Use of gene motor in preparation of drug for treating sepsis, and drug

Abstract

A use of hsa_circRNA gene MOTOR in the preparation of a drug for treating sepsis, and a drug. On the basis of the gene sequence of hsa_circRNA_056558, a vector overexpressing hsa_circRNA_056558 is constructed by means of molecular cloning, the vector is transfected into cells, and exosomes generated by the cells are collected by means of ultracentrifugation, wherein the exosomes comprise MOTOR. MOTOR can enhance the function of monocytes, and is used for the immuno-modulatory treatment of sepsis.

Description

Application of MOTOR gene in preparing medicine for treating sepsis and medicine Technical Field

[0001] The present invention relates to the technical field of circRNA-encoded small peptide genes, and in particular to an application of a MOTOR gene in preparing a drug for treating sepsis and the drug. Background Art

[0002] Sepsis, a life-threatening organ dysfunction caused by a dysregulated response to infection, poses a significant threat to human health. A multicenter cross-sectional study in my country showed that sepsis patients accounted for approximately 20.6% of ICU admissions, with a 90-day mortality rate as high as 35.5%. Therefore, in-depth research on the pathogenesis of sepsis and the identification of novel therapeutic targets are both critical national and societal needs for prevention and treatment of this disease and represent a significant clinical challenge.

[0003] Immune imbalance is the fundamental mechanism of the development of sepsis. Although multi-organ damage caused by excessive inflammatory response is an important part of the pathogenesis of sepsis, the hyperinflammatory state of sepsis is often accompanied by long-term and persistent immunosuppression. Monocyte LPS tolerance is a key feature of immunosuppression in sepsis. LPS-tolerant monocytes have a reduced ability to internalize and kill pathogenic microorganisms, and cannot respond effectively during secondary infection, which increases the risk of secondary infection in sepsis patients, and secondary infection is precisely the main cause of death in sepsis patients. However, the exact mechanism of LPS tolerance in monocytes has not yet been elucidated, and there is currently a lack of molecular drugs that regulate monocyte LPS tolerance. This is a core issue that urgently needs to be tackled.

[0004] Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention proposes the use of the hsa_circRNA gene MOTOR in the preparation of a drug for the treatment of sepsis. MOTOR reverses LPS tolerance of sepsis monocytes and prolongs the survival time of mice.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present application proposes the use of a MOTOR gene or a pharmaceutical derivative thereof in the preparation of a drug for treating sepsis. The nucleotide sequence of the MOTOR gene is shown in SEQ NO.1.

[0008] Furthermore, the drug is used in a dosage form suitable for releasing the MOTOR gene or its pharmaceutically acceptable derivative in the user's peripheral blood.

[0009] The present application also provides a drug, comprising exosomes containing the MOTOR gene; the nucleotide sequence of the MOTOR gene is shown in SEQ NO.1.

[0010] The drug of the present invention can be added to conventional excipients and prepared into various pharmaceutically acceptable dosage forms, such as tablets, capsules, oral solutions, lozenges, injections, ointments, granules or various sustained-release preparations, according to conventional processes.

[0011] The carriers of the drug of the present invention are common types available in the pharmaceutical field, including: adhesives, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents or bases, etc.

[0012] Preferably, the dosage form is an injection.

[0013] Beneficial effects of the present invention:

[0014] The gene sequence of hsa_circRNA_056558 of the present application can reverse the LPS tolerance of monocytes during sepsis, promote the expression of chemokines, proinflammatory cytokines and antigen presentation genes secreted by monocytes under bacterial infection, regulate the innate and adaptive immune functions of patients with sepsis, prevent secondary infection, and improve survival time. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention will be further described below with reference to the accompanying drawings.

[0016] FIG1 is a schematic diagram of the structure of the small peptide MOTOR located in the cytoplasm of the present application;

[0017] FIG2 is a diagram showing the inhibition of inflammatory factors and chemokines expression by knocking down MOTOR in the present application;

[0018] FIG3 is a diagram showing that overexpression of MOTOR in the present application promotes the expression of inflammatory factors and chemokines;

[0019] Figure 4 is a particle size analysis diagram of the Motor exosomes of the present application. DETAILED DESCRIPTION

[0020] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0021] In this specification, reference to terms such as "one embodiment," "example," or "specific example" means that a specific feature, structure, material, or characteristic described in conjunction with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example.

