Kits, drugs and uses thereof based on circular RNA circMagi1 and / or G3BP2 protein

Abstract

The present invention belongs to the field of medicines, and specifically relates to a kit, a drug and its use based on circular RNA circMagi1 and / or G3BP2 protein. The present invention provides a new pathogenic bacteria infection marker screening marker circular RNA circMagi1 and the G3BP2 protein interacting therewith, a screening kit made with the new pathogenic bacteria infection marker, which can achieve effective and rapid screening of pathogenic bacteria Pseudomonas aeruginosa infection markers, and a drug made with a G3BP2 protein expression promoter, which can improve the body's anti-inflammatory ability to cope with Pseudomonas aeruginosa infection. The kit and drug of the present invention have good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of medicines, and in particular relates to a kit, a medicine and uses thereof based on circular RNA circMagi1 and / or G3BP2 protein. Background Art

[0002] Pseudomonas aeruginosa is an opportunistic pathogen and one of the main pathogenic bacteria causing nosocomial infections in my country. It usually infects immunocompromised patients such as diabetes, cancer, burns and blood diseases. Due to its easy colonization, difficult elimination and extensive drug resistance mechanism, the bacteria are resistant to conventional antibiotics, which increases the difficulty of treatment. At present, the diagnostic method for Pseudomonas aeruginosa infection is mainly based on positive culture and biochemical identification of Pseudomonas for diagnosis. However, the bacterial culture cycle is long, and it is difficult to achieve rapid diagnosis, which is very unfavorable for the timely treatment of infection. Therefore, it is very important to develop rapid in vitro detection reagents for infection by pathogenic bacteria such as Pseudomonas aeruginosa.

[0003] Circular RNA is a special type of non-coding RNA molecule. It is a covalently closed circular biomacromolecule without a 5' cap and a 3' poly (A) tail. Although it only accounts for 2% of the transcripts, it has a longer half-life due to its significant nuclease resistance. It is widely expressed in the heart, brain, liver, muscle, lung, cancer tissues, etc., and the expression level is closely related to the growth stage. Circular RNA is involved in many pathological and physiological processes, and can function as miRNAs sponges, protein sponges, decoys, and scaffolds, so it often plays a vital role in the occurrence and development of diseases. Studies have shown that circular RNA is involved in the regulation of innate immune defense caused by viral infection, but there is little research on circular RNA related to pathogenic bacterial infection. Haitao Yuan et al. It was disclosed that some circular RNA expressions were associated with Pseudomonas aeruginosa infection, but the circular RNA circMagi1 was not involved.

[0004] Circular RNA circMagi1 (also known as hsa_circ_0066459) is a circular RNA that plays an important role in neuronal development, regeneration, and synaptic plasticity. According to related studies, it originates from a gene that controls synapse formation and is associated with psychiatric disorders.

[0005] G3BP2 protein is well-known for its function as a key component of stress granules. It is a multifunctional protein with many functions involved in the body's physiological processes. High expression of G3BP2 protein is associated with the occurrence of various tumors such as colorectal cancer, bladder cancer, breast cancer, and non-small cell lung cancer. It can directly bind to and regulate the expression of c-Myc. In the development of various cancers, the expression abundance of G3BP2 is positively correlated with the degree of cancer pathological development. Therefore, G3BP2 can be used as a combined or independent detection indicator for tumor diagnosis and provide a new target for gene therapy. However, there are no reports on the relationship between G3BP2 and pathogenic bacterial infection. Summary of the invention

[0006] The purpose of the present invention is to provide a new circular RNA pathogenic bacterial infection marker, as well as the use of the marker and a detection reagent for a protein interacting therewith in the preparation of a kit for screening pathogenic bacterial infection, and in a drug for treating pathogenic bacterial infection.

[0007] The technical solution of the present invention includes:

[0008] Use of a reagent for detecting the expression level of circular RNA circMagi1, G3BP2 protein and / or a complex of circular RNA circMagi1 and G3BP2 protein in the preparation of a kit for screening pathogenic bacterial infection, wherein the nucleotide sequence of the circular RNA circMagi1 is shown in SEQ ID NO.1 or SEQ ID NO.5;

[0009] The amino acid sequence of the G3BP2 protein is shown in SEQ ID NO. 4.

[0010] Furthermore, the reagent for detecting the expression level of circular RNA circMagi1 is an RNA sequencing reagent or a PCR detection reagent.

[0011] Furthermore, the reagent for detecting the expression level of G3BP2 protein is an enzyme-linked immunosorbent assay reagent, a Western blot reagent, a protein spectrum assay reagent or a protein chip assay reagent.

[0012] Furthermore, the reagent for detecting the expression level of the complex of circular RNA circMagi1 and G3BP2 protein is an RNA sequencing reagent, a PCR detection reagent, an enzyme-linked immunosorbent assay reagent, a Western blot reagent, a protein spectrum assay reagent or a protein chip assay reagent.

[0013] Furthermore, the pathogenic bacteria is Pseudomonas aeruginosa.

[0014] Furthermore, the reagent is a reagent for detecting the expression level of RNA circMagi1, G3BP2 protein and / or the complex of circular RNA circMagi1 and G3BP2 protein in lung tissue or bronchoalveolar lavage fluid.

