Application of circular RNA in the preparation of drugs for preventing and treating radiation-induced liver fibrosis

Abstract

The present invention discloses the application of circular RNA in the preparation of drugs for preventing and treating radiation-induced liver fibrosis, and belongs to the field of biomedical technology. The nucleotide sequence of the circular RNA is shown in SEQ ID NO: 1; by upregulating the expression level of the circular RNA, the effect of preventing and treating radiation-induced liver fibrosis is exerted. The present invention constructs a radiation-induced liver fibrosis model, and proves that overexpression of circular RNA circ96498 can inhibit the proliferation and activation of radiation-induced liver stellate cells, the expression of inflammatory factors and fibrosis factors, and the NF-κB and JNK / Smad2 signaling pathways of radiation-induced liver stellate cells, and exert the effect of treating radiation-induced liver fibrosis in mice. The present invention provides a new approach for the clinical prevention and treatment of radiation-induced liver fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of circular RNA in the preparation of medicines for preventing and treating radiation-induced liver fibrosis. Background Art

[0002] Radiotherapy (RT) has become an important treatment for primary hepatocellular carcinoma, but radiation-induced liver injury (RILI) caused by RT cannot be ignored. Severe cases can lead to liver failure or even death. Radiation-induced liver fibrosis (RILF) is one of the manifestations of radiation-induced liver injury, which can last for a long time and seriously affect the patient's quality of life. Therefore, finding targets that can alleviate RILF is currently a key and difficult point in the prevention and treatment of liver radiation injury.

[0003] Hepatic stellate cells (HSCs) are key effector cells in mediating the occurrence of RILF. After being stimulated by external factors, quiescent HSCs are transformed into myofibroblasts with proliferative activity, recruiting and generating a large amount of extracellular matrix components such as collagen and fibronectin at the damaged site to participate in liver repair, while excessive extracellular matrix deposition promotes the occurrence of liver fibrosis. Radiation-induced HSC activation is a complex process involving multiple cytokines and pathways, such as tumor necrosis factor α, transforming growth factor β, hedgehog pathway, NF-κB pathway, and JNK / Smad2 pathway.

[0004] Circular RNA (circRNA) is a type of gene transcription product that has no free 5' or 3' end and forms a single-stranded closed loop between nucleic acid molecules by covalent bonds. CircRNA exists in large quantities in eukaryotic cells and has certain tissue, timing and disease specificity. Many studies have also confirmed that circRNA is closely related to the occurrence and development of various diseases such as Alzheimer's disease, diabetes, and tumors, suggesting that circRNA has application value in disease diagnosis and prevention. However, the role of circRNA in the occurrence and development of RILF is still unclear, and whether it can be used as a target for the prevention and treatment of RILF needs further exploration. Summary of the invention

[0005] The purpose of the present invention is to provide an application of circular RNA in the preparation of a drug for preventing and treating radiation-induced liver fibrosis, so as to solve the problems existing in the above-mentioned prior art. The present invention overexpresses circular RNA circ96498 to treat radiation-induced liver fibrosis in mice, and finds that NF-κB and JNK / Smad2 signaling pathways play an important role in the process of radiation-induced liver fibrosis, providing a new target and a new approach for preventing and treating radiation-induced liver fibrosis.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides application of circular RNA in preparing medicine for preventing and treating radiation-induced liver fibrosis. The nucleotide sequence of the circular RNA is shown in SEQ ID NO: 1.

[0008] Furthermore, by upregulating the expression level of the circular RNA, the effect of preventing and treating radiation-induced liver fibrosis is exerted.

[0009] Furthermore, the circular RNA is inserted into an overexpression plasmid to exert the effect of preventing and treating radiation-induced liver fibrosis.

[0010] The present invention also provides the use of a circular RNA gene expression promoter in the preparation of a drug for preventing and treating radiation-induced liver fibrosis, wherein the promoter comprises a nucleotide sequence as shown in SEQ ID NO: 1.

[0011] Furthermore, the promoter exerts the effect of preventing and treating radiation-induced liver fibrosis by inhibiting the proliferation of hepatic stellate cells, the secretion of inflammatory factors and fibrosis factors, and the NF-κB and JNK / Smad2 signaling pathways of hepatic stellate cells.

[0012] Furthermore, the inflammatory factors include TNF-α, IL-6 and IL-1β; the fibrosis factors include α-SMA and collagen1.

[0013] The present invention also provides a drug for preventing and treating radiation-induced liver fibrosis, wherein the drug comprises a circular RNA overexpression plasmid, and the nucleotide sequence of the circular RNA is shown in SEQ ID NO:1.

[0014] Furthermore, the medicine also includes pharmaceutically acceptable excipients; the excipients include one or more of diluents, binders, disintegrants, lubricants and flavoring agents.

[0015] Furthermore, the dosage form of the drug is tablets, capsules, granules, injections or sprays.

[0016] Furthermore, the drug is taken orally or parenterally.

