MiR-140-5p for inhibiting expression of B7-H4 in macrophages and expression of miR-140-5p in liver with schistosomiasis japonicas

Abstract

The invention discloses miR-140-5p for inhibiting expression of B7-H4 in macrophages and expression of the miR-140-5p in liver with schistosomiasis japonicum, and belongs to the technical field of prevention and treatment of schistosomiasis japonicum. A miR-140-5p simulant inhibits expression of B7-H4 protein in a macrophage RAW264.7 cell strain; the miR-140-5p inhibitor is used for promoting the expression of the B7-H4 protein in a macrophage RAW264.7 cell strain; the STAT5 siRNA inhibits the activity of an expression vector containing a miR-140-5p promoter, and further inhibits the expression of B7-H4 protein in liver tissues of schistosoma japonicum katsurada. The miR-140-5p simulant or inhibitor or STAT5 siRNA is adopted to regulate the inhibition expression or promotion expression of B7-H4 protein in the schistosoma japonicum, and the application prospect of preparing the liver medicine or food for treating or improving the schistosoma japonicum is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of prevention and treatment of schistosomiasis japonicum, and in particular to miR-140-5p that inhibits B7-H4 expression in macrophages and its expression in the liver of schistosomiasis japonicum patients. Background Art

[0002] Schistosomiasis japonicum is a zoonotic parasitic disease that seriously endangers human health. It is mainly prevalent in the middle and lower reaches of the Yangtze River and 12 provinces, municipalities and autonomous regions to its south in China. Schistosomiasis japonicum infects the host through cercariae that invade the skin. During the development of schistosomiasis japonicum, cercariae mature into adults in the portal vein system and then migrate in pairs to the mesenteric veins, where they mate and lay eggs in the blood vessels of the intestinal wall. Therefore, infection with schistosomiasis japonicum can cause portal hypertension syndrome, liver fibrosis, and ulceration and necrosis of intestinal mucosal tissue.

[0003] At present, praziquantel is mainly used clinically as the first choice drug for the prevention and treatment of Japanese schistosomiasis. However, as a broad-spectrum anti-insect drug, praziquantel still has problems such as large side effects, low solubility, and low efficacy against schistosomiasis. In addition, long-term and large-scale use has caused the emergence of drug resistance in schistosomiasis. Therefore, the development of new anti-schistosomiasis drugs and the formulation and implementation of new and more effective prevention and control methods are particularly important and urgent.

[0004] Macrophages are important immune cells that participate in the formation of Schistosoma japonicum egg granulomas and the development of liver fibrosis. MicroRNA, as an endogenous non-coding RNA, often negatively regulates the expression of target genes at the post-transcriptional level. It can play an important role in a variety of physiological and pathological processes and participates in the regulation of gene expression related to growth, development, differentiation, proliferation, death, metabolism and environmental response. The expression of miR-140-5p in mice infected with Schistosoma japonicum and the relationship between miR-140-5p and B7-H4 expression in mice infected with Schistosoma japonicum have not been reported. Summary of the invention

[0005] Technical problem to be solved: In view of the problems existing in the prior art, the present invention provides a miR-140-5p that inhibits the expression of B7-H4 in macrophages and its expression in the liver of Schistosoma japonicum. A miR-140-5p mimic or inhibitor, or STAT5 siRNA, is used to regulate the inhibitory expression or promoted expression of B7-H4 protein in Schistosoma japonicum, so as to prepare a drug or food for treating or improving the liver of Schistosoma japonicum.

[0006] Technical solution: The miR-140-5p mimic or inhibitor described in the present invention expresses B7-H4 in macrophages, and the miR-140-5p mimic inhibits B7-H4 protein expression in the macrophage RAW264.7 cell line; the miR-140-5p inhibitor promotes B7-H4 protein expression in the macrophage RAW264.7 cell line.

[0007] Preferably, the miR-140-5p mimic inhibits the luciferase activity of an expression vector containing the wild-type B7-H4 3'UTR; and the miR-140-5p inhibitor promotes the luciferase activity of an expression vector containing the wild-type B7-H4 3'UTR.

[0008] Preferably, the miR-140-5p mimetic or inhibitor does not have the ability to regulate the luciferase activity of an expression vector containing a mutant B7-H4 3'UTR.

[0009] The present invention also discloses the expression of miR-140-5p in the liver of Schistosoma japonicum, and the miR-140-5p inhibits the expression of B7-H4 protein in the liver tissue of Schistosoma japonicum.

