Application of circulating miRNA composition in preparation of detection product for immune checkpoint inhibitor-associated pneumonia

Abstract

The invention discloses an application of a circulating miRNA (micro Ribonucleic Acid) composition in preparation of a detection product for immune checkpoint inhibitor associated pneumonia. The circulating miRNA is a combination of a serum exosome miR-193a-5p, a serum miR-193a-5p and a serum miR-378a-3p. The combined diagnosis of the three miRNAs can distinguish immune checkpoint inhibitor-associated pneumonia patients and healthy people, and the sensitivity and the specificity are high. Compared with a clinical conventional immune checkpoint inhibitor-related pneumonia marker lymphocyte, the circulating miRNA composition provided by the invention has a better diagnosis effect. The circulating miRNA composition provided by the invention can be used for rapidly and reliably identifying epidemic checkpoint inhibitor-related pneumonia patients, and has high clinical application value and wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection, and particularly to the application of a circulating miRNA composition in the preparation of a detection product for immune checkpoint inhibitor-related pneumonitis. Background Art

[0002] Immune checkpoint inhibitor-related pneumonitis (CIP) is a common and fatal immune-related adverse reaction. Research shows that the fatality rate of CIP can be as high as 26.3%. In clinical studies, due to the exclusion of patients with various risk factors, the incidence of CIP is not high, at 3% - 5%, but in real-world diagnoses, the incidence of CIP can be as high as 19%. The emergence of new immunotherapeutic drugs and the exploration of different combination regimens have led to higher incidence, severity, and mortality of CIP, which has become the most common treatment-related severe condition among cancer patients.

[0003] CIP occurs from several days to several years after drug administration, can occur throughout the entire treatment process, and even appears after the treatment ends. CIP lacks typical clinical symptoms, its imaging manifestations are diverse, and it also lacks specificity. The diagnosis of CIP requires comprehensive consideration of the patient's medical history, symptoms, imaging, and tests, and multidisciplinary consultations are also needed. For patients who still cannot be clearly diagnosed, auxiliary diagnoses such as alveolar lavage and lung biopsy are required. However, the pathological features of CIP are also not specific. It can be seen that the diagnosis of CIP faces great challenges. Most cases of CIP are reversible, and the key lies in early identification and early treatment. Missed diagnosis or delayed treatment of CIP patients will lead to further deterioration of the disease. However, blindly treating CIP may also affect the efficacy of immunotherapy. Therefore, exploring early diagnostic markers for CIP and thereby reducing the fatality rate of CIP and improving the prognosis of patients is an urgent problem faced by researchers.

[0004] MicroRNA (miRNA) is an endogenous non-coding RNA with a length of approximately 20-24 nucleotides. It mainly exerts post-transcriptional regulatory effects by specifically binding to the bases in the untranslated region of the target gene mRNA. Alterations in miRNA expression lead to changes in the gene profiles involved in a series of biological processes, thereby causing many human diseases. miRNAs are highly stable in human body fluids, and 10% of the circulating miRNAs are secreted by exosomes. Exosomes are an important way of intercellular communication, and various cells can secrete exosomes. Exosomes participate in many physiological processes and also play an important role in immune regulation and inflammatory responses. Compared with protein markers, miRNAs have unique advantages. For example, the combination of multiple miRNAs differentially expressed in different pathways will provide more information than protein markers and is more time-saving and labor-saving than developing specific antibodies against specific proteins. Most importantly, the rapid development of molecular biology techniques based on nucleic acid amplification technology can quickly and accurately detect the levels of miRNAs, providing a rapid, sensitive, specific, and stable detection method for the diagnosis based on the expression levels of exosomal miRNAs.

[0005] Currently, there is no miRNA detection method for CIP. Therefore, it is of great significance to develop a method that can quickly detect CIP at an early stage. Summary of the Invention

[0006] To overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides the application of a circulating miRNA composition in the preparation of a detection product for immune checkpoint inhibitor-related pneumonia.

[0007] The first object of the present invention is to provide the application of a circulating miRNA composition in the preparation of a detection product for immune checkpoint inhibitor-related pneumonia.

[0008] The second object of the present invention is to provide the application of a reagent for detecting a circulating miRNA composition in the preparation of a detection product for immune checkpoint inhibitor-related pneumonia.