[0017] The present invention discloses the following technical effects:

[0018] The present invention constructs a radiation-induced liver fibrosis model and proves that overexpression of circular RNA circ96498 can inhibit the proliferation and activation of radiation-induced hepatic stellate cells, the expression of inflammatory factors and fibrotic factors, and the NF-κB and JNK / Smad2 signaling pathways of radiation-induced hepatic stellate cells, thereby playing a therapeutic effect on radiation-induced liver fibrosis in mice. The present invention provides a new approach for the clinical prevention and treatment of radiation-induced liver fibrosis.

[0019] In addition, the present invention reveals that circular RNA circ96498 regulates the activation of irradiated hepatic stellate cells by inhibiting the NF-κB and JNK / Smad2 signaling pathways of irradiated hepatic stellate cells, thereby inhibiting radiation-induced liver fibrosis, indicating that NF-κB and JNK / Smad2 signaling pathways play an important role in the process of radiation-induced liver fibrosis, providing a new target for the prevention and treatment of radiation-induced liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The results of localization of circ96498 in human hepatic stellate cells and verification of its cyclization characteristics; A: Fluorescence in situ hybridization assay to detect the localization of circ96498 in human hepatic stellate cells; Non-RILI, non-radioactive liver injury; RILI, radiation-induced liver injury; B: qRT-PCR detection of the expression of circ96498 in LX2 cells under different radiation doses; NIR, non-radiotherapy; C: Agarose gel electrophoresis to verify the cyclization characteristics of circ96498; D: Schematic diagram of the biological synthesis and structure of circ96498; **P<0.01;

[0022] Figure 2 circ96498 inhibits the proliferation of irradiated hepatic stellate cells; AB: CCK-8 assay to detect the proliferation of irradiated hepatic stellate cells LX2 after knockdown or overexpression of circ96498; CD: EdU assay to detect the proliferation of irradiated hepatic stellate cells LX2 after knockdown or overexpression of circ96498; *P<0.05; **P<0.01;

[0023] Figure 3circ96498 promotes apoptosis of irradiated hepatic stellate cells; A: Flow cytometry was used to detect apoptosis of irradiated hepatic stellate cells LX2 after knockdown of circ96498; B: Flow cytometry was used to detect apoptosis of irradiated hepatic stellate cells LX2 after overexpression of circ96498; **P<0.01;

[0024] Figure 4 circ96498 inhibits the secretion of inflammatory factors in irradiated hepatic stellate cells; A: ELISA was used to detect the expression of inflammatory factors TNF-α, IL-6 and IL-1β in the culture supernatant of irradiated hepatic stellate cells LX2 after knockdown of circ96498; B: ELISA was used to detect the expression of inflammatory factors TNF-α, IL-6 and IL-1β in the culture supernatant of irradiated hepatic stellate cells LX2 after overexpression of circ96498; *P<0.05; **P<0.01;

[0025] Figure 5 circ96498 inhibits the expression of fibrotic factors in irradiated hepatic stellate cells; A: Western Blot detection of the expression of α-SMA and collagen1, fibrotic inflammatory factors in irradiated hepatic stellate cells LX2 after knockdown or overexpression of circ96498; B: Immunofluorescence detection of the expression of α-SMA and collagen1, fibrotic inflammatory factors in irradiated hepatic stellate cells LX2 after knockdown or overexpression of circ96498;

[0026] Figure 6 circ96498 inhibits the expression of NF-κB and JNK / Smad2 signaling pathways in irradiated hepatic stellate cells; A: Western Blot detection of the expression of LX2-related signaling pathways in irradiated hepatic stellate cells after knockdown of circ96498; B: Western Blot detection of the expression of LX2-related signaling pathways in irradiated hepatic stellate cells after overexpression of circ96498;

[0027] Figure 7 Knockdown of circ96498 in hepatic stellate cells inhibited the expression of inflammatory factors in mice; A: qRT-PCR detected the mRNA expression of liver inflammatory factors Il1b, Il6 and Tnf-α after knockdown of circ96498 in hepatic stellate cells in mice; B: ELISA detected the expression of IL-1β, IL-6 and TNF-α in mouse serum after knockdown of circ96498 in hepatic stellate cells in mice; *P<0.05; **P<0.01;

[0028] Figure 8Knockdown of circ96498 inhibits radiation-induced liver fibrosis in mice; A: HE staining, immunohistochemistry and Sirius red staining were used to detect the effect of knockdown of circ96498 on radiation-induced liver fibrosis in mice; B: Immunohistochemistry and Sirius red staining statistical graph of the effect of knockdown of circ96498 on radiation-induced liver fibrosis in mice; C: ELISA detection of the expression of ALT and AST in the serum of mice after knockdown of circ96498; **P<0.01. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Unless otherwise specified, the materials, instruments and reagents used in the present invention can be obtained from commercial channels; the experimental methods used are conventional experimental methods in the art unless otherwise specified.

[0035] The nucleotide sequence of circular RNA hsa_circ_0096498 (circ96498 for short) in the following examples is shown in SEQ ID NO: 1:

[0036]

[0037] Example 1 Radiation induces upregulation of circ96498 expression in hepatic stellate cells.

