A circRNA marker for non-small cell lung cancer screening

Abstract

The present invention provides a circRNA marker for non-small cell lung cancer screening, which relates to the field of biomedical technology. The marker is hsa_circ_0016601, and the sequence number of hsa_circ_0016601 is shown in SEQ ID NO:01. First, the circRNA differential expression profile of non-small cell lung cancer tissue and adjacent normal tissue is constructed by high-throughput sequencing, and the expression of hsa_circ_0016601 in non-small cell lung cancer is detected by RT-qPCR, thereby discovering for the first time that hsa_circ_0016601 can be used as a biomarker for the diagnosis of non-small cell lung cancer, and, compared with normal people, the expression of the circRNA biomarker in the serum of clinical large-sample non-small cell lung cancer patients is increased. The above-mentioned circRNA biomarkers highly expressed in the serum of patients with non-small cell lung cancer are related to histological type, degree of differentiation, tumor size, TNM stage, distant metastasis, cell proliferation-related antigen Ki-67 expression and pleural infiltration. The biomarker provided by the present invention has an AUC of 0.810 for distinguishing serum non-small cell lung cancer patients from healthy normal persons, a sensitivity of 72% and a specificity of 77%, and a cutoff value of 2.4180.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a circRNA marker for screening non-small cell lung cancer. Background Art

[0002] Lung cancer ranks first in both morbidity and mortality. Non-small cell lung cancer (NSCLC) is the main type of primary lung cancer, including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Although with the development of multidisciplinary comprehensive treatment of lung cancer, most lung cancer patients are found to be in the late stage and the overall survival rate is still very low due to the low popularity of conventional imaging examinations and the lack of effective early diagnostic methods. Therefore, screening and early diagnosis and treatment are effective measures to reduce the mortality rate of lung cancer.

[0003] Circular RNA (circRNA) is a type of non-coding RNA (ncRNA) molecule that does not have a 5' end cap and a 3' end polyadenylic acid (polyA) tail and forms a circular structure with covalent bonds. It has the characteristics of stable structure, high abundance and tissue-specific expression. Its main function is not only to competitively bind to microRNA (miRNA) to play a post-transcriptional regulatory role; it also interacts with nuclear ribonucleoprotein or RNA polymerase II in the cell nucleus to regulate transcription; or directly binds to transcription factors to regulate classical RNA splicing, protein encoding, etc., and participates in gene expression regulation through various modes of action. With the development of high-throughput sequencing technology, more and more studies have found that the dysregulation of circRNAs is closely related to the occurrence and development of non-small cell lung cancer. Finding dysregulated circRNAs in non-small cell lung cancer and exploring their specific mechanisms of action are of great significance for the diagnosis and treatment of non-small cell lung cancer. Summary of the invention

[0004] The purpose of the present invention is to find dysregulated circRNAs in non-small cell lung cancer and explore their specific mechanism of action for the diagnosis and treatment of non-small cell lung cancer.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A circRNA marker for non-small cell lung cancer screening, wherein the marker is hsa_circ_0016601, and the sequence number of hsa_circ_0016601 is shown in SEQ ID NO:01.

[0007] The present application also provides the use of hsa_circ_0016601 as a marker in the preparation of a non-small cell lung cancer diagnostic product, and the sequence of hsa_circ_0016601 is shown in SEQ ID NO:01.

[0008] Preferably, the diagnostic product is a diagnostic reagent or a diagnostic kit.

[0009] The present application also provides an application of hsa_circ_0016601 in combination with a tumor marker for detecting non-small cell lung cancer in the blood as a marker in the preparation of a non-small cell lung cancer diagnostic product, characterized in that: the sequence of the hsa_circ_0016601 is as shown in SEQ ID NO:01, and the tumor marker for detecting non-small cell lung cancer in the blood is at least one of CEA, SCC, and Cyfra21-1.

[0010] Preferably, the tumor markers for detecting non-small cell lung cancer in the blood include CEA, SCC, and Cyfra21-1.

[0011] The present application also provides a diagnostic reagent for non-therapeutic purposes of non-small cell lung cancer, characterized in that: the reagent comprises a specific primer for the hsa_circ_0016601 biomarker,

[0012] Preferably, it comprises an upstream primer as shown in SEQ ID NO:2 and a downstream primer as shown in SEQ ID NO:3.

