Application of exosomal miRNA in predicting the efficacy of immunotherapy in patients with non-small cell lung cancer

Abstract

The present invention relates to the field of biological detection technology, and specifically to the application of exosomal miRNA in predicting the effect of immunotherapy on patients with non-small cell lung cancer. The present invention provides exosomal miRNA markers for the prognosis evaluation of immunotherapy in patients with non-small cell lung cancer, wherein the exosomal miRNA markers are selected from at least one of miR-138-5p, miR-17-5p, miR-197-3p and miR-21-5p. The present invention provides a scientific reference for whether to use PD-1 / PD-L1 inhibitors for immunotherapy and the prognosis judgment of tumors after treatment. The method for monitoring the prognosis of immunotherapy for non-small cell lung cancer developed by the present invention based on non-invasive detection technology predicts the prognosis of patients with non-small cell lung cancer by evaluating the prognostic risk of patients with non-small cell lung cancer, effectively reduces the cost of treatment, and has important application value.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and particularly to the application of exosomal miRNA in predicting the immunotherapy effect of patients with non-small cell lung cancer. Background Art

[0002] Lung cancer is one of the most lethal solid cancers worldwide, with approximately 95% of lung cancers classified as non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). As the disease progresses, the survival rate for stage IV disease gradually declines to around four months, making early intervention crucial. Systemic cellular chemotherapy has been the mainstay of treatment for advanced NSCLC, but the benefits of chemotherapy have plateaued, necessitating the exploration of new treatment modalities. Despite a deeper understanding of the role of driver mutations in NSCLC, such as epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) fusion genes, the benefits of treatment remain limited. However, the growth and spread of cancer depend not only on the characteristics of tumor cells but also on their interactions with the immune system. Following the positive results achieved with immune checkpoint inhibitors, immunotherapy has achieved breakthroughs in anti-tumor treatment.

[0003] Anti-tumor immunotherapy is a vast field, offering patients a wide range of potential therapeutic targets. With a deeper understanding of lung cancer immune escape, immune surveillance, immunoediting, and reactivation of cancer immunity, immunotherapy is slowly becoming a reliable treatment for lung cancer. Considering the heterogeneous and dynamic nature of PD-L1 expression in tumors, as well as the involvement of miRNAs in tumor development and progression, cisplatin resistance to chemotherapy, and patient sensitivity to radiotherapy and gene therapy, exosomal miRNAs are expected to become potential biomarkers for monitoring the efficacy of immunotherapy and predicting prognosis in non-small cell lung cancer, bringing benefits to patient treatment and prognosis. Summary of the Invention

[0004] The purpose of the present invention is to predict the efficacy of immune checkpoint inhibitor treatment in patients with non-small cell lung cancer.

[0005] After years of research, the inventors screened and obtained a potential RNA molecular marker combination that is closely related to non-small cell lung cancer and can be used for clinical immunotherapy efficacy monitoring and prognosis judgment. It includes four miRNAs: miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p.

[0006] The RNA molecular markers provided by the present invention, whether used alone or in combination in part or in whole, can indicate non-small cell lung cancer with high sensitivity and specificity, and have excellent immunotherapy diagnostic performance. The RNA molecular markers provided by the present invention are present in exosomes.

[0007] In a first aspect, the present invention claims protection for the use of exosomal miRNA in predicting the effect of immunotherapy on patients with non-small cell lung cancer, wherein the exosomal miRNA is selected from at least one of miR-138-5p, miR-17-5p, miR-197-3p and miR-21-5p.

[0008] The exosomal miRNA described in the present invention comes from body fluids, such as blood, urine, saliva or sputum.

[0009] In the application provided by the present invention, the primer probe combination for detecting miR-138-5p is shown in SEQ ID NO: 1-3; the primer probe combination for detecting miR-17-5p is shown in SEQ ID NO: 4-6; the primer probe combination for detecting miR-197-3p is shown in SEQ ID NO: 7-9; the primer probe combination for detecting miR-21-5p is shown in SEQ ID NO: 10-12.

