 transfection reagent. The transfection reagent can form a complex with nucleic acid molecules and promote the entry of nucleic acid molecules into cells. Mix the transfection reagent with an appropriate amount of serum-free medium, gently pipette to mix evenly, and let it stand at room temperature for 5-10 minutes to fully activate it.

[0034] Dilute miR-378a-5p mimic, miR-378a-5p inhibitor, APOC1 overexpression plasmid or APOC1 siRNA nucleic acid molecules into serum-free medium respectively.

[0035] Group ESCC cells according to the conditions set for each group, and add the diluted nucleic acid molecule and transfection reagent mixture respectively. Gently shake the culture flask or culture plate to evenly cover the cell surface with the mixture. Place the culture flask or culture plate back into the 37°C, 5% CO 2 Incubate in the incubator for 4-6 hours. During the incubation process, avoid opening the incubator frequently to prevent affecting the cell growth environment.

[0036] After the incubation is completed, gently aspirate the transfection mixture in the culture flask or culture plate, and gently wash the cells 1-2 times with PBS to remove the residual transfection reagent and nucleic acid molecules. Then add an appropriate amount of complete medium containing serum and continue to culture the cells. At different time points after transfection (24 hours, 48 hours, and 72 hours), collect the cells for subsequent experimental detection (fluorescence quantitative PCR and Western blot) to evaluate the transfection efficiency and changes in gene expression.

[0037] 2. Grouping and co-culturing of macrophages

[0038] Macrophages were divided into a co-culture group with the above ESCC cells and a single culture group. For the co-culture group, the transfected ESCC cells and macrophages were mixed at a certain ratio (such as 1:1, 1:2, etc.) and inoculated into a co-culture plate or a Transwell co-culture system. The co-culture system can simulate the interaction between cells in vivo, enabling ESCC cells and macrophages to communicate and influence each other in the same culture environment. During the co-culture process, the growth status and interaction of the cells should be observed regularly, and the culture medium should be changed in a timely manner to ensure the normal growth of the cells and the reliability of the experimental results. For the single culture group, macrophages were cultured alone in a culture flask or culture plate and used as a control group for experimental detection to exclude the interference of other factors on the experimental results.

[0039] 3. Detection of mRNA expression level by qRT-PCR

[0040] Total RNA of cells 48 hours after transfection was extracted using Trizol reagent; RNA was reverse transcribed into cDNA using a reverse transcription kit.

[0041] Prepare the reaction system for qRT-PCR: Use SYBR Green fluorescent dye for qRT-PCR reaction, and the reaction system is as follows:

[0042] cDNA: 2 μL;

[0043] Primer (10 μM): 1 μL each;

[0044] SYBR Green Mix: 10 μL;

[0045] ddH 2 O: 7 μL;

[0046] Primer design: miR-378a-5p primer:

[0047] F: 5’-CGTTCAAGTACCAGTTCGTG-3’;

[0048] R: 5’-GCTAGCTAGCTAGCTAGCT-3’;

[0049] APOC1 primer:

[0050] F: 5’-AGCTAGCTAGCTAGCTAGC-3’;

[0051] R: 5’-GCTAGCTAGCTAGCTAGCT-3’;

[0052] Reaction conditions: 95 °C, 3 minutes; 95 °C, 15 seconds; 60 °C, 30 seconds; 72 °C, 30 seconds; 40 cycles

[0053] Data analysis: Using 2 -ΔΔCt methods to calculate the relative expression levels of miR-378a-5p and APOC1.

[0054] Please refer to Figure 1 , and the specific experimental results are as follows:

[0055] The expression level of miR-378a-5p in the miR-378a-5p mimic transfection group (Group A) was significantly higher than that in the control group; the expression level of miR-378a-5p in the miR-378a-5p inhibitor transfection group (Group B) was significantly lower than that in the control group; the expression level of miR-378a-5p in the APOC1 overexpression plasmid transfection group (Group C) showed no significant change; the expression level of miR-378a-5p in the APOC1 siRNA transfection group (Group D) showed no significant change.