[0013] Compared with the prior art, this application has the following beneficial effects:

[0014] 1. In this application, high-throughput sequencing was used to construct the differential expression profile of circRNA in non-small cell lung cancer tissues and adjacent normal tissues, and RT-qPCR was used to detect the expression of hsa_circ_0016601 in non-small cell lung cancer.

[0015] 2. This application first discovered that hsa_circ_0016601 can be used as a biomarker for the diagnosis of non-small cell lung cancer.

[0016] 3. This application found that compared with normal people, the expression of this circRNA biomarker in the serum of a large clinical sample of patients with non-small cell lung cancer was increased.

[0017] 4. In this application, analysis of clinical pathological data showed that the above-mentioned circRNA biomarkers highly expressed in the serum of patients with non-small cell lung cancer were associated with histological type, degree of differentiation, tumor size, TNM stage (tumor node metastasis classification, TMN), distant metastasis, cell proliferation-related antigen Ki-67 expression and pleural infiltration.

[0018] 5. The hsa_circ_0016601 biomarker for the diagnosis of non-small cell lung cancer provided by the present invention has an AUC of 0.810 for distinguishing between non-small cell lung cancer patients and healthy normal people in serum, a sensitivity and specificity of 72% and 77% respectively, and a cutoff value of 2.4180.

[0019] 6. In this application, the hsa_circ_0016601 biomarker is combined with the tumor markers CEA, SCC, and Cyfra21-1 for detecting non-small cell lung cancer in the blood in sequence. The AUC for distinguishing between patients with non-small cell lung cancer and healthy normal people in serum can reach 0.879, the sensitivity and specificity can reach 86% and 66%, and the cutoff value is 8.2744. The results show that the hsa_circ_0016601-CEA-SCC-Cyfra21-1 combined indicator has higher diagnostic efficacy than the single hsa_circ_0016601 detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 To analyze the differential expression profile of differentially expressed circRNAs in non-small cell lung cancer tissues by sequencing;

[0021] in:

[0022] Figure 2 is the molecular characteristics of hsa_circ_0016601; among them:

[0023] Figure 2 A is hsa_circ_0016601, which is formed by reverse splicing of 9 exons 2-10 of the maternal gene DNAH14, located on chromosome 1, with a genomic sequence length of 1140bp;

[0024] Figure 2B is RT-qPCR of genomic DNA (gDNA) and complementary DNA (cDNA) of non-small cell lung cancer cell lines A549 and NCI-H1299. The results show that the back-to-back primers of hsa_circ_0016601 can only be amplified from cDNA and do not exist in gDNA; while DNAH14 can be amplified from both cDNA and gDNA by head-to-head primers; 2% agarose gel electrophoresis was performed on the PCR products amplified by back-to-back primers, and the electrophoresis bands were single and bright, and the size was consistent with the predicted size of the target gene product;

[0025] Figure 2 C is Sanger sequencing, the sequencing results match the hsa_circ_0016601 molecular sequence in the circbank database, and the hsa_circ_0016601 cyclization site sequence is clear;

[0026] Figure 2 D is the prediction using circular RNA interactome that hsa_circ_0016601 may play different biological functions by binding to different miRNAs or RNA-binding proteins (RBPs);

[0027] Figure 3 To evaluate the molecular characteristics and methodological aspects of serum hsa_circ_0016601 in non-small cell lung cancer;

[0028] Figure 3 A is a schematic diagram of RT-qPCR detection of the expression of hsa_circ_0016601 and DNAH14 mRNA in A549 and NCI-H266 cells treated with RNase R;

[0029] Figure 3 B is a schematic diagram of RT-qPCR detection of the expression of hsa_circ_0016601 and DNAH14 mRNA in A549 cells treated with actinomycin D at different time points;

[0030] Figure 3 C is serum samples from 20 healthy controls randomly mixed in equal amounts and placed at room temperature for 24 hours;

[0031] Figure 3 D is another group of healthy controls with random equal mixed samples that were repeatedly frozen and thawed 0, 1, 3, 5, and 10 times;

[0032] Figure 4 is the expression of serum hsa_circ_0016601 in non-small cell lung cancer; among them:

[0033] Figure 4 A is a schematic diagram of the expression of serum in 132 patients with non-small cell lung cancer and 119 healthy controls detected by RT-qPCR;

[0034] Figure 4 B is a schematic diagram of detecting the expression level of hsa_circ_0016601 between the TNM stage I-II group and the stage III-IV group of the above-mentioned non-small cell lung cancer patients;

