Application of METTL3 in the preparation of drugs for preventing and treating radiation-induced lung injury

Abstract

The present invention relates to the application of METTL3 in the preparation of drugs for preventing and treating radiation-induced lung injury. The present invention discovers that METTL3 knockdown has a preventive and therapeutic effect on radiation injury of human lung cells and lung tissues in animal models, can inhibit the epithelial-mesenchymal transition process during the occurrence and development of radiation-induced lung injury, and enhance the antioxidant and post-radiation cell proliferation abilities of lung cells. Therefore, the preventive and therapeutic drugs of the present invention are designed with METTL3 as the target gene, and have important significance and application value in dealing with nuclear and radiation accidents, nuclear emergencies, and reducing the lung radiation injury of tumor radiotherapy patients, etc.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of METTL3 in the preparation of drugs for preventing and treating radiation-induced lung injury. Background Art

[0002] Radiation-induced lung injury (RILI) is a common complication after radiotherapy for thoracic tumors and preconditioning for bone marrow transplantation. In addition, since the lung is an organ sensitive to radiation, accidental nuclear radiation accidents or nuclear radiation injuries in wartime usually also affect the lung, resulting in radiation-induced lung injury. In tumor radiotherapy, a considerable number of patients will have varying degrees of radiation-induced lung injury reactions. Radiation-induced lung injury is more severe in patients with thoracic malignancies such as lung cancer, breast cancer, and esophageal cancer during radiotherapy. The initial stage of radiation-induced lung injury is mainly exudative inflammation, and it gradually develops into chronic inflammation in the later stage and can persist for several months. Some patients may develop pulmonary fibrosis. From the specific manifestations of the injury, the early stage shows radiation pneumonia, and the clinical manifestations are dyspnea, cough, low fever, chest discomfort, etc. The later stage shows chronic radiation-induced pulmonary fibrosis. The clinical manifestations are progressive dyspnea, persistent dry cough, symptoms and signs of cor pulmonale, and severe cases develop into chronic respiratory failure. Currently, it is considered that radiation-induced lung injury is a complex and dynamic reaction process involving multiple cells, multiple factors, and multiple genes. Clinically, there are no very effective means and drugs for the prevention and treatment of radiation-induced lung injury. Generally, symptomatic treatment is used, such as oxygen inhalation, cough suppression, and antiasthmatic treatment. This kind of injury is difficult to cure, causing the patient's lung injury to not heal for a long time and may eventually develop into canceration, seriously affecting the patient's quality of life. Currently, relevant studies have pointed out that radioprotective agents can be used for the prevention or treatment of radiation-induced lung injury. For example, amifostine is the only radioprotective agent currently approved by the FDA for clinical use. Devine et al. compared the effects of amifostine (injectable amifostine) on the radiotherapy toxicity and efficacy of non-small cell lung cancer patients receiving chemoradiotherapy or radiotherapy alone in 16 clinical studies. The results showed that amifostine could reduce the risk of acute lung toxicity after radiotherapy by 44% (P = 0.0001) and did not affect the efficacy. However, due to its obvious side effects such as hypotension, severe nausea, and poor tolerance (especially intravenous administration), its widespread use in clinical practice is limited.

[0003] Epithelial-to-mesenchymal transition (EMT) is a process in which epithelial cells gradually lose their epithelial characteristics and acquire mesenchymal fibroblast-like characteristics [Lamouille S et al. Nat Rev Mol Cell Biol. 2014; 15(3): 178-196.]. Continuous inflammation induced by ionizing radiation leads to damage of lung epithelial cells, causing the occurrence of EMT, and ultimately leading to the development of pulmonary fibrosis [Hanania AN et al. Chest. 2019; 156(1): 150-162.]. Therefore, understanding the occurrence and development mechanism of EMT is of great significance for the prevention and (or) treatment of radiation-induced lung injury. Although some progress has been made in the prevention and treatment of radiation-induced lung injury in recent years, many problems still exist. Therefore, finding new drugs for the prevention and treatment of radiation-induced lung injury is of practical significance.

