LU500371B1 - Application of GAS5 in the diagnosis of severe asthma - Google Patents

Abstract

The invention discloses an application of GAS5 in the diagnosis of severe asthma. The invention firstly discovers that GAS5 is remarkably increased in severe asthma, and whether a subject suffers from severe asthma can be judged by detecting the expression level of GAS5; The invention also discloses that that expression level of the GAS5 is remarkably reduced after the glucocorticoid drugs are applied to the severe asthma sample, and whether the subject suffer from severe asthma can be judged by detecting the expression level of the GAS5 before and after the glucocorticoid drugs are applied, so that the problem of individual difference is solved.

Description

DESCRIPTION Application of GASS in the diagnosis of severe asthma

TECHNICAL FIELD The invention belongs to the field of biomedicine and relates to the application of GASS in the diagnosis of severe asthma.

BACKGROUND Bronchial asthma (asthma for short) is a chronic inflammatory disease of the airway with heterogeneous characteristics in etiology, pathogenesis, clinical manifestations and results (De Ferrari, L.; Chiappori, A.; Bagnasco, D, et al, Molecular phenotyping and biomarker development: are we on our way towards targeted therapy for severe asthma? Expert review of respiratory medicine 2016, 10 (1), 29-38.). Severe asthma, a subtype of asthma, is the main cause of disability and death of asthma. Epidemiological survey in China showed that severe asthma among people over 14 years old accounted for about

5.99% of asthmatic patients. Although the proportion was not high, it led to frequent emergency treatment or hospitalization of patients, seriously affected their quality of life and physical and mental health, and occupied huge medical resources (Asthma Group, Respiratory Branch of Chinese Medical Association, China Asthma Alliance. Chinese Experts Consensus on the Diagnosis and Management of Severe Asthma. Chinese journal of tuberculosis and respiration. 2017; 40(11): 813-829). Based on the 2014 ERS/ATS guidelines and China’s expert consensus (Bel, E. H.; Sousa, A.; Fleming, L.,. et al, Diagnosis and definition of severe refractory asthma: an international consensus statement from the Innovative Medicine Initiative (IMI). Thorax 2011, 66 (10), 910-7), severe asthma refers to the asthma patient who cannot be controlled until after the treatment according to the Grade 4 or 5 recommended by the Global Initiative for Asthma (GINA) or cannot be controlled under the above treatment when the asthma 1s diagnosed clearly. It 1s also excluded that many diseases such as chronic obstructive pulmonary disease, bronchitis, tumor, interstitial lung disease, gastroesophageal reflux disease and the like which may lead to similar symptoms, diseases closely related to asthma such as aspirin-aggravated respiratory system diseases and allergic bronchopulmonary aspergillosis, diseases affecting the severity of asthma such as sleep-related respiratory diseases, chronic rhinitis, gastroesophageal reflux and heart diseases should also be taken into account in the diagnosis of severe asthma. The pathogenesis of severe asthma 1s different from that of common asthma,Severe asthma shows more complex and diverse inflammatory reactions, a higher degree of neutrophil participation, more severe lung function loss and airway remodeling, the reason of which 1s still not completely clear.

Glucocorticoid resistance is an important feature of severe asthma. When glucocorticoids work normally, glucocorticoid-activated glucocorticoid receptor(GR) is phosphorylated into GR®*™!! and transported to the nucleus. The DNA binding domain of GR“!!! dimer binds to glucocorticoid response element (GRE), which activates glucocorticoid effector genes to promote the transcription of anti-inflammatory genes and inhibit the transcription of inflammatory genes to exert anti-inflammatory effects (Petta, I.; Dejager, L.; Ballegeer, M., et al,. The Interactome of the Glucocorticoid Receptor and Its Influence on the Actions of Glucocorticoids in Combatting Inflammatory and Infectious Diseases. Microbiology and molecular biology reviews: MMBR 2016, 80 (2), 495-522). At present, the mechanism of glucocorticoid resistance in severe asthma is still unclear.