[0038] The paracancerous tissues of 20 patients with hepatocellular carcinoma who underwent radiotherapy in Nanfang Hospital of Southern Medical University from 2018 to 2022 were collected before radiotherapy. The total radiotherapy dose was 40-60Gy, and the number of fractions was 5-20. All patients underwent CT and MRI examinations before and after radiotherapy. According to whether radiation liver injury occurred on MRI imaging, they were divided into non-radioactive liver injury group (Non-RILI group, n=10) and radiation liver injury group (RILI group, n=10). Fluorescence in situ hybridization (FISH) was used to detect the expression of circ96498 and α-SMA in the two groups of samples, and the colocalization of circ96498 and hepatic stellate cells was evaluated. The results showed that the expression of circ96498 and α-SMA in the RILI group was significantly upregulated, and the two had good colocalization, indicating that the expression of circ96498 was significantly increased in RILI patients and specifically expressed in hepatic stellate cells ( Figure 1 (A).

[0039] Different radiation doses of 0, 2, 4, 6, 8, and 10 Gy were used to irradiate human hepatic stellate cells LX2. qRT-PCR was used to detect the expression of circ96498 in LX2 24 hours later. The results showed that circ96498 was upregulated in LX2 after irradiation, and the upregulation was most significant at 8 Gy, indicating that radiation can induce upregulation of circ96498 in hepatic stellate cells ( Figure 1 In addition, agarose gel electrophoresis and Sanger sequencing also confirmed the circularization properties of circ96498 ( Figure 1 (C, D).

[0040] The detection methods of the above-mentioned fluorescence in situ hybridization and qRT-PCR are as follows:

[0041] (1) Fluorescence in situ hybridization

[0042] ① Slice and bake: The collected specimens were fixed with 4% paraformaldehyde for 48 hours, and then the tissues were dehydrated and embedded in paraffin. Then the slices were sliced ​​and placed in a 65℃ oven for 2 hours before the experiment;

[0043] ② Dewaxing and alcohol gradient dehydration: xylene I 10 min → xylene II 10 min → xylene III 10 min → 100% ethanol I 5 min → 100% ethanol II 5 min → 90% ethanol 5 min → 80% ethanol 5 min → 70% ethanol 5 min → PBS soaking 5 min;

[0044] ③ High-pressure antigen repair: Pour the prepared 1× citric acid buffer into the box, cover the slide, and then put it into the pressure cooker. Add a small amount of citric acid buffer to the pressure cooker. When the water boils, cover the pot, and stop heating after the jet continues for 5 minutes. Take out the slide box and cool it at room temperature;

[0045] ④ Proteinase K treatment: shake off excess liquid on the tissue, add an appropriate amount of proteinase K working solution to the tissue, and place it at 37°C for 25 minutes. Wash with PBS for 3 times, 100 rpm / min, 5 minutes each time;

[0046] ⑤ Pre-hybridization working solution treatment: shake off the excess liquid on the slice, add pre-hybridization working solution to cover the tissue, incubate in a 42℃ wet box for 2 hours, cover with a special cover glass for in situ hybridization. Wash with PBS for 3 times, 100 rpm / min, 5 minutes each time;

[0047] ⑥ Hybridization 1: shake off excess liquid on the slice, add hybridization working solution, probe concentration is 1:5 (use 100ug / ml salmon sperm DNA super hybridization solution to dilute the probe), cover with in situ hybridization slide, hybridize overnight at 42℃; remove the cover glass, wash with PBS 3 times, 100rpm / min, 5min each time;

[0048] ⑦ Hybridization 2: Gently shake dry the slices, add preheated probe mixture 2 hybridization solution (60 μL), place horizontally in a wet box at 40°C for hybridization for 45 minutes; shake and wash with PBS 3 times, 100 rpm / min, 5 minutes each time;

[0049] ⑧Signal hybridization: Gently shake dry the slices, add preheated signal probe hybridization solution (60 μL), place horizontally in a wet box at 40°C for hybridization for 45 min; shake and wash with PBS 3 times, 100 rpm / min, 5 min each time;

[0050] ⑨DAPI counterstaining of nuclei: add DAPI staining solution to the slices and incubate in the dark for 5 minutes. Wash with PBS for 3 times at 100 rpm / min, 5 minutes each time. After washing, add anti-fluorescence quenching sealing agent to seal the slices;

[0051] ⑩ Microscopic examination and photography: Observe and collect images under a Nikon upright fluorescence microscope.

