A molecular marker and kit for non-invasive early diagnosis and drug efficacy evaluation of lung adenocarcinoma

Abstract

This invention discloses a molecular biomarker and kit for non-invasive early diagnosis and drug efficacy evaluation of lung adenocarcinoma. Specifically, this invention discloses the application of CpG islands of the PRSS3 gene as molecular biomarkers and / or substances for detecting the methylation level of said molecular biomarkers in the preparation of products for the treatment, diagnosis, or prognostic evaluation of non-small cell lung cancer. The molecular biomarker and its primers for detecting methylation status or level of this invention are characterized by high sensitivity and specificity; the kit based on detecting DNA sequence methylation within the PRSS3 gene can specifically detect PRSS3 gene-related DNA methylation levels, and can also specifically and quantitatively detect PRSS3 gene-related DNA methylation levels; the detection results can provide an evaluation method for monitoring the condition and individualized medication of non-small cell lung cancer patients, early tumor diagnosis, and prognostic assessment of drug treatment.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology and relates to a molecular biomarker and kit for non-invasive early diagnosis and drug efficacy evaluation of lung adenocarcinoma. Specifically, it relates to a potential biomarker and kit for non-invasive early diagnosis and drug efficacy evaluation of lung adenocarcinoma, especially for evaluating apparent drug efficacy, and the application of this molecular biomarker in the preparation of formulations for the diagnosis, treatment and prognosis of non-small cell lung cancer. Background Technology

[0002] Non-small cell lung cancer (NSCLC) is one of the most common and prevalent cancers in my country, and its mortality rate ranks among the highest of all malignant tumors worldwide and in my country (Bray F, Jemal A, Grey N, Ferlay J, Forman D. Global cancer transitions according to the Human Development Index (2008-2030): a population-based study[J]. Lancet Oncol 2012; 13:790-801.). NSCLC is a highly heterogeneous lung adenocarcinoma caused by genetic and epigenetic alterations (Chen Z, Fillmore CM, Hammerman PS, Kim CF, Wong KK. Non-small-cell lung cancers: a heterogeneous set of diseases. Nature reviews Cancer. 2014; 14:535-546.). The heterogeneity of NSCLC is considered a major obstacle to biomarker-driven therapeutic interventions and early diagnosis, resulting in a far from satisfactory 5-year survival rate for all NSCLC patients. Therefore, identifying early molecular targets for lung cancer is of significant clinical and scientific value for the accurate diagnosis and personalized treatment of tumors.

[0003] Further investigation into the tumor genome of lung cancer revealed that tumor heterogeneity can occur through co-genomic alterations in oncogenic drivers and tumor suppressor genes (TSGs), rather than a single-genome-driven model defining NSCLC tumorigenesis and molecular classification. Non-genetic heterogeneity through epigenomic and transcriptomic alterations has demonstrated the potential for developing novel biomarkers and effective targeted therapies. In particular, epigenetics, through modifications such as DNA methylation and histones, regulates cell and tissue-specific gene expression. Unlike gene mutations, epigenetic alterations lead to progressive and reversible phenotypic changes. This characteristic opens new avenues for the prevention and treatment of some diseases, especially tumors, and is of great significance for the research and development of demethylating drugs for tumor therapy. Abnormal changes in gene expression and function caused by abnormal hypermethylation of CpG islands in the promoter region of tumor suppressor genes and abnormal histone modifications are not only used as epigenetic markers and targets for molecular diagnosis, treatment and prognostic assessment of tumors, but are also considered to be one of the main factors causing disease and the formation of treatment resistance (Oh JH, Jung SH, Hong SJ, and Rhyu MG: DNA Methylation as Surrogate Marker For Gastric Cancer. J Cancer Prev 20:172-178, 2015.).

[0004] DNA methylation is a type of phenotypic modification closely related to cancer development. In particular, hypermethylation of the promoter in CpG island regions can lead to transcriptional silencing of tumor suppressor genes, thereby affecting the tumorigenesis process. Since DNA methylation is found in almost all cancers, and often occurs in precancerous or early stages of cancer, it holds promise as an ideal biomarker for early cancer diagnosis. Detecting the methylation status of specific genes can effectively improve the diagnostic efficacy of lung cancer, and related methylation detection is gradually moving from research to clinical applications.

[0005] Currently, precision medicine, represented by targeted therapy for lung cancer, is developing rapidly, but it still faces many problems and challenges. Precision medicine needs more specific biomarkers for the early diagnosis of lung cancer. Therefore, screening lung cancer biomarkers with high specificity and sensitivity to guide early diagnosis and evaluate efficacy is of great clinical significance for the precision treatment of lung cancer. Summary of the Invention

[0006] The purpose of this invention is to provide a highly sensitive and specific molecular biomarker for the diagnosis and detection of non-small cell lung cancer, and / or a primer pair for detecting the methylation level of this molecular biomarker. The technical problem to be solved is not limited to the described technical subject matter; other technical subjects not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0007] To achieve the above objectives, the present invention first provides the application of molecular markers and / or substances for detecting the methylation level of said molecular markers in the preparation of products for the diagnosis and / or prognostic assessment of lung squamous cell carcinoma and lung adenocarcinoma, and non-small cell lung cancer, wherein said molecular markers are DNA molecules with nucleotide sequences of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 and / or SEQ ID No. 4.

[0008] Furthermore, the molecular markers are the CpG islands (CpGI_1-4) of the four PRSS3 genes identified in this invention, wherein:

[0009] CpGI_1, as a molecular marker M1, has a DNA sequence located at the transcription start site of the PRSS3 splice variant 1 gene, from +21 bp to +452 bp, and its nucleotide sequence is shown in SEQ ID No. 1.

[0010] CpGI_2, as a molecular marker M2, has a DNA sequence located at the transcription start site of the PRSS3 splice variant 1 gene from +428bp to +840bp, and its nucleotide sequence is shown in SEQ ID No. 2.

[0011] CpGI_3, as the molecular marker M3, is a DNA sequence located at the transcription start site of the PRSS3 splice variant 2 gene from -355bp to +126bp, and its nucleotide sequence is shown in SEQ ID No. 3.

[0012] CpGI_4, as the molecular marker M4, is a DNA sequence located from 386 bp (+386 bp) to 540 bp (+540 bp) downstream of the transcription start site of the PRSS3 splice variant 1 gene, and its nucleotide sequence is shown in SEQ ID No. 4.

[0013] Furthermore, the substance used to detect the methylation level of the molecular marker can be a substance used to detect the methylation level of the CpG islands (CpGI_1-4) of the PRSS3 gene.

[0014] In the above applications, the substance may be a primer pair used to detect the methylation level of the molecular marker.

[0015] In the above applications, the primer pair may be at least one of the following:

[0016] A1) A primer pair consisting of primer F1 and primer R1; wherein primer F1 is a single-stranded DNA molecule as shown in SEQ ID No. 5; and primer R1 is a single-stranded DNA molecule as shown in SEQ ID No. 6.

[0017] A2) A primer pair consisting of primer F2 and primer R2; wherein primer F2 is a single-stranded DNA molecule as shown in SEQ ID No. 7; and primer R2 is a single-stranded DNA molecule as shown in SEQ ID No. 8.

[0018] A3) A primer pair consisting of primer F3 and primer R3; wherein primer F3 is the single-stranded DNA molecule shown in SEQ ID No. 9; and primer R3 is the single-stranded DNA molecule shown in SEQ ID No. 10.

[0019] A4) A primer pair consisting of primer F4 and primer R4; wherein primer F4 is the single-stranded DNA molecule shown in SEQ ID No. 11; and primer R4 is the single-stranded DNA molecule shown in SEQ ID No. 12.