[0015] The present invention also provides a kit for screening pathogenic bacterial infection, which comprises reagents for detecting the expression level of circular RNA circMagi1, G3BP2 protein and / or the complex of circular RNA circMagi1 and G3BP2 protein.

[0016] Furthermore, the reagent for detecting the expression level of circular RNA circMagi1 is an RNA sequencing reagent or a PCR detection reagent;

[0017] The reagent for detecting the expression level of G3BP2 protein is an enzyme-linked immunosorbent assay reagent, a Western blot reagent, a protein spectrum assay reagent or a protein chip assay reagent;

[0018] The reagent for detecting the expression level of the complex of circular RNA circMagi1 and G3BP2 protein is an RNA sequencing reagent, a PCR detection reagent, an enzyme-linked immunosorbent assay reagent, a Western blot reagent, a protein spectrum assay reagent or a protein chip assay reagent.

[0019] Furthermore, the pathogenic bacteria is Pseudomonas aeruginosa.

[0020] Furthermore, the reagent is a reagent for detecting the expression level of RNA circMagi1, G3BP2 protein and / or the complex of circular RNA circMagi1 and G3BP2 protein in lung tissue or bronchoalveolar lavage fluid.

[0021] The present invention also provides a use of a G3BP2 protein expression promoter in the preparation of a drug for resisting pathogenic bacterial infection, wherein the G3BP2 protein expression promoter comprises a plasmid or a lentivirus that overexpresses the G3BP2 protein;

[0022] The amino acid sequence of the G3BP2 protein is shown in SEQ ID NO. 4;

[0023] The pathogenic bacteria is Pseudomonas aeruginosa;

[0024] The drug is an anti-inflammatory drug.

[0025] Finally, the present invention provides a drug for resisting pathogenic bacterial infection, which is a preparation prepared by using a G3BP2 protein expression promoter as an active ingredient and adding pharmaceutically acceptable excipients;

[0026] The preparation is an oral preparation, a nasal preparation or an injectable preparation;

[0027] The pathogenic bacteria is Pseudomonas aeruginosa.

[0028] The key of the present invention is that the expression levels of circular RNA circMagi1 and the G3BP2 protein interacting therewith in mouse lung tissue and human bronchoalveolar lavage fluid are significantly correlated with infection with Pseudomonas aeruginosa, so the expression levels of circular RNA circMagi1 and the G3BP2 protein interacting therewith in lung tissue or bronchoalveolar lavage fluid can be used as a basis for judging infection with Pseudomonas aeruginosa. Detecting the expression level of circular RNA circMagi1, or G3BP2 protein, or the complex of circular RNA circMagi1 and G3BP2 protein in lung tissue and bronchoalveolar lavage fluid as an auxiliary diagnosis method, combined with the currently commonly used clinical diagnosis methods such as clinical manifestations and blood routine examinations, can make infection with Pseudomonas aeruginosa be diagnosed quickly and accurately. As for the specific means of detecting the expression level of circular RNA circMagi1 and the G3BP2 protein interacting therewith in human lung tissue and bronchoalveolar lavage fluid, various means disclosed in the prior art can be used. The embodiment of the present invention specifically uses a PCR detection method for circular RNA circMagi1 detection and a Western Blot method for G3BP2 protein detection, but is not limited to this method. Any method that can detect the expression level of circular RNA circMagi1 and G3BP2 protein in human lung tissue and bronchoalveolar lavage fluid can be used for screening for Pseudomonas aeruginosa infection.

[0029] The key to the present invention is that it is determined that circular RNA circMagi1 regulates immune defense to respond to pathogen infection through a signal axis. G3BP2 protein plays an important role in resisting Pseudomonas aeruginosa infection. When G3BP2 expression is increased, it is beneficial to improve the body's anti-inflammatory ability to respond to Pseudomonas aeruginosa infection. The G3BP2 protein expression promoter is made into a drug against Pseudomonas aeruginosa infection, which has practical promotion and application value.

[0030] The present invention provides a new pathogenic bacteria infection marker screening marker and a new pathogenic bacteria infection marker screening kit, which can realize the effective and rapid screening of pathogenic bacteria Pseudomonas aeruginosa infection markers and have good application prospects.

[0031] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0032] The above contents of the present invention are further described in detail below through specific implementation methods. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the structure of circular RNA circMagi1.

[0034] Figure 2 Verification of the circular structure of circular RNA circMagi1, where A: Actinomycin D treatment verifies the stability of the circMagi1 circular structure; B: RNase R treatment verifies the stability of the circMagi1 circular structure.

[0035] Figure 3The subcellular localization results of circular RNA circMagi1, where A: Cytoplasmic and nuclear RNA separation detection showed that circMagi1 was mainly expressed in the cytoplasm; B: RNA fluorescence in situ hybridization verified that circMagi1 was mainly distributed in the iBMDM cytoplasm, where DAPI is the nuclear staining result, circMagi1 is the circular RNA circMagi1 staining result, and Merge is the merged image after cytoplasmic and nuclear staining.