[0004] N 6 -methyladenosine (m 6 A) is a reversible RNA methylation form discovered by scientists for the first time in recent years, that is, the methylation modification of the 6th nitrogen atom of adenine in RNA molecules [Meyer KD et al. Cell. 2012; 149(7): 1635-1646.]. 6 A methylation modification is mainly determined by three types of proteases [Oerum S et al. Nucleic Acids Res. 2021; 49(13): 7239-7255.]. The first type is m 6 A methyltransferase, also known as the encoder (Writers), the genes it encodes are called writing genes, and the complex they form will promote m 6 A methylation group is written into RNA, including METTL3, METTL14, METTL16 and WATP. The second type is m 6 A demethylase, also known as Eraser, is a proteinase that removes mRNA from RNA. 6 A methylation group, thereby affecting the pathophysiological process of the disease. Common ones include FTO and ALKBH5. The third category is readers, whose encoded genes are called reading genes, which can be used with m 6 A methylation site binds and reads information, including two subtypes, YTHDFs and YTHDCs. Reading gene recognition RNA methylation modification information, and participating in downstream RNA translation, degradation and other processes, thereby exerting its specific biological function. 6A is involved in regulating the physiological and pathological processes of various diseases, including DNA damage response, heat shock response, T cell homeostasis, tumorigenesis, metastasis, and adipogenesis [Deng X et al. Cell Res. 2018; 28(5): 507-517.]. However, the role of METTL3 in the occurrence and development of radiation-induced lung injury has not been reported, and whether METTL3 is involved in regulating EMT in radiation-induced lung injury remains unclear. Therefore, more research and exploration are needed on the application of METTL3 in the prevention and treatment of radiation-induced lung injury. Summary of the Invention

[0005] To solve the above technical problems, through the study of the correlation between the expression of METTL3 gene and radiation-induced lung injury, the present invention discovers that METTL3 small interfering RNA or METTL3 adeno-associated virus has a crucial preventive and therapeutic effect on radiation injury of human lung cells and lung tissues of animal models. Therefore, a preventive and therapeutic drug related to radiation-induced lung injury is provided to inhibit the epithelial-mesenchymal transition process during the occurrence and development of radiation-induced lung injury and enhance the antioxidant and post-radiation cell proliferation abilities of lung cells.

[0006] The first object of the present invention is to provide the use of an antagonist of METTL3 (methyltransferase-like protein 3) in the preparation of a preventive and therapeutic drug for radiation-induced lung injury. 6 A methyltransferase-like protein 3) in the preparation of a preventive and therapeutic drug for radiation-induced lung injury.

[0007] Further, the preventive and therapeutic drug for radiation-induced lung injury reduces the expression of METTL3.

[0008] Further, the antagonist includes, but is not limited to, antibodies, siRNA, shRNA, sequences encapsulated by lentivirus, etc.

[0009] The second object of the present invention is to provide a preventive and therapeutic drug for radiation-induced lung injury, which is designed targeting METTL3 and reduces the expression of METTL3.

[0010] Further, the drug includes antibodies, siRNA, shRNA, or lentivirus that knock down the expression of METTL3.

[0011] Further, the drug also includes pharmaceutical excipients, such as pharmaceutically acceptable salts, excipients, carriers, etc. Specifically, the pharmaceutical excipients are selected from any one or more of biocompatible high molecular polymers, mixtures or copolymers of high molecular polymers, such as any one or more of copolymers of polylactic acid, polyglycolic acid and glycolic acid, copolymers of p-carboxyphenylpropane and sebacic acid, or ethylene vinyl acetate copolymer.

[0012] Further, the dosage form of the drug is selected from water extracts, powders, lotions, tinctures, oils, or aerosols.

[0013] The target provided by the present invention can also be used for drug screening. For example, the expression level of METTL3 is detected by a METTL3 detection reagent, and drugs for preventing and treating radiation-induced lung injury are screened according to the degree of reduction in the expression level.

[0014] By means of the above solution, the present invention has at least the following advantages:

[0015] The METTL3 small interfering RNA or METTL3 adeno-associated virus of the present invention has a preventive and therapeutic effect on radiation-induced injury of human lung cells and lung tissues of animal models. It can inhibit the epithelial-mesenchymal transition process during the occurrence and development of radiation-induced lung injury and enhance the antioxidant capacity and post-radiation cell proliferation ability of cells. The METTL3 small interfering RNA, the METTL3 adeno-associated virus, and drugs including the METTL3 small interfering RNA or the METTL3 adeno-associated virus can be used for the prevention and treatment of human acute lung radiation injury, and have important significance and application value in dealing with nuclear and radiation accidents, nuclear emergencies, and reducing the lung radiation injury of tumor radiotherapy patients, etc.

[0016] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it according to the content of the specification, the following takes the preferred embodiments of the present invention and combines with detailed drawings to illustrate as follows. Brief Description of the Drawings

[0017] In order to make the content of the present invention easier to be clearly understood, the present invention will be further described in detail below according to the specific embodiments of the present invention and in combination with the drawings.