[0020] A5) A primer pair consisting of primer F5 and primer R5; wherein primer F5 is a single-stranded DNA molecule as shown in SEQ ID No. 13; and primer R5 is a single-stranded DNA molecule as shown in SEQ ID No. 14.

[0021] Primers F1 and R1 were used to detect the methylation level of the molecular marker M1 (SEQ ID No. 1).

[0022] Primers F2 and R2 were used to detect the methylation level of molecular marker M2 (SEQ ID No. 2).

[0023] Primers F3 and R3 were used to detect the methylation level of the molecular marker M3 (SEQ ID No. 3).

[0024] Primers F4, R4, F5, and R5 were used to detect the methylation level of the molecular marker M4 (SEQ ID No. 4).

[0025] The molecular markers and / or primer pairs are also within the scope of protection of this invention.

[0026] The present invention also provides reagents or kits containing the primer pairs.

[0027] Furthermore, the kit also includes Taq DNA polymerase, dNTPs, PCR buffer, and Mg2+ required for PCR amplification. 2+ One or more of them.

[0028] Furthermore, the kit also includes a bisulfite, specifically sodium bisulfite (NaHSO3).

[0029] Furthermore, the kit may also include a DNA methylation positive control and a negative control.

[0030] Furthermore, the kit may also include an internal reference gene detection reagent, which may be the GAPDH gene and / or the β-actin gene, but is not limited thereto.

[0031] The various reagent components of the kit may be present in separate containers, or may be pre-assembled into a reagent mixture, either wholly or partially.

[0032] The present invention also provides any of the following applications of the primer pair:

[0033] B1) The application of the primer pair in detecting the methylation level of the molecular marker;

[0034] B2) The application of the primer pair in the preparation of products for detecting the methylation level of the molecular marker;

[0035] B3) The application of the primer pair in the preparation of products for screening non-small cell lung cancer;

[0036] B4) The application of the primer pair in the preparation of products for differentiating between squamous cell carcinoma and adenocarcinoma of the lung;

[0037] B5) The use of the primer pair in the preparation of products for the diagnosis and / or prognostic assessment of non-small cell lung cancer;

[0038] B6) The application of the primer pair described therein in the preparation of products for efficacy evaluation of non-small cell lung cancer.

[0039] In the above applications, the product may be a reagent or a reagent kit.

[0040] The present invention also provides the use of DNA methylation inhibitors that inhibit or reduce the methylation level of said molecular markers in the preparation of medicaments for the treatment or prevention of non-small cell lung cancer.

[0041] The drug may be an epigenetic drug (epigenetic drug).

[0042] The DNA methylation inhibitor may be 5-aza-2'-deoxycytidine (5-Aza) or 5-azacytidine (5-Azacytidine), but is not limited thereto;

[0043] In this article, non-small cell lung cancer (NSCLC) may refer to lung adenocarcinoma or lung squamous cell carcinoma.

[0044] This invention also provides a method for detecting the methylation level of the PRSS3 gene promoter, the method comprising the following steps:

[0045] C1) Extract DNA from the sample to be tested;

[0046] C2) The DNA is treated with bisulfite to obtain sulfidated DNA;

[0047] C3) Using the sulfurized DNA as a template, PCR amplification was performed using the primer pair to obtain the PCR amplification product;

[0048] C4) The methylation of the PCR amplification product was detected.

[0049] In the above method, the sample to be tested may be cells, tissues, and / or bronchoalveolar lavage fluid.

[0050] In one embodiment of the present invention, the method may specifically be the MassArray system methylation detection method (hereinafter referred to as the MassArray method).

[0051] Furthermore, the primer pairs in the MassArray method may be primer pairs consisting of primer F1 (SEQ ID No. 5) and primer R1 (SEQ ID No. 6), primer pairs consisting of primer F2 (SEQ ID No. 7) and primer R2 (SEQ ID No. 8), and / or primer pairs consisting of primer F3 (SEQ ID No. 9) and primer R3 (SEQ ID No. 10).

[0052] Furthermore, the PCR amplification reaction components described in C3) include upstream primer F, downstream primer R, DNA template, PCR amplification enzyme, dNTPs, 10×PCR Buffer, and ddH2O.

[0053] Further, the PCR amplification reaction conditions described in C3) are: 94℃ for 4 min; 94℃ for 20 sec, 56℃ for 30 sec, 72℃ for 1 min, 45 cycles; 72℃ for 3 min.

[0054] C2) The bisulfite is sodium bisulfite (NaHSO3).

[0055] In the above method, C4) the methylation detection of the PCR amplification product further includes an agarose gel electrophoresis detection step, a shrimp alkaline phosphatase (SAP) reaction step, a T-digestion / RNase A digestion reaction step, a resin purification step, and a chip spotting step.

[0056] Further, the SAP reaction system (7.00 μL) consists of: 1.70 μL RNase-free ddH2O, 0.30 μL SAP enzyme, and 5.00 μL PCR amplification product; the SAP reaction conditions are: 37℃ for 20 min, and 85℃ for 5 min.

[0057] Test results were obtained using EpiTYPER. TM The software acquires raw data, which is calculated based on the area comparison of peaks containing G and peaks containing A to determine the degree of methylation and demethylation of the sample under test, representing a relative quantitative ratio between the two. If the detection value in the table is 0.43, it means that the methylation degree of the corresponding methylation site in the sample is 43%. If the methylation percentage is ≥60%, the site is considered "hypermethylated"; if the methylation percentage is 20-60% (excluding endpoints), it is considered "partially methylated"; if the methylation percentage is ≤20%, it is considered "unmethylated".

[0058] In one embodiment of the present invention, the method may specifically be a methylation-specific qPCR detection method (MS-qPCR method for short).

[0059] Furthermore, in the above method, the primer pair may be a primer pair composed of primer F4 (SEQ ID No. 11) and primer R4 (SEQ ID No. 12) (methylated primer); and a primer pair composed of primer F5 (SEQ ID No. 13) and primer R5 (SEQ ID No. 14) (unmethylated primer).

[0060] Using the aforementioned methylation primers, MSP amplification was performed using sulfur-modified DNA as a template. If the PRSS3 gene was methylated, a 152 bp fragment was obtained; if the PRSS3 gene was not methylated, no amplification product was produced. Therefore, this invention refers to these primers as methylation primers. The methylation primers have an upstream primer with a Tm of 65.42 and a GC content of 60%, and a downstream primer with a Tm of 64.96 and a GC content of 48.15%.

[0061] Using this unmethylated primer, MSP amplification was performed with sulfur-modified DNA as a template. If the PRSS3 gene was not methylated, a 155 bp fragment was obtained; if the PRSS3 gene was methylated, no amplification product was produced. Therefore, this primer is referred to as an unmethylated primer. The unmethylated primer has a Tm of 62.78 and a GC content of 50% for the upstream primer, and a Tm of 59.32 and a GC content of 31.03% for the downstream primer.

[0062] If both fragments of 152bp and 155bp are present in the PCR amplification product, it indicates that the PRSS3 gene has undergone partial methylation.

[0063] Further, the PCR amplification reaction system (20.00 μL) consists of: 7 μL ddH2O, 10 μL 2×SYBR-GreenMaster qPCR Mix, 0.5 μL primer F (10 nmol / μL), 0.5 μL primer R (10 nmol / μL), and 2.00 μL DNA template.

[0064] Furthermore, the PCR amplification reaction conditions are as follows: 50℃ for 2 min, 95℃ for 10 min, 95℃ for 15 s; 60℃ for 1 min; 40 cycles, collecting fluorescence; 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s, 60℃ for 15 s, to prepare a melting curve.