[0052] (2) qRT-PCR

[0053] ① Extract total RNA from cells: 1) Discard the supernatant, wash once with PBS, add 1mL Trizol reagent to the cells in the 6-well plate, let stand on ice for 5 minutes, and then collect into a 1.5mL EP tube. 2) Add 200μL of chloroform (1 / 5 the amount of Trizol reagent) to the EP tube, flip the EP tube upside down 5-6 times to mix it thoroughly, let stand at room temperature for 5 minutes, and after the liquid in the tube is separated, put it into a 4℃ centrifuge for centrifugation at 12000rpm / min for 15min. 3) After the centrifugation, the mixed solution can be seen to be separated into layers, the upper layer is a colorless aqueous phase containing total RNA, and the lower layer is a light red phenol-chloroform phase. Collect the upper layer of the EP tube into a new EP tube, add an equal volume of isopropanol, flip the EP tube upside down 5-6 times, and let it stand at room temperature for 10min. Then centrifuge at 4℃, 12000rpm / min, 10min. 4) Remove the supernatant, add 1 mL of 75% ethanol (prepared with DEPC water) to the tube to wash the RNA precipitate, and then place it in a 4°C centrifuge at 12,000 rpm / min for 5 minutes. Repeat this step twice. 5) Discard the supernatant, place it in a clean bench to dry thoroughly, and finally add 20-30 μL of pre-cooled DEPC water to dissolve it.

[0054] ② Determination of RNA concentration: Use Nanodrop 2000 software to determine the extracted RNA concentration, keep experimental records, and store the samples at -80℃ for later use or immediately proceed with subsequent processing.

[0055] ③Reverse transcription of mRNA:

[0056] HiScript III All-in-one RT SuperMix Perfect for qPCR Kit (purchased from Nanjing Novozyme Biotechnology) was used to reverse transcribe mRNA into cDNA, and the reaction system was prepared according to Table 1 for mRNA reverse transcription.

[0057] Table 1 mRNA reverse transcription reaction loading system

[0058]

[0059] After the above system is mixed, it is centrifuged instantly and the reaction program on the machine is: 50℃15min, 85℃5s.

[0060] ④qRT-PCR

[0061] Primer sequences of relevant genes were designed (see Table 2), and the reaction system was prepared according to the instructions of Taq Pro Universal SYBR qPCR MasterMix Kit (purchased from Nanjing Novozyme Biotechnology) (see Table 3), and amplification was performed using a real-time fluorescence quantitative PCR instrument Q6.

[0062] Table 2 Primer sequences of related genes

[0063]

[0064] Table 3 mRNA qRT-PCR reaction sample addition system

[0065]

[0066] After the above system is mixed, centrifuge it instantly, and the reaction program on the machine is: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 60℃ annealing and extension for 34s and data collection, 40 cycles; melting curve analysis is performed after the cycle. Melting curve acquisition program: 95℃15s, 60℃1min, 95℃30s, 60℃15s, and melting curve data are acquired.

[0067] ⑤Analysis of experimental results

[0068] After the reaction, confirm the amplification curve and melting curve of qRT-PCR, collect the Ct value of each template, and use 2 -△△Ct The mRNA was calculated using GAPDH as an internal reference.

[0069] Example 2 circ96498 inhibits the proliferation of irradiated hepatic stellate cells

[0070] The siRNA si-circ96498 or control si-circNC (both purchased from Guangzhou Ruibo Biotechnology) was transfected into hepatic stellate cells LX2, and 8Gy irradiation was performed 24 hours later. Then, the cell proliferation ability of irradiated hepatic stellate cells was detected by CCK-8 and EdU experiments. The results showed that si-circ96498 could significantly enhance the cell proliferation ability of irradiated hepatic stellate cells ( Figure 2 At the same time, the circ96498 overexpression plasmid circ96498-OE or the control circNC-OE (both purchased from Guangzhou Jisai Biotechnology) was transfected into the hepatic stellate cells LX2, and 8Gy irradiation was performed 24 hours later. Then, the cell proliferation ability of irradiated hepatic stellate cells was detected by CCK-8 and EdU experiments. The results showed that circ96498-OE could significantly inhibit the cell proliferation ability of irradiated hepatic stellate cells ( Figure 2 ). In summary, up-regulating the expression of circ96498 can significantly inhibit the proliferation of irradiated hepatic stellate cells.

[0071] The detection methods of the above CCK-8 and EdU experiments are as follows:

[0072] (1) CCK-8

[0073] ① Hepatic stellate cells were seeded into 96-well plates (3000 cells / well) and given corresponding treatments;

[0074] ② The cell proliferation activity was detected using CCK-8 kit (purchased from Japan Tongren) at 6, 24, 48, 72, 96, and 120 h after cell irradiation;

[0075] ③ At the set time point, remove the culture medium in the wells, wash once with PBS, then add 100 μL of freshly prepared solution (containing 10 μL CCK-8 reagent and 90 μL DMEM culture medium) to each well and continue culturing for 2 hours;

[0076] ④ Use an ELISA reader to read the absorbance at 450nm and draw a cell proliferation curve.

[0077] (2)EdU

[0078] The EdU detection kit (purchased from Guangzhou Ruibo Biotechnology) was used to detect cell proliferation activity.

