Molecular marker for lung cancer and application thereof

Abstract

An application of a reagent for detecting the expression level of a molecular marker in a biological sample in preparing a product for diagnosing lung cancer in a subject, wherein the molecular marker includes a combination of any two or three of COL1A1, PCDH18 and TUBB4A. The reagent is used for detecting mRNA level by a method of real-time fluorescent quantitative reverse transcription polymerase chain reaction, polymerase chain reaction, nuclease protection assay, in situ hybridisation, nucleic acid microarray, serial analysis of gene expression, Western blot, enzyme-linked immunosorbent assay, radioimmunoassay, radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation, complement fixation, fluorescence-activated cell sorting, mass analysis or protein microarray. The reagent comprises a primer, a probe and an antibody and the product comprises a kit, nucleic acid membrane strip, a preparation and a chip. The lung cancer can be lung squamous cell carcinoma.

Description

The present invention belongs to the field of biomedicine, and particularly relates to a molecular marker for lung cancer and an application thereof. Background Lung cancer is the most common malignant tumor in the world, and is also a leading cause of cancer deaths, which imposes a great threat to human health and life. Worldwide statistics in 2013 shows that in the United States, nearly 230,000 new cases of lung cancer are expected to occur, and nearly 160,000 people die of lung cancer, accounting for 28% and 26% of all cancer deaths in men and women, respectively. The five-year survival rate for lung cancer in the United States during 2002-2008 is 16%, of which the five-year survival rate is 52% for patients with focal lesions, 25% for patients with local lymph node metastasis, and only 4% for patients with hematologic distant metastasis (see Jemal A, Siegel R, Xu J Q, et al. Cancerstatistics [J]. Ca Cancer J Clin, 2013, 52(5):1-24.). In recent years, although clinical diagnosis and treatment technology of lung cancer has been improved and enhanced, effective diagnostic methods for lung cancer are still lacked. At present, main diagnostic methods of lung cancer are as follows: (I) imaging methods: such as chest X-ray and low-dose spiral CT. However, early lung cancer is difficult to be detected on by chest X-ray; small pulmonary nodules can be detected by low-dose spiral CT, but the false positive rate is as high as 96.4%, which brings unnecessary psychological burden to examined subjects. At the same time, chest X-ray and low-dose spiral CT should not be used frequently due to radiation. In addition, the imaging methods are often influenced by a device, a physician's experience in film reading, and effective film reading time. (2) Cvtological methods: such as sputum cytology, bronchoscopic brush or biopsy, and bronchoalveolar lavage fluid cytology. Sputum cytology and bronchoscopic brush or biopsy have a low sensitivity to peripheral lung cancer. At the same time, bronchoscopic brush or biopsy and bronchoalveolar lavage fluid cytology are complicated in operation and make the examined subjects not very comfortable. (3) Common markers include carcinoembryonic antigen (CEA), squamous cell carcinoma associated antigen (SCC-Ag), cytokeratin 19 fragment (CYFRA21-1), carbohydrate antigen 125 (CA125), carbohydrate antigen 153 (CA153), carbohydrate antigen 199 (CA199), neuron-specific enolase (NSE), tissue polypeptide antigen (TPA), etc. Among which, neuron-specific enolase (NSE): Enolase is a glycolytic enzyme commonly found in mammalian tissues in the form of a series of isoenzyme dimers (aa, aP, PP and yy). NSE is an isoenzyme containing y-subunit, which exists in neuroendocrine cells and neurogenic tumors (such as small cell lung cancer, neuroblastoma and intestinal cancer). Small cell lung cancer (SCLC) is a tumor originating from neuroendocrine cells, so NSE is closely-related to SCLC and is a marker of small cell lung cancer. Positive rate of NSE in patients with SCLC is about 60%-80%, and the positive rate in patients with non-small cell lung cancer is <20%. Therefore, NSE facilitates the diagnosis of SCLC and the differential diagnosis between SCLC and NSCLC. NSE is also an effective indicator for observation and follow-up of chemotherapy effect of lung cancer; NSE level will be decreased after chemotherapy reaction, and can reach a normal level after complete remission of the disease. Cytokeratin 19 fragment (CK19, CYFRA21-1): CYFRA21-1 is one of the soluble components of epithelial keratin with a molecular weight of about 30 kDa, which is a very good marker for differentiating benign and malignant lung diseases, especially for detecting lung squamous cell carcinoma. CYFRA21-1 is a relatively new