[0010] In the application provided by the present invention, the expression level of one or more RNA molecular markers selected from miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p is detected, and the critical value of the molecular marker is determined by the ROC curve. Patients whose expression level of the RNA molecular marker is higher than the critical value are classified into the high-level molecular expression group, and patients whose expression level of the RNA molecular marker is lower than the critical value are classified into the low-level expression group. The immune efficacy is compared between the groups to determine the immunotherapy effect and / or prognosis of the non-small cell lung cancer patients, and / or the risk of recurrence of non-small cell lung cancer after treatment.

[0011] In the application provided by the present invention, the immunotherapy is treatment with anti-PD-1 antibodies; and the non-small cell lung cancer patient is a patient with advanced non-small cell lung cancer.

[0012] In the second aspect, the present invention claims protection for a primer probe combination for detecting exosomal miR-138-5p, miR-17-5p, miR-197-3p or miR-21-5p, wherein the primer probe combination for detecting miR-138-5p is shown as SEQ ID NO: 1-3; the primer probe combination for detecting miR-17-5p is shown as SEQ ID NO: 4-6; the primer probe combination for detecting miR-197-3p is shown as SEQ ID NO: 7-9; the primer probe combination for detecting miR-21-5p is shown as SEQ ID NO: 10-12.

[0013] In a third aspect, the present invention seeks protection for a reagent or kit for detecting the therapeutic effect of anti-PD-1 antibody treatment on patients with advanced non-small cell lung cancer, comprising a primer-probe combination for detecting exosomal miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p.

[0014] The reagent or kit provided by the present invention contains the above-mentioned primer-probe combination, and also contains primer-probe combinations for detecting internal reference genes and external reference genes; preferably, the internal reference gene is U6, and the external reference gene is cel-miR-39.

[0015] According to the understanding of those skilled in the art, the present invention claims protection for the use of exosomal miR-138-5p, miR-17-5p, miR-197-3p and / or miR-21-5pRNA or the above-mentioned reagents or kits in the diagnosis of immune efficacy, prognosis, efficacy monitoring and / or recurrence monitoring of non-small cell lung cancer.

[0016] In a fourth aspect, the present invention claims protection for a method for detecting the therapeutic effect, prognosis, efficacy monitoring and / or recurrence monitoring of anti-PD-1 antibody treatment on patients with advanced non-small cell lung cancer for non-diagnostic purposes. Specifically, the method provided by the present invention uses the above-mentioned reagents or kits to detect the expression levels of one or more RNA molecular markers selected from miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p.

[0017] In the method provided by the present invention, the expression level of one or more RNA molecular markers selected from miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p is detected, and the critical value of the molecular marker is determined by the ROC curve. Patients whose expression level of the RNA molecular marker is higher than the critical value are classified into the high-level molecular expression group, and patients whose expression level of the RNA molecular marker is lower than the critical value are classified into the low-level expression group. The immune efficacy is compared between the groups to determine the therapeutic effect and / or prognosis of the non-small cell lung cancer.

[0018] As a specific embodiment of the present invention, the present invention provides a method for diagnosing, prognosing, monitoring therapeutic efficacy and / or monitoring recurrence of non-small cell lung cancer, comprising the following steps:

[0019] (1) Collect body fluid samples from the subject to be tested, such as blood, urine, sputum and saliva, preferably plasma;

[0020] (2) isolating exosomes from the above-mentioned body fluid samples;

[0021] (3) Extraction of exosomal RNA;

[0022] (4) Detecting one or more RNAs selected from miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p.

[0023] In one embodiment, the detection in step (4) includes the steps of reverse transcription and quantitative PCR. Preferably, the reagent used in the quantitative PCR is the reagent described in the second aspect above.

[0024] In another embodiment, step (4) further includes the step of normalizing the expression level using an internal reference gene (e.g., U6) and an external reference gene (e.g., cel-miR-39); preferably, the expression level value of the normalized molecular marker is further subjected to logistic regression processing to obtain an output value, and the critical value of the molecular marker is determined by the ROC curve, and patients whose expression level of the RNA molecular marker is higher than the critical value are classified into the high-level molecular expression group, and patients whose expression level of the RNA molecular marker is lower than the critical value are classified into the low-level expression group, and a comparison of the immune efficacy between the groups is performed.