[0036] Figure 4 The expression changes of circular RNA circMagi1 during acute lung infection, where A: circMagi1 expression at 24 h after infection; B: circMagi1 expression at various time points after acute lung infection.

[0037] Figure 5 These are the experimental results of circular RNA circMagi1 binding to proteins, where A: RNA Pulldown silver staining gel; B: Enrichment analysis of the results of mass spectrometry detection using the KOBAS database; C: RNA Pulldown combined with RIP detection; D: RIP sample plus Trizol to extract RNA and then use qPCR to detect the enrichment level of circMagi1.

[0038] Figure 6 These are the experimental results of circMagi1 binding to G3BP2 protein to enhance its protein stability, including: A: circMagi1 affects G3BP2 protein stability through the proteasome pathway; B: circMagi1 inhibits G3BP2 protein ubiquitination detection; C: circMagi1 enhances G3BP2 protein stability by inhibiting K48 ubiquitination; D: G3BP2 protein expression was detected in mouse lung tissue at different infection times, with 4-5 mice in each group, and 2 mice were randomly selected from each group for detection; E: Quantitative analysis of G3BP2 protein expression.

[0039] Figure 7 These are the experimental results of circular RNA circMagi1 participating in the regulation of the cytokine signaling axis through G3BP2, where A: cell interference efficiency detection; B: cytokine detection; Vehicle+sh-NC: iBMDM control strain; circMagi1+sh-NC: iBMDM dual-stable control strain; circMagi1+sh-G3BP2: iBMDM dual-stable strain.

[0040] Figure 8The expression changes of human circular RNA circMagi1 in the patient's bronchoalveolar lavage fluid, including: A: Sequence alignment of human and mouse circMagi1; B: Expression detection of human circMagi1 (hsa_circ_0066459) in the patient's bronchoalveolar lavage fluid.

[0041] Fig. 9 The results of ROC curve analysis were clinically verified. DETAILED DESCRIPTION

[0042] The reagents and materials used in the specific embodiments of the present invention are all commercially available unless otherwise specified.

[0043] Example 1 Pathogenic bacterial infection diagnostic kit for detecting circular RNA circMagi1

[0044] The circular RNA circ_0066459 (hsa_circ_0066459) detected by the kit of this example is human circular RNA circMagi1, and its nucleotide sequence is shown in SEQ ID NO.1. The details are as follows:

[0045] GCACAACCCGATCTCCCAGAGAAGGAGAAGTGCCTGGCGTGGACTATAACTTTCTGACTGTGAAGGAGTTCTTGGACCTCGAGCAGAGTGGGACTCTTCTGGAAGTCGGCACCTATGAAGGAAACTATTATGGGACACCCAAGCCTCCTAGCCAGCCAGTCAGT GGGAAAGTGATCACGACGGATGCCTTGCACAGCCTTCAGTCTGGCTCTAAGCAGTCGACCCCGAAGCGAACCAAGTCCTACAATGATATGCAAAATGCTGGCATAGTCCACGCGGAATGAGGAGGAGGATGACGTTCCTGAAATGAACAGCAGCTTTACAG.

[0046] The specific components of the PCR detection kit used to detect the above circular RNA circ_0066459 are as follows:

[0047]

[0048] The nucleotide sequences of the above primers are as follows:

[0049] PCR Forward Primer is shown in SEQ ID NO. 2:

[0050] ACCCCGAAGCGAACCAAGTCCT.

[0051] PCR Reverse Primer is shown in SEQ ID NO. 3:

[0052] CGGGTTGTGCCTGTAAAGCTGC.

[0053] The method of using the above kit is: take lung tissue or alveolar lavage fluid, and use the above kit to detect the expression level of RNA circ_0066459 according to the existing PCR detection method.

[0054] Example 2 Pathogenic bacteria infection diagnostic kit for detecting protein G3BP2

[0055] The amino acid sequence of the protein G3BP2 detected by the kit of this embodiment is shown in SEQ ID NO. 4:

[0056] MVMEKPSPLLVGREFVRQYYTLLNKAPEYLHRFYGRNSSYVHGGVDASGKPQEAVYGQNDIHHKVLSLNFSECHTKIRHVDAHATLSDGVVVQVMGLLSNSGQPERKFMQTFVLAPEGSV PNKFYVHNDMFRYEDEVFGDSEPELDEESEDEVEEEQEDRQPSPEPVQENANSAYYDAHPVTNGIEEPLEESSHEPEPEPESETKTEELKPQVEEKHLEELEEKSATPPPAEPASLPQEPP KAFSWASVTSKNLPPSGTVSSSGIPPHVKAPVSQPRVDAKPEVQSQPPRVREQRPRERPGFPPRGPRPGRGDMEQNDSDNRRIIRYPDSHQLFVGNLPHDIDENELKEFFMSFGNVVELR INTKGVGGKLPNFGFVVFDDSEPVQRILIAKPIMFRGEVRLNVEEKKTRAARERETRGGGDDRRDIRRNDRGPGGPRGIVGGGMMRDRDGRGPPPRGGMTQKLGSGRGTGQMEGRFTGQRR

[0057] The detection reagent used to detect the above protein G3BP2 is the reagent required for detecting G3BP2 by Western Blot technology; or the reagent required for protein spectrum, protein chip and ELISA kit.