[0022] The present invention uses the following steps to verify the effect of MOTOR on monocyte LPS tolerance during sepsis. The specific steps are shown in Figures 1-4;

[0023] Example 1 MOTOR is a circRNA with coding ability that can translate a small peptide of 137aa

[0024] 1.1 MOTOR was identified as a circRNA by PCR and Sanger sequencing. Bioinformatics was used to predict the coding capacity of MOTOR. Western blot and immunofluorescence techniques were used to verify the coding capacity and subcellular localization of MOTOR. It was found that MOTOR can translate into a small peptide localized in the cytoplasm (see Figure 1).

[0025] 1.1.1 PCR and Sanger sequencing experiments

[0026] (1) RNA extraction:

[0027] a) Prepare the necessary reagents and consumables: Trizol; chloroform; isopropanol; DEPC water; RNase-free pipette tips of different sizes; 1.5 mL RNase-free EP tubing

[0028] b) The specific operation is as follows: human mononuclear THP1 cells are collected and placed in EP tubes. After adding 1 ml of Trizol to each EP tube, the cells are repeatedly pipetted until no cell pellet is visible. The centrifuge is precooled to 4°C, 0.2 ml of chloroform is added to each EP tube, vortexed for 15 seconds, and allowed to stand at room temperature for 5 minutes. The centrifugation condition is then set to 12,000 rcf. Centrifuge for 15 minutes; after centrifugation, it is divided into three layers, the upper layer is colorless aqueous RNA, the middle layer and the lower layer are organic phenol chloroform layers, carefully remove the EP tube, vertically aspirate 0.4 ml of the supernatant, add 0.5 ml of isopropanol, invert and mix ten times, and let stand at room temperature for 10 minutes; set the centrifugation conditions to 12000 rcf for 10 minutes; then discard the supernatant, add 75% ethanol prepared with DEPC water to each EP tube, invert and mix; set the centrifugation conditions to 7500 rcf for 5 minutes, remove and discard the supernatant; centrifuge for 15 seconds, discard the supernatant, and retain the RNA precipitate; then add 30 ul of DEPC water to dissolve the precipitate and use a spectrophotometer to measure the RNA concentration, and then use DEPC water to adjust the concentration to 1 ng / ul.

[0029] (2) Reverse transcription PCR: Use reverse transcription kit (PrimeScript TM Reverse transcription-PCR was performed on 500 ng of RNA using RT reagent Kit with gDNA Eraser, TAKARA, RR047A).

[0030] (3) PCR: Dilute the cDNA obtained by reverse transcription in the previous step three times with RNase-free water and prepare the system according to the following:

[0031] The PCR primer sequences are as follows:

[0032] After the PCR system was configured, the reaction was carried out using a Bio-Rad PCR instrument, and the PCR products were sent to Shanghai Bioengineering for Sanger sequencing (Figure 1).

[0033] 1.1.2 Western Blot Experiment

[0034] (1) Protein extraction experiment:

[0035] a) Prepare the necessary reagents and consumables: RIPA protein lysis buffer; Cocktail protease inhibitor; sterilized 1.5ml EP tube

[0036] b) Specific steps are as follows: Transfect the FLAG-MOTOR plasmid into cells in a 12-well plate, add 100 μl of RIPA and Cocktail inhibitor, and incubate on ice for 10 minutes. Centrifuge at 12,000 rpm, 4°C, for 20 minutes. Carefully aspirate the supernatant, which is the desired protein. Add 5x loading buffer and heat at 100°C for 5 minutes.