[0010] Preferably, the STAT5 siRNA inhibits the activity of an expression vector containing the miR-140-5p promoter.

[0011] Preferably, the Schistosoma japonicum liver tissue is selected from 6-8 week old C57BL / 6 experimental mice, and the Schistosoma japonicum cercariae infection is carried out according to the operating procedures.

[0012] Compared with the prior art, the present invention has at least the following outstanding advantages: The miR-140-5p of the present invention inhibits the expression of B7-H4 in macrophages and its expression in the liver of Schistosoma japonicum. A miR-140-5p mimic or inhibitor, or STAT5 siRNA is used to regulate the inhibitory expression or promoted expression of B7-H4 protein in Schistosoma japonicum, so as to prepare a medicine or food for treating or improving the liver of Schistosoma japonicum. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The expression results of miR-140 in the liver tissue of mice infected with Schistosoma japonicum in the embodiment of the present invention are shown; wherein, N represents uninfected normal mice, N12W represents mice infected with Schistosoma japonicum for 12 weeks, and each group has at least 3 mice; when the mice were infected with Schistosoma japonicum for 12 weeks, the expression of miR-140 in the liver tissue of the mice was significantly lower than that of the uninfected normal mice, and the difference between the groups was statistically significant (compared with the N group, * P<0.05).

[0014] Figure 2 The results of B7-H4 expression in the liver tissue of mice infected with Schistosoma japonicum in the examples of the present invention; wherein NL represents uninfected normal mice, Sl represents mice infected with Schistosoma japonicum for 12 weeks, and each group includes 3 mice; when mice were infected with Schistosoma japonicum for 12 weeks, the expression level of B7-H4 protein in the liver tissue of mice was significantly higher than that of uninfected normal mice.

[0015] Figure 3 The results of miR-140 inhibiting B7-H4 protein expression in macrophages in the embodiments of the present invention are shown; wherein lane 1 represents the untransfected cell sample group, lane 2 represents the cell sample transfected with the disordered control (negative control) mimic, lane 3 represents the cell sample transfected with the mimic of miR-140; lane 4 represents the sample transfected with the inhibitor of disordered control (negative control); lane 5 represents the cell sample transfected with the inhibitor of miR-140; compared with the negative control group, the miR-140 mimic can inhibit the expression of B7-H4 protein in the cells, while the miR-140 inhibitor promotes the expression of B7-H4 protein in the cells; whether the cells are transfected with the inhibitor or the mimic of disordered control (negative control), the expression level of B7-H4 protein in the cells is similar to that of the untransfected group, and the change is not obvious.

[0016] Figure 4 The results of miR-140 inhibiting wild-type B7-H4 3'UTR activity expression in macrophages in the embodiments of the present invention are shown in Figure 2. All groups of cells were transfected with expression vectors of wild-type B7-H4 3'UTR, lane 1 is a cell sample transfected with a disordered control (negative control) mimic, lane 2 is a cell sample transfected with a miR-140 mimic; lane 3 is a sample transfected with an inhibitor of a disordered control (negative control); lane 4 is a cell sample transfected with a miR-140 inhibitor; compared with the negative control group, miR-140 mimics can inhibit the activity of wild-type B7-H4 3'UTR in cells, while miR-140 inhibitors promote the activity of wild-type B7-H4 3'UTR in cells; compared with the respective negative control groups, *** P < 0.0001, ** P<0.01.

[0017] Figure 5The results of the embodiment of the present invention show that miR-140 does not affect the activity expression of mutant B7-H4 3'UTR in macrophages; all groups of cells were transfected with expression vectors of mutant B7-H4 3'UTR, lane 1 is a cell sample transfected with a disordered control (negative control) mimic, lane 2 is a cell sample transfected with a miR-140 mimic; lane 3 is a sample transfected with an inhibitor of disordered control (negative control); lane 4 is a cell sample transfected with a miR-140 inhibitor; compared with the negative control group, miR-140 mimics have no effect on the activity of mutant B7-H4 3'UTR, and miR-140 inhibitors have no effect on the activity of mutant B7-H4 3'UTR in cells; compared with the respective negative control groups, ns P>0.05.