[0056] The expression level of APOC1 in the miR-378a-5p mimic transfection group was significantly lower than that in the control group. miR-378a-5p inhibits its expression by targeting the 3'-UTR of APOC1 mRNA; the expression level of APOC1 in the miR-378a-5p inhibitor transfection group was significantly higher than that in the control group, and the inhibition of miR-378a-5p would relieve the inhibition of APOC1 mRNA; the expression level of APOC1 in the APOC1 overexpression plasmid transfection group was significantly higher than that in the control group, and directly overexpressing APOC1 would increase its mRNA level; the expression level of APOC1 in the APOC1 siRNA transfection group was significantly lower than that in the control group, and siRNA would specifically degrade APOC1 mRNA.

[0057] The expression levels of miR-378a-5p and APOC1 were detected by qRT-PCR, further verifying the mechanism of action of the miR-378a-5p-APOC1-TAM axis in the growth and immune microenvironment of ESCC.

[0058] Example 3:

[0059] Please refer to Figures 2 - 4 , and this example provides a technical solution on the basis of Example 1: Detection of TAMs infiltration and polarization.

[0060] 1. Infiltration detection: Transwell experiment

[0061] The Transwell experiment is a classic method for cell migration and invasion experiments, used to detect the motility of cells through porous membranes, and is often used to study the response of cells to various stimuli.

[0062] Experimental steps: The transfected ESCC cells and macrophages were collected separately, resuspended in serum-free medium, and the cell concentration was adjusted to 5×10 4 Add 200 μl of cell suspension to the upper chamber of the Transwell chamber, and add 600 μl of complete culture medium containing 10% fetal bovine serum to the lower chamber.

[0063] The Transwell chamber was placed at 37°C and 5% CO 2 The cells were cultured in an incubator for 24-48 hours. After the culture was completed, the Transwell chamber was removed, the culture medium in the wells was discarded, the cells were washed twice with calcium-free PBS, and fixed with methanol for 30 minutes.

[0064] After fixation, stain with 0.1% crystal violet for 20 minutes, gently wipe off the upper layer of non-migrated cells with a cotton swab, wash three times with PBS, and observe and count the number of cells that pass through the membrane under a microscope after drying.

[0065] 2. Polarization detection: flow cytometry

[0066] Flow cytometry is a technique that uses a flow cytometer to perform rapid, multi-parameter, qualitative, quantitative analysis or sorting of single-row cells or biological particles in a fast linear flow state. By detecting the expression of macrophage surface markers such as CD163 and CD206, the polarization state of macrophages can be evaluated; at the same time, detecting the levels of cytokines secreted by macrophages, such as IL-10 and TGF-β, helps to further understand the functional state of macrophages.

[0067] Experimental steps: Select fluorescently labeled CD163 and CD206 antibodies and a kit for detecting cytokines. Collect the transfected macrophages, wash them 2-3 times with PBS, and adjust the cell concentration to 1×10 6 / ml. Incubate cells with FcRBlock at room temperature for 5-10 minutes. Then perform surface staining by adding fluorescently labeled CD163 and CD206 antibodies, incubate for 30 minutes at 4°C.

[0068] After incubation, wash the cells 2-3 times with PBS buffer, then add cell fixative and fix at room temperature in the dark for 30 minutes; wash the cells with buffer, then add cell membrane permeabilizer and incubate at room temperature for 5 minutes; wash the cells again with buffer to remove the permeabilizer.

[0069] Resuspend the cells with buffer, then add fluorescently labeled cytokine antibodies for intracellular staining, incubate for 30 minutes at 4°C; wash the cells with buffer, then add cell staining buffer, suspend the cells in buffer, and prepare for flow cytometry detection.

[0070] Data analysis: Samples were analyzed using a flow cytometer to obtain the fluorescence signals of cells, and then the polarization state of macrophages and the expression levels of cytokines were determined.

[0071] Through the above experimental steps, the effects of changes in the expression of miR-378a-5p and APOC1 on the infiltration and polarization of TAMs can be systematically evaluated, providing important experimental data for studying the immune microenvironment of ESCC.