[0025] Among them, patients with non-small cell lung cancer in the low-level miR-138-5p expression group had good immunotherapy efficacy and / or low risk of recurrence after treatment; while patients in the high-level miR-17-5p, miR-197-3p, and miR-21-5p expression group had good immunotherapy efficacy and / or low risk of recurrence after treatment.

[0026] The beneficial effects of the present invention are:

[0027] The above-mentioned exosomal RNA markers and detection methods provided by the present invention realize a non-invasive detection method based on patient plasma. They are fast and efficient, have good patient compliance, and provide extremely high sensitivity and specificity for anti-tumor immunotherapy of non-small cell lung cancer. They are potential biomarkers for monitoring the efficacy of immunotherapy of non-small cell lung cancer and judging the prognosis, bringing good news for the treatment and prognosis of patients.

[0028] The present invention provides a scientific reference for whether to use PD-1 / PD-L1 inhibitors for immunotherapy and tumor prognosis judgment.

[0029] The present invention is a method for diagnosing non-small cell lung cancer and monitoring the prognosis of immunotherapy developed based on non-invasive detection technology. By assessing the prognostic risk of non-small cell lung cancer patients, the prognosis of the patients can be predicted, effectively reducing treatment costs, and having important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The performance of exosomal miR-138-5p in predicting immune response in patients with non-small cell lung cancer was demonstrated.

[0031] Figure 2 The performance of exosomal miR-17-5p in predicting immune response in patients with non-small cell lung cancer was demonstrated.

[0032] Figure 3 The performance of exosomal miR-197-3p in predicting immune response in patients with non-small cell lung cancer was demonstrated.

[0033] Figure 4 The performance of exosomal miR-21-5p in predicting immune response in patients with non-small cell lung cancer was demonstrated.

[0034] Figure 5 The performance of exosomal miR-138-5p, miR-17-5p, and miR-197-3p in jointly predicting the efficacy of immune responses in patients with non-small cell lung cancer was shown.

[0035] Figure 6 The performance of exosomal miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p in jointly predicting the efficacy of immune responses in patients with non-small cell lung cancer was shown.

[0036] Figure 7 A schematic diagram of the correlation between exosomal miR-138-5p, miR-17-5p, and miR-197-3p and overall survival is shown.

[0037] Figure 8 A schematic diagram of the correlation between exosomal miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p and overall survival is shown. DETAILED DESCRIPTION

[0038] The technical problem to be solved by the present invention is how to predict the efficacy and prognosis of immune checkpoint inhibitor treatment in patients with non-small cell lung cancer.

[0039] The present invention can be further understood by the examples, however, it is to be understood that these examples do not limit the present invention. Changes of the present invention now known or further developed are considered to fall within the scope of the present invention described herein and hereinafter claimed.

[0040] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0041] The term "non-small cell lung cancer" in the present invention can refer to squamous cell carcinoma (SCC), adenocarcinoma, and large cell carcinoma. The term "RNA molecular marker" is an RNA molecule that is associated with a specific disease, indication, or trait and can be used to indicate the specific disease, indication, or trait. In the present invention, the RNA molecular marker is used to indicate non-small cell lung cancer and can therefore be used as a non-small cell lung cancer RNA molecular marker.

[0042] The KM method, or product-limit method, is the most commonly used method for survival analysis today. It was proposed by Kaplan and Meier in 1958, hence the name Kaplan-Meier method, often abbreviated as KM method. Based on the cut-off value obtained in step 3.1, all samples are divided into two categories for survival probability analysis.

[0043] The ROC (Receiver Operating Characteristic) curve can only divide the variables into two categories for scoring based on the boundary value: the disease progression group and the disease non-progression group.

[0044] Progression-free survival (PFS) is defined as the time from the start of immunotherapy to disease progression or death.

[0045] Example 1 Screening of miRNAs associated with non-small cell lung cancer

[0046] This example provides a preliminary screening of exosomal miRNA markers associated with non-small cell lung cancer, the steps are as follows:

[0047] 1.1. The NCBI PUBMED database was used to screen exosomal miRNAs related to non-small cell lung cancer patients compared with the control group, and 10 miRNA molecular markers with significant differential expression were initially obtained, as shown in Table 1.