[0018] Figure 6 The CHIP experiment results of the binding of the transcription factor STAT5 to the miR-140 promoter in macrophages in the embodiment of the present invention are shown in FIG. 1 ; wherein lane 1 (Marker) is a DNA molecular weight standard, lane 2 uses an anti-H3 antibody, lanes 6 / 10 use an anti-STAT5 antibody, lanes 3 / 7 / 11 (IgG) use IgG as a negative control, lanes 4 / 8 / 12 (Input) are positive controls, and lanes 5 / 9 / 13 (ddH2O) are used directly for PCR experiments using ddH2O as a template; at the same time, lanes 2 / 3 / 4 / 5 was identified by PCR using RPL30 primers. This group of experiments was mainly used to verify the accuracy and reliability of the experimental operation; lanes 6 / 7 / 8 / 9 were identified by PCR using the first pair of primers targeting the STAT5 binding site on the promoter, and lanes 10 / 11 / 12 / 13 were identified by PCR using the second pair of primers targeting the STAT5 binding site on the promoter. The above two groups of data were used to identify whether STAT5 could bind to the STAT5 binding site at the -456 / -446bp position of the miR-140 promoter.

[0019] Figure 7 The results of 1L-10 inhibiting the activity of miR-140 promoter through STAT5 in the embodiment of the present invention; when the STAT5 binding site at the -456 / -446bp position of the miR-140 promoter exists, IL-10 can inhibit the promoter activity. Compared with cells without IL-10 treatment, the promoter activity in cells treated with IL-10 is significantly reduced. * P<0.05; when the STAT5 binding site sequence at the -456 / -446bp position of the miR-140 promoter was mutated, IL-10 could not bind to the promoter through STAT5 and thus affect the promoter activity. Compared with the untreated group, ns P>0.05.

[0020] Figure 8 The results of 1L-10 inhibiting miR-140 expression through STAT5 in the present invention; when cells were only treated with IL-10, compared with the untreated group, IL-10 significantly inhibited miR-140 expression, ** P<0.01; when cells were transfected with negative small interfering RNA (NC) and then treated with IL-10, IL-10 still significantly inhibited the expression of miR-140, and the difference between the groups was statistically significant. # P<0.05; when cells were transfected with STAT5 small interfering RNA (S) and then treated with IL-10, IL-10 could not affect the expression of miR-140, and there was no statistically significant difference between the groups. ns P>0.05; at the same time, compared with the non-transfected cells, there was no significant difference in the expression of miR-140 in the cells treated with IL-10 after transfection with negative small interfering RNA (NC). ns P>0.05; however, compared with the negative small interfering RNA (NC), when the cells were transfected with STAT5 small interfering RNA (S) and then treated with IL-10, the expression of miR-140 was upregulated. $$ P<0.01. DETAILED DESCRIPTION

[0021] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the following will be combined with the attached Figures 1 to 8 The technical solutions of the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.

[0022] Example 1: A miR-140-5p mimic or inhibitor described in the present invention expresses B7-H4 in macrophages. The miR-140-5p mimic inhibits B7-H4 protein expression in macrophage RAW264.7 cell line; The miR-140-5p inhibitor promotes B7-H4 protein expression in macrophage RAW264.7 cell line. The miR-140-5p mimic can inhibit the luciferase activity of an expression vector containing a wild-type B7-H4 3'UTR, and the miR-140-5p inhibitor can promote the luciferase activity of an expression vector containing a wild-type B7-H4 3'UTR. The miR-140-5p mimic or inhibitor does not have the ability to regulate the luciferase activity of an expression vector containing a mutant B7-H4 3'UTR.

[0023] Example 2: The present invention also discloses the expression of miR-140-5p in the liver of Schistosoma japonicum, and miR-140-5p inhibits the expression of B7-H4 protein in the liver tissue of Schistosoma japonicum; the activity of the expression vector containing the miR-140-5p promoter is inhibited by STAT5 siRNA, so as to inhibit the expression of B7-H4 protein in Schistosoma japonicum, and prepare a medicine or food for treating or improving the liver of Schistosoma japonicum. The Schistosoma japonicum liver tissue used in the present invention is selected from 6-8 week old C57BL / 6 experimental mice of Nantong University Animal Center, and the Schistosoma japonicum cercariae infection is carried out according to the operating procedures.