[0035] Figure 4 C is a schematic diagram of RT-qPCR detection of 27 paired serum preoperative and postoperative samples of non-small cell lung cancer to evaluate hsa_circ_0016601 as a prognostic indicator;

[0036] Figure 5 To evaluate the diagnostic efficacy of serum hsa_circ_0016601 in non-small cell lung cancer;

[0037] Figure 5 A is a schematic diagram of ROC curves for serum hsa_circ_0016601, CEA, SCC, and Cyfra21-1 expression levels in 132 patients with non-small cell lung cancer and 119 healthy controls;

[0038] Figure 5 B is a schematic diagram of the ROC curve of the combined diagnosis of hsa_circ_0016601 and CEA, SCC, and Cyfra21-1;

[0039] Figure 5 C is a schematic diagram of the ROC curve for the combined diagnosis of hsa_circ_0016601 and CEA, SCC, and Cyfra21-1;

[0040] Figure 5 D is a schematic diagram of the ROC curve comparison of serum hsa_circ_0016601 in combination with non-small cell lung cancer tumor markers CEA, SCC and Cyfra21-1 and hsa_circ_0016601 alone. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with specific embodiments.

[0042] A circRNA marker for non-small cell lung cancer screening, wherein the marker is hsa_circ_0016601, and the sequence number of hsa_circ_0016601 is shown in SEQ ID NO:01.

[0043] Use of hsa_circ_0016601 as a marker in the preparation of a non-small cell lung cancer diagnostic product, wherein the sequence of hsa_circ_0016601 is shown in SEQ ID NO:01.

[0044] The diagnostic product is a diagnostic reagent or a diagnostic kit.

[0045] Use of hsa_circ_0016601 in combination with a tumor marker for detecting non-small cell lung cancer in the blood as a marker in the preparation of a non-small cell lung cancer diagnostic product, wherein the sequence of hsa_circ_0016601 is as shown in SEQ ID NO:01, and the tumor marker for detecting non-small cell lung cancer in the blood is at least one of CEA, SCC, and Cyfra21-1. Preferably, the tumor marker for detecting non-small cell lung cancer in the blood includes CEA, SCC, and Cyfra21-1.

[0046] A diagnostic reagent for non-therapeutic purposes of non-small cell lung cancer, the reagent comprising specific primers for circRNA biomarkers, including an upstream primer as shown in SEQ ID NO: 2 and a downstream primer as shown in SEQ ID NO: 3.

[0047] The following is an explanation of the above content in combination with specific verification experiments:

[0048] Embodiment 1:

[0049] Sample collection:

[0050] The serum samples in this example were from patients with non-small cell lung cancer (stage I-IV) or physical examination populations who were treated in the Affiliated Hospital of Nantong University during 2016-2021, including 132 patients with non-small cell lung cancer and 119 healthy controls. The 3 pairs of non-small cell lung cancer tissue samples were from the Department of Thoracic Surgery of the Affiliated Hospital of Nantong University and the Affiliated Cancer Hospital of Nantong University. All subjects included in the study have signed informed consent forms;

[0051] The clinical pathological data of the above 132 patients with non-small cell lung cancer were sorted out, and they were divided into hsa_circ_0016601 high expression group and hsa_circ_0016601 low expression group according to the median expression level of hsa_circ_0016601 in serum non-small cell lung cancer. The correlation between the expression level of hsa_circ_0016601 and the clinical pathological parameters of patients was analyzed. The results showed that high expression of hsa_circ_0016601 in the serum of patients with non-small cell lung cancer was related to histological type, degree of differentiation, tumor size, TNM stage, distant metastasis, Ki-67 expression and pleural infiltration, as shown in Table 1;

[0052] Table 1 shows the correlation between serum hsa_circ_0016601 and clinical pathological parameters:

[0053]

[0054]

[0055] Statistical analysis were carried out using Pearson χ2 test.

[0056] 2. circRNA high-throughput sequencing:

[0057] First, the ribosomal RNA was removed from the extracted total RNA, and then purified with RNase R to remove linear RNA, and then the sequencing library was prepared. Figure 1 shown.