[0058] The method of using the above kit is: take lung tissue or alveolar lavage fluid, and use the above kit to detect the expression level of protein G3BP2 according to the existing Western Blot method, protein spectrum method, protein chip method or ELISA method.

[0059] Example 3 Pathogenic bacterial infection diagnostic kit for detecting circular RNA circMagi1

[0060] The circular RNA circMagi1 (mmu_circ_0001496) detected by the kit of this example is a mouse circular RNA circMagi1, and its nucleotide sequence is shown in SEQ ID NO.5. The details are as follows:

[0061] GCACAACCCGGTCTCCCAGAGAAGGAGAAGTGCCTGGTGTGGATTACAGCTTTCTGACTGTGAAGGAGTTCTTGGACCTCGAGCAGAGCGGGACCCTGTTGGAAGTCGGCACCTATGAAGGAAACTATTATGGGACACCCAAACCTCCTAGCCAGCCAGTCAGT GGGAAAGTGATCACGACGGATGCCTTGCACAGCCTGCAGTCTGGCTCCAAGCAGTCGACCCCTAAGCGAACAAAGTCCTACAATGATATGCAAAATGCTGGCATAGTCCACCCGGAGAATGAGGAGGAGGAGGATGTCCCTGAAATGAACAGTAGCTTTACAG.

[0062] The specific components of the PCR detection kit for detecting the above circular RNA circMagi1 are as follows:

[0063]

[0064] The nucleotide sequences of the above primers are as follows:

[0065] PCR Forward Primer is shown in SEQ ID NO. 6:

[0066] ACCCCTAAGCGAACAAAGTCCT.

[0067] PCR Reverse Primer is shown in SEQ ID NO. 7:

[0068] CGGGTTGTGCCTGTAAAGCTAC.

[0069] The method of using the above kit is: take lung tissue or alveolar lavage fluid, and use the above kit to detect the expression level of mmu_circ_0001496 or has_circ_0066459 according to the existing PCR detection method.

[0070] The beneficial effects of the present invention are further illustrated by experimental examples below:

[0071] It should be noted that the sequence of the circular RNA has_circ_0066459 in the experimental part of the clinical sample in Experimental Example 2 is human circMagi1. The sequence of the circular RNA circMagi1 in the animal experimental part in Experimental Examples 1 and 2 is mouse circMagi1. According to common sense in the field, the conclusions obtained from the study of mouse sequences are also applicable to human sequences.

[0072] Among them, human circMagi1 and its corresponding primers are shown in Example 1, and mouse circMagi1 and its corresponding primers are shown in Example 3. The primers for the control mouse linearMagi1, the internal reference gene GAPDH, and the _G3BP2 gene and cytokines used in the experiment are as follows:

[0073]

[0074] Experimental Example 1 Structural confirmation and localization of circular RNA circMagi1

[0075] 1. Experimental Methods

[0076] 1 Validation of the circular structure of circular RNA circMagi1

[0077] 1.1 RNase R digestion experiment

[0078] RNA was extracted from lung tissue of mice in good growth condition and divided into two parts: one for RNase R treatment and the other for digestion buffer only as a control, then reverse transcribed into cDNA and tested by qPCR. RNase R treatment conditions: 2μg total RNA was mixed with 5 U / μg RNase R (Epicenter® Technologies) and incubated at 37℃ for 20 minutes.

[0079] 1.2 Actinomycin D treatment experiment

[0080] Mouse alveolar epithelial cells (MLE-12 cells) were exposed to DMEF / 12 medium containing 2 μg / mL actinomycin D for 2, 4, 8, or 12 hours, and the cells were collected. Cell RNA was extracted and then reverse transcribed into cDNA for qPCR detection.

[0081] 2. Subcellular localization of circular RNA circMagi1

[0082] 2.1 Cytoplasm and nuclear separation experiment

[0083] Mouse alveolar epithelial cells (MLE-12 cells) in good growth condition were obtained, and the cytoplasm and nucleus of MLE-12 cells were extracted respectively according to the instructions of the Cytoplasmic and Nuclear RNA Purification Kit of Norgen Company. The RNA extracted was reverse transcribed into cDNA, and then qPCR detection was performed.

[0084] 2.2 RNA fluorescence in situ hybridization experiment

[0085] Immortalized macrophages (iBMDM cells) with good growth status were taken, and the cytoplasm and nucleus of iBMDM cells were extracted respectively according to the instructions of the Cytoplasmic and Nuclear RNA Purification Kit of Norgen Company, and then RNA fluorescence in situ hybridization was performed according to the instructions of the Fluorescent In Situ Hybridization Kit of Ribo Company.

[0086] The cDNA synthesis and qPCR detection involved in the above experiments were carried out according to the following steps:

[0087] A: cDNA synthesis

[0088] (1) Using PrimeScript TM RT reagent Kit with gDNA Eraser removes genomic DNA mixed in total RNA. The system is as follows:

[0089]

[0090] The above system was reacted at 42℃ for 2 min, and then quickly transferred to ice to continue the following reaction:

[0091]

[0092] The above system was added in a total of 10 μl, reacted at 37°C for 15 min, reacted at 85°C for 5 s, and then placed on ice.