[0024] The test steps and methods of a specific embodiment of the present invention are as follows: 1. Experimental mice infected with Schistosoma japonicum cercariae: The experimental mice were 6-8 week old C57BL / 6 mice purchased from the Animal Center of Nantong University. They were infected with schistosome cercariae according to the operating procedures. The infection cycle was 12 weeks. The experimental mice were intraperitoneally injected with IL-10 neutralizing antibodies and IgG neutralizing antibodies at intervals of two days. After 7 injections, the liver tissues of the experimental mice were taken for experiments.

[0025] (II) RAW264.7 cell recovery and culture: The temperature of the water bath was controlled at 37°C to thaw RAW264.7 cells within 1 min. The RAW264.7 cells were transferred to a pre-prepared centrifuge tube containing 15 mL of complete medium. The cells were centrifuged at 4°C and 100 g (RCF) for 5 min. After centrifugation, the supernatant was discarded and 3 mL of complete medium was added to the precipitate to resuspend the cells and mix well. The cell medium was changed and the cells were passaged according to the cell status.

[0026] 3. Passaging and cryopreservation of RAW264.7 cells: After the above-mentioned revived RAW264.7 cells were cultured for 24 hours, the old culture medium was discarded, 1 mL of PBS was added for washing, the supernatant was discarded by centrifugation, and the attached cells were suspended by blowing, and centrifuged at 4°C and 100g (RCF) for 5 minutes, the supernatant was discarded, and then 4 mL of complete culture medium was added for blowing and mixing, 2 mL of the mixture was transferred to another culture bottle containing 2 mL of complete culture medium, and the mixture was placed in an incubator after mixing; the other 2 mL of the mixture was transferred to a 15 mL centrifuge tube, 2 mL of complete culture medium was added to the centrifuge tube, and the tube was centrifuged at 4°C and 100g (RCF) for 5 minutes. After the centrifugation, the supernatant was discarded, 900 μL of serum and 100 μL of DMSO were added to the precipitate, the cell precipitate was resuspended, and the mixture was transferred to a disposable cryovial after mixing, and the cryovial was placed in a refrigerator and first stored at 4°C for 30 minutes, and then stored at -80°C.

[0027] 4. Cell transfection: RAW264.7 cells were cultured at (4-5) × 10 5 The cells / well density was spread flat in a six-well plate, and 2 mL of complete medium was added to the incubator for 24 hours. When the cell density reached 80%, transfection could be performed. 200 μL of basal medium was added to a 1.5 mL EP tube, and then 5 μL of the mimic or inhibitor was added and vortexed. 200 μL of basal medium was added to another 1.5 mL EP tube, and then 4 μL of INTERFERin transfection reagent was added and vortexed. After standing for 5 minutes, the basal medium containing the mimic or inhibitor was transferred to the EP tube containing the INTERFERin transfection reagent, vortexed, centrifuged and stood for 15 minutes. During this period, the complete medium in the six-well plate was discarded and replaced with a new complete medium, and the transfection reagent after standing was evenly added to the six-well plate, and the new medium was replaced after waiting for 5~6 hours to continue culturing; 12 hours after transfection, IL-10 was added to treat RAW264.7 cells for 24 hours according to the experimental requirements.

[0028] (V) Extraction of RAW264.7 cell protein: The RAW264.7 cells in the six-well plate were blown with culture medium, and all the RAW264.7 cell culture medium was recovered into EP tubes, centrifuged at low speed for 5 minutes at 4°C, and then washed with PBS, and then centrifuged at low speed for 5 minutes at 4°C. After centrifugation, the supernatant was discarded. The lysis solution was prepared in a ratio of protein lysis solution (RIPA): phosphatase inhibitor (P53): protease inhibitor (PMSF) = 100:1:1, and the lysis solution was added to the cell pellet to resuspend the cells and blow mixed. The ultrasonic crusher was turned on and washed with distilled water and wiped dry. Each EP tube was ultrasonically crushed for 10 seconds. After ultrasonic treatment, the pellet was discarded after high-speed centrifugation at 4°C, and the supernatant was taken to detect the protein concentration. After the detection, 80μL 2×loading buffer was added to each EP tube, and then placed in a 100°C metal pot for 10 minutes to denature the protein. Then high-speed centrifugation was performed for 5 minutes, and the supernatant was taken and added to a new EP tube and stored at -20°C.