tumor marker; in clinical practice, sandwich enzyme-linked immunosorbent assay is used in combination with two specific monoclonal antibodies (KS19.1 and BM19.21) to detect CYFRA21-1 in serum. CYFRA21-1 exists in cytoplasm of tumor cells of epithelial origin and can be released into serum after tumor cell necrosis. Immunohistochemical studies show that lung cancer is rich in CYFRA21-1, and CYFRA21-1 is the most sensitive tumor marker of NSCLC. Because CYFRA21-1 represents only one fragment of cytokeratin 19, CYFRA21-1 has a higher specificity than tissue polypeptide antigen (TPA). Studies show that the positive rate of CYFRA21-1 in lung squamous cell carcinoma can be as high as 80%, and CYFRA21-1 has a high specificity for the diagnosis of lung cancer; the concentration of CYFRA21-1 in the blood of people with benign lung diseases and healthy people is very low, regardless of gender, age and smoking habit. The sensitivity of CYFRA21-1 is correlated with histological type of a tumor, for example, the highest sensitivity is 76.5% for lung squamous cell carcinoma, 47.8% for adenocarcinoma and only 42.1% for small cell lung cancer. The content of CYFRA21-1 in serum is positively correlated with the course of lung squamous cell carcinoma; according to TNM stage of lung cancer, the sensitivity for patients in stages I-IV is 60.0%, 88.8%, 80% and 100%, respectively. (3) Progastrin-releasing peptide (ProGRP): ProGRP is a relatively stable precursor of the hormone gastrin-releasing peptide (GRP). Human GRP mainly exists in gastrointestinal tract, respiratory tract and central nervous system. Some studies suggest that GRP is released by tumor cells of small cell lung cancer, and the grow th of SCLC cells may be stimulated by GRP. As a marker of SCLC, ProGRP has a high sensitivity (75.5%) and a high specificity (97%). A good correlation is also found between ProGRP level and therapeutic reaction; in patients with tumors completely disappear after treatment, the ProGRP level will drop to a normal range, and even cannot be detected for more than one month. Domestic and foreign studies show that the sensitivity, specificity and reliability of ProGRP in detecting small cell lung cancer is superior to those of NSE; ProGRP has high sensitivity and specificity, with a positive predictive value and a negative predictive value above 90%; ProGRP has a higher positive rate for limitedstage lesions than NSE, which increases the possibility of early diagnosis to a certain extent; in addition, ProGRP provides valuable information on chemotherapy reaction, curative effect evaluation, disease course monitoring and prognosis. (4) Carcinoembiyonic antigen (CEA): CEA is a tumor antigen which exists in various tumor cells, and CEA can be directly produced by lung cancer cells. CEA is the earliest found tumor marker of lung cancer, and is also the most representative tumor marker in the diagnosis of lung cancer at present; the increase of CEA is relatively obvious in adenocarcinoma and squamous cell carcinoma. Serum CEA level in patients with primary lung cancer is significantly increased, the positive rate in diagnosis of lung cancer is 40%-60%, and the positive rate and specificity are high especially in lung adenocarcinoma. In addition, tire positive rate of CEA in patients with early lung cancer is low, and is significantly increased in the middle and late stages of the tumor. Therefore, the therapeutic reaction and prognosis of a patient can be better reflected by the dynamic change of CEA level. However, the sensitivity and specificity of the tumor markers are limited when used for detection individually. A more accurate and sensitive technology need to be developed in order to make a rapid and accurate diagnosis of lung cancer. Summary The present invention discloses a bioniarker which can be used for diagnosing lung cancer, wherein the biomarker includes PCDH18, CDKN2A and / or H0XA9. Herein, the term "and / or" used in phrases such as "A and / or B" is intended to cover both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). The term "biomarker" refers to biomolecules that exist in an individual in various concentrations and can be used for predicting a cancer state of the individual. The biomarker may include but not be limited to a nucleic acid, a protein and a variant or fragment thereof. The biomarker may be DNA containing all or part of a nucleotide sequence encoding the biomarker or a complement of such sequence. The biomarker of nucleic acid that can be used in the present invention is deemed to include DNA and RNA containing all or part of a nucleotide sequence for any purpose. In the present invention, the biomarker such as PCDH18 (gene ID: 54510), CDKN2A (gene ID: 1029) and HOXA9 (gene ID: 3205) includes a gene, a protein encoded by the gene, as well as