[0093] B. qPCR reaction

[0094] (1) Reaction system

[0095]

[0096] (2) Two-step PCR amplification standard procedure:

[0097] Stage 1: Preincubation

[0098] 95℃ 30 s

[0099] Stage 2: 2 Step Amplification (45 cycles)

[0100] 95℃ 5 s

[0101] 60℃ 30 s

[0102] Stage 3: Melting

[0103] 95℃ 10 s

[0104] 65℃ 60 s

[0105] 97℃ 1 s

[0106] 2. Experimental Results

[0107] circMagi1 (circ-0001496) is formed by reverse splicing of the exon element of Magi1 gene ( Figure 1 ). RNase R digestion experiments and actinomycin D treatment experiments determined that circ-0001496 was resistant to RNase R, had a longer half-life, and had circular RNA characteristics, thus confirming that it was a circular RNA ( Figure 2 ), and then used cytoplasmic nuclear separation experiments and RNA fluorescence in situ hybridization technology to determine that it mainly exists in the cytoplasm ( Figure 3 ).

[0108] The above experimental results show that there is a structurally stable circular RNA circMagi1 in lung tissue. Circular RNA circMagi1 mainly exists in the cytoplasm of cells.

[0109] Experimental Example 2 Functional study of circular RNA circMagi1

[0110] 1. Correlation between circMagi1 and pathogenic bacterial infection

[0111] 1.1 Construction of infection animal model

[0112] The mice were infected by instilling PAO1 Pseudomonas aeruginosa liquid at the root of the tongue. The specific method was as follows: the mice were anesthetized by intraperitoneal injection of 10% chloral hydrate, and the injection volume was adjusted according to the weight of the mice; the anesthetized mice were placed on the tongue drop table, and the tongue of the mice was gently pinched out with plastic tweezers, and then the tongue of the mice was fixed with hands disinfected with alcohol; the epiglottis cartilage of the mice was found and its opening and closing were observed to be normal; 3×10 6 Insert the prepared PAO1 Pseudomonas aeruginosa solution (CFU / 50 μL / mouse) into the pipette tip near the upper palate and accurately drip the solution onto the epiglottis cartilage; observe the mouse's choking until the solution is completely choked into the lungs.

[0113] 1.2 Lung tissue testing

[0114] The infected mice were routinely fed for different periods of time, and lung tissues were taken for qPCR detection of circMagi1 expression, while lung tissues of healthy mice were used as controls. The specific method was as follows: the mice were killed by cervical dislocation, the mouse chest was wiped with alcohol cotton balls, the lung tissues were taken out, and placed on a clean culture dish; the excess blood clots and trachea on the lung tissue were removed with scissors and tweezers; the lung tissue obtained was quickly frozen in liquid nitrogen, and the lung tissue was divided into two parts after grinding, one part was frozen at -80℃ for standby use, and 500 μL of Trizol was added to the other part, total RNA was extracted, and the expression content of circMagi1 was detected by qPCR after reverse transcription, wherein the method of extracting RNA and performing qPCR detection after reverse transcription was the same as that in Experimental Example 1.

[0115] 1.3 Relationship between circMagi1 expression in lung tissue and PAO1 infection

[0116] Lung tissue samples at different time points of the constructed mouse Pseudomonas aeruginosa acute lung infection model were collected, and the expression of circMagi1 was detected by qPCR. Figure 4 .

[0117] Figure 4 The results showed that the expression of circMagi1 was negatively correlated with the process of host immune response to infection, and its expression level showed a trend of first decreasing and then recovering. That is, in the early stage of the immune response, the expression of circMagi1 decreased, and in the later stage of the immune response (i.e., recovery), the expression of circMagi1 returned to the initial stage. This suggests that in Pseudomonas aeruginosa infection, the host may activate the host's immune response by degrading circRNAs.

[0118] 2. Interaction between circMagi1 and proteins

[0119] 2.1 RNA pulldown and RIP detection to identify interacting proteins of circMagi1

[0120] (1) A stable iBMDM cell line overexpressing circMagi1 was constructed by packaging lentivirus and infecting iBMDM cells. The circMagi1 probe (P-circMagi1 probe) synthesized by Ruibo Biotechnology, which can specifically bind to the reverse cleavage site of circMagi1, and the P-NC probe used as a control were co-incubated with commercial streptavidin-labeled magnetic beads, and then co-incubated with iBMDM cell lysates overexpressing circMagi1, and the magnetic bead-circMagi1-protein complex was isolated and prepared as a WB sample;

[0121] (2) SDS polyacrylamide gel was used to separate circMagi1-binding proteins in WB samples, and the differential proteins were visualized by silver staining. Then, the differential gel strips were cut and sent to Novogene for protein spectrum identification and analysis. The protein expression abundance was ranked and screened, and 13 candidate targets were obtained.