[0048] Table 1 Preliminary list of miRNA molecular markers

[0049]

[0050]

[0051] Example 2 Screening of differentially expressed exosomal miRNAs

[0052] This example provides screening for differentially expressed exosomal miRNAs, and the steps are as follows:

[0053] 2.1. Sample collection and data organization

[0054] This study was a retrospective, non-interventional clinical study. This study was approved by the Ethics Committee of the Cancer Hospital, Chinese Academy of Medical Sciences (approval number: 19 / 147-1925) and conducted in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all participants.

[0055] The present invention mainly includes patients with advanced non-small cell lung cancer who received anti-PD-1 antibody treatment in the Oncology Department of Lianyungang Hospital from June 2019 to June 2021.

[0056] Inclusion criteria are: 1) patients with clinically diagnosed localized or advanced non-small cell lung cancer; 2) patients receiving immunotherapy for the first time, but with no limit on the number of treatment lines.

[0057] Exclusion criteria included: 1) other thoracic tumors such as thymic carcinoma and pleural mesothelioma; and 2) no baseline blood sample. Patients enrolled in the study received anti-PD-1 antibody immunotherapy every three weeks (camrelizumab at 3 mg / kg or 240 mg every three weeks; sintilimab and toripalimab at 200 mg every three weeks). Patients typically underwent enhanced CT of the neck, chest, and abdomen every six weeks to assess treatment efficacy, with enhanced MRI of the head performed as needed to further evaluate efficacy.

[0058] The above data were obtained by querying hospitalization medical records and telephone follow-up. The last follow-up time of this study was December 31, 2021.

[0059] 2.2. qPCR verification of differentially expressed exosomal miRNAs (miRNA detection system based on fluorescent quantitative PCR platform)

[0060] 2.2.1. Extraction of plasma exosomes and plasma exosome total RNA

[0061] The plasma exosomes were isolated using the commercial ExoEasy kit from Qiagen or Exosupur from Enze Kangtai. The total RNA in the isolated exosomes was extracted using the Qiagen miReasy mini kit, and the RNA concentration and quality were detected using Agilent 2100, and the RNA concentration was recorded.

[0062] 2.2.2 Reverse transcription system

[0063] TAKARA's PrimeScriptTM RT reagent Kit (Perfect Real Time) and PremixEx TaqTM (Probe qPCR) kit were used for reverse transcription and qPCR detection.

[0064] Prepare the reverse transcription reaction system according to the components listed in Table 2 (the reaction solution should be prepared on ice), and then place it in a PCR instrument for reaction. The reaction conditions are 37°C for 60 minutes, 85°C for 5 seconds, and 12°C for ∞. After the reverse transcription is completed, add 50 μL of DEPC H2O for dilution and take 3 μL as a template for PCR reaction.

[0065] Table 2 Reverse transcription reaction system

[0066]

[0067] 2.2.3 qPCR reaction system

[0068] The primers and probes for detecting exosomal miRNA molecular markers are described in Table 3 and include:

[0069] Primers and probe for detecting miR-138-5p: the upstream primer sequence shown in SEQ ID NO: 1, and the downstream primer sequence shown in SEQ ID NO: 2; the probe sequence shown in SEQ ID NO: 3;

[0070] Primers and probe for detecting miR-17-5p: the upstream primer sequence shown in SEQ ID NO: 4, and the downstream primer sequence shown in SEQ ID NO: 5; the probe sequence shown in SEQ ID NO: 6;

[0071] Primers and probe for detecting miR-197-3p: the upstream primer sequence shown in SEQ ID NO: 7, and the downstream primer sequence shown in SEQ ID NO: 8; the probe sequence shown in SEQ ID NO: 9;

[0072] Primers and probe for detecting miR-21-5p: the upstream primer sequence shown in SEQ ID NO: 10, and the downstream primer sequence shown in SEQ ID NO: 11; the probe sequence shown in SEQ ID NO: 12;