a homologue, a mutation and an isoform of the gene. The term covers a full-length, unprocessed bioniarker, as well as any form of biomarker derived from processing in cells. The term covers a naturally occurring variant of the biomarker (such as a splicing variant or an allele). The term "subject" refers to any animal (e.g., mammal), including but not limited to human beings, non-human primates, dogs, cats, rodents, etc. Further, the subject is a human subject. The terms "subject", "individual" and "patient" are used interchangeably herein. Therefore, the terms "subject", "individual" and "patient" cover individuals with cancer (e.g., lung cancer), including those individuals of candidates who have undergone or will undergo excision (surgery) to remove cancer tissue. The terms "biological sample", "sample", "specimen" and "test sample" are used interchangeably herein to refer to any material, biological fluid, tissue or cell obtained from an individual or obtained in other ways. Fluid samples include blood (including whole blood, white blood cells, peripheral blood mononuclear cells, bufiy coat, plasma and serum), sputum, tears, mucus, nasal wash, nasopharyngeal aspirates, breath, urine, semen, saliva, meningeal liquid, amniotic fluid, gland liquid, lymph fluid, mammilla aspirates, bronchus aspirates, synovia, joint aspirates, ascites, cells, cell extract and cerebrospinal fluid. If required, a sample can be a combination of samples from an individual, such as a combination of tissue and fluid samples. The term "biological sample" also includes a material containing a homogeneous solid material, e.g., a material from a stool sample, a tissue sample or tissue biopsy. The term "biological sample" also includes a material derived from tissue culture or cell culture. Any suitable method for obtaining a biological sample may be adopted; exemplary' methods include, for example, phlebotomy, swab (e.g., oral swab) and fine needle aspiration biopsy. The sample can also be collected, for example, by microdissection (e.g., laser capture microdissection (LCM) or laser microdissection (LMD)), bladder irrigation, smear (e.g., PAP smear) or catheter lavage. A "biological sample" obtained or gained from an individual includes any such sample that has been processed in any suitable manner after being obtained from an individual, for example, a freshly frozen or formalin-fixed and / or paraffin-embedded sample. Further, the sample is tissue. Further, the reagent includes a reagent used for determining expression level of the biomarker in the subject sample by quantitative PCR, NGS, Northern blot, Southern blot, microarray, SAGE, immunoassay (ELISA, EIA, agglutination test, nephelometry, turbidimetry, Western blot, immunoprecipitation, immunocytochemistry, flow cytometry and Luminex assay) or mass spectrometry. Further, the immunoassay includes ELISA, EIA, agglutination test, nephelometry, turbidimetry, Western blot, immunoprecipitation, immunocytochemistryflow cytometry and Luminex assay. Further, the reagent comprises a primer, a probe and an antibody. The "primer" refers to oligonucleotide which is hybridized with a sequence ("primer binding site") in a target nucleic acid and can be used as a point to initiate a synthesis along a complementary chain of the nucleic acid in conditions suitable for tire synthesis. The "probe" refers to a molecule which can bind to a specific sequence or subsequence or other part of another molecule. Unless otherwise noted, the term "probe" generally refers to a polynucleotide probe which can bind to another polynucleotide (usually called a "target polynucleotide") by complementary base pairing. According to the stringency of hybridization conditions, the probe can bind to the target polynucleotide which lacks complete sequence complementarity with the probe. Hie probe can be used for direct or indirect marking. A hybridization method includes but is not limited to: solution phase, solid phase, mixed phase or in situ hybridization assay. The "antibody" refers to an immunoglobulin molecule which identifies and specifically binds to a target, such as protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid ora combination thereof, by at least one antigen binding site. As used herein, the term covers complete polyclonal antibody, complete monoclonal antibody, single-chain antibody, antibody fragment (such as Fab, Fab', F(ab')2 and Fv fragments), single-chain Fv(scFv) antibody, multispecific antibody (such as bisspecific antibody), monospecific antibody, univalent antibody, chimeric antibody, humanized antibody, human antibody, fusion protein containing an antigen binding site of an antibody, and any other modified immunoglobulin molecule containing an antigen binding site, provided that the antibody exhibits a required biological binding activity. The antibody can be any one of the five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgA 1 and IgA2). Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. The antibody can be bare or conjugated with other molecules, including but not limited to toxins and radioisotopes. Further, the product comprises a kit, a nucleic acid membrane strip, a preparation and a chip. A reagent, a tool and / or an instruction for performing the method described herein can be provided in the kit. For example, the kit may comprise a reagent, a tool and an instruction for determining an appropriate therapy for a patient with cancer. The kit may comprise a reagent for collecting a tissue sample from a patient (e.g. by biopsy) and a reagent for treating the tissue sample. The kit may also comprise one or more reagents for conducting gene or gene product expression analysis, e.g., a reagent used for determining expression level of the biomarker in the subject sample by quantitative PCR, NGS, Northern blot, Southern blot, microarray, SAGE, immunoassay or mass spectrometry. The kit may also comprise an appropriate buffer solution used for the assays. The kit may also comprise a detection reagent required for any of the assays. A more detailed description of appropriate reagents and methods is provided below. The kit characterized herein may also comprise an instruction sheet which describes how to conduct the assays to measure gene or gene product expression. The instruction sheet may include a description on how to determine a reference group, including how to determine expression level of a gene or gene product marker in the reference group and how to compile expression data to establish a reference for comparison with atrial patient. The instruction sheet may also include a description for determining the gene or gene product expression in the trial patient and for comparing the expression level with the expression in the reference group in order to subsequently determine an appropriate chemotherapy for the trial patient. A method for determining the appropriate chemotherapy is described above and can be described in detail in the instruction sheet. Information material included in the kit may be descriptive, instructive, sales or other material which relates to the method described herein and / or the use of the reagent used in the method described herein. For example, the infonnation material of the kit may contain contact information, such as physical address, email address, website or telephone number, and a user of the kit can obtain a great deal of information about conducting gene expression analysis and interpreting the results, especially when the kit is applied to people who may be positive for a particular therapeutic agent. Further, the lung cancer includes lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer and small cell lung cancer, and preferably, the lung cancer is lung squamous cell carcinoma. In another aspect, the present invention provides a device suitable for diagnosing lung cancer, comprising: a) An analyzer unit containing one or more detection reagents which specifically bind the biomarker, wherein the unit is suitable for determining amount of one or more biomarkers in a sample, and b) An analyzer unit used for comparing one or more measured quantities with one or more reference quantities to diagnose lung cancer, wherein the unit comprises a database of one or more reference quantities and a computer-executed algorithm used for conducting comparison; The biomarker includes PCDH18, CDKN2A and / or H0XA9. As used herein, the term "comparison" covers comparing the quantity (expression level) of a biomarker as mentioned herein contained in a sample to be analyzed with the quantity of a suitable reference source specified elsewhere in the Description. It should be understood that the comparison used herein refers to the comparison of a corresponding parameter or value, such as the comparison of an absolute quantity with an absolute reference quantity, the comparison of a concentration with a reference concentration, or the comparison of a strength signal obtained from a test sample with the same type of strength signal from a reference sample. The comparison mentioned in step (b) of the method of the present invention can be conducted manually or by means of a computing device. For computer-aided comparison, a value of a measured quantity can be compared with a value corresponding to a suitable reference, and the value of the suitable reference is stored in a database by a computer program. Results of the comparison can be further evaluated by the computer program, i.e., a required evaluation is automatically provided in a suitable output format. The results may, preferably, be used as an aid to evaluating whether a