[0079] ① Plating: Hepatic stellate cells were seeded in 96-well plates (3000 cells / well) and treated accordingly;

[0080] ②EdU labeling: Prepare an appropriate amount of 50μM EdU culture medium at a ratio of 1000:1 of complete culture medium to EdU reagent. Discard the culture medium 48 hours after cell irradiation, wash once with PBS, add 100μL 50μM EdU culture medium to each well, and culture at 37℃ for 2 hours. Discard the culture medium, shake and wash twice with PBS (80rpm / min), 5 minutes each time;

[0081] ③Fixation: Add 50 μL 4% paraformaldehyde to each well and fix at room temperature for 30 minutes;

[0082] ④ Glycine treatment: discard the fixative, add 50 μL of 2 mg / mL glycine solution to each well, and shake slowly on a shaker (40 rpm / min) for 5 min;

[0083] ⑤ Cell permeabilization: discard the glycine solution, shake and wash with PBS (40 rpm / min) for 5 min. Add 100 μL permeabilization agent (0.5% Triton-X-100 in PBS) to each well, shake slowly on a shaker (40 rpm / min) for 10 min. Discard the permeabilization agent, shake and wash with PBS (80 rpm / min) for 5 min.

[0084] ⑥Apollo staining: Add 100 μL of 1xApollo staining solution to each well, keep away from light, shake slowly on a shaker (40 rpm / min) for 30 min at room temperature. Discard the staining solution, and wash with PBS (80 rpm / min) 3 times, 5 min each time;

[0085] ⑦DNA staining: Add 1x Hoechst33342 reaction solution to each well, keep away from light, shake slowly on a shaker (40rpm / min) for 30min at room temperature. Discard the staining solution, wash with PBS (80rpm / min) 3 times, 5min each time;

[0086] ⑧ Microscopic examination and photography: Observe and collect images under a Nikon inverted fluorescence microscope.

[0087] Example 3 circ96498 promotes apoptosis of irradiated hepatic stellate cells

[0088] According to Example 2, siRNA si-circ96498 or control si-circNC was transfected into hepatic stellate cells LX2, and 8Gy irradiation was performed 24 hours later. Flow cytometry was used to detect apoptosis of irradiated hepatic stellate cells 48 hours after irradiation. The results showed that si-circ96498 could significantly inhibit apoptosis of irradiated hepatic stellate cells ( Figure 3 A). The circ96498 overexpression plasmid circ96498-OE or the control circNC-OE was transfected into the hepatic stellate cells LX2, and 8Gy was irradiated 24 hours later. The apoptosis of irradiated hepatic stellate cells was detected by flow cytometry 48 hours after irradiation. The results showed that circ96498-OE could significantly enhance the apoptosis of irradiated hepatic stellate cells ( Figure 3 In conclusion, up-regulation of circ96498 expression can significantly promote the apoptosis of irradiated hepatic stellate cells.

[0089] The above-mentioned flow cytometry detection method for apoptosis is as follows:

[0090] The cell apoptosis detection kit (purchased from Jiangsu Keygene Biotechnology) was used for detection.

[0091] ① Hepatic stellate cells were seeded into 6-well plates and given corresponding treatments;

[0092] ② After 48 hours of cell irradiation, discard the supernatant, wash once with PBS, digest the cells with trypsin and collect them into 1.5 mL EP tubes;

[0093] ③Discard the supernatant and add 500 μL Binding Buffer to suspend the cells;

[0094] ④ Add 5μL Annexin V-APC and mix well, then add 5μL Propidium Iodide and mix well;

[0095] ⑤React at room temperature in the dark for 10 minutes;

[0096] ⑥Perform flow cytometry testing within 1 hour.

[0097] Example 4 circ96498 inhibits the secretion of inflammatory factors in irradiated hepatic stellate cells

[0098] According to Example 2, siRNA si-circ96498 or control si-circNC was transfected into hepatic stellate cells LX2, and 8Gy irradiation was performed 24 hours later. The culture supernatant of the cells was collected 48 hours after irradiation, and the expression of inflammatory factors TNF-α, IL-6 and IL-1β was detected by ELISA. The results showed that si-circ96498 could significantly promote the secretion of inflammatory factors TNF-α, IL-6 and IL-1β in irradiated hepatic stellate cells ( Figure 4 A). The circ96498 overexpression plasmid circ96498-OE or the control circNC-OE was transfected into hepatic stellate cells LX2, and 8Gy irradiation was performed 24 hours later. The culture supernatant of the cells was collected 48 hours after irradiation, and the expression of inflammatory factors TNF-α, IL-6 and IL-1β was detected by ELISA. The results showed that circ96498-OE could significantly inhibit the secretion of inflammatory factors TNF-α, IL-6 and IL-1β in irradiated hepatic stellate cells ( Figure 4 In conclusion, overexpression of circ96498 can significantly inhibit the secretion of inflammatory factors TNF-α, IL-6, and IL-1β in irradiated hepatic stellate cells.