[0029] (VI) Western blot: Clean the glass plate and dry it, fix it on the plywood, and prepare to pour the gel. Prepare the separation gel according to the formula, wait for 40 minutes for the separation gel to solidify, and then pour the stacking gel. Put the prepared gel in the electrophoresis tank, pour the newly prepared electrophoresis solution, and start adding samples. Electrophoresis at 60V for 30 minutes, then adjust the voltage to 100V, and stop electrophoresis when bromophenol blue moves to the bottom of the gel. Prepare the gel, PVDF membrane, filter paper, etc. into a "sandwich", put it in the electrophoresis tank for electrophoresis, set the voltage to 100V, and transfer the membrane for 60 minutes; after the transfer is completed, the PVDF membrane is blocked with 5% skim milk for 1 hour. After the blocking is completed, add the primary antibody for incubation and put it in a 4℃ refrigerator shaker overnight; the next day, wash the membrane 3 times with 1×TBST, each time for 15 minutes, then incubate the secondary antibody for 2 hours, and then wash the membrane 4 times with 1×TBST, each time for 15 minutes. Finally, prepare the developer for development.

[0030] 7. RNA extraction: For RNA extraction of liver tissue, the tissue was placed in an appropriate amount of TRIZOL for full homogenization and lysis, and the supernatant was collected for subsequent operations. For RNA extraction of RAW264.7 cells, the culture medium in the six-well plate was discarded, the cells in the culture medium were washed with PBS, and then 1 mL of TRIZOL was added to each well, the adherent cells were blown and the cells were collected for subsequent operations.

[0031] The lysate containing liver cells was transferred to a 1.5 mL EP tube and allowed to stand at room temperature. Then 200 μL of chloroform was added and shaken for 15 seconds. The tube was allowed to stand for 5 to 10 minutes. Then the centrifuge was precooled to 4°C and centrifuged at 12300 g (RCF) for 20 minutes. 400 μL of supernatant was transferred to a new EP tube, 400 μL of isopropanol was added and mixed, and the tube was allowed to stand at room temperature for 20 minutes. Then the tube was centrifuged at 4°C and 12300 g (RCF) for 20 minutes. After centrifugation, the supernatant was discarded. 1 mL of precooled 75% anhydrous ethanol was added to the precipitate in the EP tube. After inversion and mixing, the tube was centrifuged at 4°C for 15 minutes. The 75% anhydrous ethanol was discarded. The RNA precipitate was dried for 5 minutes. Then 30 μL of DEPC water was added to dissolve the RNA precipitate. The RNA concentration was detected after incubation at 55°C for 10 minutes.

[0032] 8. Reverse transcription experiment: Use the extracted RNA as a template and follow the reverse transcription instructions. The reverse transcription program of the PCR instrument is set to: connect at 37°C for 1 hour and connect at 84°C for 5 minutes. After reverse transcription is completed, add 90 μL of sterile distilled water to 10 μL of cDNA product for subsequent experiments.

[0033] 9. Real-time quantitative PCR (RT-qPCR): The samples were loaded according to the instructions of the real-time fluorescence quantitative PCR kit, the mixed reagents were mixed and centrifuged, and the PCR instrument program was set as follows: connection at 95°C for 30 s, connection at 95°C for 5 s → connection at 61°C for 30 s → connection at 72°C for 30 s, and the cycle was repeated 40 times; then connection at 95°C for 15 s → connection at 61°C for 30 s → connection at 95°C for 15 s.

[0034] (X) Plasmid construction: (1) Primer design: Find the 3'UTR region of B7-H4 from the TargetScan database, or find the promoter sequence of miR-140 from the gene bank, design primers respectively, and send them to Shanghai Sangon Biotechnology for synthesis. Centrifuge the synthesized primers at high speed for 5 minutes and dilute them with dd H2O according to the instructions.

[0035] (2) Extract template DNA: Collect RAW264.7 cells in the six-well plate into an EP tube by blowing and beating, centrifuge at low speed for 5 minutes, discard the supernatant after centrifugation, and collect the resuspended precipitate. Add 100μL of BufferATL to the resuspended precipitate, add 10μL of proteinase K, and then add 100μL of Buffer AL, vortex mix for 15 seconds, and place in a preheated water bath at 56℃ for 10 minutes. Then, add 50μL of anhydrous ethanol, vortex mix for 15 seconds, and place at room temperature for 3 minutes. Transfer all the liquid in the EP tube to the adsorption column, centrifuge at 6000g (RCF) for 1min, and re-place the adsorption column in a clean 2mL collection tube; add 500μL Buffer AW1, centrifuge at 6000g (RCF) for 1min, and re-place the adsorption column in a clean 2mL collection tube; add 500μL Buffer AW2, centrifuge at 6000g (RCF) for 1min, re-place the adsorption column in a clean 2mL collection tube, and centrifuge at high speed for 3min to completely dry the membrane. Re-place the adsorption column in a clean 1.5mL EP tube, add 20-100μL Buffer AE, leave at room temperature for 1~2 min, centrifuge at high speed for 5min, collect the liquid in the EP tube, and the product is the template DNA.

