Serum miRNA marker combination and kit suitable for screening early lung cancer

Abstract

The application provides a serum miRNA marker combination and kit suitable for screening early lung cancer. The application provides an early lung cancer non-invasive screening scheme which can realize early lung cancer screening, has strong diagnostic performance, is safe, convenient and easy to standardize. The performance of the application in detecting 840 serum samples is AUC=0.979, sensitivity 98.8%, specificity 97% and accuracy 98.2%, which further proves that the application can efficiently and accurately realize non-invasive screening of early lung cancer, and provides a reliable solution to solve the clinical problem of low detection rate of early lung cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a combination of serum miRNA biomarkers and kits suitable for screening early-stage lung cancer. Background Technology

[0002] Lung cancer is the leading cause of cancer-related morbidity and mortality worldwide, with non-small cell lung cancer accounting for the vast majority. Early-stage lung cancer often presents with no obvious symptoms, and most patients are diagnosed at an advanced stage, missing the optimal window for surgical treatment, resulting in an extremely low five-year survival rate. Conversely, if detected and intervened in the early stages, the five-year survival rate can be significantly increased to over 80%.

[0003] Currently, the "gold standard" for lung cancer screening is low-dose computed tomography (LDCT). However, LDCT has some limitations: it is expensive, involves radiation, has a high false positive rate, and is not widely available in some areas. Therefore, developing an accurate, convenient, and non-invasive blood test for early lung cancer screening is a major clinical problem that urgently needs to be solved.

[0004] MicroRNAs are endogenous non-coding small RNA molecules, 18-25 nucleotides in length. They are widely present in bodily fluids such as serum and plasma, and their expression exhibits tissue and disease specificity. Serum miRNAs are highly stable and easily detectable, making them ideal biomarkers for liquid biopsies. Although some studies have reported the association of certain miRNAs with lung cancer, most studies suffer from problems such as small sample sizes, insufficient validation, or insufficient ability of biomarker combinations to differentiate early-stage lung cancer. Therefore, identifying and validating a set of miRNA combinations with high sensitivity and specificity in serum of early-stage lung cancer is crucial for achieving effective early screening. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a combination and kit of serum miRNA biomarkers with excellent diagnostic performance, suitable for large-scale population screening of early lung cancer.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a combination of serum miRNA biomarkers suitable for screening early-stage lung cancer, including hsa-miR-223-5p, hsa-miR-29b-2-3p, hsa-miR-15b-5p, hsa-miR-103a-3p, hsa-miR-339-5p, hsa-miR-126-3p, hsa-miR-652-3p, hsa-miR-423-5p, hsa-miR-374a-5p, hsa- miR-142-3p, hsa-miR-106b-5p, hsa-miR-660-5p, hsa-miR-146b-5p, hsa-miR-345-5p, hsa-miR-29b-3p , hsa-miR-18a-5p, hsa-miR-126-5p, hsa-miR-151b, hsa-let-7a-5p, hsa-miR-342-3p, hsa-miR-140-3p.

[0008] Preferably, the serum miRNA marker combination is hsa-miR-223-5p, hsa-miR-15b-5p, hsa-miR-652-3p, hsa-miR-374a-5p, hsa-miR-151b, hsa-let-7a-5p, or hsa-miR-342-3p.

[0009] This invention also provides the application of the above-mentioned combination of serum miRNA biomarkers in the preparation of products for screening early lung cancer.

[0010] Preferably, the product includes reagents / kits for detecting the expression levels of the serum miRNA biomarker combination.

[0011] The present invention also provides a kit suitable for screening early lung cancer, including reagents for detecting the expression level of the above-mentioned combination of serum miRNA markers.

[0012] Preferably, the reagent comprises stem-loop reverse transcription primers, an upstream primer, a downstream primer, and a probe for detecting the serum miRNA marker combination, wherein:

[0013] The stem-loop reverse transcription primer consists of a universal stem-loop structure sequence and a short sequence complementary to the target miRNA, with the short sequence complementary to the target miRNA being 6-8 bases in length.