[0099] The detection method of the above ELISA is as follows:

[0100] TNF-α, IL-6 and IL-1β cytokine ELISA detection kits (purchased from Shanghai ELISA Biologicals) were used to detect the content of TNF-α, IL-6 and IL-1β in the cell supernatant. The specific steps are as follows:

[0101] ① Sample collection: Collect the cell supernatant, centrifuge at 2000 rpm for 10 min at room temperature, and collect the supernatant in a 1.5 mL centrifuge tube;

[0102] ② Standard sample addition: set standard sample wells and sample wells, and add 50μL of standard samples of different concentrations to each standard sample well;

[0103] ③ Sample addition: Set up blank wells (blank control wells do not contain samples and enzyme-labeled reagents, and the rest of the steps are the same) and test sample wells. First add 40 μL of sample diluent to the test sample wells on the enzyme-labeled plate, and then add 10 μL of the test sample (the final sample dilution is 5 times);

[0104] ④ Add enzyme: add 100 μL of enzyme-labeled reagent to each well, except for the blank well;

[0105] ⑤ Incubation: Seal the plate with a sealing film and incubate at 37°C for 1 hour;

[0106] ⑥ Liquid preparation: dilute the 20 times concentrated washing solution with 20 times distilled water for later use;

[0107] ⑦ Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with detergent, let it stand for 30 seconds and then discard, repeat 5 times;

[0108] ⑧ Color development: first add 50μL of color development solution A to each well, then add 50μL of color development solution B, gently shake and mix, and develop the color at 37℃ in the dark for 15min;

[0109] ⑨ Determination: Set the blank well to zero and measure the absorbance (OD value) of each well in turn at a wavelength of 450nm;

[0110] ⑩Calculation: Collect data, draw standard curves for each cytokine and generate corresponding formulas to calculate the content of each cytokine.

[0111] Example 5 circ96498 inhibits the fibrotic phenotype of irradiated hepatic stellate cells

[0112] According to Example 2, siRNA si-circ96498 or control si-circNC was transfected into hepatic stellate cells LX2, and 8Gy irradiation was performed 24 hours later. After 48 hours of irradiation, the expression of α-SMA and collagen1 proteins in irradiated hepatic stellate cells was detected by Western blot and immunofluorescence. The results showed that si-circ96498 could significantly promote the expression of fibrotic factors α-SMA and collagen1 proteins in irradiated hepatic stellate cells ( Figure 5 A and B). The circ96498 overexpression plasmid circ96498-OE or the control circNC-OE was transfected into the hepatic stellate cells LX2, and 8Gy irradiation was performed 24 hours later. After 48 hours of irradiation, the expression of α-SMA and collagen1 proteins in irradiated hepatic stellate cells was detected by Western blot or immunofluorescence. The results showed that overexpression of circ96498-OE could significantly inhibit the expression of fibrotic factors α-SMA and collagen1 proteins in irradiated hepatic stellate cells ( Figure 5 (A, B).

[0113] The above Western blot and immunofluorescence detection methods are as follows:

[0114] (1) Western blot

[0115] ① Cell lysis: Add an appropriate amount of RIPA protein lysis buffer to the cells to be tested to fully lyse them.

[0116] ② Protein concentration determination and sample preparation: Use the BCA method to detect the protein concentration, mix by pipetting according to the ratio of protein volume: 5×loading buffer = 4:1, and place the mixed sample in a 100°C metal bath and heat for 5 minutes.

[0117] ③ Electrophoresis: prepare 10% SDS-PAGE separation gel and 5% SDS-PAGE concentration gel, take 20μg protein sample for sample loading, set the electrophoresis voltage, concentration gel (upper gel) stage 60V, 20min, separation gel (lower gel) stage, 90V, 80-90min, turn off the power when bromophenol blue is about to reach the bottom of the separation gel.

[0118] ④ Transfer: After electrophoresis, set a constant current of 200 mA for 2 h at low temperature (4°C) to transfer the protein to the PVDF membrane.

[0119] ⑤ Blocking: Immerse the PVDF membrane in 5% BSA for blocking, and place it on a slow shaker (40 rpm / min) at room temperature for 1 hour.

[0120] ⑥ Primary antibody incubation: After blocking, take out the PVDF membrane and immerse it in TBST (1% TBST = 1L TBS buffer + 1mL Tween-20) solution for 5 minutes. Then, place the PVDF membrane in the prepared primary antibody solution and incubate it on a slow shaker at 4°C overnight.

[0121] ⑦ Secondary antibody incubation: The next day, take out the PVDF membrane, place it on a fast (120r / min) shaker at room temperature, wash it with TBST solution, 10min each time, repeat 3 times. Place the washed PVDF membrane in the corresponding secondary antibody solution, and shake it on a slow (40rpm / min) shaker at room temperature for 1h.

[0122] ⑧ Luminescence: After the secondary antibody incubation is completed, the PVDF membrane is washed three times with TBST solution as above, and the band image is collected using ECL chemiluminescence.

[0123] (2) Immunofluorescence

[0124] ① Plating: LX2 cells were seeded in 24-well plates. After intervention treatment, the culture medium was removed and the cells were washed once with PBS.