[0122] (3) 13 Flag-tagged target protein overexpression plasmids were constructed, and circMagi1 was co-transfected with them into 293T cells. Samples were collected 48 hours after transfection, and the magnetic beads-circMagi1-protein complex was obtained by RNA pulldown technology. The binding of circMagi1 and target protein was detected by WB; the Flag agarose beads-target protein-RNA complex was obtained by RIP technology, and the binding of target protein and circMagi1 was detected by qPCR. The specific detection method is as follows:

[0123] A: RNA Pulldown Technology

[0124] 1) Preparation of cell lysate

[0125] Discard the cell culture medium, wash the cells twice with pre-cooled PBS, and discard the supernatant; add 1 mL of cell lysis buffer (weak) (add 200 U / mL RNase Inhibitor) to each dish of cells; place at 4℃ for 10 minutes, blow the cells into a 1.5 mL EP tube, and lyse on ice for 30 minutes, mixing by inverting during the process; centrifuge at 4℃, 17000g, centrifuge for 15 minutes; aspirate the supernatant into a new EP tube and store on ice; take 50 µL of supernatant into a new EP tube, add 12.5 µL of 5×SDS protein loading buffer, and boil at 95-100℃ for 10 minutes as the input group; keep the remaining lysis buffer aside for later use;

[0126] 2) Preparation of probe-bound beads

[0127] Mix the beads by pipetting, take 20 µL of beads for each sample (corresponding to no more than 1 mL of lysis buffer), place them on a magnetic rack and remove the supernatant (you can mix them in n+1 according to the required amount); wash them 3 times with 1 mL of cell lysis buffer (weak), and remove the supernatant; add 200 µL of cell lysis buffer (weak) to the washed beads; add 20 pmol of RNA probe for every 20 µL of beads; rotate the mixer at room temperature for 20 min to ensure that the probe is fully combined with the beads; place the beads from the previous step on a magnetic rack and remove the supernatant; wash the beads once with cell lysis buffer (weak), and resuspend them with 100 uL of cell lysis buffer (weak) for every 20 µL of beads;

[0128] 3) RNA Pulldown

[0129] Pipette the cell lysate treated in step (1) into each tube of beads treated in the previous step; rotate and mix at 4°C for 4 h to allow the RNA in the cell lysate to fully bind to the probe on the beads; place the sample on a magnetic rack, discard the supernatant, add 1 mL of cell lysis buffer (weak) to wash, and repeat this step 5 times; aspirate the supernatant to obtain the protein-RNA-Beads complex, add 30 µL of 1 × SDS protein loading buffer to resuspend, heat at 95-100°C for 10 min, and store after cooling.

[0130] B. RIP technology

[0131] 1) Cell lysis

[0132] Complete step (1) according to the RNA pulldown procedure; take 50 µL of cell lysate into a new EP tube and add 500 µL Trizol as the input group;

[0133] 2) Beads-protein interaction

[0134] Vortex at low speed to mix the anti-Flag tag beads (using Flag tag protein as an example); take 20 µL beads for every 1 mL cell lysate, centrifuge for 5-10 seconds, and remove the supernatant; wash 3 times with 1 mL cell lysate (weak), and remove the supernatant; pipette 1 mL cell lysate into each tube of beads treated in the previous step; rotate and mix overnight at 4°C to allow the Flag tag protein in the cell lysate to fully bind to the antibody on the beads; centrifuge the sample for 5-10 seconds, carefully remove the supernatant, and then add 1 mL cell lysate (weak) to wash, and repeat this step 5 times; divide the sample into two parts as needed, discard the supernatant of 1 / 10, add 30 µL of 1× SDS protein loading buffer to resuspend, heat at 100°C for 10 min to elute the protein for IP binding detection; discard the supernatant of 9 / 10, add 500 µL Trizol to extract RNA;

[0135] 3) Extraction of RNA bound to proteins

[0136] Extract RNA from the samples that were added with Trizol in the previous step according to the steps; after reverse transcription, use qPCR to jointly detect and analyze with the Input group.

[0137] C. Western Blot (WB) detection

[0138] 1) Protein sample preparation is the same as RNA pulldown;

[0139] 2) Determination of protein concentration by BCA method

[0140] a. Preparation of protein standards

[0141] Use PBS solution to completely dissolve the protein standard, take 10 µL and dilute to 100 µL, so that the final concentration is 0.5 mg / mL. The diluted 0.5 mg / mL protein standard can be divided into aliquots and placed in a -20℃ refrigerator for long-term storage;

[0142] b. Preparation of BCA working solution

[0143] According to the number of samples, prepare an appropriate amount of BCA working solution at a ratio of 50:1 for each sample with 200 µL of reagent A: reagent B, mix thoroughly, and prepare fresh for each experiment;

[0144] c. Protein concentration determination

[0145] ① Pipette 20, 16, 12, 8, 4, 2, 1, and 0 µL of protein standard dilution into a 96-well plate, and add PBS to each well to make up to 20 µL, so that the final concentration of the standard is 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.025, and 0 mg / mL, respectively;

[0146] ② Add an appropriate volume of sample to a 96-well plate. If the sample is less than 20 µL, add PBS to make it up to 20 µL and make duplicate wells.