[0058] 3. Use the circbank database to query the nucleotide sequence information of hsa_circ_0016601. The sequence information is referenced Figure 2 A, specifically expressed as follows:

[0059]

[0060] The primer sequences of hsa_circ_0016601, DNAH14, 18S and GAPDH were designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. The primer sequences are as follows:

[0061] Upstream primer of hsa_circ_0016601: 5'-AGCAAGCTACCCAGGCATT-3'

[0062] Downstream primer of hsa_circ_0016601: 5'-AAAGGAACTGGCTTTAGAAAAGT-3'

[0063] Upstream primer of DNAH14: 5'-ACCTTCACCCACCATGTGAT-3'

[0064] Downstream primer of DNAH14: 5'-GAGCCCTTAAACGCTCTCCT-3'

[0065] 18S upstream primer: 5'-CGCTCGCTCCTCTCCTACTT-3'

[0066] 18S downstream primer: 5'-CGGGTTGGTTTTGATCTGATAA-3'

[0067] Upstream primer for GAPDH: 5'-AGAAGGCTGGGGCTCATTTG-3'

[0068] Downstream primer of GAPDH: 5'-GCAGGAGGCATTGCTGATGAT-3'

[0069] 4. Agarose gel electrophoresis:

[0070] Prepare electrophoresis solution: dilute 50×TAE buffer with ultrapure water at 1:49 to 1×TAE; prepare agarose gel: take an appropriate amount of 1×TAE, add 2%-5% agarose powder to it, place the mixed solution in a microwave oven and heat for 1 minute, take it out after bubbling and boiling, and place it in the microwave oven again after boiling stops, repeat three times until the agarose powder is completely dissolved and the solution becomes clear. Immediately add gel red dye, blow or shake to mix well, the ratio of dye to solution is 10000:1;

[0071] Pour the solution into the gel tank while it is still hot, with a thickness of about 0.5cm, insert a comb, and wait for the gel to solidify for about 0.5h; after the gel solidifies, remove the comb, pour the electrophoresis solution (i.e. diluted 1×TAE buffer) into the electrophoresis tank, and place the entire gel in, making sure that the sample well is close to the negative electrode and the electrophoresis solution soaks the gel block; add samples: add 6μL directly to the marker, and the test group is a mixture of 5μL sample and 1μL loading buffer; electrophoresis: constant voltage 110V, 20min. The electrophoresis results are as follows: Figure 2 As shown in B.

[0072] 5. The looping sequence at the junction of hsa_circ_0016601 was verified by Sanger sequencing. Figure 2 As shown in C.

[0073] 6. Use circularRNAinteractome (https: / / circinteractome.nia.nih.gov / ) to predict miRNAs or RNA-binding proteins that hsa_circ_0016601 may bind to, such as Figure 2 As shown in D.

[0074] 7. RNase R enzyme digestion:

[0075] The RNase R enzyme reaction system is as follows:

[0076]

[0077] The RNase R enzyme reaction procedure is as follows:

[0078]

[0079] The results are as follows Figure 3 As shown in A.

[0080] 8. Actinomycin D:

[0081] Use complete culture medium to dilute the 1 mg / mL actinomycin D solution to 2.5 μg / ml for later use; add the complete culture medium containing actinomycin D to the pre-laid six-well plate, collect cells at 0, 5, 10, 15, 20, and 25 hours, extract RNA, reverse transcribe, and perform RT-qPCR. The results are shown in Figure 3 As shown in B.

[0082] 9. Place at room temperature:

[0083] See also Figure 3C. Serum samples from 20 healthy controls were randomly mixed in equal amounts into one portion and mixed evenly to prepare 5 tubes of homogenized mixed serum; "0", "6", "12", "18", and "24" were marked on the tube caps, and the numbers on the tube caps corresponded to the time (h) that the serum was placed at room temperature; 1 mL TRIzol LS Reagent was directly added to tube "0", and after sufficient lysis, it was placed in a -80℃ refrigerator for collection; the remaining tubes were left still, and 1 mL TRIzol LS Reagent was added to the corresponding EP tubes at 6h, 12h, 18h, and 24h, respectively, and after sufficient lysis, it was placed in a -80℃ refrigerator for collection; RNA extraction, reverse transcription, and RT-qPCR were performed on the five tubes of mixed serum at the same time, and the differences in molecular Ct values ​​were compared.