How to predict and restore the sensitivity of patients to glucocorticoid has always been a difficult problem and hot spot in the study of severe asthma.

Long non-coding RNA (IncRNA) is a group of RNA with length more than 200 nucleotides and no protein-encoding function.

Although IncRNA does not encode protein, it participates in the physiological process of many cells.

At present, the research on LncRNA and bronchial asthma is rare, and the research on severe asthma is even less (Li, X.; Wu, Z.; Fu, X.,. et al, IncRNAs: insights into their function and mechanics in underlying disorders.

Mutation research.

Reviews in mutation research 2014, 762, 1-21). As a growth inhibitor, GASS has been investigated for its role in various tumors such as ovarian cancer, non-small cell lung cancer, and myocardial apoptosis, but it has few studies related to asthma, especially severe asthma (Cao, Q.; Wang, N.; Qi, J, et al, Long noncoding RNAGASS acts as a tumor suppressor in bladder transitional cell carcinoma via regulation of chemokine (CC motif) ligand 1 expression.

Molecular medicine reports 2016, 13 (1), 27-34). At present, there are no mature methods and biomarkers for assessing the sensitivity of patients with asthma to glucocorticoids, and finding biomarkers that can assess the sensitivity of glucocorticoids is of great significance for the early diagnosis and treatment of patients with severe asthma.

Based on the above description, we intended to deeply clarify the role and specific regulatory mechanism of GASS in hormone resistance of severe asthma through in vivo and in vitro experiments, to verify whether it can be used as a biomarker of severe asthma.

SUMMARY In order to make up for the defects of the prior art, the invention aims to provide a marker related to severe asthma, and whether a subject suffers from severe asthma can be accurately judged by using the marker, so that corresponding prevention and treatment measures are taken aiming at high risk and low risk, and the quality of life is improved.

In order to achieve the purpose, the invention adopts the following technical scheme: The present invention provides the application of a reagent for detecting GASS in a sample for the preparation of a product for the diagnosis of severe asthma.

A "sample" in the present invention is a sample comprising a cell or cellular material from which a nucleic acid, polypeptide, or other analyte can be obtained.

Examples of biological samples include, without limitation, urine, blood, serum, plasma, cerebrospinal fluid, pleural fluid, bronchial lavage, sputum, peritoneal fluid, bladder 1rrigation, secretions, oral irrigation, swabs, isolated cells, tissue samples, touch preparations, and fine needle aspirates.

Preferably, the sample 1s human blood.

Further, the reagent includes a reagent for detecting GASS by a nucleic acid sequencing technique, a nucleic acid hybridization technique, and a nucleic acid amplification technique.

Illustrative non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, strand terminator (Sanger) sequencing and dye terminator sequencing.

One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because RNA is less stable in cells and more susceptible to nuclease attack in experiments.

Another illustrative non-limiting example of a nucleic acid sequencing technique includes next generation sequencing (deep sequencing/high-throughput sequencing), the high- throughput sequencing technique being a single molecular cluster-based synthetic-while- sequencing technique based on proprietary reversible stopping chemical reaction principles. During sequencing, random fragments of genomic DNA are attached to the surface of optically transparent glass, and after the DNA fragments are subjected to extension and bridge amplification, hundreds of millions of clusters are formed on the surface of the glass, wherein each cluster is a single-molecule cluster with thousands of identical templates; and then the template DNA to be tested is sequenced by utilizing four special deoxynucleotide with fluorescent groups through a reversible synthesis-while- sequencing technology.