[0009] The third object of the present invention is to provide a kit for detecting immune checkpoint inhibitor-related pneumonia.

[0010] The present invention claims the following:

[0011] The application of a circulating miRNA composition in the preparation of a detection product for immune checkpoint inhibitor-related pneumonia, wherein the circulating miRNA composition is miR-193a-5p and miR-378a-3p.

[0012] Use of a reagent for detecting a circulating miRNA composition in the preparation of a detection product for immune checkpoint inhibitor-related pneumonia, wherein the circulating miRNA composition is miR-193a-5p and miR-378a-3p.

[0013] The nucleotide sequence of the miR-193a-5p is as shown in SEQ ID NO: 1, and the nucleotide sequence of the miR-378a-3p is as shown in SEQ ID NO: 2.

[0014] Preferably, the circulating miRNA composition is serum exosomal miR-193a-5p, serum miR-193a-5p and serum miR-378a-3p.

[0015] Preferably, the detection reagent for miR-193a-5p is a primer with a nucleotide sequence as shown in SEQ ID NO: 3, and the detection reagent for miR-378a-3p is a primer with a nucleotide sequence as shown in SEQ ID NO: 4.

[0016] A kit for detecting immune checkpoint inhibitor-related pneumonia, the kit contains a reagent for detecting a circulating miRNA composition, and the circulating miRNA composition is miR-193a-5p and miR-378a-3p.

[0017] The nucleotide sequence of the miR-193a-5p is as shown in SEQ ID NO: 1, and the nucleotide sequence of the miR-378a-3p is as shown in SEQ ID NO: 2.

[0018] Preferably, the circulating miRNA composition is serum exosomal miR-193a-5p, serum miR-193a-5p and serum miR-378a-3p.

[0019] Preferably, the detection reagent for miR-193a-5p is a primer with a nucleotide sequence as shown in SEQ ID NO: 3, and the detection reagent for miR-378a-3p is a primer with a nucleotide sequence as shown in SEQ ID NO: 4.

[0020] Preferably, the kit further contains a serum RNA extraction reagent, a serum exosomal RNA extraction reagent, a reverse transcription reagent and / or a real-time fluorescence quantitative PCR detection reagent.

[0021] A method for detecting immune checkpoint inhibitor-related pneumonia, the method comprises the following steps:

[0022] S1. Obtain the relative expression levels of serum exosomal miR-193a-5p, serum miR-193a-5p, and serum miR-378a-3p in the subject.

[0023] S2. Substitute the relative expression levels into the formula for calculation. The formula is: miRNAsig = -4.343 + (0.786 × serum exosomal miR-193a-5p) + (0.349 × serum miR-193a-5p) + (0.711 × serum miR-378a-3p).

[0024] S3. Compare the value obtained after calculation with the threshold value of -0.795. If the value obtained after calculation is greater than -0.795, the subject is a patient with immune checkpoint inhibitor-related pneumonia.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention discloses the application of a circulating miRNA composition in the preparation of a detection product for immune checkpoint inhibitor-related pneumonia. The circulating miRNA is a combination of serum exosomal miR-193a-5p, serum miR-193a-5p, and serum miR-378a-3p. Using the combined diagnosis of 3 miRNAs can distinguish patients with immune checkpoint inhibitor-related pneumonia from healthy people, with high sensitivity and specificity. Compared with the conventional immune checkpoint inhibitor-related pneumonia marker lymphocytes in clinical practice, the circulating miRNA composition of the present invention has a better diagnostic effect. Using the circulating miRNA composition of the present invention, patients with immune checkpoint inhibitor-related pneumonia can be quickly and reliably identified, which has high clinical application value and broad application prospects. Description of the Drawings

[0027] Figure 1 It is a flowchart of the method for detecting the expression levels of serum exosomal miR-193a-5p, serum miR-193a-5p, and serum miR-378a-3p by RT-qPCR method.

[0028] Figure 2 It is a receiver operating characteristic (ROC) curve of the CIP diagnostic model constructed based on serum exosomal miR-193a-5p, serum miR-193a-5p, and serum miR-378a-3p.