[0018] Figure 1 It is the Western blot identification result after transfection of BEAS-2B cells with METTL3 small interfering RNA in Example 1 of the present invention and the Western blot identification result after intravenous injection of AAV9-shMETTL3 into mice via the tail vein.

[0019] Figure 2 It is the cell morphology before and after radiation and the expression of epithelial marker E-cadherin and mesenchymal markers α-SMA and Vimentin after transfection of BEAS-2B cells with METTL3 small interfering RNA in Example 2 of the present invention;

[0020] Figure 3 It is the free radical level before and after radiation of cells after transfection of BEAS-2B cells with METTL3 small interfering RNA in Example 2 of the present invention;

[0021] Figure 4 It is the colony formation rate before and after radiation of cells after transfection of BEAS-2B cells with METTL3 small interfering RNA in Example 2 of the present invention;

[0022] Figure 5 This is the H&E staining result of METTL3 adeno-associated virus shMETTL3 in alleviating radiation-induced lung injury in mice in Example 3 of the present invention;

[0023] Figure 6 This is that METTL3 adeno-associated virus shMETTL3 in Example 3 of the present invention reduces the occurrence of radiation-induced pulmonary epithelial-mesenchymal transition in mice. Detailed implementation manners

[0024] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the illustrated embodiments are not intended to limit the present invention.

[0025] Example 1 Construction of METTL3 small interfering RNA and adeno-associated virus and verification of expression after infection

[0026] In this example, human METTL3 small interfering RNA (siRNA-METTL3) was directly purchased from Snata Cruz Company (product number: sc-92172, production batch number: K1021). Mouse AAV9-shMETTL3 was synthesized by Shandong Weizhen Biotechnology Co., Ltd., and the shRNA sequence is as follows:

[0027] Table 1 Target sequences of mouse METTL3 shRNA

[0028]

[0029] Human lung epithelial cells BEAS-2B in the exponential growth phase were inoculated into 6-well plates at a density of 10.0×10 4 cells / well. After the cells adhered and grew, 6 μL of 10 μM METTL3 small interfering RNA and its control (siRNA-NC) were transfected into each well, and the cells were continuously cultured in an incubator at 37°C and 5% CO2 for 48 h. The cells were collected, lysed with RIPA, and then detected by Western blot.

[0030] As Figure 1 shown in A, the expression level of METTL3 in the cells transfected with the siRNA-METTL3 group was significantly lower than that in the siRNA-NC group, indicating that siRNA-METTL3 can significantly reduce the expression level of METTL3 in lung cells.

[0031] Male C57 mice aged 6-8 weeks were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., and 200 μL of 5×10 12For AAV9-shMETTL3 and AAV9-shNC at vp / mL, after two weeks, lung tissues were collected. After lysing cells with RIPA, Western blot detection was performed.

[0032] As Figure 1 shown in B, in the lung tissues of mice in the AAV9-shMETTL3 group injected via the tail vein, the expression level of METTL3 was significantly lower than that in the AAV9-shNC group, indicating that AAV9-shMETTL3 can significantly reduce the expression level of METTL3 in mouse lung tissues.

[0033] Example 2 Anti-radiation effect test of METTL3 small interfering RNA on lung cells

[0034] 1. MTLL3 small interfering RNA inhibits epithelial-mesenchymal transition induced by ionizing radiation

[0035] Human lung epithelial cells BEAS-2B in the exponential growth phase were seeded in 6-well plates for culture. After 24 h, METTL3 small interfering RNA and its control were transfected. After 48 h, 0 Gy and 10 Gy of X-ray irradiation were given, and the morphological changes of cells were observed under the microscope after 72 h. The expressions of epithelial marker E-cadherin and mesenchymal markers α-SMA and Vimentin were detected by Western Blot and immunofluorescence.

[0036] As Figure 2 shown in A, the results showed that after irradiation with 10 Gy, compared with the control group, most of the lung epithelial cells with knocked-down METTL3 changed from the swollen, elongated morphology with extended pseudopods to a cubical appearance, and knocking down METTL3 could inhibit the occurrence of ionizing radiation-induced epithelial-mesenchymal transition in lung epithelial cells ( Figure 2 B, Figure 2 C).