Nucleic acid hybridization techniques in the present invention include, but are not limited to, in situ hybridization (ISH), microarray and Southern or Northern blot. In situ hybridization (ISH) is a hybridization using a labeled complementary DNA or RNA strand as a probe to locate a specific DNA or RNA sequence in a portion or slice of tissue (in situ) or, 1f the tissue is small enough, throughout the tissue (whole-tissue embedded ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and localize mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole-tissue embedding. Sample cells and tissues are typically processed to immobilize the target transcript in situ and increase access to the probe. The probes hybridize to the target sequence at elevated temperature and excess probes are washed away. Autoradiography, fluorescence microscopy, or immunohistochemistry were used, respectively, to locate and quantitate radiolabeled, fluorescent, or antigen-labeled base-

labeled probes in the tissue. The ISH may also use two or more probes labeled with radioactive or other non-radioactive markers to simultaneously detect two or more transcripts.

Southern and Northern blot were used to detect specific DNA or RNA sequence, respectively. The DNA or RNA extracted from the sample was cleaved, separated by electrophoresis on a matrix gel, and transferred to a membrane filter. The filter-bound DNA or RNA is hybridized to a labeled probe complementary to the sequence of interest. Hybridization probes bound to the filter are detected. A variation of this procedure is a reverse Northern blot in which the substrate nucleic acid immobilized to the membrane is a collection of isolated DNA fragments and the probe is RNA extracted from and labeled with tissue.

The present invention can amplify nucleic acid before or simultaneously with detection. Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence- based amplification (NASBA). Those of ordinary skill in the art will recognize that some amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while others amplify RNA directly (e.g., TMA and NASBA).

Typically, PCR increases the copy number of the target nucleic acid sequence in an exponential manner using multiple cycles of denaturation, annealing of the primer pair to the opposite strand, and primer extension, RT-PCR uses reverse transcriptase (RT) to prepare complementary DNA(cDNA) from mRNA, which is then amplified by PCR to produce multiple copies of DNA; TMA self-catalytically synthesizes multiple copies of a target nucleic acid sequence under substantially constant temperature, ionic strength, and pH conditions, wherein multiple RNA copies of the target sequence themselves catalytically generate additional copies, TMA optionally including the application of blocking, moieties, stopping, and other modifying moieties to improve sensitivity and accuracy of the TMA process; LCR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid.

The DNA oligonucleotides are covalently linked through a DNA ligase in repeated multiple cycles of thermal denaturation, hybridization, and ligation to produce a detectable double- stranded ligated oligonucleotide product; SDA uses multiple cycles of primer sequence annealing of the opposite strand to the target sequence, primer extension in the presence of dNTPaS to produce a double-stranded hemiphosphorothioated primer extension product, endonuclease-mediated cleavage with a semi-modified restriction endonuclease recognition site, and polymerase-mediated extension from the 3' end of the cleavage to displace the existing strand and to produce the strand for the next round of primer annealing, cleavage, and strand displacement to cause geometric amplification of the product.

The non-amplified or amplify nucleic acids of that present invention may be detect by any conventional means.

Further, the reagent comprises: Probes that specifically recognize GASS; or Primers for specific amplification of GASS .

A "probe" in that present invention refer to a molecule that is capable of binding to a particular sequence or subsequence or other portion of another molecule. Unless otherwise indicate, "probe" generally refers to a polynucleotide probe capable of bin to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks full sequence complementarity with the probe. The probes may be labeled directly or indirectly, and their range includes primers. Hybridization method, including but not limit to: solution phase, solid phase, mixed phase or in situ hybridization assay.

As the probe, a labeled probe that labels a polynucleotide for cancer detection with fluorescence labeling, radiolabeling, biotin labeling, or the like may be used. Labeling methods for polynucleotides are well known. The presence or absence of the test nucleic acid in the sample can be checked by immobilizing the test nucleic acid or an amplification product thereof, hybridizing with a labeled probe, washing, and then determining a label bound to the solid phase. Alternatively, it is also possible to immobilize a polynucleotide for cancer detection, hybridize a test nucleic acid with it, and then detect the test nucleic acid bound to the solid phase using a labeled probe or the like. In this case, the polynucleotide for cancer detection bound to the solid phase is also referred to as a probe. Methods for determining a test nucleic acid using a polynucleotide probe are also known in the art. The method may be performed by contacting a polynucleotide probe with a test nucleic acid at or near Tm (preferably within+4°C) in a buffer for hybridization, washing, and then determining the hybridized labeled probe or the template nucleic acid bound to the solid phase probe.