[0125] ②Fixation: Add 0.5mL 4% paraformaldehyde fixative and fix for 30min;

[0126] ③ Washing: discard the fixative and wash with PBS three times, shaking slowly on a shaker (40 rpm / min), 5 min each time;

[0127] ④ Blocking: Add 0.5 mL 5% BSA for blocking and leave at room temperature for 1 hour;

[0128] ⑤ Apply primary antibody: discard the blocking solution, add 0.2mL of the diluted primary antibody, and shake slowly (40rpm / min) at 4℃ overnight;

[0129] ⑥ Apply secondary antibody: discard the primary antibody, wash with PBS 3 times, shake slowly on a shaker (40rpm / min), 5min each time; add 0.2mL of the secondary antibody diluted in proportion, shake slowly on a shaker (40rpm / min) at room temperature and away from light for 1h;

[0130] ⑦ Nuclear staining: Recover the secondary antibody; wash with PBS three times, shake slowly on a shaker (40 rpm / min), 5 min each time; add 0.2 mL of nuclear staining solution (DAPI), shake slowly on a shaker (40 rpm / min) for 5 min at room temperature and away from light;

[0131] ⑧Washing: discard the nuclear staining solution, wash with PBS three times, shake slowly on a shaker (40 rpm / min), 5 min each time;

[0132] ⑨ Taking photos: Use an inverted fluorescence microscope IX73 to observe and take fluorescent photos.

[0133] Example 6 circ96498 inhibits NF-κB and JNK / Smad2 signaling pathways in irradiated hepatic stellate cells

[0134] According to Example 2, siRNA si-circ96498 or control si-circNC was transfected into hepatic stellate cells LX2, and 8Gy irradiation was performed 24 hours later. After 48 hours of irradiation, the expression of NF-κB and JNK / Smad2 signaling pathway-related molecules in irradiated hepatic stellate cells was detected by Western blot. The results showed that si-circ96498 could significantly activate the NF-κB and JNK / Smad2 signaling pathways in irradiated hepatic stellate cells ( Figure 6 A). The circ96498 overexpression plasmid circ96498-OE or the control circNC-OE was transfected into hepatic stellate cells LX2, and 8Gy irradiation was performed 24 hours later. After 48 hours of irradiation, the expression of molecules related to the NF-κB and JNK / Smad2 signaling pathways in irradiated hepatic stellate cells was detected by Western blot. The results showed that circ96498-OE could significantly inhibit the NF-κB and JNK / Smad2 signaling pathways in irradiated hepatic stellate cells ( Figure 6 (middle B).

[0135] Example 7 In vivo experiments demonstrate that circ96498 inhibits radiation-induced liver fibrosis in mice

[0136] Female C57BL / 6 mice aged 6-8 weeks were randomly divided into non-radiotherapy group (NIR, n=6), radiotherapy group (IR+vector, n=6) and radiotherapy + knockdown of circ96498 group (IR+sh-circ96498, n=6). One week before radiotherapy, mice were injected with AAV-Gfap-shcirc96498 (purchased from Shanghai Hanheng Biotechnology) into the tail vein to specifically knock out the expression of circ96498 in mouse hepatic stellate cells, or injected with blank vector (vector) as a control. The right liver lobe of mice was irradiated with X-rays (8Gy×5, once a day) to construct a radiation liver fibrosis model. The mice were killed 4 weeks after the last irradiation, and the right liver tissue and serum of the mice were collected to evaluate the radiation liver fibrosis of the mice.

[0137] The right liver tissue of mice was ground and tissue RNA was extracted. The expression of inflammatory factors Il1b, Il6 and Tnf-α mRNA was detected by qRT-PCR. The results showed that radiotherapy upregulated the expression of inflammatory factors Il1b, Il6 and Tnf-α mRNA, while knocking down circ96498 in hepatic stellate cells further upregulated the expression of inflammatory factors Il1b, Il6 and Tnf-α mRNA ( Figure 7 A). The above qRT-PCR detection method is shown in Example 1, wherein the sequences of the primers used are shown in Table 4 below:

[0138] Table 4 Primer sequences of related genes

[0139]

[0140]

[0141] Mouse serum was collected and the expression of inflammatory factors IL-1β, IL-6 and TNF-α was detected by ELISA. The results showed that radiotherapy upregulated the expression of inflammatory factors IL-1β, IL-6 and TNF-α, while knocking down circ96498 in hepatic stellate cells further upregulated the expression of inflammatory factors IL-1β, IL-6 and TNF-α ( Figure 7 In summary, knockdown of circ96498 expression in hepatic stellate cells in vivo promotes the formation of the hepatic inflammatory environment and aggravates liver inflammation.

[0142] The right liver tissues of mice were taken for HE, immunohistochemistry and Sirius red staining to evaluate the fibrosis of the mouse liver. The results showed that radiotherapy promoted the expression of α-SMA in the mouse liver and aggravated its liver fibrosis, while knocking down circ96498 in hepatic stellate cells further upregulated the expression of α-SMA and aggravated fibrosis ( Figure 8In addition, ELISA detection of the expression of ALT and AST in mouse serum revealed that radiotherapy upregulated the expression levels of ALT and AST in mouse serum, leading to liver damage, while knockdown of circ96498 in hepatic stellate cells further increased the expression levels of ALT and AST in serum, further aggravating liver damage ( Figure 8 (middle B).