[0014] The upstream primer is a miRNA-specific primer that binds partially complementary to the original miRNA sequence;

[0015] The downstream primer is a primer with a sequence complementary to the universal sequence of stem-loop reverse transcription primers, which is suitable for amplifying cDNA of all different miRNAs;

[0016] The probe is modified with a fluorescent group at its 5' end and a quenching group at its 3' end.

[0017] More preferably, the 5' end fluorescent group is FAM, and the 3' end quenching group is MGB.

[0018] Preferably, the kit uses the following logistic regression formula to determine the test results:

[0019] in Ct = Ct value of the detection marker - Ct value of the internal reference; when Y ≥ 0.5, it is considered positive; when Y < 0.5, it is considered negative.

[0020] Preferably, hsa-miR-106b-5p is used as an internal reference.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention provides a non-invasive screening method for early lung cancer that is effective, safe, convenient, and easy to standardize. Using this invention to test 840 serum samples, the results showed an AUC of 0.979, sensitivity of 98.8%, specificity of 97%, and accuracy of 98.2%, further demonstrating that this invention can efficiently and accurately achieve non-invasive screening for early lung cancer, providing a reliable solution to the clinical challenge of low early lung cancer detection rates. Attached Figure Description

[0023] Figure 1 The subject working curves for the expression levels of hsa-miR-223-5p in the serum of lung cancer patients and healthy individuals provided in the embodiments of the present invention.

[0024] Figure 2 The subject working curves for the expression levels of hsa-miR-15b-5p in the serum of lung cancer patients and healthy individuals provided in this embodiment of the invention.

[0025] Figure 3 The subject working curves for the expression levels of hsa-miR-652-3p in the serum of lung cancer patients and healthy individuals provided in this embodiment of the invention.

[0026] Figure 4 The subject working curves for the expression levels of hsa-miR-374a-5p in the serum of lung cancer patients and healthy individuals provided in the embodiments of the present invention.

[0027] Figure 5The subject working curves for hsa-miR-151b expression levels in the serum of lung cancer patients and healthy individuals provided in this embodiment of the invention.

[0028] Figure 6 The present invention provides subject working curves for the expression levels of hsa-let-7a-5p in the serum of lung cancer patients and healthy individuals.

[0029] Figure 7 The subject operating curves for hsa-miR-342-3p expression levels in the serum of lung cancer patients and healthy individuals provided in this embodiment of the invention.

[0030] Figure 8 The present invention provides receiver operating procedure curves for the expression levels of seven biomarker combinations (hsa-miR-223-5p, hsa-miR-15b-5p, hsa-miR-652-3p, hsa-miR-374a-5p, hsa-miR-151b, hsa-let-7a-5p, and hsa-miR-342-3p) in the serum of lung cancer patients and healthy individuals. Detailed Implementation

[0031] The present invention will now be described in detail with reference to specific embodiments. The following specific embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way.

[0032] Example

[0033] I. High-throughput sequencing for screening and discovery of differentially expressed miRNA biomarkers

[0034] Differentially expressed miRNAs were identified through high-throughput sequencing. The high-throughput sequencing process included the following steps: total RNA was extracted from the samples and small RNA fragments were enriched; small RNA sequencing libraries were constructed through reverse transcription, end repair, and adapter ligation; the libraries were amplified by PCR and then sequenced to obtain raw sequencing data; bioinformatics analysis was used to screen for differentially expressed miRNA sequences in cancer samples and normal samples, providing candidate molecules for the subsequent construction of cancer detection combinations. The miRNA sequences are detailed in Table 1.

[0035] Table 1. Differentially expressed miRNAs and their sequences

[0036]

[0037] II. Extraction of miRNA from serum samples

[0038] Serum samples were extracted using the Human Serum / Plasma miRNA Nucleic Acid Extraction and Purification Kit (Guangdong-Shenzhen Medical Device Registration No. 20240191) from Merrick (Shenzhen) Biotechnology Co., Ltd. The steps are as follows:

[0039] 1. Transfer 100 μL of sample to a 1.5 mL RNase-free centrifuge tube.