The size of the polynucleotide used as the probe is preferably 18 or more nucleotides, more preferably 20 or more nucleotides, and the full length or less of the coding region. When use as a primer, that polynucleotide 1s preferably 18 or more nucleotide in size and 50 or less nucleotides in size. These probes have a base sequence complementary to a specific base sequence of the target gene. Here, the so-called "complementary", as long as it is hybrid, may not be completely complementary. These polynucleotides generally have 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% homology to the specific base sequence. These probes may be DNA or RNA, and may be polynucleotides in which a part or all of the nucleotides are replaced with artificial nucleic acids such as PN, LNA, ENA, GNA, and TNA.

The term "primer" in the present invention refers to an oligonucleotide, whether naturally occurring or synthetically produced in a purified restriction digest, which can serve as a starting point for synthesis when placed under conditions that induce synthesis of a primer extension product complementary to a nucleic acid strand, 1.e., in the presence of a nucleotide and an inducer such as a DNA polymerase and at a suitable temperature and pH. The primers may be single-stranded or double-stranded and must be long enough to induce synthesis of the desired extension product in the presence of an inducer. The exact length of the primer depends on a number of factors including temperature, primer source and method of use. For example, for diagnostic applications, oligonucleotide primers typically contain 15-25 or more nucleotides, depending on the complexity of the target sequence, although it may contain fewer nucleotides. Factor involved in determine that appropriate length of the primer will be readily apparent to those skilled in the art.

Furthermore, GAS5 was significantly up-regulated in severe asthma when compared to normal asthma in the absence of glucocorticoids.

Furthermore, GASS was significantly down-regulated in severe asthma after administration of glucocorticoids.

The glucocorticoid drug include, but are not limited to, prednisone, methylprednisolone, betamethasone, beclomethasone propionate, diprospan, prednisolone, hydrocortisone, dexamethasone.

Further, the glucocorticoids are administered in vitro after collection of samples.

In a particular embodiment of the invention, the glucocorticoid drug is administered by adding the glucocorticoid drug to the PBMC isolated from the blood sample.

In a specific embodiment of the present invention, the glucocorticoid drug is dexamethasone.

The present invention provides a product for diagnosing severe asthma comprising a reagent for detecting GASS in a sample.

Further, the product reagent comprises: Probes that specifically recognize GASS; or Primers for specific amplification of GASS . Further, that product also include glucocorticoid drugs include, but not limited to, prednisone, methylprednisolone, betamethasone, beclomethasone propionate, diprospan, prednisolone, hydrocortisone, dexamethasone.

In a specific embodiment of the invention, the glucocorticoid drug is dexamethasone.

It will be understood by those skilled in the art that one inventive point of the present invention is to find that after in vitro intervention with glucocorticoid (administration of dexamethasone), the expression of GASS in severe asthma is significantly down-

regulated, while that in normal asthma is significantly up-regulated.

Therefore, glucocorticoids are indispensable as an effective product to exclude individual differences in the diagnosis of severe asthma.

Further, the product comprises a chip and a kit.

The kit of the present invention may also include instructions for use of the kit, which describe how the kit is used for detection, and how the results of the detection are used to judge disease development and select a treatment regimen.

The components of the kit may be packaged in aqueous media or in lyophilized form.

Suitable containers in a kit generally include at least one vial, test tube, flask, PET bottle, syringe, or other container in which a component may be placed, and preferably, may be appropriately aliquoted.

Where more than one component is present in the kit, a second, third or other additional container will also typically be included in the kit in which the additional components are placed separately.