subject should undergo an image-based diagnostic evaluation as described elsewhere herein. For example, the results of the comparison can be provided in terms of raw data (absolute or relative quantities), and in terms of indicators in the form of words, phrases, symbols or numerical values for some circumstances, which may indicate a specific diagnosis. Therefore, reference quantities should be selected such that differences or similarities in the quantities compared allow the diagnosis of the present invention to be performed. Further, the lung cancer includes lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer and small cell lung cancer. Further, the lung cancer is lung squamous cell carcinoma. In another aspect, the present invention provides an application of the biomarker in preparing the product for diagnosing lung cancer, wherein the biomarker includes PCDH18, CDKN2A and / or H0XA9. Further, the lung cancer includes lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer and small cell lung cancer. Further, the lung cancer is lung squamous cell carcinoma. In another aspect, the present invention provides an application of the biomarker in preparing the product for diagnosing lung cancer, wherein the biomarker includes PCDH18, CDKN2A and / or H0XA9. Further, the lung cancer includes lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer and small cell lung cancer. Further, the lung cancer is lung squamous cell carcinoma. In another aspect, the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a PCDH 18 promotor, a CDKN2A inhibitor and / or a H0XA9 inhibitor. In some embodiments, the pharmaceutical composition also comprises a pharmaceutically acceptable buffer solution, carrier or excipient. "Pharmaceutically acceptable" means a non-toxic material that does not reduce an active component. Such pharmaceutically acceptable buffer solution, carrier or excipient is known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R. Gemiaro, Mack Publishing Company (1990); and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Pharmaceutical Press (2000)). The term "buffer solution" is intended to refer to an aqueous solution containing an acidbase mixture for the purpose of pH stabilization. Examples of the buffer solution are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazole lactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES. The carrier of the present invention includes antimicrobial agent, isotonic reagent, antioxidant, local anesthetic, suspension agent, dispersing agent, emulsifying agent, chelating agent, thickening agent or solubilizing agent. The excipient may be one or more of the following: a carbohydrate, a polymer, a lipid and a mineral. Examples of the carbohydrate include lactose, sucrose, mannitol and cyclodextrin, which are added to the composition, for example, to facilitate freeze-drying. Examples of tire polymer are starch hydrolyzed to varying degrees, cellulose ether, cellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, propyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, poly sulphonate, polyethylene glycol / polyethylenc oxide, polyethylene oxide / polypropylene oxide copolymer, polyvinyl alcohol / polyvinyl acetate, poly(lactic acid), poly(glycolic acid) or copolymers thereof having various compositions, and polyvinylpyrrolidone (all of which have different molecular weights), which are added to the composition, for example, to control viscosity, achieve bioadhesion, or protect an active component from chemical and proteolytic degradation. Examples of the lipid are fatty acid, phosphatide, monoglyceride, diglyceride, triglyceride, ceramide, sphingolipids and glycolipids (both with different acyl chain lengths and saturation), egg lecithin, soybean lecithin, and hydrogenated lecithin and soybean lecithin, which are added to the composition for similar reasons as the polymer. Examples of the mineral are talc, magnesia, zinc oxide and titanium oxide, which are added to the composition to achieve the benefits such as reduced liquid accumulation or favorable pigment properties. In another aspect, the present invention provides an application of the pharmaceutical composition in preparing a drug for preventing or treating lung cancer. Further, the lung cancer includes lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer and small cell lung cancer. Further, the lung cancer is lung squamous cell carcinoma. In another aspect, the present invention provides an application of the biomarker in screening a drug candidate for preventing or treating lung cancer, wherein the biomarker includes PCDH18, CDKN2A and / or H0XA9. Further, the lung cancer includes lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer and small cell lung cancer. Further, the lung cancer is lung squamous cell