[0040] 2. Add 200 μL of lysis buffer and 10 μL of proteinase K respectively, vortex thoroughly to mix, and then centrifuge briefly.

[0041] 3. After standing at 3.65℃ for 10 minutes, cool to room temperature.

[0042] 4. Insert the elution column into the collection tube for later use.

[0043] 5. Add 600 μL of anhydrous ethanol to the 1.5 mL RNase-free centrifuge tube from step 3, vortex to mix, centrifuge briefly, transfer 650 μL of the mixture to the elution column from step 4, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0044] 6. Recycle the elution column, add all the remaining mixture to the elution column from step 4, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0045] 7. Place the elution column back into the collection tube, add 500 μL of washing buffer 1, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0046] 8. Place the elution column back into the collection tube, add 500µL of washing buffer 2, centrifuge at 12,000rpm for 30sec, and discard the filtrate.

[0047] 9. Repeat step 8 once.

[0048] 10. Place the elution column back into the collection tube and centrifuge at 12,000 rpm for 2 min to remove residual liquid.

[0049] 11. Place the elution column into a new 1.5 mL RNase-free centrifuge tube, add 60 µL of elution buffer to the center of the elution column, and incubate at room temperature for 2 min.

[0050] Centrifuge at 12,000 rpm for 1 min. Collect the filtrate, which is the total RNA solution.

[0051] III. Sample Nucleic Acid Reverse Transcription

[0052] 1. The preparation of reverse transcription reagents is shown in Table 2.

[0053] Table 2. Reverse transcription reagents and dosage

[0054]

[0055] The reverse transcription primers are shown in Table 3.

[0056] Table 3. Reverse transcription primers and sequences

[0057]

[0058] 2. RNA sample loading

[0059] 1) Take 2 μL of the extracted miRNA and add it to the well of the pre-amplified 8 μL reverse transcription reaction solution.

[0060] 2) After adding the samples, seal the 96-well plate, briefly centrifuge using a plate centrifuge, vortex for 10-20 seconds to mix, and then centrifuge again. After centrifugation, visually confirm that there is liquid in each well and that the liquid level is consistent.

[0061] 3. Reverse transcription on the machine

[0062] 1) Turn on the PCR instrument, place the 96-well plate containing the samples, and start the instrument according to the reverse transcription reaction program.

[0063] 2) The reverse transcription procedure is shown in Table 4.

[0064] Table 4. Reverse transcription program settings

[0065]

[0066] IV. cDNA Amplification

[0067] 1. The preparation of the amplification reaction solution is shown in Table 5.

[0068] Table 5. Components and dosage of amplification reaction solution

[0069]

[0070] The upstream primer, downstream primer, and probe sequences are shown in Table 6.

[0071] Table 6. Upstream primer, downstream primer, and probe sequences

[0072]

[0073] 2. Sample addition: Add 2 μL of the corresponding target reverse transcription product to the prepared 18 μL amplification reaction solution. After adding the sample, cap the tube or seal the 96-well plate, mix well, and centrifuge.

[0074] 3. PCR amplification

[0075] The amplification reaction procedure is shown in Table 7.

[0076] Table 7. Amplification Reaction Procedure

[0077]

[0078] V. Results Analysis

[0079] After the reaction is complete, the amplification instrument automatically saves the results. In the parameter settings, the amplification curve algorithm is set to the absolute fluorescence method, and the automatic threshold is selected.

[0080] reagent kit judgment criteria

[0081] Based on the Logistic regression equation, seven indicators were fitted. The performance of the fitted indicators was better than that of individual indicators. Through data analysis, a logistic regression judgment model was obtained, and the logistic regression judgment formula is as follows:

[0082]

[0083] Ct calculation method = Ct value of detected marker - Ct value of internal reference

[0084] When Y ≥ 0.5, it is considered positive; when Y < 0.5, it is considered negative.