[0029] Figure 3 It is a comparison of the diagnostic effects between the model constructed based on serum exosomal miR-193a-5p, serum miR-193a-5p, and serum miR-378a-3p and the clinically commonly used lymphocyte levels. Detailed Embodiments

[0030] The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0031] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0032] Example 1 Primer Design

[0033] I. Experimental Method

[0034] Log in to the website http: / / www.mirbase.org / index.shtml to obtain the mature sequences of hsa-miR-193a-5p and hsa-miR-378a-3p: miRBase:>hsa-miR-193a-5p MIMAT0004614 and hsa-miR-378a-3p MIMAT0000732, so as to design primers with high sensitivity, good specificity, and high amplification efficiency.

[0035] The mature sequence of hsa-miR-193a-5p: 5’-UGGGUCUUUGCGGGCGAGAUGA-3’ (SEQ ID NO: 1);

[0036] The mature sequence of hsa-miR-378a-3p: 5’-ACUGGACUUGGAGUCAGAAGGC-3’ (SEQ ID NO: 2).

[0037] *Explanation of SEQ ID NO: 1-2 in the sequence listing of the specification: According to the editing rules of the WIPO Sequence software, the nucleotide sequence must only contain the symbols listed in "Part 1 of Annex I of WIPOST.26". The base "t" is "u" in the RNA sequence. Therefore, SEQ ID NO: 1-2 in the specification of the present invention is substantially the same as SEQ ID NO: 1-2 in the sequence listing.

[0038] II. Experimental Results

[0039] RT-qPCR specific primers for detecting miR-193a-5p and miR-378a-3p with strong specificity, high sensitivity, and high amplification efficiency were determined:

[0040] miR-193a-5p: 5’-TGGGTCTTTGCGGGCG-3’ (SEQ ID NO: 3);

[0041] miR-378a-3p: 5'-CGCGACTGGACTTGGAGTCA-3' (SEQ ID NO: 4).

[0042] Kit for Detecting Immune Checkpoint Inhibitor-Related Pneumonia in Example 2

[0043] I. Composition

[0044] (1) RT-qPCR specific primers for detecting miR-193a-5p and miR-378a-3p in Example 1;

[0045] (2) U6 Forward Primer, U6 Reverse Primer and mRQ 3' Prime in the miRNA FirstStrand Synthesis Kit from Takara;

[0046] (3) ddH2O, Premix Ex Taq TM ;

[0047] (4) miRNA FirstStrand Synthesis Kit from Takara.

[0048] II. Usage Method

[0049] Figure 1 The following is the usage method of the kit of the present invention, and the specific usage steps are as follows:

[0050] 1. Extraction of exosomes from the serum to be tested

[0051] (1) Collect the serum, centrifuge at 300×g for 10 min, remove the remaining cells after centrifugation, and collect the supernatant;

[0052] (2) Transfer the supernatant to a new centrifuge tube, centrifuge at 14000×g for 30 min, remove cells, bacteria, apoptotic bodies, etc. after centrifugation, and collect the supernatant;

[0053] (3) Transfer the supernatant to an ultracentrifuge tube, ultracentrifuge at 100000×g for 90 min, discard the supernatant, add sterile PBS to resuspend the precipitate, obtain serum exosomes, and store them at -80°C for standby or use immediately.

[0054] 2. Extraction of RNA from serum and serum exosomes

[0055] (1) Extract serum and serum exosome samples, add Trizol to the samples to make up to 1 mL, and let it stand at 15 - 30°C for 5 min.

[0056] (2) Add 200 μL of chloroform, vortex for 15 s, let stand at room temperature for 5 min, and then centrifuge at 12,000×g at 4 °C for 15 min. After centrifugation, there are three layers. Collect the upper colorless and transparent RNA layer for later use;

[0057] (3) Transfer the liquid in the RNA layer to a new RNase-free EP tube, add isopropanol to precipitate the RNA molecules. The volume of isopropanol is the same as that of the supernatant in step (2). Let stand at 15 - 30 °C for 5 min, and then centrifuge at 12,000×g at 4 °C for 10 min. A gelatinous precipitate appears at the bottom of the centrifuge tube;

[0058] (4) Discard the supernatant, add 1 mL of 75% ethanol solution (v / v) for washing. After vortexing, centrifuge at 12,000×g at 4 °C for 5 min; Repeat the washing once, and then invert the EP tube on the filter paper until the ethanol has completely evaporated;

[0059] (5) Add 15 μL of DEPC water to dissolve the RNA precipitate, pipette to blow the precipitate, let stand at 55 - 60 °C for 10 min to promote RNA dissolution, and use a Nano Drop2000 ultra-micro spectrophotometer to measure the quality and concentration of RNA, thus obtaining serum RNA and serum exosome RNA.

