Application of lncRNA biomarkers in the diagnosis of esophageal squamous cell carcinoma

Abstract

The present invention discloses the application of lncRNA biomarkers in the diagnosis of esophageal squamous cell carcinoma, which relates to the technical field of esophageal squamous cell carcinoma diagnosis. Esophageal squamous cell carcinoma markers include LINC00482. The lncRNA markers screened by the present invention have the technical advantages of high sensitivity and strong specificity in the diagnosis of esophageal squamous cell carcinoma, and have good diagnostic characteristics. They are of great significance for early diagnosis, early intervention, and in-depth understanding of the pathological mechanism of esophageal squamous cell carcinoma. In addition, the biomarker-assisted diagnosis or diagnosis method of esophageal squamous cell carcinoma proposed by the present invention has the technical advantages of simple operation, low price, and short detection cycle, and can be used for the detection needs of various samples such as blood and tissue.

Description

Technical Field

[0001] The present invention relates to the technical field of esophageal squamous cell carcinoma diagnosis, and in particular to the application of lncRNA biomarkers in the diagnosis of esophageal squamous cell carcinoma. Background Art

[0002] Esophageal squamous cell carcinoma (ESCC) is a highly aggressive and lethal digestive tract malignancy that originates from human esophageal epithelial cells. Despite ongoing efforts by clinicians and researchers to improve outcomes, the incidence of this deadly malignancy continues to rise rapidly, with an overall 5-year survival rate remaining below 25%. Therefore, early diagnosis and prompt treatment are crucial for improving the prognosis of ESCC. However, due to the lack of obvious symptoms in the early stages of the disease, many patients are diagnosed at a late stage. Although noninvasive tests for cancer-related biomarkers (such as alpha-fetoprotein and prostate-specific antigen) have long been widely used as complementary diagnostic methods for specific cancer types, no biomarker with ideal sensitivity and specificity is currently available for ESCC. Although the use of targeted cancer therapy has become an unstoppable trend, it is still in its infancy, particularly in the treatment of ESCC. Therefore, sensitive and specific biomarkers, as well as candidate therapeutic targets, are urgently needed for the clinical management of ESCC.

[0003] Long noncoding RNAs (lncRNAs) are a group of RNA transcripts exceeding 200 nucleotides in length that are implicated in a variety of physiological and pathological processes, including carcinogenesis. When they were first discovered, the conventional hypothesis was that these transcripts were largely "transcriptional noise," unable to encode and translate proteins. In reality, while most lncRNAs lack open reading frames and do not direct protein synthesis, they nonetheless play crucial roles in complex gene regulatory networks, including as molecular signals, decoys, guides, and scaffolds. These regulatory actions occur at the epigenetic, transcriptional, and post-transcriptional levels, leading to alterations in cellular state and function. It is estimated that lncRNAs far outnumber mRNAs and other noncoding RNAs, such as microRNAs. LncRNAs are not rare molecules; rather, they represent the largest class of transcripts in mammals. Unfortunately, most of their regulatory mechanisms remain incompletely understood. Accumulating evidence indicates that aberrant lncRNA expression contributes to the development and progression of human cancers. For example, HOTAIR is upregulated in breast cancer cells and promotes metastasis by epigenetically silencing target genes in the HoxD cluster. In contrast, some lncRNAs act as protective measures against malignant transformation of normal cells. Notably, some lncRNAs not only exhibit cell / tumor-specific expression but also are stably present in the blood and have the potential to serve as key predictive biomarkers for cancer development, metastasis, treatment response, recurrence, and survival.

[0004] Currently, there is no set of diagnostic biomarkers with ideal sensitivity and specificity for esophageal squamous cell carcinoma. Therefore, it is of great significance to explore the molecular characteristics of lncRNA in esophageal squamous cell carcinoma and find lncRNA biomarkers that can be used for early diagnosis of esophageal squamous cell carcinoma.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The present invention aims to provide an application of lncRNA biomarkers in the diagnosis of esophageal squamous cell carcinoma to address the deficiencies of existing biomarker applications for esophageal squamous cell carcinoma diagnosis. Existing technologies for diagnosing esophageal squamous cell carcinoma have the following deficiencies: (1) Imaging signs of esophageal squamous cell carcinoma are limited by tumor size and lack high sensitivity and specificity; (2) Serum tumor markers such as squamous cell carcinoma antigen and carcinoembryonic antigen have low specificity and accuracy; and (3) Histopathology, the "gold standard" for tumor diagnosis, is an invasive examination that causes certain damage to the body, and pathological biopsy still has limitations due to tumor heterogeneity.