[0085] VI. Testing of the Samples to be Tested

[0086] Using the kit containing the above miRNA combination, 840 samples were tested, including 560 positive samples and 280 negative samples. The sample testing procedure was carried out according to steps one through five above. Serum samples were tested using the detection kit. Performance analysis showed that the AUC of hsa-miR-223-5p alone was 0.587. Figure 1 As shown; using hsa-miR-15b-5p alone, the AUC was 0.677, as... Figure 2 As shown; using hsa-miR-652-3p alone, the AUC was 0.653, as... Figure 3 As shown; using hsa-miR-374a-5p alone, the AUC was 0.609, as... Figure 4 As shown; using hsa-miR-151b alone, the AUC was 0.683, as... Figure 5 As shown; using HSA-LET-7A-5P alone, the AUC was 0.918, as... Figure 6 As shown; using hsa-miR-342-3p alone, the AUC was 0.5, as... Figure 7 As shown; the results of the combined detection using 7 biomarkers were 98.8% sensitivity, 97% specificity, 98.2% accuracy, and AUC=0.979. Figure 8 As shown.

[0087] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A combination of serum miRNA biomarkers suitable for screening early-stage lung cancer, including hsa-miR-223-5p, hsa-miR-29b-2-3p, hsa-miR-15b-5p, hsa-miR-103a-3p, hsa-miR-339-5p, hsa-miR-126-3p, hsa-miR-652-3p, hsa-miR-423-5p, hsa-miR-374a-5p, hsa-mi R-142-3p, hsa-miR-106b-5p, hsa-miR-660-5p, hsa-miR-146b-5p, hsa-miR-345-5p, hsa-miR-29b-3p, hsa-miR-18a-5p, hsa-miR-126-5p, hsa-miR-151b, hsa-let-7a-5p, hsa-miR-342-3p, hsa-miR-140-3p.

2. The serum miRNA biomarker combination according to claim 1, characterized in that, The serum miRNA marker combination is hsa-miR-223-5p, hsa-miR-15b-5p, hsa-miR-652-3p, hsa-miR-374a-5p, hsa-miR-151b, hsa-let-7a-5p, and hsa-miR-342-3p.

3. The use of the serum miRNA biomarker combination according to claim 1 or 2 in the preparation of products for screening early lung cancer.

4. The application according to claim 3, characterized in that, The product includes reagents / kits for detecting the expression levels of the serum miRNA biomarker combination.

5. A kit for screening early lung cancer, comprising a reagent for detecting the expression level of the combination of serum miRNA markers as described in claim 1 or 2.

6. The reagent kit according to claim 5, characterized in that, The reagents include stem-loop reverse transcription primers, an upstream primer, a downstream primer, and a probe for detecting the serum miRNA biomarker combination, wherein: The stem-loop reverse transcription primer consists of a universal stem-loop structure sequence and a short sequence complementary to the target miRNA, with the short sequence complementary to the target miRNA being 6-8 bases in length. The upstream primer is a miRNA-specific primer that binds partially complementary to the original miRNA sequence; The downstream primer is a primer with a sequence complementary to the universal sequence of stem-loop reverse transcription primers, which is suitable for amplifying cDNA of all different miRNAs; The probe is modified with a fluorescent group at its 5' end and a quenching group at its 3' end.

7. The reagent kit according to claim 6, characterized in that, The 5' end fluorescent group is preferably FAM, and the 3' end quenching group is preferably MGB.

8. The reagent kit according to claim 5, characterized in that, The kit uses the following logistic regression formula to determine the test results: ； in Ct = Ct value of the detection marker - Ct value of the internal reference; when Y ≥ 0.5, it is considered positive; when Y < 0.5, it is considered negative.

9. The reagent kit according to claim 8, characterized in that, hsa-miR-106b-5p was used as an internal reference.

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