[0060] 3. cDNA synthesis

[0061] Refer to the operation manual of miRNA FirstStrand Synthesis of Takara company to reverse transcribe the extracted serum RNA and serum exosome RNA, thus synthesizing the required cDNA.

[0062] 4. RT-qPCR reaction

[0063] The RT-qPCR reaction system and amplification program are shown in Table 1 and Table 2 respectively.

[0064] Table 1 RT-qPCR reaction system

[0065]

[0066] Table 2 RT-qPCR amplification program

[0067]

[0068] 5. Analysis of amplification products

[0069] Set three replicates for each sample, read the Ct values of each sample, and calculate the expression levels of miR-193a-5p and miR-378a-3p corresponding to each sample through 2 -ΔΔCt Calculate the expression levels of miR-193a-5p and miR-378a-3p corresponding to each sample.

[0070] 6. Statistical analysis and determination of the diagnostic cut-off value

[0071] Using MedCalc 19.6.0 statistical analysis software, analyze and process the relative expression levels of serum exosomal miR-193a-5p, serum miR-193a-5p, and serum miR-378a-3p obtained through calculation. Use Logistic regression to construct a multivariate model based on the relative expression levels of serum exosomal miR-193a-5p, serum miR-193a-5p, and serum miR-378a-3p. The model formula is: -ΔΔCt Calculated

[0072] miRNAsig = -4.343 + (0.786 × serum exosomal miR-193a-5p) + (0.349 × serum miR-193a-5p) + (0.711 × serum miR-378a-3p);

[0073] At the same time, determine -0.795 as the best diagnostic criterion. That is, when initially judging that the subject is a CIP patient and needs close monitoring or imaging examination, the relative expression level of the model constructed based on serum exosomal miR-193a-5p, serum miR-193a-5p, and serum miR-378a-3p is greater than -0.795.

[0074] Analysis of the diagnostic effect in Example 3

[0075] I. Experimental method

[0076] Collect serum samples from 55 patients who have received immunotherapy without developing CIP, 23 patients with pulmonary infections, and 26 CIP patients, and use the kit in Example 2 for detection.

[0077] II. Experimental results

[0078] Figure 2 The receiver operating characteristic (ROC) curve for the combined diagnosis of CIP by serum exosomal miR-193a-5p, serum miR-193a-5p, and serum miR-378a-3p can be used to evaluate the diagnostic effect of the combined diagnosis of CIP by serum exosomal miR-193a-5p, serum miR-193a-5p, and serum miR-378a-3p. The results show that the area under the receiver operating characteristic curve (AUC) for the model constructed based on serum exosomal miR-193a-5p, serum miR-193a-5p, and serum miR-378a-3p in diagnosing CIP is 0.816, the 95% confidence interval (95% CI) is 0.698 - 0.935, the standard error is 0.061, and P = 0.0002; when the critical value is -0.795, the sensitivity is 60% and the specificity is 89.5%.

[0079] Example 4 Comparison of Diagnostic Effects

[0080] I. Experimental Method

[0081] During the CIP diagnosis process, the lymphocyte level is often detected as an auxiliary diagnostic indicator. The peripheral blood specimens of patients are obtained by peripheral vein puncture blood collection method, and the lymphocyte level is detected by an automatic blood cell analyzer.

[0082] The expression levels of serum exosomal miR-193a-5p, serum miR-193a-5p and serum miR-378a-3p of the subjects are detected using the kit of Example 2, and the relative expression levels are calculated using the miRNAsig model.

[0083] Operation of the combined diagnosis of the two:

[0084] The receiver operating characteristic curve analysis of the combined diagnosis of multiple indicators is realized using SPSS software.