[0143] The above immunohistochemical detection method is as follows:

[0144] ① Slice and bake: The collected specimens were fixed with 4% paraformaldehyde for 48 hours, and then the tissues were dehydrated and embedded in paraffin. Then the slices were sliced ​​and placed in a 65℃ oven for 2 hours before the experiment;

[0145] ② Dewaxing and alcohol gradient dehydration: xylene I 10 min → xylene II 10 min → xylene III 10 min → 100% ethanol I 5 min → 100% ethanol II 5 min → 90% ethanol 5 min → 80% ethanol 5 min → 70% ethanol 5 min → PBS soaking 5 min;

[0146] ③ High-pressure antigen repair: Pour the prepared 1× citric acid buffer into the box, cover the slide, and then put it into the pressure cooker. Add a small amount of citric acid buffer to the pressure cooker. When the water boils, cover the pot, and stop heating after the jet continues for 5 minutes. Take out the slide box and cool it at room temperature;

[0147] ④ Blocking: Treat the slide tissue with 3% hydrogen peroxide before blocking. Drop hydrogen peroxide on the tissue and incubate for 15 minutes. Then wash the slide slowly with PBS for 5 minutes each time, for a total of 3 times. Gently dry the slide, add goat serum and incubate the slices for 30 minutes.

[0148] ⑤ Primary antibody incubation: Gently shake the slide dry, add an appropriate volume of primary antibody, and incubate overnight at 4°C;

[0149] ⑥ Secondary antibody incubation: The next day, warm the slides at room temperature for 30 minutes. Wash with PBS 3 times, 100 rpm / min, 5 minutes each time. Add an appropriate volume of secondary antibody and incubate at room temperature for 1 hour; then wash with PBS 3 times;

[0150] ⑦DAB color development: Gently shake the slide dry, add DAB color developer on the tissue, immediately start timing and observe the tissue staining reaction under the microscope, put the slide into PBS to stop the color development according to the staining situation, and record the staining time. The slides that need to compare the staining intensity should be stained at a unified time;

[0151] ⑧Hematoxylin re-staining and blueing: After color development, place the slide in a basin and rinse with running water for 5 minutes, then immerse in hematoxylin staining for 2 minutes, then rinse with running water for 2 minutes, then differentiate with 1% hydrochloric acid alcohol for 6 seconds, and then rinse with running water for 10 minutes;

[0152] ⑨ Dehydration and sealing: 75% ethanol 5 min → 85% ethanol 5 min → 95% ethanol 5 min → 100% ethanol I 5 min → 100% ethanol II 5 min → xylene I 5 min → xylene II 5 min. After natural drying, seal the slides with neutral gum.

[0153] ⑩Microscopic examination: Observe and take photos using an upright microscope.

[0154] In summary, knockdown of circ96498 in hepatic stellate cells promoted radiation-induced liver injury and radiation-induced liver fibrosis in mice. Consistent with the experimental results at the cell level, it was confirmed that circ96498 negatively regulated the progression of radiation-induced liver injury and radiation-induced liver fibrosis, and that the progression of radiation-induced liver injury and radiation-induced liver fibrosis could be prevented by upregulating the expression level of circ96498, providing a new important target and new approach for the prevention and treatment of radiation-induced liver injury and radiation-induced liver fibrosis.

[0155] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of circular RNA in the preparation of a drug for preventing and treating radiation-induced liver fibrosis, characterized in that: The nucleotide sequence of the circular RNA is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: By up-regulating the expression level of the circular RNA, the effect of preventing and treating radiation-induced liver fibrosis is exerted.

3. The use according to claim 2, characterized in that: The circular RNA is inserted into an overexpression plasmid to exert the effect of preventing and treating radiation-induced liver fibrosis.

4. Use of a circular RNA gene expression promoter in the preparation of a drug for preventing and treating radiation-induced liver fibrosis, characterized in that: The nucleotide sequence of the promoter is shown in SEQ ID NO:

1.

5. The use according to claim 4, characterized in that: The promoter exerts the effect of preventing and treating radiation-induced liver fibrosis by inhibiting the proliferation of hepatic stellate cells, the secretion of inflammatory factors and fibrosis factors, and the NF-κB and JNK / Smad2 signaling pathways of hepatic stellate cells.

6. The use according to claim 5, characterized in that: The inflammatory factors include TNF-α, IL-6 and IL-1β; the fibrosis factors include α-SMA and collagen 1.

7. A drug for preventing and treating radiation-induced liver fibrosis, characterized in that: The drug comprises a circular RNA overexpression plasmid, and the nucleotide sequence of the circular RNA is shown in SEQ ID NO:

1.

8. The drug according to claim 7, characterized in that The medicine also includes pharmaceutically acceptable excipients; the excipients include one or more of diluents, binders, disintegrants, lubricants and flavoring agents.

9. The drug according to claim 7, characterized in that The dosage form of the medicine is tablet, capsule, granule, injection or spray.

10. The drug according to claim 7, characterized in that The drug can be taken orally or parenterally.

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