[0073] The primers used for the internal reference gene U6 are: the upstream primer sequence shown in SEQ ID NO: 13, and the downstream primer sequence shown in SEQ ID NO: 14; the probe sequence is shown in SEQ ID NO: 15;

[0074] The primers for the external reference gene cel-miR-39 are: the upstream primer sequence shown in SEQ ID NO: 16, and the downstream primer sequence shown in SEQ ID NO: 17; the probe sequence is shown in SEQ ID NO: 18;

[0075] Table 3 Primer probe sequences

[0076]

[0077]

[0078] Prepare a qPCR reaction system using the following components in Table 4 (prepare the reaction solution on ice), including a no-template control as a negative control. Then, place the reaction in a real-time fluorescence PCR instrument (ABI7500) and perform amplification according to the reaction conditions in Table 5.

[0079] Table 4 qPCR reaction system

[0080]

[0081] Table 5 qPCR reaction conditions

[0082]

[0083] 2.2.4. QPCR verification of differentially expressed exosomal miRNA results

[0084] Using a miRNA detection system based on a fluorescence quantitative PCR platform, perform QPCR verification of differential expression detection on the plasma exosome RNA extracted in step 1.1 according to steps 2.2.2-2.2.3.

[0085] The Ct values ​​of the ten initial miRNA molecular markers and the internal and external reference genes in Example 1 were detected respectively. Further, according to the Ct values ​​of all target RNAs and the Ct values ​​of the reference genes RNA, U6 and Cel-39-3p and the relative quantitative formula 2 -ΔΔCt By calculating the fold change of relative expression, the relative expression of each RNA molecular marker relative to the reference gene can be known. Finally, four miRNA molecular markers with significant differential expression were obtained, as shown in Table 6.

[0086] Table 6 qPCR reaction conditions

[0087]

[0088] Example 3 Analysis of plasma exosome RNA molecular markers to predict the efficacy of immunotherapy for non-small cell lung cancer

[0089] This example analyzes the results of predicting the immune efficacy of non-small cell lung cancer using plasma exosome RNA molecular markers.

[0090] 3.1. Obtaining the CUT-off value by ROC characteristic curve analysis

[0091] The CT values ​​of the significantly different miRNA molecular markers (miR-138-5p, miR-17-5p, miR-197-3p, miR-21-5p) obtained in Example 2 were calculated using the relative quantitative formula 2. -ΔΔCt By calculating the fold change in relative expression of RNA markers, we can determine the relative expression of each RNA marker relative to the reference gene. Based on this, we further employed logistic regression training (using the R language glm function) and used receiver operating characteristic curves and area under the curve (using the R language roc function) to evaluate the accuracy of the aforementioned RNA markers, used alone or in combination, in detecting the efficacy of immunotherapy in non-small cell lung cancer.

[0092] The Receiver Operating Characteristic (ROC) curve can classify a variable into two categories based on a cutoff value. The ROC curve uses sensitivity on the y-axis and I-specificity on the x-axis, with different cutoff values ​​generating different points.

[0093] (1) Results of separate detection of miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p

[0094] like Figure 1 、 Figure 2 、 Figure 3 and Figure 4 As shown, when each RNA marker was used alone, t-test analysis using R language, based on the test results in 2.2.4, yielded p ≤ 0.05, indicating that the expression of each RNA marker was significantly associated with non-small cell lung cancer. The AUCs for miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p in diagnosing immunotherapy efficacy in non-small cell lung cancer were 0.47, 0.76, 0.77, and 0.71, respectively, demonstrating good sensitivity and specificity.

[0095] (2) Combined detection of miR-138-5p, miR-17-5p, and miR-197-3p

[0096] like Figure 5 As shown, when miR-138-5p, miR-17-5p, and miR-197-3p were combined, a t-test analysis using R language based on the detection results of 2.2.4 showed p ≤ 0.05, indicating that the combined expression of miR-138-5p, miR-17-5p, and miR-197-3p was significantly correlated with the immune response to non-small cell lung cancer. Combined marker detection demonstrated better sensitivity and specificity (AUC = 0.8523).