However, different combinations of components may be contained in one vial.

The kit of the present invention will also generally comprise a container for containing the reactants, sealed for commercial sale.

Such containers may include injection or blow molded plastic containers in which the desired vials may be retained.

The invention provides a detection system for severe asthma, comprising: 1) means for determining a characteristic value of marker GASS in a sample; 2) means for comparing marker feature values in a sample; 3) data storage media.

A data storage medium in the present invention stores a data set, and the term "data set" refers to a data set that is physically and/or logically set.

Thus, the set of data may be embodied in a single data storage medium or in physically separate data storage media operatively connected to each other. Preferably, that data set is implement into a database. Thus, a database as used herein contains a collection of data on a suitable storage medium. Furthermore, the database preferably further comprises a database management system. The database management system is preferably an internet based hierarchical database management system or an object facing database management system. Further, the database may be a federated database or an integrated database. More preferably, the database will be implemented as a distributed (federated) system, such as a Client-Server-System. More preferably, the database is constructed to allow a search algorithm to compare the test data set to the data set containing the data set. In particular, by using such algorithms, a database can be searched (i.e., a query search) for similar or identical data sets indicative of asthma or its severity. Thus, if the same or similar data sets could be identified in the data set, the test data set would be associated with asthma or its severity. As a result, the information obtained from the data set may be used to diagnose asthma or severity thereof based on the test data set obtained from the subject. More preferably, the data set comprises characteristic values of any one set of included markers, such as characteristic data of common asthma and severe asthma, and characteristic data of markers before and after dexamethasone administration.

The term "data storage medium" includes a data storage medium or a cloud disk based on a single physical entity such as a CD, CD-ROM, hard disk, optical storage medium or magnetic disk. Furthermore, the term also includes a data storage medium consisting of physically separated entities operatively connected to each other in a manner that provides the aforementioned data set, preferably in a suitable manner for a query search.

The "system" in the present invention relates to different tools operatively connected to each other.

The tools may be embodied in a single device or may be physically separate devices operatively connected to each other.

The tool for comparing the marker feature values preferably operates based on an algorithm for comparison.

The data storage medium preferably contain a set or database of data as described above, wherein each set of store data sets 1s indicative of asthma or severity thereof.

Thus, the system of the present invention allows for the identification of whether a data set stored in a data storage medium contains a test data set.

As a result, the system of the present invention can be used as a diagnostic tool for diagnosing asthma or severity thereof.

In a preferred embodiment of the system, a means for determining a characteristic value of a sample marker is included.

The term "means for determining a characteristic value of a marker" preferably relates to, for example, a nucleic acid amplification device, a nucleic acid hybridization device for determining a marker.

The present invention provides a method for judging severe asthma, which diagnoses severe asthma by detecting the expression level of GASS, as a preferred embodiment, when directly comparing the expression level of GASS in a sample, if the expression level of GASS is significantly increased as compared with normal asthma, the subject suffers from severe asthma; As a more preferred embodiment, when a glucocorticoid such as dexamethasone is administered, the subject suffers from severe asthma if the expression level of GASS is significantly down-regulated as compared to pre- administration.

Further, the glucocorticoid drug is administered in vitro.

Further, the glucocorticoids were administered by extracting PBMC from the samples for culture and then adding the glucocorticoids. The invention has the advantages and beneficial effects that: According to the invention, the expression of the GASS gene is firstly found to be related to severe asthma, and whether a subject suffers from severe asthma can be judged by detecting the expression level of GASS in the subject. The present invention provides a novel means for diagnosing severe asthma in which a subject suffers from severe asthma when the expression level of GASS is significantly increased as compared with normal asthma. The invention provides a new method for diagnosing severe asthma, when the expression level of GASS is remarkably reduced after glucocorticoid in vitro intervention, a subject suffers from severe asthma, and the method is adopted for diagnosis, thereby solving the problem caused by individual difference.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graph showing the changes of GASS expression in PBMC after in vitro dexamethasone intervention. SA: Severe asthma; SA+D: dexamethasone intervention for severe asthma; MA: common asthma; MA+D: dexamethasone intervention for common asthma; ***: P<0.001. Figure 2 is a comparison of the expression levels of GR*?%® in PBMC after in vitro dexamethasone intervention. Fig,3 is a diagram for comparing the expression levels of GASS in the lung tissue of mice in group; Control group: mice in the normal control group; SA: mice in the severe asthma group; SA+D: mice in the dexamethasone group. ***: P<0.001.