[0036] (3) PCR: The extracted template DNA is used for PCR amplification; the mixed reagents are added and then centrifuged to mix; the PCR instrument is set up as follows: connect at 98°C for 10 seconds → connect at 58°C for 30 seconds → connect at 72°C for 2 minutes, and cycle 30 times; connect at 72°C for another 10 minutes, and then at 4°C forever.

[0037] (4) The PCR product was A-tailed and subjected to agarose gel electrophoresis. The product was then recovered using a gel recovery kit.

[0038] (5) The gel-recovered product was ligated to the pMD19-T vector and placed in a PCR instrument at 16°C for 3 h.

[0039] (6) Transform the gene into competent cells and culture at 37°C.

[0040] (7) Verification: Amplify a single colony and perform PCR verification and sequencing verification.

[0041] (8) Perform double restriction digestion on the plasmid with the correct sequencing result and the psiCHECK2 vector (for the construction of plasmid related to the 3'UTR region) or the pGL3-Enhancer vector (for the construction of plasmid related to the miR 140 promoter). After detection by agarose gel electrophoresis, recover the target band and the expression vector by gel electrophoresis. Connect the two and place them in a PCR instrument at 16°C overnight.

[0042] (9) Transform the ligation product, pick a single colony and shake it, perform PCR on the bacterial solution, extract the plasmid from the positive bacteria, verify it with double enzyme digestion, and send it for sequencing.

[0043] (XI) Dual luciferase reporter gene assay analysis: (1) Pretreatment: First place the AP solution and STOP solution at room temperature, and dilute the 5× lysis solution into 1× lysis solution for later use.

[0044] (2) Discard the culture medium in the 24-well plate, add PBS for washing, add 150 μL of 1× lysis buffer to each well, and place on a shaker at room temperature for 15 min.

[0045] (3) During this time, turn on the GloMax 20 / 20 chemiluminescence analyzer, prepare EP tubes, and add 10 μL AP solution to each tube.

[0046] (4) Pipette 10 μL of lysate into the EP tube containing AP solution and test on the machine; after stopping, add 10 μL of STOP solution and detect the fluorescence activity.

[0047] (XII) ChIP: (1) Culture 4×10 cells in a 10 cm culture dish in advance 6 RAW264.7 cells were cultured the next day, 37% formaldehyde was added and placed on a shaker at room temperature for 10 min, then glycine was added to terminate the reaction, and the cells were washed twice with PBS. The cells were collected in a centrifuge tube using a cell scraper and centrifuged at 4°C and 200g (RCF) for 5 min to collect the precipitate.

[0048] (2) Add 1 mL of 1× buffer A + DTT + PIC, incubate on ice for 10 min, centrifuge at 800 g (RCF) at 4°C for 5 min, and collect the precipitate.

[0049] (3) Add 1 mL of pre-cooled buffer B + DTT, centrifuge at 4°C and 800 g (RCF) for 5 min, collect the precipitate, and resuspend it in 100 μL of buffer B + DTT.

[0050] (4) Add 0.5 μL micrococcal nuclease and incubate in a water bath at 37°C for 20 min. Then add 12 μL 0.5 M EDTA to terminate the digestion. Centrifuge at 4°C and 15,000 g (RCF) for 1 min to collect the precipitate. Resuspend the precipitate with 100 μL 1× ChIP+PIC and incubate on ice for 10 min.

[0051] (5) Perform ultrasonic treatment at a power of 100 W for 20 s, repeat the treatment three times, centrifuge at 4°C and 8500 g (RCF) for 10 min, and collect the supernatant in another EP tube.

[0052] (6) Pipette 10 μg of chromatin DNA, fill it up to 500 μL with 1× ChIP buffer, add 0.2 μL PIC, aspirate 10 μL of sample as input control, and store it at -20°C.

[0053] (7) The remaining samples in the EP tube were divided into three parts, defined as H3 group, IgG group and anti-STAT5 group, and antibodies were added to each group and incubated overnight at 4°C.