[0147] ③ Add 200 µL of the prepared BCA working solution to each well and place at 37°C for 20-30 min or at room temperature for 2 h;

[0148] ④ Determine A562 using an ELISA reader;

[0149] ⑤ Make a standard curve and calculate the protein concentration of the sample based on the sample volume used;

[0150] d. Take 10-20 µg of protein sample, add 5× protein loading buffer containing β-mercaptoethanol, and boil at 100℃ for 5-10 min to denature the protein;

[0151] 3) Use a 10 µL pipette or sample loading needle to add samples and pre-stained markers to the electrophoresis gel in order. Adjust the voltage to 80 V. After 30 minutes, when the upper layer of compressed gel has run out, adjust the voltage to 120 V and continue electrophoresis for 1 hour. Stop electrophoresis when the bromophenol blue indicator is about 1 cm from the bottom edge of the gel;

[0152] 4) Take out the electrophoresis gel and transfer it to the transfer buffer for soaking. Cut the protein gel of the target size according to the Marker indication and cut off the upper right corner as a mark; take out the PVDF membrane and cut it to the size of the target protein gel, and also cut off the upper right corner, and soak it in methanol for 15 seconds for activation;

[0153] 5) Soak the transfer clip, sponge and filter paper in the transfer solution, and clamp the protein glue and PVDF membrane in the order of blackboard, sponge, filter paper, protein glue, PVDF membrane, filter paper, sponge and whiteboard from bottom to top, and remove the bubbles; this step is carried out in the transfer solution environment;

[0154] 6) Place the transfer clip into the electrophoresis tank, put a biological ice box in the gap, and pour the transfer solution to fill the electrophoresis tank;

[0155] 7) Electrophoresis at 100 V in an ice-water bath. Adjust the time according to the concentration of the separation gel and the size of the target protein.

[0156] 8) After electrophoresis, remove the PVDF membrane with tweezers, transfer it to the blocking solution, and block it at 70-80 rpm for 1 h at room temperature;

[0157] 9) After blocking, seal the PVDF membrane and the corresponding primary antibody dilution solution in a hybridization bag and incubate in a 4°C refrigerator overnight;

[0158] 10) After the primary antibody incubation, recover the primary antibody as needed and wash the PVDF membrane with TBST solution three times, 10 minutes each time;

[0159] 11) Transfer the washed PVDF membrane to the corresponding secondary antibody dilution solution and incubate at room temperature at 70-80 rpm for 2 h;

[0160] 12) Wash the PVDF membrane three times with TBST solution and then expose it to light. Then, silver stain it.

[0161] D. qPCR detection was the same as in Experimental Example 1.

[0162] 2.2 Analysis of circMagi1 and its interacting proteins

[0163] RNA pulldown and RIP were used to search for circMagi1's interacting proteins. It was found that there were 13 proteins interacting with circMagi1, among which circMagi1 had the strongest binding ability with G3BP2 protein ( Figure 5 ).

[0164] 3. Effect of circMagi1 binding to G3BP2 protein on the stability of G3BP2 protein

[0165] 3.1 Methods

[0166] 1) By packaging lentivirus and infecting iBMDM cells, a stable iBMDM cell line overexpressing circMagi1 was constructed, and the lentiviral empty plasmid was packaged and then infected with iBMDM. The iBMDM cells obtained after puromycin (puro) selection were used as controls. Both cells were treated with actinomycin ketone (CHX) at a final concentration of 10μg / mL, and MG132 and 3-MA inhibitors were added at a final concentration of 10μM for treatment. After 2 hours, the cells were collected and the levels of GAPDH protein and G3BP2 protein were detected by WB;

[0167] 2) G3BP2, circMagi1 and ubiquitin UB overexpression plasmids were co-transfected into 293T cells. 293T cells transfected with empty plasmids and 293T cells co-transfected with G3BP2 and ubiquitin UB overexpression plasmids were used as controls. Samples were collected 48 hours after transfection, and the ubiquitination of G3BP2 and K48 ubiquitination were verified by WB.

[0168] 3) Take the frozen lung tissues at different infection time points under "1.2 Lung Tissue Detection", add WB lysis buffer for lysis, perform protein quantification after ultrasound, randomly select two samples from each group, detect the expression of G3BP2 protein by WB, and use Image J for quantification.

[0169] 3.2 Results

[0170] Results Figure 6 ,from Figure 6 As shown in A~C, circMagi1 can enhance the stability of G3BP2 protein by inhibiting K48 ubiquitination of G3BP2 protein after binding to G3BP2 protein. Figure 6 As can be seen from D~E: The expression of G3BP2 protein is also negatively correlated with the process of host immune response to infection, and its expression level also shows a trend of first decreasing and then recovering. That is, in the early stage of the immune response, the expression level of G3BP2 protein is reduced, and only after recovery from the infection does the expression level of G3BP2 protein return to the level before infection.

[0171] 4. Experiments on circular RNA circMagi1 regulating the cytokine signaling axis through G3BP2

[0172] 4.1 Methods

[0173] (1) An iBMDM stable strain that overexpressed circMagi1 and reduced / weakened G3BP2 expression (referred to as the iBMDM stable strain) was constructed by packaging lentivirus and infecting cells, and the construction of the stable strain was successfully detected by qPCR. At the same time, an iBMDM stable strain that only overexpressed circMagi1 (referred to as the iBMDM stable control strain) and iBMDM cells infected with lentiviral empty vector plasmids were used as controls;

[0174] (2) The cell line constructed in step (1) was infected with the PAO1 strain at an MOI of 20. After 6 hours of infection, total cell RNA was collected and the expression levels of relevant cytokines were detected.