DESCRIPTION OF THE INVENTION The invention is described in further detail below with reference to the accompanying drawings and embodiments. The following embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention. Experimental methods for which specific conditions are not indicated in the examples are usually performed under conventional conditions or as recommended by the manufacturer. Embodiment 1 screening for genetic markers associated with asthma

1. Research subjects a. Severe asthma group: five cases. The diagnostic criteria of severe asthma were according to the 2017 edition of Chinese experts’ consensus on the diagnosis and treatment of severe asthma. The specific inclusion criteria were as follows: (1) There was no acute exacerbation of asthma within one month before inclusion; (2) No hospitalization due to asthma in the past one month; (3)FEV1>45% estimated value, and FEV1>55% estimated value after use of bronchodilator; @ Prednisolone dose < 20mg, daily; (5) No upper or lower respiratory tract infection in recent one month; (6) Non- smoking patients. b. Normal asthma group: 20 cases. The diagnostic criteria of asthma were according to the 2016 edition of bronchial asthma prevention and treatment guidelines in China. Positive bronchodilation test (FEV1% increase > 12% and FEV1 absolute value increase > 200ml), or positive bronchial provocation test, or average daily diurnal variation rate of peak expiratory flow (PEF) > 10%, or PEF weekly variation rate > 20%, common asthma patients are asthma patients who fail to meet the above critical criteria, and smokers are excluded. c. Normal control group: 20 healthy volunteers, non-smokers, with no history of asthma and other allergic and immune system diseases. The subjects of the above three groups were matched in age and gender. All the subjects were required to exclude the coinfection, pulmonary embolism, COPD, pulmonary tuberculosis, hematological diseases, malignant tumors and liver dysfunction. The routine blood test, liver function, renal function, blood glucose, blood lipid and ECG were performed to exclude the basic diseases.

2. Sample collection and PBMC separation In the morning, on an empty stomach, 10ml of peripheral blood was drawn out with a heparin anticoagulation tube at room temperature. The blood was immediately separated by Ficoll, and centrifuged at 2000g for 15min under the condition of 4°C. The separated PBMC were washed twice with PBS, and then cultured for 24h at 37°C and 5% CO» incubator in RPMI 1640 medium flask containing 10% serum with 10ul of phytohemagglutinin. Then 100nmol/L dexamethasone was added into the flask for 24 h.

3. Bronchial epithelial cell culture Human bronchial epithelial cell line (16HBE, Shanghai Yaji Biotechnology Co., Ltd.) was purchased and the cells were inoculated into 6-well plates and cultured in a cell incubator at 37°C and 5% CO». The medium was changed every day for 1-4 days after inoculation, and every 2-3 days thereafter. When the cell growth reached 90% saturation, it was passaged. During passage, the spear head sucked out the culture medium, and PBS was used for washing twice. After the 0.25% trypsin/EDTA was added for digestion at

37°C for about Smin, most of the cells were rounded, suspended and debossed under the microscope, and then RPMI 1640 medium containing serum was added to stop digestion. The spear head was repeatedly blown and punched with six-well plate to make the cells debossed and uniformly suspended, The cells were centrifuged by a cell suspension centrifuge at 1000r/min for 10min, the supernatant was discarded, and the cells were suspended by RPMI 1640 medium.