[0007] The present invention is achieved in that:

[0008] The present invention provides a reagent for detecting esophageal squamous cell carcinoma markers for use in preparing esophageal squamous cell carcinoma auxiliary diagnosis or diagnostic products or esophageal squamous cell carcinoma efficacy monitoring products, wherein the esophageal squamous cell carcinoma lncRNA marker is selected from any one of (1)-(2):

[0009] (1) LINC00482;

[0010] (2) LINC00482, and at least one marker selected from the group consisting of XIST, TMLHE-AS1, AP001437.2, AC087672.2, AL353608.2, and AL445588.1.

[0011] The present invention has the following beneficial effects:

[0012] The lncRNA markers screened in this study have the technical advantages of high sensitivity and specificity for diagnosing esophageal squamous cell carcinoma, and possess excellent diagnostic properties, which are of great significance for early diagnosis, early intervention, and a deeper understanding of the pathological mechanisms of esophageal squamous cell carcinoma. Furthermore, the biomarker-assisted diagnosis or diagnosis of esophageal squamous cell carcinoma proposed in this study has the technical advantages of being simple to use, low-cost, and having a short detection cycle, and can be applied to the testing needs of a variety of samples, including blood and tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0014] Figure 1 The present invention uses linear regression analysis to construct a diagnostic model for esophageal squamous cell carcinoma, and the optimal λ value is obtained after 10 cross-validations;

[0015] Figure 2 The box plots show the expression levels of the seven biomarkers XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2 and AL445588.1 in the blood of esophageal squamous cell carcinoma patients (case) and normal subjects (control);

[0016] Figure 3 Schematic diagram of the ROC curves of the seven biomarkers of the present invention, XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2 and AL445588.1, for the diagnosis of esophageal squamous cell carcinoma;

[0017] Figure 4 Schematic diagram of the ROC curves for the diagnostic performance analysis of esophageal squamous cell carcinoma using seven biomarkers (XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2, and AL445588.1) alone;

[0018] Figure 5 The results of the diagnostic performance analysis of the pairwise combinations of XIST and LINC00482; XIST and TMLHE-AS1; XIST and AP001437.2; XIST and AL353608.2; XIST and AC087672.2; and XIST and AL445588.1 for esophageal squamous cell carcinoma are shown;

[0019] Figure 6 Diagnostic performance analysis results of pairwise combinations of LINC00482 and TMLHE-AS1; LINC00482 and AP001437.2; LINC00482 and AL353608.2; LINC00482 and AC087672.2; LINC00482 and AL445588.1; and AC087672.2 and AL445588.1 for esophageal squamous cell carcinoma;

[0020] Figure 7 Diagram showing the diagnostic performance analysis results of pairwise combinations of TMLHE-AS1 and AP001437.2; TMLHE-AS1 and AL353608.2; TMLHE-AS1 and AC087672.2; TMLHE-AS1 and AL445588.1; AP001437.2 and AL353608.2; AP001437.2 and AC087672.2; AP001437.2 and AL445588.1; AL353608.2 and AC087672.2; AL353608.2 and AL445588.1;

[0021] Figure 8Diagram showing the diagnostic performance analysis results of three-way combinations of markers for esophageal squamous cell carcinoma: XIST, LINC00482 and TMLHE-AS1; XIST, LINC00482 and AP001437.2; XIST, LINC00482 and AL353608.2; XIST, LINC00482 and AC087672.2; XIST, LINC00482 and AL445588.1; LINC00482, AC087672.2 and TMLHE-AS1; LINC00482, AC087672.2 and AP001437.2; LINC00482, AC087672.2 and AL353608.2; and LINC00482, AC087672.2 and AL445588.1;

[0022] Figure 9 Diagram showing the diagnostic performance analysis results of the four-by-four combinations of XIST, LINC00482, AP001437.2, and AC087672.2; TMLHE-AS1, LINC00482, AP001437.2, and AC087672.2; XIST, LINC00482, TMLHE-AS1, and AP001437.2; and XIST, LINC00482, TMLHE-AS1, and AL353608.2 for esophageal squamous cell carcinoma.