[0084] 10. Repeated freezing and thawing:

[0085] See also Figure 3 D. Prepare 5 tubes of homogenized mixed serum at room temperature. Mark "0", "1", "3", "5", and "10" on the tube caps. The numbers on the tube caps correspond to the number of freeze-thaw cycles of the serum. Add 1 mL of TRIzol LS Reagent directly to tube "0". After sufficient lysis, place it in a -80℃ refrigerator for collection. Place the remaining tubes of mixed serum in a -80℃ refrigerator until completely solidified, and then place it on ice to slowly thaw. This is one freeze-thaw cycle. Add TRIzol LS Reagent to serums "1", "3", "5", and "10" after 1, 3, 5, and 10 times of "freezing" and "thawing", respectively. After sufficient lysis, place it in a -80℃ refrigerator for collection. After adding TRIzol LS Reagent to all five tubes of mixed serum, RNA extraction, reverse transcription, and RT-qPCR can be started. The five tubes should be operated simultaneously to reduce batch errors.

[0086] 11. Cell culture:

[0087] Non-small cell lung cancer cell lines A549, NCI-H1299, and NCI-H226 were cultured in RPIM-1640 medium containing 10% FBS at 37°C and 5% CO2, and passaged every 2-3 days. Cells in the logarithmic growth phase were used for the experiment. Figure 2 B and 3A,

[0088] 12. Extraction of total RNA from serum:

[0089] Add 1 mL TRIzol LS Reagent to 300 μL serum sample, mix well and lyse on ice for 5 min; add 200 μL chloroform, vortex mix for 15 s and let stand on ice for 2-3 min; centrifuge at 12000 r / min for 15 min at 4°C in a low-temperature ultracentrifuge; the sample after centrifugation is divided into three layers, and about 500 μL of the upper aqueous phase is transferred to 1.5 mL EP tube, and add 500μL isopropanol to the tube to precipitate RNA; invert to mix, and let it stand on ice for 15min; use a low-temperature ultracentrifuge at 4℃ and a speed of 12000r / min for 10min; after centrifugation, a gelatinous precipitate can be seen at the bottom of the tube, discard the supernatant and keep the precipitate, add 1mL75% ethanol to the tube for washing, and gently invert the EP tube to wash the precipitate; use a low-temperature ultracentrifuge at 4℃ and a speed of 7500r / min for 10min; turn on the oven to preheat, 50℃; after centrifugation, discard the supernatant and keep the precipitate, and try to absorb the residual liquid in the EP tube; open the EP tube cap and place it in a 50℃ oven for 5-10min until the precipitate becomes transparent; add 15μL DEPC water, blow with a gun tip to mix thoroughly, and place it in a 60℃ oven for incubation for 5-10min to obtain the RNA stock solution; use Nanodrop to detect the concentration and purity of RNA.

[0090] 13. Reverse transcription:

[0091] The serum RNA reverse transcription reaction system is as follows:

[0092]

[0093] The reverse transcription reaction procedure is as follows:

[0094]

[0095]

[0096] 14.RT-qPCR:

[0097] RT-qPCR was used to detect the expression of hsa_circ_0016601 in serum of 132 patients with non-small cell lung cancer and 119 healthy controls. The results showed that the expression level of hsa_circ_0016601 in serum patients with non-small cell lung cancer was significantly increased. Figure 4 As shown in A;

[0098] RT-qPCR was used to detect the expression level of hsa_circ_0016601 between the above-mentioned non-small cell lung cancer patients with TNM stage I-II and stage III-IV. The results showed that the expression level of hsa_circ_0016601 in serum non-small cell lung cancer patients with TNM stage III-IV was significantly increased. Figure 4 As shown in B;

[0099] RT-qPCR was used to detect 27 pairs of matched serum non-small cell lung cancer pre- and post-operative samples. The results showed that the expression level of serum non-small cell lung cancer patients after surgery was significantly reduced, such as Figure 4 As shown in C;

[0100] The RT-qPCR amplification system is as follows:

[0101]

[0102] The RT-qPCR amplification procedure is as follows:

[0103]

[0104]

[0105] 15. Serum samples from 132 patients with non-small cell lung cancer and 119 healthy controls were analyzed.

[0106] ROC curve analysis was performed on the expression levels of hsa_circ_0016601, CEA, SCC, and Cyfra21-1. The results showed that the area under the ROC curve (AUC) of hsa_circ_0016601 was 0.810. Compared with the traditional tumor markers CEA, SCC, and Cyfra21-1 for non-small cell lung cancer detection, the AUC values ​​were high, indicating that the diagnostic accuracy was higher. The AUC value of combined diagnosis increased, indicating that the accuracy of combined diagnosis was higher. Figure 5 As shown in A-5D.