4. Establishment of model of hormone insensitive asthma mice Six-week-old SPF grade BALB/c female mice were selected, with eight animals in each group. Asthma group: that is, OVA+LPS group. On the 1% and 14" days, respectively, mice were intraperitoneally injected with 0.1mL sensitization solution (PBS containing pg of OVA plus +2.25mg of aluminum hydroxide). From the 28th day, use 1% OVA solution ultrasonic atomization excitation for 3 consecutive days, 20 minutes each time, once a day. On the 27% day, 10 pg (60 uL) of LPS solution was was dripped through the nose. Positive control group: that is: OVA+LPS+ DXM group. The OV A-sensitized,- stimulated and LPS-stimulated mice were co-modeled and dexamethasone (5 mg/kg/d) was injected subcutaneously into the mice on the 29" and 30" days. Normal control group: mice were sensitized and stimulated with PBS. Mouse lung tissue samples were collected on day 31 (24h after the last stimulate) and stored in liquid nitrogen.

5. Detection of GASS expression level and transcription level of related genes by qRT-

PCR Lysing with 1 mL of Trizol reagent, transferring the Trizol lysate into EP tube, and standing at room temperature of 15-30°C for 5 min; In the above EP tube, chloroform was added in an amount of 0.2ml per 1 mL of Trizol, the cap of the EP tube was closed,

the EP tube was shaken with force for 15s in the hand, the EP tube was placed at room temperature (15-30°C) for 2-3min, and then 12,000 g (2-8°C) was centrifuged for 15min; The upper aqueous phase was removed and placed in a new EP tube, 2-propanol was added in an amount of 0.5ml per 1 mL of Trizol. After standing at room temperature (15— 30°C) for 10min, 12,000g (2-8°C) was centrifuged for 10min to discard the supernatant. After being washed with 1 mL of 75% ethanol for every 1 mL of Trizol, the samples were vortex-mixed, centrifuged at 7,500 g (2-8°C) for Smin, and the supernatant was discarded. Allowing the precipitated RNA to dry naturally at room temperature; RNA precipitate was dissolved in Rnase-free water. Determination of RNA concentration, use UV spectrophotometer to detect OD280 and OD260 for RNA purity determination. The cDNA was synthesized using cDNA Synthesis Kit (Vazyme) and the specific parameters for quantitative PCR experiment were set according to the instructions of commercial kits. The PCR reaction mix (operated on ice) was prepared using qPCR SYBR Green Master Mix(Vazyme). The amplified gene primers for human (or mouse) were designed and synthesized by Gene Pharma, with U6 as the internal reference. Performing transient centrifugation on the eight headers to ensure that all reaction solutions are at the bottom of the reaction hole, performing PCR reaction by using a standard three-step procedure, and performing melting curve analysis; Quantitative PCR data analysis.

6. Western blotting was used to detect the protein expression level.

Cells or lung tissue were homogenized on ice with 0.5ml of buffer A (10mM HEPES pH