[0065] DNA samples were treated with bisulfite, converting unmethylated cytosine to uracil, while methylated cytosine remained unchanged. Changes in DNA methylation levels were then detected using PCR to determine the presence or absence of CpG island methylation in the gene. The amplified products were analyzed using DNA agarose gel electrophoresis, with gel scanning revealing the results.

[0066] Furthermore, the bisulfite may be sodium bisulfite (NaHSO3).

[0067] Furthermore, the method for interpreting the results of methylation detection of the PCR amplification products is as follows:

[0068] The first method is to determine whether the desired product is obtained:

[0069] Amplification using methylating primers (F4 and R4) yielded a 152bp amplification product, while amplification using non-methylating primers (F5 and R5) produced no 155bp amplification product, which was interpreted as "hypermethylation" (relatively high methylation level). Amplification using both methylating and non-methylating primers produced 152bp amplification products, which was interpreted as "partial methylation" (relatively low methylation level).

[0070] Amplification with unmethylated primers yielded a 155bp amplification product, while amplification with methylated primers did not produce a 155bp amplification product. This sample was interpreted as "unmethylated".

[0071] The second method involves determining the result using methylation-specific quantitative PCR (MS-qPCR).

[0072] Through 2 -ΔΔCtMethods: (Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) ​​for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002 May 1; 30(9):e36.). Ct values ​​obtained by amplification using methylated primer pairs and Ct values ​​obtained by amplification using unmethylated primer pairs were homogenized and presented as folds of the Ct values ​​amplified by β-actin.

[0073] The purpose of the above applications and methods may be for disease diagnosis, disease prognosis and / or disease treatment, or they may be for non-disease diagnosis, non-disease prognosis and non-disease treatment purposes; their direct purpose may be to obtain information on intermediate results of disease diagnosis, disease prognosis and / or disease treatment, or their direct purpose may be for non-disease diagnosis, non-disease prognosis and / or non-disease treatment purposes.

[0074] This invention reveals that human trypsinogen 3 (PRSS3) splice variants exhibit differential expression in NSCLC, regulated by CpG island hypermethylation (iCpGIme) within the PRSS3 gene. In particular, iCpGIme epigenetically silences the tumor suppressor gene PRSS3 splice variant 3 (PRSS3-V3), which contributes to lung cancer development. Therefore, iCpGIme can serve as a molecular marker and drug target for early diagnosis, treatment, and prognostic detection of non-small cell lung cancer, especially for molecular subtyping of adenocarcinoma and analysis of tumor heterogeneity in non-small cell lung cancer.

[0075] Through extensive and in-depth research, the inventors unexpectedly discovered hypermethylation (iCpGIme) of CpG islands (iCpGI) within the PRSS3 gene in lung adenocarcinoma tissue and bronchoalveolar lavage fluid samples. Based on this, they developed a molecular marker for the diagnosis and detection of non-small cell lung cancer. Hypermethylation of CpG islands (iCpGIme) within the PRSS3 gene, as a molecular marker, can be applied to the non-invasive prediction or early diagnosis of lung adenocarcinoma. Furthermore, hypermethylated CpG islands within the PRSS3 gene can serve as drug targets, especially potential targets for epigenetic drugs, in the screening and development of drugs for non-small cell lung cancer.

[0076] This invention provides a molecular marker for the diagnosis and detection of non-small cell lung cancer, and primer pairs for detecting the methylation status or level of this molecular marker (PRSS3 gene CpG island). These markers are characterized by high sensitivity and specificity. The kit based on the detection of DNA sequence methylation within the PRSS3 gene can specifically detect the PRSS3 gene-related DNA methylation level, and can also perform specific and quantitative detection of the PRSS3 gene-related DNA methylation level. The detection results can provide an evaluation method for monitoring the condition and individualized medication of non-small cell lung cancer patients, early tumor diagnosis, and prognostic assessment of drug treatment. Attached Figure Description

[0077] Figure 1 To screen PRSS3 for DNA methylation regulatory sites in non-small cell lung cancer. Figure 1 A is a schematic diagram of the PRSS3CpG site and primers; Figure 1 B and Figure 1 C is based on the CCLE dataset ( Figure 1 B) Aggregated heatmaps of the correlation between CpG site methylation and splicing variant expression in LUAD (n=315) tissues from the TCGA database. The correlation was used as a distance function to visualize the data for lung cancer cell lines (B) Figure 1 B) or lung cancer tissue specimen ( Figure 1 Heatmap clustering analysis was performed on the methylation of CpG islands in C). ICpGs were ranked according to the correlation between methylation level and PRSS3 splicing variant mRNA expression level. DNA methylation and mRNA expression levels were normalized, with Mean = 0 and SD = 1. Figure 1 D represents the correlation analysis between PRSS3 splicing variant mRNA expression level and CpG site DNA methylation, *p<0.05, **p<0.01.

[0078] Figure 2 To investigate the epigenetic regulation of PRSS3 and its splicing variants in non-small cell lung cancer. Figure 2 A is a visualization clustering heatmap analysis of PRSS3 methylation status in NSCLC cells detected by MassARRAY; Figure 2 B represents the methylation level of PRSS3 iCpGI in NSCLC cells analyzed by MSP-qPCR. In vitro methylated DNA (IVD) and normal blood lymphocyte DNA (NL) served as positive and negative controls, respectively. Figure 2 C represents the analysis of PRSS3iCpGI methylation status in tumors and adjacent normal tissues of NSCLC patients (n=13) using MSP-qPCR. The frequencies of methylated (M) and unmethylated (UM) samples are expressed as a percentage of cases. Figure 2D and 2E are for RT-qPCR detection of PRSS3 (5 μM, 96 h) in 5-Aza-CR treated NSCLC cell lines. Figure 2 D) and its splicing variants PRSS3-V1~V3 ( Figure 2 The expression of E); Figure 2 F represents the changes in PRSS3 iCpGI methylation in 5-Aza-CR treated NSCLC cell lines (5 μM, 96 h) detected by MassARRAY, *p<0.05, **p<0.01.

[0079] Figure 3 This study aimed to detect somatic methylation of the PRSS3 gene in bronchoalveolar lavage fluid (BALF). Figure 3 A represents the expression of PRSS3-V1 to V3 in BALF from patients with chronic pneumonia, lung adenocarcinoma, and lung squamous cell carcinoma, analyzed by RT-qPCR. Figure 3 B represents the MSP-qPCR analysis of the methylation status of BALF specimens. Figure 3 C is a normalized stacked bar graph representing the percentage of PRSS3 gene somatic methylation relative to PRSS3-V1~V3 expression in BALF; Figure 3 D represents Spearman and Pearson correlation analyses of the relationship between gene somatic methylation and PRSS3 splicing variant expression in BALF specimens, *p<0.05, **p<0.01. Detailed Implementation

[0080] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0081] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0082] Example 1: Discovery of molecular markers for early diagnosis and prognostic detection of non-small cell lung cancer

[0083] In this embodiment, clustering heatmaps were used to analyze the methylation status of CpG islands and CpG sites. Person and Spearman coefficients were used to analyze the correlation between different CpG sites and the mRNA expression of different PRSS3 splicing variants.

[0084] The raw data were obtained from the Cancer Cell Line Database (CCLE, http: / / www.broadinstitute.org / ccle) and the Cancer Genome Database (TCGA, https: / / www.cancer.gov / ). Raw DNA methylation and TCGA RNA sequencing data were downloaded for sample analysis (sample size: CCLE lung cancer cell line n = 187; TCGA database lung adenocarcinoma n = 315). The CCLE database recorded three CpG islands for PRSS3, while the TCGA database recorded six CpG sites for PRSS3. Figure 1 A).