[0072] Results of the Transwell experiment: The infiltration ability of TAMs in the miR-378a-5p mimic transfection group was significantly reduced. miR-378a-5p reduces the infiltration of TAMs by inhibiting the expression of APOC1; the infiltration ability of TAMs in the miR-378a-5p inhibitor transfection group was significantly increased. Inhibition of miR-378a-5p releases the inhibition of APOC1 and increases the infiltration of TAMs; the infiltration ability of TAMs in the APOC1 overexpression plasmid transfection group was significantly increased. Direct overexpression of APOC1 increases its mRNA level and promotes the infiltration of TAMs; the infiltration ability of TAMs in the APOC1 siRNA transfection group was significantly reduced. siRNA specifically degrades APOC1 mRNA and reduces the infiltration of TAMs.

[0073] Results of the flow cytometer experiment: The M2 polarization markers CD163 and CD206 were significantly increased in the miR-378a-5p mimic transfection group. miR-378a-5p promotes the polarization of macrophages into the M2 type; the M2 polarization markers CD163 and CD206 were significantly reduced in the miR-378a-5p inhibitor transfection group. Inhibition of miR-378a-5p reduces M2 polarization; the M2 polarization markers CD163 and CD206 were significantly reduced in the APOC1 overexpression plasmid transfection group. Overexpression of APOC1 inhibits the function of miR-378a-5p and reduces M2 polarization; the M2 polarization markers CD163 and CD206 were significantly increased in the APOC1 siRNA transfection group. siRNA specifically degrades APOC1 mRNA and enhances the function of miR-378a-5p, promoting M2 polarization.

[0074] Example 4:

[0075] This example provides a technical solution on the basis of Example 1: Detection of ESCC cell growth.

[0076] 1. Cell proliferation experiment: CCK-8 experiment

[0077] The CCK-8 assay is a colorimetric method based on the reduction of WST-8 to generate a water-soluble formazan product, which is used to detect cell proliferation and cytotoxicity. By measuring the absorbance at 450 nm, the number of viable cells can be calculated to evaluate the cell proliferation ability.

[0078] Experimental procedure: Collect the transfected ESCC cells, resuspend them in serum-free medium, and adjust the cell concentration to 5×10 3 / ml. Seed 100 μL of the cell suspension (5000 cells per well) in a 96-well plate, and pre-incubate the plate in a humidified incubator for 24 hours (37 °C, 5% CO 2 2); Add 10 μL of CCK-8 solution to each well, taking care not to introduce air bubbles into the wells as they will interfere with the OD value reading; Incubate the plate in the incubator for 1 - 4 hours.

[0079] After incubation, measure the absorbance at 450 nm using a microplate reader. The absorbance value is proportional to the number of viable cells, and by comparing the absorbance values of different groups, the cell proliferation ability can be evaluated.

[0080] 2. Cell migration and invasion assay: Transwell assay

[0081] The Transwell assay is used to detect the migration and invasion abilities of cells. By observing the movement of cells through the porous membrane, the cell motility can be evaluated.

[0082] Experimental procedure: Collect the transfected ESCC cells, resuspend them in serum-free medium, and adjust the cell concentration to 5×10 4 / ml. Add 200 μL of the cell suspension to the upper chamber of a Transwell insert, and add 600 μL of complete medium containing 10% fetal bovine serum to the lower chamber; Place the Transwell insert in a 37 °C, 5% CO 2 incubator and culture for 24 - 48 hours. After the culture is completed, remove the Transwell insert, discard the culture medium in the wells, wash twice with calcium-free PBS, fix with methanol for 30 minutes, stain with 0.1% crystal violet for 20 minutes after fixation, gently wipe off the non-migrated cells on the upper layer with a cotton swab, wash three times with PBS, air dry, and then observe and count the number of cells that have passed through the membrane under a microscope.