[0037] 2. MTLL3 small interfering RNA reduces free radicals caused by radiation

[0038] Human lung epithelial cells BEAS-2B in the exponential growth phase were seeded in 6-well plates for culture. After 24 h, METTL3 small interfering RNA and its control were transfected. After 48 h, 0 Gy and 10 Gy of X-ray irradiation were given. Cells were collected 0.5 h after irradiation and suspended in diluted DCFH-DA (DCFH-DA was diluted 1:1000 with serum-free medium to a final concentration of 10 μmol / L), and incubated in a 37 °C cell culture incubator for 20 minutes. Invert and mix well every 3 - 5 minutes to allow the probe to fully contact the cells. Wash the cells three times with serum-free cell culture medium to fully remove DCFH-DA that did not enter the cells. The same number of cells were seeded in 96-well plates and detected using an enzyme-labeled instrument.

[0039] As Figure 3 shown (P < 0.05), the results showed that knockdown of METTL3 significantly reduced the free radical level in irradiated cells.

[0040] 3. MTLL3 small interfering RNA alleviated radiation-induced growth inhibition

[0041] Human normal lung epithelial cells BEAS-2B were transfected with METTL3 small interfering RNA and its control for 48 h. After being irradiated with 0 Gy, 0.5 Gy, and 1 Gy of X-rays respectively, and cultured for 10 - 14 days, cell colonies containing more than 50 cells were counted under a microscope to detect the effect of METTL3 knockdown on the colony formation rate of irradiated BEAS-2B cells.

[0042] As Figure 4 shown (P < 0.05), the results showed that knockdown of METTL3 significantly increased the survival rate of BEAS-2B cells after radiation.

[0043] Example 3 Anti-radiation effect test of METTL3 small interfering RNA on mouse lung tissue

[0044] Male C57 mice aged 6 - 8 weeks were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. After anesthesia by intraperitoneal injection of 4% chloral hydrate at 1% of the body weight of each mouse, small animal positioning was performed using a small animal precision irradiator, and the right lung was precisely irradiated with an irradiation aperture of 10 mm to achieve a prescription dose of 20 Gy for 100% of the volume, establishing a mouse model of radioactive lung injury. Then the mice were randomly divided into 2 groups (10 mice in each group):

[0045] 1) The mice were given a tail vein injection of 200 μL of 5×10 12 vp / mL AAV9-shNC;

[0046] 2) The mice were given a tail vein injection of 200 μL of 5×10 12 vp / mL AAV9-shMETTL3.

[0047] At 8 and 16 weeks after irradiation, the C57 mice were sacrificed by cervical dislocation. The right lung tissue was taken, rinsed repeatedly with normal saline, and then paraffin sections were made and stained with H&E for observation.

[0048] As Figure 5 shown (P < 0.01), the results showed that: knockdown of METTL3 could reduce the occurrence of radioactive lung injury and maintain the normal physiological function of the lung.

[0049] The right lung tissue was minced and homogenized with a tissue homogenizer. After taking the supernatant, Western blot was performed to detect the expression of epithelial marker E-cadherin and mesenchymal index α-SMA.

[0050] As Figure 6 shown, the results indicate that knocking down METTL3 can inhibit the occurrence of ionizing radiation-induced epithelial-mesenchymal transition in mouse lungs.

[0051] In summary, the application of METTL3 of the present invention and the discovery of drugs show that knocking down METTL3 has a preventive and therapeutic effect on radiation damage to human lung cells and lung tissues of animal models, can inhibit the epithelial-mesenchymal transition process during the occurrence and development of radioactive lung injury, and can enhance the antioxidant capacity of cells and cell proliferation ability after radiation. Small interfering RNA of METTL3, adeno-associated virus of METTL3, or drugs including small interfering RNA of METTL3 and adeno-associated virus of METTL3 can be used for the prevention and treatment of human acute lung radiation injury, and have important significance and application value in dealing with nuclear and radiation accidents, nuclear emergencies, and reducing the lung radiation injury of tumor radiotherapy patients.

[0052] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. Use of an antagonist of METTL3 in the preparation of a drug for preventing and treating radiation-induced lung injury, characterized in that: The antagonist of METTL3 is siRNA; the siRNA is Snata Cruz sc-92172.

2. The application according to claim 1, wherein: The drug for preventing and treating radiation-induced lung injury further comprises pharmaceutical excipients.

3. The application according to claim 2, wherein: The pharmaceutical excipients include carriers.

4. The application according to claim 1, wherein: The dosage form of the drug for preventing and treating radiation-induced lung injury is selected from infusion, powder, lotion, tincture, oil or aerosol.

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