[0085] (1) Define variables and establish a table: For example, in the first column (testa) is the value of lymphocytes; in the second column (testb) is the value of the miRNAsig model, which is a numerical variable; disease is the disease state, 0 means disease-free, 1 means diseased, that is, suffering from CIP;

[0086] (2) Import the data into Medcalc;

[0087] (3) Logistic regression

[0088] Click Statistics—Regression—Logistic regression. In the Logistic regression dialog box, select Disease in the Dependent Variable box, and select testa and testb in the first and second rows of the Independent Variable box respectively. In the Logistic Regression result dialog box, Save predicted probabilities, and the probabilities are saved in the dataset;

[0089] (4) At this time, there is an additional LOGREGR_Pred1 in the data frame, that is, the predicted probability values generated by testa and testb through logistic, which comprehensively reflects the diagnostic ability of testa and testb. The receiver operating characteristic curve drawn with LOGREGR_Pred1 is the combined diagnosis of the two.

[0090] The diagnostic effects of the three biomarkers are compared using the ROC curve.

[0091] II. Experimental Results

[0092] Figure 3 The receiver operating characteristic curves of the miRNAsig model constructed with serum exosomal miR-193a-5p, serum miR-193a-5p, and serum miR-378a-3p and the diagnostic effect of the commonly used lymphocyte level in clinic. The results show that the area under the curve with the absolute value of lymphocytes as the biomarker is 0.804, the standard error is 0.051, and the 95% confidence interval is 0.704 - 0.903.

[0093] The area under the curve of the miRNAsig model is 0.816, the 95% confidence interval is 0.698 - 0.935, and the standard error is 0.061.

[0094] The area under the curve of the combination of the miRNAsig model and the absolute value of lymphocytes is 0.916, the standard error is 0.037, and the 95% confidence interval is 0.844 - 0.988.

[0095] It can be seen from Figure 3 that the miRNAsig model has a high diagnostic effect, with the area under the curve value of 0.816, higher than that of the commonly used lymphocytes in clinic. The area under the curve of the combined diagnosis of multiple indicators is 0.916, greater than that of other single indicator detections.

[0096] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of a cyclic miRNA composition in the preparation of a detection product for immune checkpoint inhibitor-related pneumonia, characterized in that, The circulating miRNA composition is miR-193a-5p and miR-378a-3p.

2. Use of a reagent for detecting a circulating miRNA composition in the preparation of a detection product for immune checkpoint inhibitor-related pneumonia, characterized in that, The circulating miRNA composition is miR-193a-5p and miR-378a-3p.

3. The application according to claim 1 or 2, characterized in that, The nucleotide sequence of the miR-193a-5p is as shown in SEQ ID NO: 1, and the nucleotide sequence of the miR-378a-3p is as shown in SEQ ID NO:

2.

4. The application according to claim 1 or 2, characterized in that, The circulating miRNA composition is serum exosomal miR-193a-5p, serum miR-193a-5p, and serum miR-378a-3p.

5. The application according to claim 2, wherein The detection reagent for miR-193a-5p is a primer with a nucleotide sequence as shown in SEQ ID NO: 3, and the detection reagent for miR-378a-3p is a primer with a nucleotide sequence as shown in SEQ ID NO:

4.

6. A kit for detecting immune checkpoint inhibitor-related pneumonia, characterized in that, The kit contains reagents for detecting the circulating miRNA composition, and the circulating miRNA composition is miR-193a-5p and miR-378a-3p.

7. The kit according to claim 6, wherein The nucleotide sequence of the miR-193a-5p is as shown in SEQ ID NO: 1, and the nucleotide sequence of the miR-378a-3p is as shown in SEQ ID NO:

2.

8. The kit according to claim 6, wherein The circulating miRNA composition is serum exosomal miR-193a-5p, serum miR-193a-5p, and serum miR-378a-3p.

9. The kit according to claim 6, wherein The detection reagent for miR-193a-5p is a primer with a nucleotide sequence as shown in SEQ ID NO: 3, and the detection reagent for miR-378a-3p is a primer with a nucleotide sequence as shown in SEQ ID NO:

4.

10. The kit according to claim 6, wherein The kit further contains a serum RNA extraction reagent, a serum exosomal RNA extraction reagent, a reverse transcription reagent, and / or a real-time fluorescence quantitative PCR detection reagent.

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