[0085] Clustering heatmap method for analyzing CpG islands ( Figure 1 B) and CpG sites ( Figure 1 C) Methylation status. First, the raw data is standardized and dimensionless. In this case, the extreme value method is used, with the formula X = (X - Min) / (Max - Min) to ensure that the maximum value of the standardized data is 1 and the minimum value is 0. For example, the closer the standardized value is to 1, the more methylated PRSS3 is.

[0086] Person coefficient and Spearman coefficient for different CpG sites Figure 1 D) Analyze the correlation between mRNA expression and different splicing variants of PRSS3. The closer the correlation coefficient is to 1 or -1, the stronger the correlation; the closer the correlation coefficient is to 0, the weaker the correlation.

[0087] The results show:

[0088] First, cluster heatmap analysis using CCLE data was performed on the PRSS3 methylation status of the three CpG islands (defined as CpGI_1–3). The analysis revealed that PRSS3 exhibited high methylation in the upstream CpG island (CpGI_1), while the downstream CpG islands (CpGI_2 and_3) showed differential methylation. Further analysis showed that although PRSS3 expression was negatively correlated with the methylation of all three CpGIs in lung cancer cell lines, PRSS3-SVs expression showed varying degrees of correlation with CpGI methylation. Specifically, PRSS3-V1 was negatively correlated with the methylation of CpGI_1–3. PRSS3-V2 was negatively correlated with CpGI_2 and 3, while PRSS3-V3 was negatively correlated with CpGI_3. Figure 1 B). Further analysis was performed on PRSS3 methylation data from the TCGA-LUAD dataset, distributed across six CpG sites (defined as CpG sites A to F) ranging from -170 to 34654 nt to cTSS. Figure 1C), revealing methylation patterns associated with hypermethylation at upstream CpG sites (CpG site A) and differential intragene methylation (such as hypomethylation at CpG sites B and C) and frequently altered CpG sites (CpG sites E, D, and F). Figure 1 Analysis of TCGA-LUAD methylation data and PRSS3-SVs expression showed that sites E and D were negatively correlated with PRSS3-V1 / V3 expression, and sites D, E, and F were negatively correlated with PRSS3-V2 expression. Figure 1 D). The results showed that this region, including sites B, C, D, and E, termed the genomic CpG island (iCpGI), is likely a differentially methylated region (DMR region) specifically involved in the epigenetic regulation of PRSS3 splice variant expression in lung cancer. Figure 1 A).

[0089] Four CpG islands (CpGI_1-4) of the PRSS3 gene were identified as potential molecular markers, as shown below:

[0090] CpGI_1, as a molecular marker M1, has a DNA sequence located at the transcription start site of the PRSS3 splice variant 1 gene, from +21 bp to +452 bp, and its nucleotide sequence is shown in SEQ ID No. 1.

[0091] CpGI_2, as a molecular marker M2, has a DNA sequence located at the transcription start site of the PRSS3 splice variant 1 gene from +428bp to +840bp, and its nucleotide sequence is shown in SEQ ID No. 2.

[0092] CpGI_3, as the molecular marker M3, is a DNA sequence located at the transcription start site of the PRSS3 splice variant 2 gene from -355bp to +126bp, and its nucleotide sequence is shown in SEQ ID No. 3.

[0093] CpGI_4, as the molecular marker M4, is a DNA sequence located from 386 bp (+386 bp) to 540 bp (+540 bp) downstream of the transcription start site of the PRSS3 splice variant 1 gene, and its nucleotide sequence is shown in SEQ ID No. 4.

[0094] Example 2: MassARRAY detection of PRSS3 gene DNA methylation status in non-small cell lung cancer tissue cells

[0095] 1. Design and synthesis of primers for detecting PRSS3 gene methylation

[0096] The PRSS3 gene sequence to be tested was retrieved and downloaded (GenBank Accession No. NC_000009.12). Based on the database analysis results in Example 1, the target sequence was input into EpiDesigner software for primer design. A suitable primer design scheme was selected and determined based on the software results. Primers were synthesized, each with a 5OD value.

[0097] The specific sequences of the primers for methylation detection are as follows:

[0098] Primer set 1 covers the DNA sequence (SEQ ID No. 1) located at the transcription start site of the PRSS3 splice variant 1 gene, from +21 bp to +452 bp.

[0099] Primer set 1 consists of F1 and R1, and its sequence is as follows:

[0100] Upstream primer F1: 5'-aggaagagagATTTTTAAGGGAAGGTTAAGGGTTT-3' (SEQ ID No. 5),

[0101] Downstream primer R1: 5'-cagtaatacgactcactatagggagaaggctCCTAAAAAAACCTCCATCCCTATC-3' (SEQ ID No. 6).

[0102] Primers F1 and R1 were used to detect the methylation level of the molecular marker M1 (SEQ ID No. 1).

[0103] Primer set 2 covers the DNA sequence (SEQ ID No. 2) located at the transcription start site of the PRSS3 splice variant 1 gene from +428 bp to +840 bp.

[0104] Primer set 2 consists of F2 and R2, and its sequence is as follows:

[0105] Upstream primer F2: 5'-aggaagagagGGATAGGGATGGAGGTTTTTTTAG-3' (SEQ ID No. 7),

[0106] Downstream primer R2: 5'-cagtaatacgactcactatagggagaaggctTAAATTCCAAACTTCCTAACCTCAA-3' (SEQ ID No. 8).

[0107] Primers F2 and R2 were used to detect the methylation level of molecular marker M2 (SEQ ID No. 2).

[0108] Primer set 3 covers the DNA sequence (SEQ ID No. 3) located at the transcription start site of the PRSS3 splice variant 2 gene from -355 bp to +126 bp.

[0109] Primer set 3 consists of F3 and R3, and its sequence is as follows:

[0110] Upstream primer F3: 5'-aggaagagagTGGTAAGAATGGTAGGGTATGTGTT-3' (SEQ ID No. 9),

[0111] Downstream primer R3: 5'-cagtaatacgactcactatagggagaaggctTTAAAAAAACTCCTAAACACCCAAA-3' (SEQ ID No. 10).

[0112] Primers F3 and R3 were used to detect the methylation level of the molecular marker M3 (SEQ ID No. 3).

[0113] All primers were synthesized by BGI Genomics (Beijing).

[0114] Using the methylated primers (i.e., primer sets 1-3) described above, PCR amplification was performed using sulfur-modified DNA as a template. Primer set 1 yielded a 432 bp fragment. The upstream primer of the methylated primer had a Tm of 59.92 and a GC content of 32%, while the downstream primer had a Tm of 60.39 and a GC content of 42%. Primer set 2 yielded a 413 bp fragment. The upstream primer of the methylated primer had a Tm of 60.39 and a GC content of 42%, while the downstream primer had a Tm of 59.48 and a GC content of 32%. Primer set 3 yielded a 481 bp fragment. The upstream primer of the methylated primer had a Tm of 57.45 and a GC content of 35%, while the downstream primer had a Tm of 59.42 and a GC content of 28%. Methylation was detected using the Agena MassArray system. The test results were obtained using EpiTYPER™ software. The raw data was calculated by comparing the areas of the G-peak and the A-peak to determine the degree of methylation and demethylation of the sample, representing a relative quantitative ratio. If the value in the table is 0.43, it means the methylation degree of the corresponding methylation site in the sample is 43%. If the methylation percentage is ≥60%, the site is considered "hypermethylated"; if the methylation percentage is 20–60% (excluding endpoints), it is considered "partially methylated"; and if the methylation percentage is ≤20%, it is considered "unmethylated".