[0023] Figure 10 Diagram showing the diagnostic performance analysis results of five-to-five combinations of markers for esophageal squamous cell carcinoma: XIST, LINC00482, TMLHE-AS1, AP001437.2, and AC087672.2; LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, and AC087672.2; XIST, LINC00482, TMLHE-AS1, AP001437.2, and AL353608.2; and XIST, LINC00482, TMLHE-AS1, AP001437.2, and AL445588.1.

[0024] Figure 11 This is the analysis result of the diagnostic performance of the combination of six markers, including XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, and AC087672.2, for esophageal squamous cell carcinoma. DETAILED DESCRIPTION

[0025] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0026] The term "biomarker" or "marker" broadly refers to any detectable compound or cell present in or derived from a sample, such as a distinguishing molecule or distinguishing fragment of a protein, peptide, proteoglycan, glycoprotein, lipoprotein, cell, or any of the above. For example, detection of a specific antibody or binding to a specific antibody can indicate the presence of a specific antigen (e.g., protein) in a sample. Here, a distinguishing molecule or fragment is a molecule or fragment that indicates the presence or abundance of the compound or cell identified above when detected. Biomarkers can, for example, be separated from a sample, measured directly in a sample, or detected or determined in a sample. Biomarkers can, for example, be functional, partially functional, or non-functional.

[0027] Specifically, a tumor marker is a biomarker found in samples such as serum, urine, and tissue from cancer patients. Its presence indicates the possible presence of tumor cells. Tumor markers are used to detect these markers for early diagnosis, disease progression monitoring, and treatment efficacy assessment. Because the presence and concentration of tumor markers are related to factors such as tumor type, location, size, and malignancy, tumor marker detection allows for early diagnosis and disease monitoring, improving the success rate of cancer treatment and patient survival. Cell surface markers are substances such as proteins and carbohydrates found on the surface of tumor cells. Changes in these markers can reflect the growth status and characteristics of tumor cells and are the most common type of tumor marker. Intracellular markers are substances such as proteins and RNA found within tumor cells.

[0028] The term "sample" refers to a biological specimen obtained or derived from an individual of interest. The source of a biological specimen may include fresh, frozen, and / or preserved organ or tissue samples, solid tissues derived from biopsies or priming, blood, or any blood fraction. The term "sample" includes biological specimens that have been manipulated in any way after their acquisition, such as by treatment with reagents, stabilization, enrichment for specific components (e.g., proteins or polynucleotides), or embedding in a semisolid or solid matrix for sectioning.

[0029] In a first aspect, the present invention provides a reagent for detecting an esophageal squamous cell carcinoma marker for use in preparing an auxiliary diagnosis or diagnostic product for esophageal squamous cell carcinoma or a product for monitoring the efficacy of esophageal squamous cell carcinoma, wherein the esophageal squamous cell carcinoma lncRNA marker is selected from any one of (1) to (2):

[0030] (1) LINC00482;

[0031] (2) LINC00482, and at least one marker selected from the group consisting of XIST, TMLHE-AS1, AP001437.2, AC087672.2, AL353608.2, and AL445588.1.

[0032] The present invention conducted a comprehensive transcriptomic analysis of whole blood samples from 80 patients with esophageal squamous cell carcinoma and 40 normal subjects, characterized the differences in lncRNA expression in the whole blood of patients and normal subjects, and identified a group of potential esophageal squamous cell carcinoma biomarkers through linear regression analysis.

[0033] The combined diagnosis of the above 7 markers has an extremely high AUC value and high diagnostic specificity and sensitivity.