[0107] 16. Statistical data analysis:

[0108] GraphPad Prism 8.0 and SPSS 26.0 were used for statistical analysis. The Student's t test was used for comparison of two groups of data. When there were more than two groups of data, one-way analysis of variance was used, and the chi-square test was used. 2 The correlation between hsa_circ_0016601 and clinical pathological parameters was tested and analyzed. The ROC curve was established to analyze the diagnostic value, and the Youden index (Youden index = specificity + sensitivity - 1) was calculated to evaluate the authenticity of the screening test. The data in the figure are the statistical results of three independent repeated experiments, expressed as mean ± standard deviation (SD). * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001;

[0109] According to the above statistical methods, the sensitivity (SEN), specificity (SPE), overall accuracy (ACCU), positive predictive value (PPV) and negative predictive value (NPV) of the single diagnostic model and the combined diagnostic model were analyzed in this application. The results are as follows Figure 4 , Figure 5 As shown in Table 2 below;

[0110] Table 2 shows the combined detection of serum hsa_circ_0016601, CEA, SCC and Cyfra21-1 expression levels for the diagnosis of non-small cell lung cancer patients:

[0111]

[0112]

[0113] SEN, sensitivity; SPE, specificity; ACCU, overalllaccuracy; PPV, positive predictive value; NPV, negative predictive value.

[0114] In summary:

[0115] In this application, the differential expression profile of circRNA in non-small cell lung cancer tissue and adjacent normal tissue was first constructed by high-throughput sequencing, and the expression of hsa_circ_0016601 in non-small cell lung cancer was detected by RT-qPCR, thereby discovering for the first time that hsa_circ_0016601 can be used as a biomarker for the diagnosis of non-small cell lung cancer, and that the expression of this circRNA biomarker in the serum of a large clinical sample of non-small cell lung cancer patients is increased compared with that of normal people. Analysis of clinical pathological data showed that the above-mentioned circRNA biomarkers highly expressed in the serum of patients with non-small cell lung cancer were related to histological type, degree of differentiation, tumor size, TNM stage (tumornode metastasis classification, TMN), distant metastasis, expression of cell proliferation-related antigen Ki-67, and pleural infiltration. The hsa_circ_0016601 biomarker for the diagnosis of non-small cell lung cancer provided by the present invention has an AUC of 0.810 for distinguishing patients with non-small cell lung cancer and healthy normal people in serum, a sensitivity and specificity of 72% and 77% respectively, and a cutoff value of 2.4180. The hsa_circ_0016601 biomarker is sequentially combined with the tumor markers CEA, SCC, and Cyfra21-1 for detecting non-small cell lung cancer in blood, and the AUC for distinguishing patients with non-small cell lung cancer and healthy normal people in serum can reach 0.879, the sensitivity and specificity can reach 86% and 66%, and the cutoff value is 8.2744. The results show that the hsa_circ_0016601-CEA-SCC-Cyfra21-1 combined index has a higher diagnostic efficacy than the single hsa_circ_0016601 detection.

Claims

1. Use of a reagent for detecting the expression level of hsa_circ_0016601 in the preparation of a diagnostic product for non-small cell lung cancer, characterized in that: The sequence of hsa_circ_0016601 is shown in SEQ ID NO:

01.

2. Use of the reagent for detecting the expression level of hsa_circ_0016601 according to claim 1 in the preparation of a diagnostic product for non-small cell lung cancer, characterized in that: The diagnostic product is a diagnostic reagent or a diagnostic kit.

3. Use of a reagent for detecting the expression level of hsa_circ_0016601 and a tumor marker for non-small cell lung cancer in the blood in the preparation of a non-small cell lung cancer diagnostic product, characterized in that: the sequence of hsa_circ_0016601 is as shown in SEQ ID NO: 01, and the tumor marker for non-small cell lung cancer detected in the blood is at least one of CEA, SCC, and Cyfra21-1.

4. Use of the reagent for detecting the expression level of hsa_circ_0016601 and tumor markers of non-small cell lung cancer in blood according to claim 3 in the preparation of a diagnostic product for non-small cell lung cancer, characterized in that: The tumor markers for detecting non-small cell lung cancer in the blood are CEA, SCC, and Cyfra21-1.

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