Kit for simultaneously detecting four tumor markers of lung cancer and application

Abstract

The invention relates to the technical field of auxiliary diagnosis of lung cancer, in particular to a kit for simultaneously detecting four tumor markers of lung cancer and application. The four mi RNA markers related to the lung cancer are obtained through online database screening, clinical sample inspection proves that the four mi RNAs can be used as lung cancer auxiliary diagnosis markers, auxiliary detection of the lung cancer can be rapidly achieved by analyzing the expression quantity of the four mi RNAs, and reference is provided for early diagnosis and early intervention treatment of lung cancer patients.

Description

Technical Field

[0001] The present invention relates to the technical field of lung cancer auxiliary diagnosis, and particularly relates to a kit for simultaneously detecting four kinds of lung cancer tumor markers and its application. Background Art

[0002] Lung cancer is one of the most common malignant tumors globally, and its mortality rate remains high. Early diagnosis and treatment are the keys to improving the survival rate of patients. Existing lung cancer diagnosis methods mostly rely on multidisciplinary cooperation, combining clinical manifestations, imaging techniques, pathological examinations, and molecular biological analyses. Such methods not only have high costs but also have the defect of poor diagnosis.

[0003] miRNAs are a class of non-coding RNAs molecules encoded by endogenous genes with a length of about 18 - 22 nucleotides, which participate in post-transcriptional gene expression regulation in cells. Currently, although the pathogenesis of lung cancer has not been fully clarified, many studies have found that miRNAs are abnormally expressed in lung cancer and participate in the biological regulation of lung cancer, playing the role of oncogenes or tumor suppressor genes, and are expected to become new biomarkers. Summary of the Invention

[0004] Aiming at the technical problems existing in the prior art, the present invention provides a kit for simultaneously detecting four kinds of lung cancer tumor markers and its application. The present invention screens four miRNA markers related to lung cancer through an online database, and through clinical sample tests, it is confirmed that these four miRNAs can be used as lung cancer auxiliary diagnosis markers. By analyzing their expression levels, the auxiliary detection of lung cancer can be quickly realized, providing a reference for the early diagnosis and early intervention treatment of lung cancer patients.

[0005] One object of the present invention is to provide miRNA biomarkers for lung cancer auxiliary diagnosis, and the miRNA biomarkers are one or more selected from hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and / or hsa-miR-491-5p.

[0006] Preferably, the miRNA biomarkers are a combination selected from hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and hsa-miR-491-5p.

[0007] Another object of the present invention is to provide the application of miRNAs as diagnostic markers in the preparation of a kit for auxiliary diagnosis and / or evaluation of lung cancer, and the miRNAs are one or more selected from hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and / or hsa-miR-491-5p.

[0008] Preferably, the expression level of the above miRNA in lung cancer patients is detected to assist in the diagnosis and / or evaluation of lung cancer.

[0009] One object of the present invention is a kit for assisting in the diagnosis and / or evaluation of lung cancer, the kit containing reagents for specifically detecting miRNA biomarkers, wherein the miRNA biomarkers are one or more selected from hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p and / or hsa-miR-491-5p.

[0010] Preferably, the kit contains reagents for specifically detecting four biomarkers selected from hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p and hsa-miR-491-5p.

[0011] Preferably, the reagents include upstream primers for specifically amplifying hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p and / or hsa-miR-491-5p, and their sequences are shown as SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.10 and / or SEQ ID NO.13 respectively.

[0012] Preferably, the reagents further include a common downstream primer for specifically amplifying hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p and / or hsa-miR-491-5p, and its sequence is shown as SEQ ID NO.4.

[0013] Preferably, the reagents further include reverse transcription primers for hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p and / or hsa-miR-491-5p, and their sequences are shown as SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.9 and / or SEQ ID NO.12 respectively.

[0014] More preferably, the sample detected in the kit is blood.