[0034] In other embodiments, those skilled in the art may use at least one of the above-mentioned eight markers for auxiliary diagnosis or diagnosis of esophageal squamous cell carcinoma, or for monitoring the efficacy of esophageal squamous cell carcinoma, for example, any one, any two, any three, any four, any five, any six or any seven of the above-mentioned seven markers.

[0035] In a preferred embodiment of the present invention, the lncRNA marker for esophageal squamous cell carcinoma is a combination of any two of the following markers:

[0036] XIST and LINC00482; LINC00482 and TMLHE-AS1; LINC00482 and AP001437.2; LINC00482 and AL353608.2; LINC00482 and AC087672.2; LINC00482 and AL445588.1.

[0037] Among them, the marker combination of XIST and LINC00482 had a relatively higher AUC value, with a confidence interval CI of 0.795-0.968. The marker combination of LINC00482 and AC087672.2 had a relatively higher AUC value (0.950), with a confidence interval CI of 0.893-1.000. The marker combination of AL353608.2 and AC087672.2 had a relatively higher AUC value (0.935), with a confidence interval CI of 0.868-1.000.

[0038] In a preferred embodiment of the present invention, the lncRNA marker for esophageal squamous cell carcinoma is a combination of any of the following three markers:

[0039] XIST, LINC00482 and TMLHE-AS1; XIST, LINC00482 and AP001437.2; XIST, LINC00482 and AL353608.2; XIST, LINC00482 and AC087672.2; XIST, LINC00482 and AL445588.1; LINC00482, AC087672.2 and TMLHE-AS1; LINC00482, AC087672.2 and AP001437.2; LINC00482, AC087672.2 and AL353608.2; LINC00482, AC087672.2 and AL445588.1.

[0040] Among them, the marker combination of LINC00482, AC087672.2 and AP001437.2 had a relatively higher AUC value (0.955) with a confidence interval CI of 0.904-1.000.

[0041] In a preferred embodiment of the present invention, the lncRNA marker for esophageal squamous cell carcinoma is a combination of any of the following markers:

[0042] XIST, LINC00482, AP001437.2, and AC087672.2; TMLHE-AS1, LINC00482, AP001437.2, and AC087672.2; XIST, LINC00482, TMLHE-AS1, and AP001437.2; XIST, LINC00482, TMLHE-AS1, and AL353608.2; XIST, LINC00482, TMLHE-AS1, AP001437.2, and AC087672.2; LINC00482 C00482, TMLHE-AS1, AP001437.2, AL353608.2, and AC087672.2; XIST, LINC00482, TMLHE-AS1, AP001437.2, and AL353608.2; XIST, LINC00482, TMLHE-AS1, AP001437.2, and AL445588.1; XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2.

[0043] Among them, the marker combination of TMLHE-AS1, LINC00482, AP001437.2, and AC087672.2 had a relatively higher AUC value (0.958) with a confidence interval of 0.908-1.000. The marker combinations of XIST, LINC00482, TMLHE-AS1, AP001437.2, and AC087672.2; and LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, and AC087672.2 had a relatively higher AUC value (0.958) with a confidence interval of 0.908-1.000. The results of the diagnostic performance analysis of the combination of six markers, XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, and AC087672.2, for esophageal squamous cell carcinoma show that the combination of these six markers has a relatively higher AUC value (0.958) with a confidence interval CI of 0.908-1.000.

[0044] In a preferred embodiment of the present invention, the lncRNA markers for esophageal squamous cell carcinoma are XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2, and AL445588.1.

[0045] In a preferred embodiment of the present invention, the reagent for detecting the esophageal squamous cell carcinoma lncRNA marker is a reagent for detecting the level of DNA, RNA, protein or cells carrying the esophageal squamous cell carcinoma lncRNA marker.

[0046] In a preferred embodiment of the present invention, the reagent for detecting esophageal squamous cell carcinoma lncRNA markers is a reagent for detecting the RNA level of esophageal squamous cell carcinoma lncRNA markers in a sample by real-time fluorescence quantitative reverse transcription polymerase chain reaction and / or nucleic acid mass spectrometry.