[0097] (3) Combined detection of miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p

[0098] like Figure 6 As shown, when miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p were used in combination, based on the detection results of 2.2.4, the detection results were analyzed by t test using R language and p≤0.05 was obtained, indicating that the combined application of the four markers was significantly correlated with the immune efficacy of non-small cell lung cancer, and the combined marker detection showed better sensitivity and specificity (AUC=0.8693).

[0099] 3.2 KM method to analyze survival probability

[0100] According to the CUT-off values ​​of different miRNAs (miR-138-5p, miR-17-5p, miR-197-3p, miR-21-5p) calculated by ROC analysis in step 3.1, patients with non-small cell lung cancer were divided into two groups for analysis, and the prognostic ability of the miRNA was verified by KM survival analysis.

[0101] (1) Based on the optimal cutoff values ​​of miR-138-5p, miR-17-5p, and miR-197-3p, the patients with non-small cell lung cancer were divided into a high baseline expression group (higher than the cutoff value) and a low baseline expression group (lower than or equal to the cutoff value). Then, a survival curve was drawn based on the progression status and progression-free survival time. The results are as follows: Figure 7 As shown in the figure, the analysis results suggested that under the same follow-up time, the progression-free survival rate of the group with high expression levels of miR-138-5p, miR-17-5p, and miR-197-3p was significantly increased (p < 0.001).

[0102] (2) Based on the optimal cutoff values ​​of the four markers miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p, the patients with non-small cell lung cancer were divided into a high baseline expression group (higher than the cutoff value) and a low baseline expression group (lower than or equal to the cutoff value). Then, a survival curve was drawn based on the progression status and progression-free survival time. The results are shown in Figure 2. Figure 8 As shown in the figure, the analysis results suggested that under the same follow-up time, the progression-free survival rate of the group with high expression levels of miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p was significantly increased (p < 0.001).

[0103] The results of the above embodiments indicate that the RNA molecular markers miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p provided by the present disclosure, whether used alone or in combination in part or in whole, can effectively serve as markers for predicting or assisting in predicting the efficacy of clinical immunotherapy for non-small cell lung cancer; secondly, they can also effectively serve as markers for predicting or assisting in predicting the length of progression-free survival after immunotherapy for non-small cell lung cancer.

[0104] The experiments of the present invention have shown that plasma exosome RNA may have the potential to predict the efficacy of immunotherapy for non-small cell lung cancer, and PD-L1 inhibitors may enhance the anti-tumor effect of immunotherapy by relieving the body's immunosuppressive state.

[0105] The experiments of the present invention have shown that plasma exosome RNA may also be used as a certain intervention for the clinical treatment of non-small cell lung cancer, such as immunotherapy, medication, surgery, chemotherapy, etc.

[0106] The above embodiments are only used to understand the method and core concept of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications will also fall within the scope of protection of the present invention. Sequence Listing <110> Lianyungang First People's Hospital Beijing Enze Kangtai Biotechnology Co., Ltd. <120> Application of exosomal miRNA in predicting the efficacy of immunotherapy in patients with non-small cell lung cancer <130> KHP221117332.2 <160> 19 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty one <212> DNA <213> Artificial Sequence <400> 1 cgtagctggt gttgtgaatc a 21 <210> 2 <211> 50 <212> DNA <213> Artificial Sequence <400> 2 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgaccggcct 50 <210> 3 <211> twenty two <212> DNA <213> Artificial Sequence <400> 3 ttcgcactgg atacgaccgg cc 22 <210> 4 <211> twenty three <212> DNA <213> Artificial Sequence <400> 4 tcgcactgga tacgacctac ctg 23 <210> 5 <211> 50 <212> DNA <213> Artificial Sequence <400> 5 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacctacct 50 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 tgccaaagtg cttacagtgc 20 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <400> 7 ccgcttcacc accttctc 18 <210> 8 <211> 50 <212> DNA <213> Artificial Sequence <400> 8 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgctggg 50 <210> 9 <211> twenty two <212> DNA <213> Artificial Sequence <400> 9 ttcgcactgg atacgacgct gg 22 <210> 10 <211> twenty one <212> DNA <213> Artificial Sequence <400> 10 gcgcgtagct tatcagactg a 21 <210> 11 <211> 50 <212> DNA <213> Artificial Sequence <400> 11 gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactcaaca 50 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <400> 12 ttcgcactgg atacgactca acatca 26 <210> 13 <211> 17 <212> DNA <213> Artificial Sequence <400> 13 ctcgcttcgg cagcaca 17 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 aacgcttcac gaatttgcgt 20 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <400> 15 agaagattag catggcccct gcgca 25 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <400> 16 cgctcaccgg gtgtaaatc 19 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <400> 17 cgctcaccgg gtgtaaatc 19 <210> 18 <211> twenty four <212> DNA <213> Artificial Sequence <400> 18 attcgcactg gatacgacca agct 24 <210> 19 <211> 16 <212> DNA <213> Artificial Sequence <400> 19 gtgcagggtc cgaggt 16