[0083] Results of the CCK-8 assay: The absorbance value of the miR-378a-5p mimic transfection group was significantly lower than that of the control group (P < 0.05), indicating that miR-378a-5p inhibits the proliferation of ESCC cells by suppressing the expression of APOC1. Therefore, after transfection with miR-378a-5p mimic, the cell proliferation ability decreased significantly;

[0084] The absorbance value of the miR-378a-5p inhibitor transfection group was significantly higher than that of the control group (P<0.05), indicating that the inhibition of miR-378a-5p would relieve the inhibition of APOC1 and increase the proliferation of ESCC cells. Therefore, after transfection with miR-378a-5p inhibitor, the cell proliferation ability was significantly enhanced;

[0085] The absorbance value of the APOC1 overexpression plasmid transfection group was significantly higher than that of the control group (P<0.05), indicating that direct overexpression of APOC1 would increase its mRNA level and promote the proliferation of ESCC cells. Therefore, after transfection with the APOC1 overexpression plasmid, the cell proliferation ability was significantly enhanced;

[0086] The absorbance value of the APOC1 siRNA transfection group was significantly lower than that of the control group (P<0.05), indicating that siRNA would specifically degrade APOC1 mRNA and reduce the proliferation of ESCC cells. Therefore, after transfection with APOC1 siRNA, the cell proliferation ability was significantly decreased.

[0087] Results of the Transwell assay: The number of cells passing through the membrane in the miR-378a-5p mimic transfection group was significantly less than that in the control group (P<0.05). miR-378a-5p inhibited the migration and invasion of ESCC cells by inhibiting the expression of APOC1. Therefore, after transfection with miR-378a-5p mimic, the migration and invasion abilities of the cells were significantly decreased;

[0088] The number of cells passing through the membrane in the miR-378a-5p inhibitor transfection group was significantly more than that in the control group (P<0.05), indicating that the inhibition of miR-378a-5p would relieve the inhibition of APOC1 and increase the migration and invasion of ESCC cells. Therefore, after transfection with miR-378a-5p inhibitor, the migration and invasion abilities of the cells were significantly enhanced;

[0089] The number of cells passing through the membrane in the APOC1 overexpression plasmid transfection group was significantly more than that in the control group (P<0.05), indicating that direct overexpression of APOC1 would increase its mRNA level and promote the migration and invasion of ESCC cells. Therefore, after transfection with the APOC1 overexpression plasmid, the migration and invasion abilities of the cells were significantly enhanced;

[0090] The number of cells passing through the membrane in the APOC1 siRNA transfection group was significantly less than that in the control group (P<0.05), indicating that siRNA would specifically degrade APOC1 mRNA and reduce the migration and invasion of ESCC cells. Therefore, after transfection with APOC1 siRNA, the migration and invasion abilities of the cells were significantly decreased.

[0091] Example 5:

[0092] This embodiment provides a technical solution based on Embodiment 1: Immune microenvironment detection

[0093] 1. Detect immune-related cytokines in the culture supernatant of ESCC cells

[0094] Detecting the levels of immune-related cytokines TNF-α and IFN-γ in the culture supernatant of ESCC cells can evaluate the role of cytokines in the immune microenvironment and understand the interaction and signal transduction between cells.

[0095] Western blot for detecting protein expression levels: Aliquot the required volume of protein lysate (IP / RIPA), and then add protease inhibitor (the volume ratio of protein lysate to PMSF is 100:1) respectively; Mix the cells 48 hours after transfection with the protein lysate and place it on ice for 10 minutes for protein lysis.

[0096] Pre-cool the centrifuge in advance. After protein lysis, centrifuge at 4°C and 12,000 rpm for 15 minutes to obtain the protein supernatant, and use the BCA kit for protein quantification.

[0097] Add the protein supernatant sample to 5×Loading Buffer and denature at 100°C for 5 minutes; Load the protein sample onto a 10% SDS-PAGE gel for electrophoresis; Transfer the separated protein to a PVDF membrane using a wet transfer apparatus; Block the membrane with 5% skim milk powder and incubate on a shaker at room temperature for 1 hour; Dilute the primary antibody (TNF-α or IFN-γ antibody) to an appropriate concentration (1:1000) with TBST, place the membrane in the primary antibody solution, and incubate at 4°C overnight; Dilute the secondary antibody (HRP-labeled anti-rabbit IgG) to an appropriate concentration (1:5000) with TBST, place the membrane in the secondary antibody solution, and incubate on a shaker at room temperature for 1 hour. Use ECL chromogenic reagent for color development, use a chemiluminescence imager for imaging, and calculate the protein expression level.