[0047] In a preferred embodiment of the present invention, the reagent for detecting the lncRNA marker for esophageal squamous cell carcinoma is a reagent for detecting the protein level of the lncRNA marker for esophageal squamous cell carcinoma in a sample by ELISA, Western blotting and / or Elisopt method, or a reagent for detecting the level of cells carrying the lncRNA marker for esophageal squamous cell carcinoma in a sample by TUNEL detection and / or flow cytometry.

[0048] In a preferred embodiment of the present invention, the product is selected from a reagent, a kit or a chip.

[0049] A chip, also known as a suspension array or liquid array, includes a carrier and nucleic acid molecules (such as primers and / or probes) and / or antibodies bound to the carrier surface.

[0050] The chip is, for example, a microfluidic chip, which is selected from a PDMS chip or a metal droplet generator of a T-shaped chip, a flow focusing chip or a coaxial flow chip, or a PMMA microfluidic chip.

[0051] In a preferred embodiment of the present invention, the product includes probes and / or primers for detecting lncRNA markers of esophageal squamous cell carcinoma.

[0052] In a preferred embodiment of the present invention, the product further comprises a reverse transcriptase. In one embodiment, the reverse transcriptase is present in the form of a reverse transcriptase mix. In a preferred embodiment of the present invention, the product further comprises a DNA polymerase, such as selected from a hot start DNA polymerase, such as Tth DNA polymerase, Taq DNA polymerase, etc.

[0053] The auxiliary diagnosis or diagnostic product (such as a kit) provided by the present invention may optionally include any reagents and / or consumables acceptable in the art for PCR reaction or for preparing a PCR reaction system. Specific examples may include, but are not limited to, one or more of dNTPs, salts or salt solutions, negative controls, positive controls, blank controls, calibrators, and PCR reaction vessels.

[0054] Furthermore, the kit includes at least one of a buffer, a detection reagent, a diluent, and a washing solution (or rinse solution), but is not limited thereto. In one embodiment, the kit further includes a color development solution, a stop solution, an RNA extraction reagent, and the like.

[0055] In a preferred embodiment of the present invention, the auxiliary diagnosis or diagnostic use of the auxiliary diagnosis or diagnostic product for esophageal squamous cell carcinoma includes: if the level of the esophageal squamous cell carcinoma lncRNA marker in the test sample is higher than the level of the esophageal squamous cell carcinoma lncRNA marker in the healthy sample, it is judged that the test sample is at risk of esophageal squamous cell carcinoma.

[0056] The efficacy monitoring uses of the esophageal squamous cell carcinoma efficacy monitoring product include: if within one or more esophageal squamous cell carcinoma treatment cycles, the level of the esophageal squamous cell carcinoma lncRNA marker in the late stage of treatment of the test sample is higher than the level of the esophageal squamous cell carcinoma lncRNA marker in the early stage of esophageal squamous cell carcinoma treatment, it is judged that the esophageal squamous cell carcinoma efficacy is poor, the esophageal squamous cell carcinoma may be resistant to the treatment method, and the treatment plan needs to be adjusted.

[0057] In a preferred embodiment of the present invention, the test sample is selected from esophageal cancer surgical tissue specimens, formalin-fixed esophageal cancer tissue or adjacent tissue, paraffin-embedded esophageal cancer tissue or adjacent tissue, blood, serum or plasma.

[0058] In a preferred embodiment of the present invention, the exosomes are derived from serum, plasma or urine.

[0059] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0060] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0061] The following experimental screening identified lncRNA in the blood as a diagnostic marker for esophageal squamous cell carcinoma, which can be used for the differential diagnosis of esophageal squamous cell carcinoma. This experiment was approved by the Ethics Committee of West China Hospital, Sichuan University. All subjects participated voluntarily, were verbally informed of the purpose of the experiment, and signed informed consent.

[0062] Subjects: 80 patients with esophageal squamous cell carcinoma and 40 healthy controls were enrolled at West China Hospital. Detailed sociodemographic information, including gender and age, was recorded. Subjects in each group were matched for age, gender, and education level.