[0054] (8) Add 30 μL of ChIP-grade protein G agarose beads to each EP tube, incubate at 4°C for 2 h, centrifuge at 4°C and 7000g (RCF) for 1 min, remove the supernatant, add 1 mL of low-salt rinse solution, incubate at 4°C for 5 min, centrifuge again to remove the supernatant, and repeat the process 3 times. Then, add 1 mL of high-salt rinse solution, incubate at 4°C for 5 min, centrifuge again to remove the supernatant, add 150 μL of 1× ChIP elution solution, leave the input group at room temperature for 2 h, and incubate the other two groups at 65°C for 30 min.

[0055] (9) Centrifuge at 4°C and 8500g (RCF) for 1 min, take the supernatant, add 6 μL 5M NaCl and 2 μL proteinase K to each EP tube, and incubate at 65°C for 2 h. Purify DNA according to the instructions and perform PCR detection.

[0056] Analysis of test results: Figure 1~2It can be seen that 12 weeks after the experimental mice were infected with Schistosoma japonicum, the expression of miR-140 in the liver tissue of the experimental mice decreased, while the expression of B7-H4 increased.

[0057] Depend on Figure 3 It can be seen that in the macrophage RAW264.7 cell line, miR-140-5p mimics inhibited the expression of B7-H4 protein, while miR-140-5p inhibitor promoted the expression of B7-H4 protein.

[0058] Depend on Figures 4 and 5 It can be seen that when plasmids containing wild-type or mutant B7-H4 3'UTR regions were constructed and transfected into RAW264.7 cell lines, it can be seen that miR-140-5p mimics inhibited the activity of B7-H4 3'UTR region, while miR-140-5p inhibitors promoted the activity of B7-H4 3'UTR region. Neither miR-140-5p mimics nor inhibitors had any regulatory effect on the activity of mutant B7-H4 3'UTR region.

[0059] Depend on Figure 6 It can be seen that the transcription factors regulating miR-140 were analyzed, and STAT5 was screened and confirmed to bind to the miR-140 promoter through CHIP experiments. Plasmids containing wild-type or mutant STAT5 binding site sequences of the miR-140 promoter were constructed and transfected into RAW264.7 cells. When the STAT5 binding site existed, IL-10 could inhibit the activity of the miR-140 promoter. However, when the STAT5 binding site on the promoter sequence was mutated, IL-10 had no obvious regulatory effect on the activity of the miR-140 promoter, such as Figure 7 At the same time, IL-10 can inhibit the transcriptional expression of miR-140. When STAT5 siRNA was added, the inhibitory effect of IL-10 on miR-140 was significantly weakened, as shown in Figure 8 shown.

[0060] The above are preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A miR-140-5p mimic or inhibitor of B7-H4 expression in macrophages, characterized in that The miR-140-5p mimic inhibits the expression of B7-H4 protein in the macrophage RAW264.7 cell line; the miR-140-5p inhibitor promotes the expression of B7-H4 protein in the macrophage RAW264.7 cell line.

2. The miR-140-5p mimic or inhibitor according to claim 1 is characterized in that B7-H4 expression in macrophages The miR-140-5p mimic inhibits the luciferase activity of the expression vector containing the wild-type B7-H4 3'UTR; the miR-140-5p inhibitor promotes the luciferase activity of the expression vector containing the wild-type B7-H4 3'UTR.

3. The miR-140-5p mimic or inhibitor according to claim 1 is characterized in that B7-H4 expression in macrophages The miR-140-5p mimics or inhibitors have no ability to regulate the luciferase activity of the expression vector containing the mutant B7-H4 3'UTR.

4. An expression of miR-140-5p in the liver of Schistosoma japonicum infection, characterized in that: The miR-140-5p inhibits the expression of B7-H4 protein in liver tissue of Schistosoma japonicum.

5. The expression of miR-140-5p in the liver of Schistosoma japonicum infection according to claim 4, characterized in that STAT5 siRNA inhibited the activity of the expression vector containing the miR-140-5p promoter.

6. The expression of miR-140-5p in the liver of Schistosoma japonicum infection according to claim 4, characterized in that The liver tissue of Schistosoma japonicum was selected from C57BL / 6 experimental mice aged 6 to 8 weeks, and the Schistosoma japonicum cercariae were infected according to the operating procedures.

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