Claims

1. Use of an exosomal miRNA combination in the preparation of a product for predicting the efficacy of PD-1 antibody treatment in patients with advanced non-small cell lung cancer, wherein the exosomal miRNA combination comprises miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p; The application is: detecting the exosomal miRNA combination, and the non-small cell lung cancer patients in the group with low expression of miR-138-5p and high expression of miR-17-5p, miR-197-3p, and miR-21-5p have good immunotherapy efficacy and / or low risk of recurrence after treatment.

2. The use according to claim 1, characterized in that The primer probe combination for detecting miR-138-5p is shown in SEQ ID NOs: 1-3; the primer probe combination for detecting miR-17-5p is shown in SEQ ID NOs: 4-6; the primer probe combination for detecting miR-197-3p is shown in SEQ ID NOs: 7-9; and the primer probe combination for detecting miR-21-5p is shown in SEQ ID NOs: 10-12.

3. The use according to claim 2, characterized in that The expression levels of multiple RNA molecular markers selected from miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p were detected, and the critical value of the molecular marker was determined by the ROC curve. Patients whose expression levels of RNA molecular markers were higher than the critical value were classified as the high-level molecular expression group, and patients whose expression levels of RNA molecular markers were lower than the critical value were classified as the low-level expression group. The immune efficacy was compared between the groups.

4. Use of a primer-probe combination for detecting exosomal miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p in the preparation of a product for predicting the therapeutic effect of PD-1 antibodies in patients with non-small cell lung cancer, characterized in that: The primer probe combination for detecting miR-138-5p is shown in SEQ ID NOs: 1-3; the primer probe combination for detecting miR-17-5p is shown in SEQ ID NOs: 4-6; the primer probe combination for detecting miR-197-3p is shown in SEQ ID NOs: 7-9; and the primer probe combination for detecting miR-21-5p is shown in SEQ ID NOs: 10-12.

5. A reagent or kit for detecting the therapeutic effect of anti-PD-1 antibody treatment on patients with advanced non-small cell lung cancer, characterized in that: Contains primer-probe combinations for detecting plasma exosomal miR-138-5p, miR-17-5p, miR-197-3p, and miR-21-5p; The primer probe combination for detecting miR-138-5p is shown in SEQ ID NOs: 1-3; the primer probe combination for detecting miR-17-5p is shown in SEQ ID NOs: 4-6; the primer probe combination for detecting miR-197-3p is shown in SEQ ID NOs: 7-9; and the primer probe combination for detecting miR-21-5p is shown in SEQ ID NOs: 10-12.

6. The reagent or kit according to claim 5, characterized in that It also contains primer probe combinations for detecting internal reference genes and external reference genes.

7. The reagent or kit according to claim 6, characterized in that The internal reference gene is U6, and the external reference gene is cel-miR-39.

8. Use of a combination of exosomal miR-138-5p, miR-17-5p, and miR-197-3p, or a combination of exosomal miR-138-5p, miR-17-5p, miR-21-5p, and miR-197-3p, or a reagent or kit according to any one of claims 6 to 7 in the preparation of a product for diagnosis, prognosis, efficacy monitoring, and / or recurrence monitoring of PD-1 antibody therapy for advanced non-small cell lung cancer.

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