[0063] Sample collection: Whole blood samples were collected from patients and healthy subjects for next-generation sequencing, and bioinformatics analysis was performed on the data.

[0064] Example 1

[0065] Screening lncRNA as a diagnostic marker for esophageal squamous cell carcinoma can be used for the differential diagnosis of esophageal squamous cell carcinoma. The specific steps are as follows:

[0066] Data preprocessing: Trim Galore (version 0.6.0) was used to detect and trim low-quality bases in the raw data. The reference genome GRCh38 was downloaded from the GENCODE database (https: / / www.gencodegenes.org / human / ). Kallisto (version 0.46.2) was used to create an index and calculate transcript expression.

[0067] Construction of an ESCC diagnostic model: The R package DESeq2 (version 1.33.1) was used to analyze differentially expressed lncRNAs in the blood of ESCC patients and healthy controls. A Padj value < 0.05 was considered statistically significant. Linear regression analysis of the differentially expressed lncRNAs was performed using the R packages survival (version 3.1-12) and glmnet (version 4.1-3), and a diagnostic model for ESCC was constructed. Gene expression levels in the blood of ESCC patients and healthy controls were statistically analyzed using the Mann-Whitney U test. A p-value < 0.05 was considered statistically significant. Box plots were generated using the R package ggplot2 (version 3.3.0). All analyses were performed in R (version 4.0.2).

[0068] By observing the abnormal lncRNA molecular characteristics of esophageal squamous cell carcinoma and constructing a diagnostic model for esophageal squamous cell carcinoma, it was found that the esophageal squamous cell carcinoma diagnostic model composed of 7 lncRNAs, including XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AL589863.2, AC245052.7, and AL357497.1, has good diagnostic value and can be used as a biomarker for the diagnosis of esophageal squamous cell carcinoma ( Figure 1 ).

[0069] Compared with normal controls, the expression levels of XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2, and AL445588.1 in the blood of patients with esophageal squamous cell carcinoma were increased ( Figure 2 ).

[0070] Example 2

[0071] Based on the results of Example 1, this example constructs an individual risk detection kit for assessing the risk of esophageal squamous cell carcinoma. The kit consists of an RNA extraction reagent and a reverse transcription reagent, and can be used to construct a detection kit for diagnosing and monitoring esophageal squamous cell carcinoma.

[0072] In this example, 40 samples from esophageal squamous cell carcinoma patients and 20 healthy controls, different from the above samples, were collected for transcriptomic sequencing analysis. Seven biomarkers (XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2, and AL445588.1) were used to analyze the diagnostic performance of esophageal squamous cell carcinoma.

[0073] The ROC curve for the diagnosis of esophageal squamous cell carcinoma showed that the diagnostic AUC value of the seven biomarkers was 0.960, with high specificity and sensitivity, and was applied to predict esophageal squamous cell carcinoma with high accuracy ( Figure 3 ).

[0074] In summary, the screened XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2, and AL445588.1 can be used as diagnostic markers for esophageal squamous cell carcinoma and for the differential diagnosis of esophageal squamous cell carcinoma. They are simple to operate, low in cost, short in detection time, and highly sensitive and specific for the auxiliary differential diagnosis of esophageal squamous cell carcinoma patients, and have important clinical significance.

[0075] Example 3

[0076] In this example, the diagnostic performance of esophageal squamous cell carcinoma was analyzed using seven biomarkers (XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2, and AL445588.1) for the samples of Example 2.

[0077] Results reference Figure 4 As shown in the figure, the results showed that the AUC of the seven biomarkers for diagnosing esophageal squamous cell carcinoma alone was greater than 0.85.

[0078] Example 4

[0079] Compared with Example 3, this example uses a combination of two biomarkers to perform diagnostic performance analysis of esophageal squamous cell carcinoma, and the samples evaluated are the samples of Example 2.