[0115] 2. Methylation detection

[0116] Let's take "primer sets 1-3" in step 1 as an example for explanation.

[0117] Test cells: A549, 801-D, NCI-H1299, NCI-H460;

[0118] All of the above-mentioned cells can be purchased from the National Biomedical Experimental Cell Resource Bank and cultured and passaged under normal conditions in the applicant's laboratory.

[0119] 2-1. Cell DNA Extraction Process

[0120] (1) Remove the culture medium from the above-mentioned test cells that are in the logarithmic growth phase and in good condition, and gently wash the cells twice with ice-cold 1×PBS.

[0121] (2) Add 1 mL of trypsin, digest for 1 min, then add 2 mL of RPMI 1640 complete culture medium to neutralize, gently pipette the suspended cells, transfer to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and collect the cells.

[0122] (3) Add 2 mL of DNA extraction solution and 100 μL of proteinase K (10 mg / mL), mix well by pipetting, and place in a 50°C constant temperature water bath for 3 h.

[0123] (4) Remove the tube, cool it to room temperature, add an equal volume of phenol / chloroform, mix by inverting the tube, centrifuge at 4200 rpm for 15 min, and carefully transfer the supernatant to a new 15 mL centrifuge tube.

[0124] (5) Add 1 / 10 volume of 7.5 mol / L ammonium acetate and 2 volumes of anhydrous ethanol, mix gently, centrifuge at 10,000 rpm for 20 min, and discard the supernatant.

[0125] (6) Add 500 μL of 70% ethanol, wash the precipitate twice, centrifuge at 13000 rpm for 5 min, discard the supernatant, and air dry.

[0126] (7) Add 100 μL of pH 8.0 TE solution to dissolve the DNA. Take 1 μL of the DNA solution and measure the DNA concentration using a NanoDrop2000c micro-nucleic acid analyzer. Store the remaining sample in a -20℃ freezer.

[0127] 2-2. Quality control of DNA samples and concentration

[0128] The DNA methylation positive and negative control samples were purchased from Zymo Research, USA, using the Human Methylated & Non-methylated DNA Set (catalog number: D5014). Human Methylated DNA is also known as invitro methylated DNA (IVD). DNA concentration and purity were determined by OD detection and gel electrophoresis; the total amount of DNA (sample to be tested, DNA methylation positive control, and negative control samples) was 3-5 μg, with a concentration greater than 50 ng / μl. The DNA samples to be tested were further treated with NaHSO3 to convert all cytosine in the unmethylated regions to thymine, while the cytosine in the methylated regions remained unchanged. This was performed using the Zymo Research EZ DNA Methylation Gold Kit (catalog number: D5006).

[0129] 2-3. Methylation Detection Procedure of Agena MassArray System

[0130] Use the Agena EpiTYPER Reagent and SpectroCHIP kit (part number: 11377D) from the USA.

[0131] (1) PCR amplification reaction

[0132] After treating the samples with NaHSO3, centrifuge briefly at low speed for later use. Prepare the PCR reaction solutions as shown in Table 1 (the reaction system preparation process should be completed on ice to prevent inactivation due to prolonged exposure to high temperature).

[0133] Table 1. Methylation detection amplification PCR reaction components in the Agena MassArray system

[0134]

[0135] (2) The PCR amplification reaction procedure is shown in Table 2.

[0136] Table 2 PCR reaction procedure

[0137]

[0138] Mix 3 μl of PCR product with 1 μl of 6*loading buffer, and detect the results by 1.5% agarose gel electrophoresis at 160V for 20 min. If the results are good, subsequent experiments can be continued.

[0139] (3) Shrimp alkaline phosphatase (SAP) reaction

[0140] The PCR amplification products were subjected to an SAP reaction to remove excess deoxyribonucleoside triphosphates (dNTPs / dUTPs). The SAP reaction system is shown in Table 3.

[0141] Table 3. MassArray system for methylation detection of SAP reaction components

[0142] reagents Final volume <![CDATA[RNase-free ddH2O]]> 1.70μL SAP enzyme 0.30μL PCR products 5.00μL Total volume 7.00μL

[0143] Add 7 μl of SAP reaction solution to each well of the 384 reaction plate, carefully cover the 384-well sealing film, and press each well firmly to prevent evaporation during the PCR program. After centrifugation, proceed with the following reaction program (Table 4).

[0144] Table 4 SAP Response Procedure

[0145] 37℃ 20min 85℃ 5min 4℃ ∞

[0146] (4) T-cell / RNase A digestion reaction

[0147] Taking advantage of RNase A's ability to specifically recognize and cleave the U 3' end of RNA, RNA fragments are cleaved into smaller fragments carrying CpG sites. The T-cleavage / RNase A digestion reaction system is shown in Table 5:

[0148] Table 5. T-shear reaction / RNase A digestion reaction components

[0149]

[0150] T-cut / RNase A digestion reaction conditions: incubation at 37°C for 3 hours.

[0151] 2-4. Resin purification

[0152] Evenly fill the 384 / 6MG Dimple plate with resin and let it air dry for 10 minutes. Add 16 μl of water to each well of the 384 sample plate. Gently place the 384 sample plate on the Dimple plate, invert it, and tap it gently to allow the resin to fall into each well of the sample plate. Seal the 384 sample plate with sealing film and place it in a centrifuge at room temperature for 30 minutes to mix thoroughly.

[0153] 2-5. Chip prototyping

[0154] Start the MassARRAY Nanodispenser RS1000 spotter and transfer the resin-purified product to a 384-well plate. On a bioarray, the spotted SpectroCHIP chip was analyzed using MALDI-TOF. The detection results were analyzed using EpiTYPER. TMThe software acquires raw data and dot plots, and checks the integrity and accuracy of the data files. The results are saved to the appropriate storage medium and submitted to the bioinformatics lab for analysis.

[0155] 2-6. Results

[0156] The results are as follows Figure 2 As shown in A and 2C, a control system was established using IVD as a methylation control, NL as a non-methylation control, and deionized water as a negative control. MassARRAY assays were performed on cell samples using methylation primers. MassARRAY analysis was used to determine the methylation of the CpG site in the PRSS3 gene locus in lung cancer cells. Figure 2 A). The results showed that A549 and 801-D cells with high PRSS3 expression exhibited hypomethylation at CpG sites in the primer set 1 region, differential methylation at CpG sites in the primer set 2 region, and hypermethylation at CpG sites in the primer set 3 region. NCI-H1299 and NCI-H460 cells with low PRSS3 expression showed the opposite methylation state. These results suggest that the differentially methylated PRSS3 region in primer set 2 may be associated with the expression of various PRSS3 splicing variants and could be a potential methylation marker region.

[0157] Example 3: Methylation-specific qPCR detection of PRSS3 gene DNA methylation status in non-small cell lung cancer tissue cells

[0158] 1. Design and synthesis of primers for PRSS3 gene methylation detection: Selecting specific CG-rich DNA regions from differentially methylated gene regions ( Figure 1 A) Multiple primer pairs were designed using Primer Express 3.0 software, and the following methylated and unmethylated primers were selected. All primers were synthesized by BGI Genomics (Beijing).

[0159] Primer set 4 covers the DNA sequence (SEQ ID No. 4) located at the transcription start site of the PRSS3 splice variant 1 gene from +386 bp to +540 bp.