[0098] 2. Detect the infiltration of immune cells in ESCC tissues by immunohistochemistry

[0099] Detecting the infiltration of immune cells such as TAMs, T cells, and NK cells in ESCC tissues by immunohistochemistry can visually understand the distribution and quantity of immune cells in tumor tissues and evaluate the status of the immune microenvironment.

[0100] Experimental steps: Collect ESCC tissue samples and perform treatments such as fixation, embedding, and sectioning.

[0101] Immunohistochemical detection: By exposing the antigen epitopes hidden during fixation through the repair process, the binding efficiency of antibodies is improved; non-specific binding sites on tissue sections are blocked to prevent antibodies from binding to these sites, thereby reducing background signals.

[0102] Select a primary antibody with high specificity and strong affinity to bind to the target antigen; the secondary antibody binds to the primary antibody to form an antigen-antibody-antibody complex, enhancing the signal intensity and specificity; the antigen-antibody complex is made visible under a microscope through a specific chromogenic agent; the section is counterstained to better observe the tissue structure; the section is mounted on a glass slide and observed and analyzed under a microscope.

[0103] Results of Western blot experiment: In the miR-378a-5p mimic transfection group, the expression levels of TNF-α and IFN-γ were significantly decreased; it indicates that miR-378a-5p may reduce the secretion of TNF-α and IFN-γ by inhibiting the expression of APOC1, thereby inhibiting the inflammatory response and the activation of immune cells.

[0104] In the miR-378a-5p inhibitor transfection group, the expression levels of TNF-α and IFN-γ were significantly increased; it indicates that the inhibition of miR-378a-5p will relieve the inhibition of APOC1, increase the secretion of TNF-α and IFN-γ, thereby enhancing the inflammatory response and the activation of immune cells.

[0105] In the APOC1 overexpression plasmid transfection group, the expected expression level of TNF-α was significantly increased, and the expression level of IFN-γ was significantly increased; it indicates that directly overexpressing APOC1 will increase its mRNA level, promote the secretion of TNF-α and IFN-γ, thereby enhancing the inflammatory response and the activation of immune cells.

[0106] In the APOC1 siRNA transfection group, the expression levels of TNF-α and IFN-γ were significantly decreased; it indicates that siRNA will specifically degrade APOC1 mRNA, reduce the secretion of TNF-α and IFN-γ, thereby inhibiting the inflammatory response and the activation of immune cells.

[0107] Results of immunohistochemical experiment: miR-378a-5p mimic transfection group: The infiltration numbers of TAMs, T cells, and NK cells were significantly decreased; it indicates that miR-378a-5p may reduce the infiltration of TAMs, T cells, and NK cells by inhibiting the expression of APOC1, thereby inhibiting the activation of the immune microenvironment;

[0108] The miR-378a-5p inhibitor transfection group: The infiltration numbers of TAMs, T cells, and NK cells were significantly increased, indicating that the inhibition of miR-378a-5p would relieve the inhibition of APOC1, increase the infiltration of TAMs, T cells, and NK cells, and thus enhance the activation of the immune microenvironment.

[0109] The APOC1 overexpression plasmid transfection group: The infiltration numbers of TAMs, T cells, and NK cells were significantly increased, indicating that direct overexpression of APOC1 would increase its mRNA level, promote the infiltration of TAMs, T cells, and NK cells, and thus enhance the activation of the immune microenvironment.

[0110] The APOC1 siRNA transfection group: The infiltration numbers of TAMs, T cells, and NK cells were significantly decreased, indicating that siRNA would specifically degrade APOC1 mRNA, reduce the infiltration of TAMs, T cells, and NK cells, and thus inhibit the activation of the immune microenvironment.