[0015] The advantages of the present invention are as follows: Four miRNA markers related to lung cancer are screened through an online database, and it is confirmed through clinical sample tests that these four miRNAs can be used as lung cancer auxiliary diagnostic markers. By analyzing their expression levels, the auxiliary detection of lung cancer can be quickly achieved, providing a reference for the early diagnosis and early intervention treatment of lung cancer patients. Description of the Drawings

[0016] Figure 1 . Expression of Four miRNAs in Lung Cancer Patients Detailed Implementation Modes

[0017] The present invention will be further described in detail below in conjunction with specific embodiments, so that those skilled in the art can understand the present invention more clearly.

[0018] The present invention screens four miRNA markers related to lung cancer through an online database, and verifies through clinical sample tests that these four miRNAs can be used as auxiliary diagnostic markers for lung cancer. By analyzing their expression levels, the auxiliary detection of lung cancer can be quickly realized, providing a reference for the early diagnosis and early intervention treatment of lung cancer patients.

[0019] One of the purposes of the present invention is to provide miRNA biomarkers for the auxiliary diagnosis of lung cancer, and the miRNA biomarkers are one or more selected from hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and / or hsa-miR-491-5p.

[0020] Preferably, the miRNA biomarkers are a combination selected from hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and hsa-miR-491-5p.

[0021] Another purpose of the present invention is to provide the application of miRNA as a diagnostic marker in the preparation of a kit for the auxiliary diagnosis and / or evaluation of lung cancer, and the miRNA is one or more selected from hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and / or hsa-miR-491-5p.

[0022] Preferably, the expression level of the above miRNA in lung cancer patients is detected to assist in the diagnosis and / or evaluation of lung cancer.

[0023] One of the purposes of the present invention is a kit for the auxiliary diagnosis and / or evaluation of lung cancer, and the kit contains reagents for specifically detecting miRNA biomarkers, wherein the miRNA biomarkers are one or more selected from hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and / or hsa-miR-491-5p.

[0024] Preferably, the kit contains reagents for specifically detecting four biomarkers selected from hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and hsa-miR-491-5p.

[0025] Preferably, the reagents include upstream primers for specifically amplifying hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and / or hsa-miR-491-5p, and their sequences are shown as SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.10, and / or SEQ ID NO.13 respectively.

[0026] Preferably, the reagents further include a common downstream primer for specifically amplifying hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and / or hsa-miR-491-5p, and its sequence is shown as SEQ ID NO.4.

[0027] Preferably, the reagents further include reverse transcription primers for hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and / or hsa-miR-491-5p, and their sequences are shown as SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.9, and / or SEQ ID NO.12 respectively.

[0028] More preferably, the sample to be detected in the kit is blood.

[0029] The following examples are only used to illustrate the present invention and do not limit the scope of the present invention. Based on the specific examples of the present invention, all other examples obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

[0030] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well-known to those skilled in the art.

[0031] Example 1 Screening of miRNA Markers for Lung Cancer

[0032] The lung cancer dataset recorded in the TCGA database was classified and screened using machine learning algorithms, and a miRNA marker database for detecting lung cancer was established. This database covered 325 lung cancer tissues and 220 healthy adjacent tissues. Algorithm analysis was performed on the differentially expressed miRNAs in the database, and miRNAs with |Fold Change| greater than 3.5 were screened. Combining the ranking, the top four miRNAs, namely hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and hsa-miR-491-5p, were finally determined. ROC curve analysis was performed on the above-screened miRNA markers, and their AUC values were calculated. The results are shown in Table 1 below.

[0033] Table 1. AUC values of four miRNA markers

[0034]

[0035] The results showed that the individual AUC values of the four miRNA markers were sufficient to serve as lung cancer diagnostic markers. When the four were combined, the diagnostic AUC value could reach 0.954, with very high diagnostic sensitivity and specificity.