[0080] Figure 5 A, B, C, D, E, and F are the results of the diagnostic performance analysis of esophageal squamous cell carcinoma using pairwise combinations of markers XIST and LINC00482; XIST and TMLHE-AS1; XIST and AP001437.2; XIST and AL353608.2; XIST and AC087672.2; and XIST and AL445588.1, respectively. Figure 5 The results showed that the marker combination of XIST and LINC00482 had a relatively higher AUC value with a confidence interval CI of 0.795-0.968.

[0081] Figure 6A, B, C, D, E, and F are the results of the diagnostic performance analysis of esophageal squamous cell carcinoma using pairwise combinations of markers LINC00482 and TMLHE-AS1; LINC00482 and AP001437.2; LINC00482 and AL353608.2; LINC00482 and AC087672.2; LINC00482 and AL445588.1; and AC087672.2 and AL445588.1, respectively. Figure 6 The results showed that the combination of LINC00482 and AC087672.2 had a relatively higher AUC value (0.950) with a confidence interval (CI) of 0.893-1.000, which was slightly lower than the combination of the seven markers in this application.

[0082] Figure 7 A, B, C, D, E, F, G, H, and I are the results of the analysis of the diagnostic performance of esophageal squamous cell carcinoma using pairwise combinations of TMLHE-AS1 and AP001437.2; TMLHE-AS1 and AL353608.2; TMLHE-AS1 and AC087672.2; TMLHE-AS1 and AL445588.1; AP001437.2 and AL353608.2; AP001437.2 and AC087672.2; AP001437.2 and AL445588.1; AL353608.2 and AC087672.2; and AL353608.2 and AL445588.1 markers. Figure 7 The results showed that the combination of AL353608.2 and AC087672.2 had a relatively higher AUC value (0.935) with a confidence interval of 0.868-1.000, which was slightly lower than the combination of the seven markers in this application.

[0083] Example 5

[0084] Compared with Example 3, this example uses a combination of multiple biomarkers to perform diagnostic performance analysis of esophageal squamous cell carcinoma, and the samples evaluated are the samples of Example 2.

[0085] Figure 8A, B, C, D, E, F, G, H, and I are the results of the analysis of the diagnostic performance of three-three combinations of markers for esophageal squamous cell carcinoma, respectively: XIST, LINC00482 and TMLHE-AS1; XIST, LINC00482 and AP001437.2; XIST, LINC00482 and AL353608.2; XIST, LINC00482 and AC087672.2; XIST, LINC00482 and AL445588.1; LINC00482, AC087672.2 and TMLHE-AS1; LINC00482, AC087672.2 and AP001437.2; LINC00482, AC087672.2 and AL353608.2; and LINC00482, AC087672.2 and AL445588.1. Figure 8 The results showed that the combination of LINC00482, AC087672.2, and AP001437.2 had a relatively higher AUC value (0.955) with a confidence interval (CI) of 0.904-1.000, slightly lower than the combination of the seven markers in this application.

[0086] Figure 9 A, B, C, and D are the diagnostic performance analysis results of the four-four combination of markers for esophageal squamous cell carcinoma, respectively: XIST, LINC00482, AP001437.2, and AC087672.2; TMLHE-AS1, LINC00482, AP001437.2, and AC087672.2; XIST, LINC00482, TMLHE-AS1, and AP001437.2; and XIST, LINC00482, TMLHE-AS1, and AL353608.2. Figure 9 The results showed that the marker combination of TMLHE-AS1, LINC00482, AP001437.2, and AC087672.2 had a relatively higher AUC value (0.958) with a confidence interval of 0.908-1.000, which was slightly lower than the combination of the seven markers in this application.

[0087] Figure 10A, B, C, and D in the figure are the diagnostic performance analysis results of the 55 combination of XIST, LINC00482, TMLHE-AS1, AP001437.2, and AC087672.2; LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, and AC087672.2; XIST, LINC00482, TMLHE-AS1, AP001437.2, and AL353608.2; and XIST, LINC00482, TMLHE-AS1, AP001437.2, and AL445588.1 markers for esophageal squamous cell carcinoma. Figure 10 The results showed that the marker combination of XIST, LINC00482, TMLHE-AS1, AP001437.2, and AC087672.2, and LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, and AC087672.2 had a relatively higher AUC value (0.958) with a confidence interval (CI) of 0.908-1.000, which was slightly lower than the combination of the seven markers in this application.