[0160] The upstream primer nucleotide sequence of the methylation primer is as follows:

[0161] F4: 5'-GGTACGCGGATAGGGAGGGGATATC-3' (SEQ ID No. 11);

[0162] The downstream primer nucleotide sequence of the methylation primer is as follows:

[0163] R4: 5'-TAATATACGCATCGATACCGCAACCCG-3' (SEQ ID No. 12);

[0164] The upstream primer nucleotide sequence of the unmethylated primer is as follows:

[0165] F5: 5'-GGGTATGTGGATAGGGAGGGGATATT-3' (SEQ ID No. 13);

[0166] The downstream primer nucleotide sequence of the unmethylated primer is as follows:

[0167] R5: 5'-AATAATATACACATCAATACCACAACCCA-3' (SEQ ID No. 14).

[0168] Using the aforementioned methylation primers (F4 and R4), MSP amplification was performed using sulfur-modified DNA as a template. If the PRSS3 gene was methylated, a 152 bp fragment was obtained; if the PRSS3 gene was not methylated, no amplification product was produced. Therefore, this invention refers to these primers as methylation primers. The methylation primers have an upstream primer with a Tm of 65.42 and a GC content of 60%, and a downstream primer with a Tm of 64.96 and a GC content of 48.15%.

[0169] Using these unmethylated primers (F5 and R5), MSP amplification was performed on sulfur-modified DNA as a template. If the PRSS3 gene was not methylated, a 155 bp fragment was obtained; if the PRSS3 gene was methylated, no amplification product was produced. Therefore, this invention refers to these primers as unmethylated primers. The unmethylated primers have a Tm of 62.78 and a GC content of 50% for the upstream primer, and a Tm of 59.32 and a GC content of 31.03% for the downstream primer.

[0170] The result interpretation method is as follows (taking primer set 4 as an example):

[0171] The first method is to determine whether the desired product is obtained:

[0172] Amplification with methylated primers yielded a 152bp amplification product, while amplification with unmethylated primers yielded no 155bp amplification product, which was interpreted as "hypermethylation" (relatively high methylation level). Amplification with both methylated and unmethylated primers yielded a 152bp amplification product, which was interpreted as "partial methylation" (relatively low methylation level).

[0173] Amplification with unmethylated primers yielded a 155bp amplification product, while amplification with methylated primers did not produce a 155bp amplification product. This sample was interpreted as "unmethylated".

[0174] The second method involves determining the result using methylation-specific quantitative PCR (MS-qPCR).

[0175] Through 2 -ΔΔCt Methods: (Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) ​​for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002 May 1; 30(9):e36.). Ct values ​​obtained by amplification using methylated primer pairs and Ct values ​​obtained by amplification using unmethylated primer pairs were homogenized and presented as folds of the Ct values ​​amplified by β-actin.

[0176] 2. Methylation detection

[0177] Let's take "primer set 4" in step 1 as an example for explanation.

[0178] Test cells: A549, 801-D, NCI-H1299, NCI-H460

[0179] All of the above-mentioned cells can be purchased from the National Biomedical Experimental Cell Resource Bank and cultured and passaged under normal conditions in the applicant's laboratory.

[0180] Test samples: Surgical samples from 13 patients with lung adenocarcinoma

[0181] The basic information of the lung adenocarcinoma patients (n=13) involved in this embodiment is shown in Table 6.

[0182] Table 6 Basic Information of Lung Adenocarcinoma Patients (n=13)

[0183]

[0184]

[0185] 2-1. Cell DNA Extraction Process

[0186] Refer to step 2-1 of Example 2.

[0187] 2-2. The positive and negative control samples for DNA methylation were Human Methylated & Non-methylated DNA Set (catalog number: D5014) purchased from Zymo Research, USA. Human Methylated DNA is also known as in vitro methylated DNA (IVD).

[0188] 2-3. Sulfidation of cell DNA samples

[0189] (1) Take 1 μg of genomic DNA (sample to be tested, DNA methylation positive control and negative control sample) and add it to a 0.2 ml centrifuge tube, and dilute it to 20 μl with ddH2O. Perform the operation using the EZ DNA Methylation Gold Kit of Zymo Research, USA (catalog number: D5006).

[0190] (2) Add 130 μl of CT solution to 20 μl of DNA sample in a centrifuge tube, place the sample in a PCR instrument, 98℃ for 10 min, 64℃ for 2.5 h, and pre-cool at 4℃ before transferring the sample into an adsorption column.

[0191] (3) Add 600 μl Binding Buffer and mix by inverting. Centrifuge at 12000 rpm for 30 s and discard the waste liquid.

[0192] (4) Add 100 μl Wash Buffer, centrifuge at 12000 rpm for 30 s, and discard the waste liquid.

[0193] (5) Add 200 μl of Desulphonation Buffer, incubate at room temperature for 20 min, centrifuge at 12000 rpm for 30 s, and discard the waste liquid. Add 200 μl of Wash Buffer, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and repeat twice.

[0194] (6) Elute the DNA with 10 μl of Elution Buffer and store at -20°C.

[0195] 2-4. MS-qPCR detection

[0196] Each PCR reaction includes a positive control, a negative control, and a template-free (water) control.

[0197] (1) MS-qPCR detection primer sequence: primer set 4

[0198] (2) qPCR amplification

[0199] The reaction system used for amplification is shown in Table 7.

[0200] Table 7. Reaction system for MS-qPCR amplification

[0201]

[0202]

[0203] Note: 2×SYBR-Green: Thermo Fisher Scientific, USA, part number 4334973.

[0204] The PCR reaction conditions were as follows: 50℃ for 2 min, 95℃ for 10 min, 95℃ for 15 s; 60℃ for 1 min; 40 cycles, fluorescence collected; 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s, 60℃ for 15 s, melting curve plotted. Experimental results are presented using 2... -ΔΔCt The analytical data (see step 1 of Example 3 for details) are analyzed, and the dissolution curve ensures the specificity of the product.

[0205] 2-5. Results

[0206] Further analysis in the M2 region revealed significant changes in the M4 region. MSP-qPCR showed that A549 and 801-D cells with high PRSS3 expression in primer group 4 were unmethylated, NCI-H1299 cells with low PRSS3 expression were partially methylated, and NCI-H460 cells with low PRSS3 expression were highly methylated. Figure 2 B). Consistent with this, methylation results of lung adenocarcinoma tissue (n=13) also showed that the primer set 4 region was highly methylated, with a methylation rate of 15.38% in normal tissue and 46.15% in tumor tissue. Figure 2 C). Methylation (%) = Number of “highly methylated” and “partially methylated” cases / Total number of cases × 100%; Non-methylation (%) = Number of “unmethylated” cases / Total number of cases × 100%.

[0207] Example 4: DNA methylation inhibitors can reverse the silencing of PRSS3 expression in non-small cell lung cancer cells.

[0208] 1. Cell RNA extraction and reverse transcription process

[0209] (1) DNA methyltransferase inhibitor 5-Aza-2′-Deoxycytidine (5-Aza-CR, 5-aza-2′-deoxycytidine, Sigma-Aldrich, USA, catalog number: 189825) addition: When the cell growth density (A549, 801-D, NCI-H41299 and NCI-H460, sourced from the National Biomedical Experimental Cell Resource Bank) reached 30%, 5 μM (final concentration) of 5-Aza was added for treatment. The culture medium was changed every 24 h, and 5-Aza (final concentration 5 μM) was added again for a total of 96 hours.

[0210] (2) Select cells in good growth condition, and administer at 1 mL / 10 6 Add Trizol reagent (Invitrogen, USA, catalog number: 15596026) to each cell. Incubate at room temperature for 10 min to allow for complete lysis, then extract with chloroform. Add 0.2 mL of chloroform to each 1 mL of Trizol.

[0211] (3) Shake violently for 15 seconds, place at room temperature for 5 minutes, centrifuge at 12000g and 4℃ for 15 minutes.