carcinoma. Description of Drawings Fig. 1 is abox-plot of PCDH18 differential expression; Fig. 2 is a box-plot of CDKN2A differential expression; Fig. 3 is abox-plot of H0XA9 differential expression; Fig. 4 is a ROC curve chart of PCDH18 for diagnosing lung squamous cell carcinoma; Fig. 5 is a ROC curve chart of CDKN2A for diagnosing lung squamous cell carcinoma; Fig. 6 is a ROC curve chart of H0XA9 for diagnosing lung squamous cell carcinoma; Fig. 7 is a ROC curve chart of PCDH18 and CDKN2A for diagnosing lung squamous cell carcinoma; Fig. 8 is a ROC curve chart of CDKN2A and H0XA9 for diagnosing lung squamous cell carcinoma; Fig. 9 is a ROC curve chart of PCDH18 and H0XA9 for diagnosing lung squamous cell carcinoma; Fig. 10 is a ROC curve chart of PCDH18+CDKN2A+HOXA9 for diagnosing lung squamous cell carcinoma. Detailed Description The present invention will be further described in detail below in combination with the drawings and the embodiments. The following embodiments are only used for illustrating the present invention, not used for limiting the scope of the present invention. Experimental methods in which specific conditions are not specified in the embodiments are carried out usually under conventional conditions or the conditions recommended by the manufacturer. Embodiment 1 Differentially expressed genes in lung squamous cell carcinoma Raw data on lung squamous cell carcinoma are downloaded from the database of Hie Cancer Genome Atlas (TCGA), and logarithm is conducted in R for subsequent analysis. After samples with incomplete clinical information are removed, the number of samples included in a TCGA cohort is para-carcinoma:carcinoma = 49:502. Differential expression analysis is conducted using a limma package in R software, and the screening criteria for differentially expressed genes are adj.Pvalue<0.01 and |log2FC|> 1. According to the criteria, 8571 differentially expressed genes are screened out in TCGA, including 5455 up-regulated differentially expressed genes and 3116 down-regulated differentially expressed genes. The expression of the genes in lung squamous cell carcinoma concerned in the present invention is shown in Figs. 1-3 and Table 1. Table 1 Data of gene differential expression Gene logFC AveExpr t P.Value adj .P. Vai PCDH18 -1.107 8.616 -6.036 0.000 0.000 CDKN2A 2.571 9.346 5.836 0.000 0.000 H0XA9 1.012 5.158 3.965 0.000 0.000 Embodiment 2 Diagnostic efficiency analysis The present invention uses R language pROC to conduct ROC diagnostic analysis on the above genes to screen out a biomarker with a diagnostic value. The larger the area under the ROC curve, the higher the diagnostic value. Data of the diagnostic efficiency of the biomarker and biomarker combination concerned in the present invention is shown in Figs. 4-10 and Table 2. Compared with an individual biomarker, a biomarker combination has a better diagnostic efficiency. The result proves that the biomarker and biomarker combination concerned in the present invention can be used for diagnosing lung squamous cell carcinoma. Table 2 Diagnostic efficiency of biomarker and biomarker combination Biomarker / biomarker combination AUC PCDH18 0.764 CDKN2A 0.733 HOXA9 0.675 PCDH18+CDKN2A 0.860 CDKN2A+HOXA9 0.821 PCDH18+HOXA9 0.864 PCDH18+CDKN2A+HOXA9 0.922 Biomarker / biomarker combination The illustration of the above embodiments is merely used for understanding the method and the core thought of tine present invention. It should be noted that forthose ordinary 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 shall also fall into the protection scope of claims of the present invention.

Claims

1. An application of a reagent for detecting expression level of a molecular marker in a biological sample in preparing a product for diagnosing lung cancer in a subject, wherein the molecular marker includes a combination of any two or three of COL1A1, PCDH18 and TUBB4A;the reagent includes a reagent used for detecting mRNA level by a method of real-time fluorescent quantitative reverse transcription polymerase chain reaction, polymerase chain reaction, nuclease protection assay, in situ hybridization, nucleic acid microarray, or serial analysis of gene expression;the reagent includes a reagent used for detecting mRNA level by a method of Western blot, enzyme-linked immunosorbent assay, radioimmunoassay, radioimmunodiffiision, immunoelectrophoresis, tissue immunostaining, immunoprecipitation, complement fixation, fluorescence-activated cell sorting, mass analysis, or protein microarray;the reagent comprises a primer, a probe and an antibody;the product comprises a kit, a nucleic acid membrane strip, a preparation and a chip.

2. The application according to claim 1, wherein the lung cancer is lung squamous cell carcinoma.

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