[0037] (2) Immunoblotting experiments

[0038] a) Gel preparation: 12% SDS-PAGE gel was prepared using a 12% PAGE gel rapid preparation kit (Bio-rad; 64484826):

[0039] 1. Take 3 ml of each Resolver A and Resolver B solution, add 3 μl of TEMED and 30 μl of 10% AP. Vortex to mix thoroughly, then inject into a glass plate, ensuring the distance between the liquid surface and the top edge of the short glass plate is 0.5 cm longer than the comb teeth. Take 1 ml of each Stack A and Resolver B solution, add 2 μl of TEMED and 10 μl of 10% AP. Vortex to mix thoroughly, then inject into a glass plate, inserting the comb teeth.

[0040] 2. After waiting for 30 minutes, the upper layer of gel solidifies and the comb teeth are removed for electrophoresis.

[0041] b) Electrophoresis:

[0042] 1. Place the solidified gel plate into the electrophoresis tank and add sufficient 1x Running buffer;

[0043] 2. Vortex the cooked protein sample to mix thoroughly, centrifuge briefly, and add 5 μL of molecular weight protein standard sample and 15 μL of protein sample to the sample wells respectively;

[0044] 3. Set the electrophoresis conditions: constant voltage 200V, 45 minutes;

[0045] c) Transfer:

[0046] 1. Cut the PVDF membrane of the corresponding size according to the target band and waking it with methanol for 10 minutes;

[0047] 2. After electrophoresis, use a gel opener to pry open the glass plate, cut the separation gel, and place it in transfer buffer;

[0048] 3. Use the sandwich method: positive electrode (white) - sponge pad - three layers of filter paper - PVDF membrane - separation gel - three layers of filter paper - sponge pad - negative electrode (black). The entire process is performed in the transfer solution, gently rolling each layer to remove bubbles.

[0049] 4. Set the transfer conditions: constant current 300mA, 60 minutes, add 1x transfer buffer; because the transfer process generates heat, the transfer tank needs to be placed in a low temperature environment.

[0050] d) Closure:

[0051] 1. Place the membrane in the pre-prepared blocking solution (2.5g bovine serum albumin + 50ml 1xTBST) and block it on a shaker at room temperature for 1 hour.

[0052] f) Immune response:

[0053] 1. Use 1xTBST to slowly wash away the surface blocking solution;

[0054] 2. Prepare the primary antibody (5 μl FLAG primary antibody + 10 ml 1xTBST) and add it to the incubation box. Allow the membrane to shake gently on the shaker in the box and incubate at 4°C overnight.

[0055] 3. Recover the primary antibody and wash the membrane three times with 1xTBST for 7 minutes each time. Then incubate with the secondary antibody at room temperature for 60 minutes. Recover the secondary antibody and wash the membrane three times with 1xTBST for 7 minutes each time. Immerse the membrane in 1xTBST solution for the next step.

[0056] g) Color reaction

[0057] 1. Open the Eblot ultra-sensitive fully automatic luminescence imaging analysis system;

[0058] 2. Mix the luminescent solution A and solution B in a ratio of 1:1, spread the solution evenly on the membrane with a pipette, and expose and take pictures.

[0059] The results showed that Flag-MOTOR could be detected, indicating that MOTOR can encode protein.

[0060] 1.1.3 Immunofluorescence staining

[0061] (1) Primary antibody incubation

[0062] Flag-MOTOR THP1 cells were collected, fixed with 4% PFA at room temperature for 1 hour, and rinsed three times with PBS, each time for 5 minutes; after blocking with Blocking medium (1% fetal bovine serum + 0.5% Tritonx-100) at room temperature for 1 hour, Flag antibody diluted 1:200 with PBS was added and incubated at 4°C overnight.

[0063] (2) Secondary antibody incubation

[0064] Rinse with PBS three times, 5 minutes each time; then dilute the corresponding secondary antibody with PBS at 1:400 (DAPI at 1:1000), incubate at room temperature for 1 hour, rinse with PBS three times, 5 minutes each time, and seal the slides with nail polish; collect images using a confocal microscope.

[0065] The results showed that Flag-MOTOR mainly existed in the cytoplasm.