[0212] (4) Transfer the colorless upper layer of the separated liquid to a new centrifuge tube, precipitate it with pre-cooled isopropanol (add 0.5 mL of isopropanol per 1 mL of Trizol), and place on ice for 20 min. Centrifuge at 12000 g, 4 °C for 10 min, and discard the supernatant.

[0213] (5) Wash the precipitate with pre-cooled 75% ethanol, add 1 mL of 75% ethanol to 1 mL of Trizol, centrifuge at 7500 g, 4 °C for 5 min, and discard the supernatant. After drying, dissolve in an appropriate amount of DEPC-H2O, confirm by 0.8% agarose gel electrophoresis, determine the RNA concentration by NanoDrop, and store at -80 °C.

[0214] (6) Take 1.0 μg of RNA and reverse transcribe it into cDNA. Use the TransScript II First-Strand cDNA Synthesis SuperMix kit (catalog number: AH301-02) produced by Beijing TransGen Biotech Co., Ltd.: Add 1 μL of Anchored Oligo(dT)20, 10 μL of 2×TS Reaction Mix and 1 μL of RT / RI Enzyme Mix to DEPC-H2O to a final volume of 20 μL. Reaction conditions: 42℃ for 30 min, 85℃ for 5 min. The cDNA obtained by reverse transcription is then stored at -20℃.

[0215] 2. qPCR

[0216] (1) qPCR primer sequences:

[0217] Primer pair 1: PRSS3 mRNA common region primer (101 bp in size), a universal primer for PRSS3 designed based on the same exon sequence of the four splice variants of PRSS3.

[0218] Upstream primer (SEQ ID NO.13): 5'-ATTCTGGCTCCCACTTCTGC-3';

[0219] Downstream primer (SEQ ID NO.14): 5'-CTCTCCCAGTCTCACCTGGA-3'.

[0220] Primer pair 2: PRSS3-V1 mRNA primers (NM_007343.4, size 124bp)

[0221] Upstream primer (SEQ ID NO.15): 5'-CTGCGAGGCGCTGGG-3';

[0222] Downstream primer: 5'-GCAGAAGTGGGAGCCAGAAT-3'.

[0223] Primer pair 3: PRSS3-V2 mRNA primers (NM_002771.4, size 129bp)

[0224] Upstream primer (SEQ ID NO.10): 5'-ATCCTTGCCTTTGTGGGAGC-3';

[0225] Downstream primer: 5'-GCAGAAGTGGGAGCCAGAAT-3'.

[0226] Primer pair 4: PRSS3-V3 mRNA primers (NM_001197097.3, size 140bp)

[0227] Upstream primer (SEQ ID NO.3): 5'-GTGCGCCATTGGTTTTCCAT-3';

[0228] Downstream primer (SEQ ID NO.4): 5'-GCAGAAGTGGGAGCCAGAAT-3'.

[0229] Primer pair 5: PRSS3-V4 mRNA primers (NM_001197098.1, size 131bp)

[0230] Upstream primer (SEQ ID NO.11): 5'-CGACTCGCATGGGACCTG-3';

[0231] Downstream primer: 5'-GCAGAAGTGGGAGCCAGAAT-3'.

[0232] The internal reference primer used for homogenization is a primer with Beta-Aactin as the internal reference.

[0233] Upstream primer (SEQ ID NO.12): 5'-TTAGTTGCGTTACACCCTTTC-3';

[0234] Downstream primer (SEQ ID NO.13): 5'-ACCTTCACCGTTCCAGTTT-3'.

[0235] (2) Reaction system for qPCR amplification

[0236] As shown in Table 8.

[0237] Table 8. Reaction system for qPCR amplification

[0238]

[0239]

[0240] Note: 2×SYBR-Green: Zymo Research, USA, product number E2004.

[0241] (3) The PCR reaction conditions are as follows:

[0242] The fluorescence was collected after 40 cycles of 50℃ for 2 min, 95℃ for 10 min, 95℃ for 15 s, and 60℃ for 1 min. A melting curve was then constructed using the same cycle. The experimental results were analyzed using 2... -ΔΔCt The analytical data and melting curves ensure the specificity of the product.

[0243] 3. MassARRAY

[0244] Refer to Example 2.

[0245] 4. Results

[0246] The use of the epigenetic drug DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza, 5-Aza-CR) can reverse the silencing of PRSS3 gene expression in non-small cell lung cancer (NSCLC) cells, indicating that the silencing of PRSS3 gene expression in NSCLC tissues is epigenetically regulated. Experiments showed that the methyltransferase inhibitor 5-Aza-CR significantly upregulated PRSS3 expression in NSCLC cells with low PRSS3 expression, while downregulated PRSS3 expression in NSCLC cells with high PRSS3 expression. Figure 2 D). Furthermore, 5-Aza-CR effectively upregulated PRSS3 splicing variants 1 and 3 and downregulated PRSS3 splicing variant 2 expression in lung cancer cells. Figure 2 E). Furthermore, MassARRAY confirmed the demethylation effect of 5-Aza-CR on A549 and NCI-H460 cells (E). Figure 2 F).

[0247] Example 5: Sensitivity and specificity of the methylated primers (F4 and R4) and unmethylated primers (F5 and R5) of the present invention in differentiating between squamous cell carcinoma and adenocarcinoma of the lung.

[0248] This embodiment utilizes methylated primers (F4 and R4) and unmethylated primers (F5 and R5) to detect the methylation level of the PRSS3 gene promoter in human pneumonia patient specimens (n=15), human lung adenocarcinoma patient specimens (n=25), and human lung squamous cell carcinoma patient specimens (n=14) derived from human bronchoalveolar lavage fluid. The steps include: (1) extracting DNA from the sample to be tested; (2) treating the DNA with bisulfite to obtain sulfurized DNA; (3) using the sulfurized DNA as a template, performing PCR amplification using the primer pairs designed in this invention to obtain PCR amplification products; (4) detecting the methylation of the PCR amplification products; (5) extracting RNA from the sample to be tested; and (6) performing RT-qPCR amplification.

[0249] The basic patient information involved in this embodiment is shown in Table 9.

[0250] Table 9 Patient Information List

[0251]

[0252]

[0253]

[0254] The result interpretation method is as follows:

[0255] The determination was made by amplification using methylation-specific quantitative PCR (MS-qPCR):

[0256] Through 2 -ΔΔCt Methods: (Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) ​​for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002 May 1; 30(9):e36.). Ct values ​​obtained by amplification using methylated primer pairs and Ct values ​​obtained by amplification using unmethylated primer pairs were homogenized and presented as methylation percentage (methylation percentage = (methylated / methylated + unmethylated) × 100%). If the methylation percentage ≥ 60%, it is considered "hypermethylated"; if the methylation percentage is 20-60% (excluding endpoints), it is considered "partially methylated"; if the methylation percentage is ≤ 20%, it is considered "unmethylated".

[0257] Person coefficient and Spearman coefficient for different CpG sites Figure 1 D) Analyze the correlation between mRNA expression and different splicing variants of PRSS3. The closer the correlation coefficient is to 1 or -1, the stronger the correlation; the closer the correlation coefficient is to 0, the weaker the correlation.

[0258] Specific steps: Refer to Examples 3 and 4

[0259] The results are shown in Table 10:

[0260] Table 10 Results of differentiating between squamous cell carcinoma and adenocarcinoma of the lung.