7.8, ImM DTT, 10mM KCI, 2mM MgCl,, 0.1M EDTA, 0.2mM NaF, 0.2mM NazVOs, 1% NP-40, 0.4mM PMSF, 1 pg/mL leucine peptide) for 30min. The homogenates were centrifuged at 2000 rpm for 30s at 4°C to remove cell debris. The supernatants were transferred into 1.5ml ice-pre-chilled Eppendorf tubes at 13,000rpm and centrifuged at 4°C for 30s. The supernatants were collected as cytoplasmic extracts. The precipitate was re-suspended in 200ul buffer C (SOMM HEPES pH 7.8, 5S0mM KCl, 300mM NaCl, 0.1M EDTA, ImM DTT, 10% glycerol, 0.2mM NaF, 0.2mM NasVO4, 0.6mM PMSF) in a cold chamber placed in a spinner for 30min, and centrifuged at 13,000rpm for Smin at 4°C. The supernatant was collected as nucleus extract. The cells or lung tissues were homogenized with RIPA buffer (5S0mmol/L Tris-HCl pH 8.0, 150mmol/L NaCl, Immol/L EDTA, 0.25% deoxycholic acid salt, 1mM Na3VO4, ImM NaF, 1ug/ml leucine peptide, 1ug/ml aprotinin, and ImM PMSF), incubated on ice for 15min, and micro- centrifuged at 4°C for 10min. The supernatant was the whole cell lysate. The protein content in the cell lysis products was determined by the BCA method. The 10-20 ug protein was separated by 5% SDS-PAGE electrophoresis and transferred to PVDF membrane, incubated with rapid blocking solution (5% BSA, TBS, 0.1% Tween-20, Beyotime) for 1h at room temperature, and then incubated with anti-GR*™*¢ and other antibodies (1:1000 dilution) in TBS of 5% BSA in a shaking table at 4°C overnight. After adequate elution, the samples were incubated with horseradish peroxidase-conjugated secondary antibodies (1:4,000 dilution, 2.5%BSA, PBS, 0.1% Tween-20)at 37°C for 1 h. Protein quantification was performed using ECL Plus (Beyotime) with GAPDH as the housekeeping protein.

7. Data analysis The SPSS 21.0 statistical software was used to analyze the measurement data. The Kolmogorov-Smirnov test was used for normality test, and the analysis of variance was used for comparison. The q test was used for pairwise comparison between the means of each group. P<0.05 indicated that the difference was statistically significant.

8. Outcome 1) After 24h in vitro dexamethasone (100nmol/L) intervention, the expression level of GASS in PBMC of patients with common asthma was significantly increased by 20 times (P<0.001, n=20), while it was decreased by 15 times in patients with severe asthma (P<0.001, n=5). Moreover, the expression level of GASS in patients with severe asthma was significantly higher than that in patients with common asthma (Figure 1). GRS"226 level in the severe asthma group was significantly higher than that in the normal asthma group and the normal control group, indicating that the change of GASS expression level was related to GR°“2?(Figure 2). 2) Compared with the severe asthma group and the control group, the expression level of GASS in the lung tissue of severe asthma mice treated with dexamethasone intervention was decreased significantly (P<0.001, n=8) (Figure 3). The above description of the embodiments is only for understanding the method of the present invention and its core idea. It should be noted that it will be apparent to those skilled in the art that various modifications and variations can be made without departing from the principles of the invention, and that such modifications and variations will fall within the scope of the appended claims.

Claims (10)

CLAIMS:

1. Application of a reagent for detecting GASS in a sample in the preparation of a product for diagnosing severe asthma.

2. An application according to claim 1, wherein the reagent comprises a reagent for detecting GASS by a sequencing technique, a nucleic acid hybridization technique, and a nucleic acid amplification technique.

3. An application according to claim 1 or 2, characterized in that GASS is significantly up-regulated in severe asthma compared to normal asthma.

4. An application according to claim 1 or 2, wherein GASS is significantly down- regulated in severe asthma after administration of glucocorticoids; preferably, the glucocorticoid drug is administered in vitro; more preferably, the glucocorticoid drug is administered by adding the glucocorticoid drug to the PBMC isolated from the blood sample.

5. An application according to claim 4, wherein the glucocorticoid drug is dexamethasone.

6. A product for diagnosing severe asthma, wherein the product comprises a reagent for detecting GASS in a sample.

7. A product of claim 6, wherein the product reagent comprises: probes that specifically recognize GASS; or primers for specific amplification of GASS .

8. A product according to claim 7, wherein the product further comprises a glucocorticoid drug, preferably, the glucocorticoid drug is dexamethasone.

9. A product according to claim 7 or 8, wherein the product comprises a chip, a kit.

10. A detection system for severe asthma comprising: 1) means for determining a characteristic value of marker GASS in a sample; 2) means for comparing marker feature values in a sample; 3) data storage media.