[0036] Example 2 Analysis of miRNA expression differences in clinical samples

[0037] Blood samples from 20 clinically diagnosed lung cancer patients and 20 healthy people during the same period were collected for standby. Total RNA of the above samples was extracted using a blood total RNA extraction kit, and cDNA was obtained by reverse transcription of the total RNA using the reverse transcription kit PrimeScript RT reagent Kit. Fluorescent quantitative PCR with specific amplification primers was used to analyze the expression differences of the miRNAs. Among them, the primer probes for miRNAs are shown in Table 2 below.

[0038] Table 2. Primer probes for miRNAs

[0039]

[0040]

[0041] Among them, the fluorescence quantitative PCR reaction system includes: 12 μL of 10×PCR Buffer, 5 μL of SYBR Green I fluorescent dye, 10 μL of dNTP, 2 μL of each upstream primer, 8 μL of downstream primer, 2 μL of Taq DNA polymerase, and 5 μL of cDNA; the fluorescence quantitative PCR reaction program is pre-denaturation at 95°C for 10 min; 45 s at 95°C, 50 s at 55°C, extension at 72°C for 45 s, for a total of 40 cycles; extension at 72°C for 5 min; preservation at 4°C. The specificity of the reaction was judged according to the melting curve, and the relative expression level of the above miRNA was calculated by the formula 2 -ΔΔCt Calculate the relative expression level of the above miRNA.

[0042] The results are shown in Figure 1 , compared with healthy people (control group), the expression levels of hsa-miR-23a-5p and hsa-miR-27a-3p were significantly up-regulated in the blood samples of 20 pairs of lung cancer patients, while the expression levels of hsa-let-7a-5p and hsa-miR-491-5p were significantly down-regulated. The diagnostic sensitivity and specificity of the combination of the four miRNAs were calculated, and the results showed that the area under the AUC curve of the four combinations was 0.916, the diagnostic sensitivity could reach 92.3%, and the specificity was as high as 90.5%. Further analysis of the experimental data confirmed that the four miRNAs have good application prospects in the diagnosis of lung cancer patients.

[0043] The function of the above embodiments is to illustrate the substantial content of the present invention, but does not limit the protection scope of the present invention. Those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and protection scope of the technical solutions of the present invention.

Claims

1. A miRNA biomarker for the auxiliary diagnosis of lung cancer, characterized in that, The miRNA biomarker is one or more selected from hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and / or hsa-miR-491-5p.

2. The miRNA biomarker according to claim 1, wherein The miRNA biomarker is a combination selected from hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and hsa-miR-491-5p.

3. Use of miRNA as a diagnostic marker in the preparation of a kit for assisting in the diagnosis and / or evaluation of lung cancer, characterized in that, The miRNA is one or more selected from hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and / or hsa-miR-491-5p.

4. The application according to claim 3, wherein By detecting the expression levels of the above miRNAs in lung cancer patients to assist in the diagnosis and / or evaluation of lung cancer.

5. A kit for assisting in the diagnosis and / or evaluation of lung cancer, characterized in that, The kit contains reagents for specifically detecting miRNA biomarkers, wherein the miRNA biomarker is one or more selected from hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and / or hsa-miR-491-5p.

6. The kit according to claim 5, characterized in that, The kit contains reagents for specifically detecting four biomarkers selected from hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and hsa-miR-491-5p.

7. The kit according to claim 5, wherein The reagent includes upstream primers for specifically amplifying hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and / or hsa-miR-491-5p, and their sequences are shown as SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.10, and / or SEQ ID NO.13 respectively.

8. The kit according to claim 5, wherein, The reagent also includes a universal downstream primer for specifically amplifying hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and / or hsa-miR-491-5p, and its sequence is shown as SEQ ID NO.

4.

9. The kit according to claim 5, wherein The reagent also includes reverse transcription primers for hsa-miR-23a-5p, hsa-let-7a-5p, hsa-miR-27a-3p, and / or hsa-miR-491-5p, and their sequences are shown as SEQ ID NO.2, SEQ ID NO.6, SEQ ID NO.9, and / or SEQ ID NO.12 respectively.

10. The kit according to claim 5, wherein, The test sample in the kit is blood.

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