[0175] 4.2 Results

[0176] Results Figure 7 ,from Figure 7 It can be seen that the expression level of G3BP2 protein in iBMDM stable strain is lower than that in iBMDM stable control strain, and there is a statistical difference. After infection with PAO1 bacteria, compared with iBMDM control strain, the expression levels of cytokines in iBMDM stable control strain and iBMDM stable strain are significantly different, among which the pro-inflammatory factors IL-1β and TNF-α of iBMDM stable control strain are significantly reduced, IL-1β of iBMDM stable strain is significantly increased, and TNF-α has no significant change; the anti-inflammatory factor IL-10 of iBMDM stable control strain is significantly increased, and IL-10 of iBMDM stable strain is significantly reduced; the chemokines CCL2 and CXCL3 of iBMDM stable control strain are significantly reduced, CCL2 of iBMDM stable strain is significantly increased, and CXCL3 has no statistically significant change. It shows that G3BP2 protein plays an important role in resisting Pseudomonas aeruginosa infection.

[0177] Compared with the iBMDM dual-stable control strain, the pro-inflammatory factors IL-1β and TNF-α of the iBMDM dual-stable strain were significantly increased, the anti-inflammatory factor IL-10 was significantly decreased, and the chemokines CCL2 and CXCL3 were significantly increased. This indicates that circular RNA circMagi1 regulates immune defense to respond to pathogen infection through the circMagi1-G3BP2-cytokine signaling axis. Increased expression of G3BP2 is beneficial to improve the body's anti-inflammatory ability to respond to Pseudomonas aeruginosa infection.

[0178] 5. Expression changes of human circular RNA circMagi1 in patients’ bronchoalveolar lavage fluid

[0179] 5.1 Methods

[0180] (1) The bronchoalveolar lavage fluid of the patients was collected and divided into a control group and an infection group according to whether the patients had lung infection. There were 10 cases in each group. There was no statistically significant difference in the general data of the two groups (P > 0.05), so they were comparable.

[0181] (2) Centrifuge at 4°C, 2000 g for 5 min, discard the supernatant, add 500 µL Trizol, and mix by pipetting;

[0182] (3) Extract RNA, reverse transcribe and use qPCR for combined detection and analysis. The qPCR detection is the same as that in Experimental Example 1.

[0183] 5.2 Results

[0184] Circular RNA has a stable structure and is highly conservative in evolution. Comparison of mouse circMagi1 and its corresponding human circular RNA circMagi1 (has_circ_0066459) sequence revealed that the sequence similarity between the two was as high as 94.26%. Considering that circMagi1 is related to pathogenic bacterial infection and immune response, bronchoalveolar lavage fluid was collected and grouped according to whether the patients were infected with Pseudomonas aeruginosa. qPCR detection was then performed. The results showed that in the infected group, the expression level of hsa_circ_0066459 was significantly downregulated compared with patients not infected with Pseudomonas aeruginosa ( P <0.001, the ROC analysis results of the infection group and the control group were AUC 0.95, specificity 100%, and sensitivity 90% ( Figures 8 and 9). This is consistent with the mouse lung infection model, that is, the expression level of circMagi1 decreased significantly during infection. This shows the relevance of circMagi1 to clinical screening applications of Pseudomonas aeruginosa infection. Since circMagi1 affects the stability of G3BP2 protein, circMagi1 decreases during Pseudomonas aeruginosa infection, and G3BP2 protein also decreases accordingly. Therefore, G3BP2 protein is also relevant to clinical screening applications of Pseudomonas aeruginosa infection and has important clinical value.

[0185] In summary, the present invention diagnoses infections caused by pathogenic bacteria such as Pseudomonas aeruginosa by detecting circular RNA circMagi1, G3BP2 or their complexes, and provides an effective basis for patients to take relevant treatment measures or make decisions; by increasing G3BP2 protein expression, the body's anti-inflammatory ability is improved to cope with Pseudomonas aeruginosa infection. The detection of circular RNA circMagi1, G3BP2 or their complexes is made into a kit for screening pathogenic bacterial infection, and the G3BP2 protein expression promoter is made into a drug for resisting pathogenic bacterial infection, which has good clinical application prospects.

Claims

1. Use of a reagent for detecting the expression level of circular RNA circMagi1 in preparing a kit for screening Pseudomonas aeruginosa infection, characterized in that: The nucleotide sequence of the circular RNA circMagi1 is shown as SEQ ID NO.1 or SEQ ID NO.

5.

2. The use according to claim 1, characterized in that The reagent for detecting the expression level of circular RNA circMagi1 is an RNA sequencing reagent or a PCR detection reagent.

3. The use according to claim 1, characterized in that The reagent is a reagent for detecting the expression level of RNA circMagi1 in lung tissue or bronchoalveolar lavage fluid.

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