[0088] Figure 11 This is the analysis result of the diagnostic performance of the combination of six markers, XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, and AC087672.2, for esophageal squamous cell carcinoma. Figure 11 The results showed that the combination of these six markers had a relatively higher AUC value (0.958) with a confidence interval CI of 0.908-1.000.

[0089] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting esophageal squamous cell carcinoma markers in the preparation of an auxiliary diagnosis or diagnostic product for esophageal squamous cell carcinoma, characterized in that: The lncRNA marker for esophageal squamous cell carcinoma is selected from any one of (1)-(2): (1) LINC00482; (2) LINC00482, and at least one selected from the following markers: XIST, TMLHE-AS1, AP001437.2, AC087672.2, AL353608.2, and AL445588.1; the reagent for detecting the esophageal squamous cell carcinoma marker is a reagent for detecting the RNA level of the esophageal squamous cell carcinoma lncRNA marker.

2. The use according to claim 1, characterized in that The esophageal squamous cell carcinoma lncRNA marker is any combination of the following two markers: XIST and LINC00482; LINC00482 and TMLHE-AS1; LINC00482 and AP001437.2; LINC00482 and AL353608.2; LINC00482 and AC087672.2; LINC00482 and AL445588.

1.

3. The use according to claim 1, characterized in that The esophageal squamous cell carcinoma lncRNA marker is a combination of any of the following three markers: XIST, LINC00482 and TMLHE-AS1; XIST, LINC00482 and AP001437.2; XIST, LINC00482 and AL353608.2; XIST, LINC00482 and AC087672.2; XIST, LINC00482 and AL445588.1; LINC00482, AC087672.2 and TMLHE-AS1; LINC00482, AC087672.2 and AP001437.2; LINC00482, AC087672.2 and AL353608.2; LINC00482, AC087672.2 and AL445588.

1.

4. The use according to claim 1, characterized in that The esophageal squamous cell carcinoma lncRNA marker is a combination of any of the following markers: XIST, LINC00482, AP001437.2, and AC087672.2; TMLHE-AS1, LINC00482, AP001437.2, and AC087672.2; XIST, LINC00482, TMLHE-AS1, and AP001437.2; XIST, LINC00482, TMLHE-AS1, and AL353608.2; XIST, LINC00482, TMLHE-AS1, AP001437.2, and AC087672.2; LINC00482 C00482, TMLHE-AS1, AP001437.2, AL353608.2, and AC087672.2; XIST, LINC00482, TMLHE-AS1, AP001437.2, and AL353608.2; XIST, LINC00482, TMLHE-AS1, AP001437.2, and AL445588.1; XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, and AC087672.

2.

5. The use according to claim 1, characterized in that The lncRNA markers for esophageal squamous cell carcinoma are XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2 and AL445588.

1.

6. The use according to claim 1, characterized in that The product is selected from a reagent, a kit or a chip.

7. The use according to claim 6, characterized in that The reagent for detecting esophageal squamous cell carcinoma markers is a reagent for detecting the RNA level of the esophageal squamous cell carcinoma lncRNA marker in a sample by real-time fluorescence quantitative reverse transcription polymerase chain reaction and / or nucleic acid mass spectrometry.

8. The use according to claim 1, characterized in that The auxiliary diagnosis or diagnostic use of the auxiliary diagnosis or diagnostic product for esophageal squamous cell carcinoma includes: if the level of the esophageal squamous cell carcinoma lncRNA marker in the test sample is higher than the level of the esophageal squamous cell carcinoma lncRNA marker in the healthy sample, it is judged that the test sample has a risk of esophageal squamous cell carcinoma.

9. The use according to claim 8, characterized in that The test sample is selected from esophageal cancer surgical tissue specimens, formalin-fixed esophageal cancer tissue or adjacent cancer tissue, paraffin-embedded esophageal cancer tissue or adjacent cancer tissue, blood, serum or plasma.

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