[0261]

[0262]

[0263] The expression of PRSS3 splice variants and the methylation status of the gene somatic cells were analyzed in 54 lavage fluid samples (BALF, including 15 patients with chronic pneumonia, 25 patients with lung adenocarcinoma, and 14 patients with lung squamous cell carcinoma) using RT-qPCR and MS-qPCR methods. PRSS3-V1 expression was significantly increased in lung squamous cell carcinoma, PRSS3-V2 expression was significantly increased in lung adenocarcinoma, while PRSS3-V3 expression was significantly decreased in both lung squamous cell carcinoma and lung adenocarcinoma tissues. Figure 3A). The methylation level of the PRSS3 genotype was further determined in BALF samples, with PRSS3 iCpGI hypomethylation observed in 100% of chronic pneumonia (15 / 15) and squamous cell carcinoma of the lung (14 / 14), and iCpGI hypermethylation in 72% (18 / 25) of lung adenocarcinoma. Figure 3 (B and Table 10).

[0264] The BALF samples showed differential correlations between gene somatic methylation and the expression of various PRSS3 splice variants. Figure 3 C), but Spearman correlation analysis showed that, compared with BALF samples from patients with chronic pneumonia, genotype methylation was significantly associated with the expression of PRSS3-V1 and V3 in BALF samples from patients with squamous cell carcinoma and adenocarcinoma of the lung. Figure 3 D). In BALF samples from lung adenocarcinoma patients, hypermethylation of the gene body was more significantly associated with low expression of PRSS3-V3. Figure 3 The results (C and 3D) confirmed the epigenetic silencing of PRSS3-V3 in lung adenocarcinoma, suggesting that methylation of PRSS3 in this region may be a diagnostic marker for lung adenocarcinoma.

[0265] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. SEQUENCE LISTING <110> Beijing Chest Hospital, Capital Medical University Beijing Tuberculosis and Thoracic Tumor Research Institute <120> A molecular biomarker and reagent kit for non-invasive early diagnosis and efficacy evaluation of lung adenocarcinoma. <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 432 <212> DNA <213> Artificial sequence <400> 1 atctccaagg gaaggtcaag ggcctggagg atgtgcggac ctgacgacag atgccccgca 60 cgctggccgg gaccgggaag ggcggtcaag tgtggaaagg gtctggcggc tgccaggcct 120 ggcagagtgg agcggggcgg ggcgcagcgg ggcggggcgg gcctggagct gcacccgctt 180 ctgggtggac gcacttggcg agcggcgcgg gatgcagacg gctgcgaggc gctgggcaca 240 ggtcagacgt cagtacccgc agggggcttg aaactggagg agggctcgaa gggagaggga 300 gccccgccaa ggagcggggc tgtgatggag agggggttcc gactcgcatg ggacctgcgg 360 gggagggtac gcggacaggg aggggatacc gactgggagg ggctcaggga cagggatgga 420 ggctcctcta gg 432 <210> 2 <211> 413 <212> DNA <213> Artificial sequence <400> 2 ggacagggat ggaggctcct ctaggggagg acgggagggg atggagggcc ctggtgtcgc 60 agaagcccac ctggggcccc ctccgggctg cggcaccgat gcgcacacta ctcccaccgc 120 ccccgagtgc ctatgtccgg ctggccgcgg ccctggaatg aatattgctc agtcccccgc 180 gagtcaggtc tgccgcgttg cagggtgagg ggaaggtgtg aagccccggg cctccgtctg 240 ccccgtgagt ccgggaacgc gcgcccccgt ggatgccacc tggcccctga gctgtgtcca 300 gtcacagctc acatagctct gggcactggt accccgactg cctttccttg ttagctgcga 360 tacacaaata catgagccag atcctttcct gaggccagga agcctggaat cta 413 <210> 3 <211> 481 <212> DNA <213> Artificial sequence <400> 3 ttaaaaggac tcctagacac ccaggccatt ctccctcctt ctaaccctca tctcctcggc 60 ctacgaaagc ccagctgtgt gatccgggga ttggtttccc ctgtcttccc catcccagca 120 tccttccagg aaatagccag tctccctctc tgctctcaga aggcaagttt ccttatcacc 180 tgtgaatcac aaacccacag agtggccaaa cgtagccgag ctgatgcaag accctggcaa 240 ggggaaagct gcaggtgtgt ttgtgctggg aagaactgtg accctcacct cacagacacc 300 tcctctcctg attcttggaa gggtataagg acaggccttc caccaccagt caggcacact 360 ctaccaccat gaatccattc ctgatccttg cctttgtggg agctgctggc gagtttcatg 420 acctgcctca ggccccaccc accccctttc ctggcagaca catgccctgc cattcttgcc 480 a 481 <210> 4 <211> 155 <212> DNA <213> Artificial sequence <400> 4 gggtacgcgg acagggaggg gataccgact gggaggggct cagggacagg gatggaggct 60 cctctagggg aggacgggag gggatggagg gccctggtgt cgcagaagcc cacctggggc 120 cccctccggg ctgcggcacc gatgcgcaca ctact 155 <210> 5 <211> 35 <212> DNA <213> Artificial sequence <400> 5 aggaagagag atttttaagg gaaggttaag ggttt 35 <210> 6 <211> 55 <212> DNA <213> Artificial sequence <400> 6 cagtaatacg actcactata gggagaaggc tcctaaaaaa acctccatcc ctatc 55 <210> 7 <211> 34 <212> DNA <213> Artificial sequence <400> 7 aggaagagag ggataggggat ggaggttttt ttag 34 <210> 8 <211> 56 <212> DNA <213> Artificial sequence <400> 8 cagtaatacg actcactata gggagaaggc ttaaattcca aacttcctaa cctcaa 56 <210> 9 <211> 35 <212> DNA <213> Artificial sequence <400> 9 aggaagagag tggtaagaat ggtagggtat gtgtt 35 <210> 10 <211> 56 <212> DNA <213> Artificial sequence <400> 10 cagtaatacg actcactata gggagaaggc tttaaaaaaa ctcctaaaca cccaaa 56 <210> 11 <211> 25 <212> DNA <213> Artificial sequence <400> 11 ggtacgcgga tagggagggg atatc 25 <210> 12 <211> 27 <212> DNA <213> Artificial sequence <400> 12 taatatacgc atcgataccg caacccg 27 <210> 13 <211> 26 <212> DNA <213> Artificial sequence <400> 13 gggtatgtgg atagggaggg gatatt 26 <210> 14 <211> 29 <212> DNA <213> Artificial sequence <400> 14 aataatatac acatcaatac cacaaccca 29

Claims

1. The application of a substance for detecting the methylation level of molecular markers in the preparation of diagnostic products for differentiating between squamous cell carcinoma and adenocarcinoma of the lung, characterized in that, The molecular marker is a DNA molecule with the nucleotide sequence SEQ ID No. 4; the substance is a primer pair for detecting the methylation level of the molecular marker, and the primer pair is A1) and A2) as described below. A1) A primer pair consisting of primer F4 and primer R4; wherein primer F4 is the single-stranded DNA molecule shown in SEQ ID No. 11; and primer R4 is the single-stranded DNA molecule shown in SEQ ID No.

12. A2) A primer pair consisting of primer F5 and primer R5; wherein primer F5 is a single-stranded DNA molecule as shown in SEQ ID No. 13; and primer R5 is a single-stranded DNA molecule as shown in SEQ ID No.

14.

2. Any of the following applications of the primer pair described in claim 1: B1) Use of the primer pair described in claim 1 in the preparation of products for screening for squamous cell carcinoma and adenocarcinoma of the lung; B2) Use of the primer pair described in claim 1 in the preparation of a product for differentiating between squamous cell carcinoma and adenocarcinoma of the lung; B3) Use of the primer pair described in claim 1 in the preparation of products for the diagnosis of squamous cell carcinoma and adenocarcinoma of the lung.

3. The application according to claim 2, characterized in that, The product is a reagent or kit.

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