[0066] Example 2 Knockdown of MOTOR inhibits the release of chemokines and proinflammatory cytokines by monocytes stimulated by LPS

[0067] 2.1 Human THP1 monocytic cells were cultured in vitro and siRNAs targeting the MOTOR circRNA were ordered. These siRNAs and control siRNAs were transfected into the cells using the NEON electroporation system. 1 μg / ml LPS was added 24 hours after transfection, and the cells were harvested 24 hours after LPS treatment. Next-generation sequencing and qPCR were used to analyze the expression of cytokines and antigen presentation-related factors, revealing that knockdown of MOTOR significantly inhibited their expression (see Figure 2).

[0068] 2.1.1 Next-generation sequencing and analysis

[0069] (1) Collect cells, centrifuge at 500 rcf for 5 min at room temperature, discard the culture medium, wash the cell pellet with PBS, and add 1 ml of Trizol.

[0070] (2) The Trizol sample containing cells was quickly frozen in liquid nitrogen for 30 seconds, placed on dry ice and sent to the company for next-generation sequencing.

[0071] (3) After the company returns the sequencing data, analyze the expression of chemokines and pro-inflammatory factors, use FPKM and z-score algorithms for normalization, and make a heat map to reflect the expression differences of related genes.

[0072] 2.1.2 qPCR

[0073] (1) Prepare the qPCR system as follows:

[0074] After qPCR detection, the CT values ​​obtained were analyzed and the expression levels of the corresponding chemokines and pro-inflammatory factors were calculated.

[0075] The results showed that knocking down MOTOR using siRNA could significantly reduce the expression levels of chemokines and pro-inflammatory factors in both next-generation sequencing and qPCR detection.

[0076] Example 3 Overexpression of MOTOR promotes the release of chemokines and proinflammatory cytokines by monocytes under LPS stimulation

[0077] MOTOR was overexpressed in monocytes by lentiviral infection, and the effect of MOTOR overexpression on cytokine release from monocytes under LPS stimulation was detected by qPCR. It was found that MOTOR overexpression promoted the response of monocytes to LPS (see Figure 3).

[0078] 3.1 Lentiviral packaging and purification

[0079] (1) HEK293T cells were co-transfected with vsvg, rev, gag and Flag-MOTOR plasmids; the nucleic acid sequence of the biological factor MOTOR encoded by the MOTOR plasmid was shown in SEQ NO.1.

[0080] (2) 48 hours after transfection, collect the supernatant from the culture dish and replace it with fresh DMEM containing 1% FBS. 96 hours later, collect the supernatant for the second time.

[0081] (3) Add 1 / 5 volume of virus concentrate, mix well, and let stand at 4°C overnight.

[0082] (4) Centrifuge the supernatant containing the virus concentrate at 4°C, 11,000 rcf for 30 minutes. Discard the supernatant and the precipitate is the virus particles. Resuspend the virus in 1.5 ml of DMEM medium and store in a -80°C refrigerator.

[0083] 3.2 Lentiviral infection

[0084] (1) Take 2x10 5 THP1 cells were placed in a 12-well plate, 500 μl of virus solution was added, and the plate was cultured in a cell culture incubator.

[0085] (2) One week after infection, puromycin was added for selection, and the selected cells were designated as MOTOR overexpressing cells (MOTOR OE).

[0086] 3.3 Detection of chemokines and proinflammatory cytokines

[0087] (1) MOTOR OE and control cells were stimulated with 1 μg / ml LPS. After 24 h, the cells were collected by centrifugation and RNA was extracted.

[0088] (2) Detection of chemokine and proinflammatory cytokine expression using qPCR

[0089] The results showed that overexpression of MOTOR could significantly increase the expression of chemokines and pro-inflammatory cytokines.

[0090] Example 4: MOTOR-containing exosomes prolong the survival time of septic mice

[0091] Collecting exosomes overexpressing MOTOR and injecting them into septic mice via tail vein injection can significantly prolong the survival time of septic mice.