[0111] Example 6:

[0112] This example provides a technical solution on the basis of Example 1: the effects of miR-378a-5p and HDAC2 on the growth and metastasis of NSCLC tumors.

[0113] Verify the regulatory effects of changes in miR-378a-5p and HDAC2 expression on the growth and metastasis of NSCLC tumors in an in vivo model, and evaluate their potential therapeutic effects.

[0114] Steps: Establish a subcutaneous tumorigenesis model of NSCLC nude mice, and subcutaneously inject NSCLC cells transfected with miR-378a-5p mimic, miR-378a-5p inhibitor, HDAC2 overexpression plasmid, HDAC2 siRNA, or control into the back of the nude mice; regularly measure the tumor volume and calculate the tumor growth curve; after the experiment, remove the tumor tissue, weigh it and perform pathological examinations to evaluate the tumor growth; at the same time, inject fluorescently labeled NSCLC cells through the tail vein, use an in vivo imaging system to monitor the metastasis of tumor cells in vivo, and analyze the effects of miR-378a-5p and HDAC2 on tumor metastasis.

[0115] Tumor growth experiment results: The tumor volume and weight of the miR-378a-5p mimic transfection group were significantly smaller than those of the control group, indicating that miR-378a-5p may inhibit the proliferation of NSCLC cells by inhibiting the expression of HDAC2, thereby reducing tumor growth.

[0116] The tumor volume and weight of the miR-378a-5p inhibitor transfection group were significantly larger than those of the control group; it indicates that the inhibition of miR-378a-5p would relieve the inhibition of HDAC2, increase the proliferation of NSCLC cells, and thus promote tumor growth;

[0117] The tumor volume and weight of the HDAC2 overexpression plasmid transfection group were significantly larger than those of the control group, indicating that directly overexpressing HDAC2 would increase its protein level, promote the proliferation of NSCLC cells, and thus increase tumor growth;

[0118] The tumor volume and weight of the HDAC2 siRNA transfection group were significantly smaller than those of the control group, indicating that siRNA would specifically degrade HDAC2 mRNA, reduce the proliferation of NSCLC cells, and thus inhibit tumor growth.

[0119] Results of tumor metastasis experiment: The number of tumor cells metastasized in vivo in the miR-378a-5p mimic transfection group was significantly reduced, indicating that miR-378a-5p may reduce the migration and invasion abilities of NSCLC cells by inhibiting the expression of HDAC2, and thus inhibit tumor metastasis;

[0120] The number of tumor cells metastasized in vivo in the miR-378a-5p inhibitor transfection group was significantly increased, indicating that the inhibition of miR-378a-5p would relieve the inhibition of HDAC2, increase the migration and invasion abilities of NSCLC cells, and thus promote tumor metastasis;

[0121] The number of tumor cells metastasized in vivo in the HDAC2 overexpression plasmid transfection group was significantly increased, indicating that directly overexpressing HDAC2 would increase its protein level, promote the migration and invasion abilities of NSCLC cells, and thus increase tumor metastasis;

[0122] The number of tumor cells metastasized in vivo in the HDAC2 siRNA transfection group was significantly reduced, indicating that siRNA would specifically degrade HDAC2 mRNA, reduce the migration and invasion abilities of NSCLC cells, and thus inhibit tumor metastasis.

[0123] Only some exemplary embodiments of the present invention are described above by way of illustration. Undoubtedly, for those of ordinary skill in the art, the described embodiments can be modified in various different ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the protection scope of the claims of the present invention.

Claims

1. The application of miR-378a in thoracic tumors is characterized by: Including the application of miR-378a in the preparation of anti-thoracic tumor preparations.

2. The use of miR-378a in breast tumors according to claim 1, characterized in that: The miR-378a is miR-378a-5p.

3. The use of miR-378a in breast tumors according to claim 1, characterized in that: The chest tumors are lung cancer and esophageal cancer.

4. The use of miR-378a in breast tumors according to claim 3, characterized in that: The lung cancer includes non-small cell lung cancer.

5. The use of miR-378a in breast tumors according to claim 3, characterized in that: The esophageal cancer includes esophageal squamous cell carcinoma.

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