[0092] 4.1 Extraction of exosomes containing MOTOR

[0093] (1) Collect the supernatant of 293T cells and 293T cells overexpressing MOTOR, centrifuge at 500g for 5 min, discard the precipitate, and retain the supernatant; centrifuge at 2000g for 5 min, discard the precipitate, and retain the supernatant; centrifuge at 12000g for 30 min, discard the precipitate, and retain the supernatant; centrifuge at 200000g for 90 min, discard the supernatant, and retain the precipitate; resuspend the precipitate with PBS, centrifuge at 200000g for 90 min, discard the supernatant, retain the precipitate, and resuspend with 200ul of PBS.

[0094] (2) Negative staining and electron microscopy

[0095] 4.2 Inject MOTOR-containing exosomes and control exosomes into septic mice and observe the survival time of the mice

[0096] 4.2.1 Establishment of a mouse model of sepsis using cecum ligation and puncture (CLP)

[0097] (1) C57BL / 6 wild-type mice (male, 6-7 weeks, 20-29 g) were anesthetized and cut open about 0.5 cm below the xiphoid process in the midline of the abdomen. The cecum was probed and slowly pulled out of the abdominal cavity. The cecum was ligated twice in the middle of the ligated segment. After confirming that there was no obvious bleeding, the cecum was returned to the abdomen without any bleeding. After the operation, 1 ml of 0.9% normal saline was subcutaneously administered for anti-shock treatment. The mice were allowed to eat and drink freely after the operation. The room temperature was kept constant.

[0098] (2) Dilute the exosomes with PBS to 1x10 per 200 μl. 9 At 24h, 48h and 72h after sepsis modeling, 200 μl of exosome solution was injected into mice via tail vein injection.

[0099] (3) The time of the first exosome injection was marked as 0 h, and the survival status of the mice was observed and recorded every 24 h.

[0100] The results showed that injection of exosomes containing MOTOR could significantly prolong the survival time of septic mice.

[0101] Figure 1 shows that MOTOR is a circRNA with coding ability; A. Schematic diagram of MOTOR and first-generation sequencing of the junction site; B. Bioinformatics predicted RNA coding ability. CDR1as and HOTAIR are classic circRNAs and lncRNAs that have been proven not to encode proteins but to function in the form of RNA. Actin is a clear protein-coding gene; C. Western blot shows that Flag antibody can detect Flag-MOTOR; D. Flag-MOTOR immunofluorescence. Figure 2 shows that knocking down MOTOR inhibits the expression of chemokines and antigen presentation-related molecules. RNA-seq (A) and qPCR (B) show the expression of chemokines and cellular antigen presentation-related molecules after knocking down MOTOR.

[0102] Figure 3 shows that overexpression of MOTOR promotes the chemokine analysis of monocytes under LPS stimulation. A. The expression level of MOTOR after overexpression of MOTOR; B. qRT-PCR detection of the mRNA levels of CCL2, CCL3, and CCL4 under LPS stimulation after overexpression of MOTOR.

[0103] Figure 4 shows that exosomes containing MOTOR promote the survival time of septic mice. A and B. NTA and electron microscopy analysis showed that exosomes were extracted by this method; C. Exosomes containing MOTOR were injected into mice via tail vein injection, and it was found that the survival time of septic mice was significantly increased.

[0104] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.

Claims

1. Use of the MOTOR gene or its pharmaceutical derivatives in the preparation of a drug for treating sepsis, characterized in that: The nucleotide sequence of the MOTOR gene is shown in SEQ NO.

1.

2. The use according to claim 1, characterized in that: The drug is used in a dosage form suitable for releasing the MOTOR gene or its pharmaceutically acceptable derivative in the user's peripheral blood.

3. The use according to claim 2, characterized in that: The dosage forms are tablets, capsules, oral liquids, lozenges, aerosols, injections, ointments, and granules.

4. The use according to claim 3, characterized in that: The dosage form is an injection.

5. A drug for preventing or treating sepsis, characterized in that: The drug includes exosomes containing the MOTOR gene; the nucleotide sequence of the MOTOR gene is shown in SEQ NO.

1.

6. The drug according to claim 5, wherein the dosage form of the drug is tablets, capsules, liquid medicines, lozenges, aerosols, injections, ointments, or granules.

7. The drug according to claim 6, characterized in that The dosage form is an injection.

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