Application of lncRNA biomarker in diagnosis of esophageal squamous carcinoma

Abstract

The invention discloses application of an lncRNA biomarker in esophageal squamous cell carcinoma diagnosis, and relates to the technical field of esophageal squamous cell carcinoma diagnosis. The esophageal squamous cell carcinoma marker comprises LINC00482. The lncRNA marker screened by the invention has the technical advantages of high esophageal squamous cell carcinoma diagnosis sensitivity and strong specificity, has good diagnosis characteristics, and has important significance on early diagnosis, early intervention, deep understanding of the pathological mechanism of esophageal squamous cell carcinoma and the like. In addition, the method for auxiliary diagnosis or diagnosis of esophageal squamous cell carcinoma by using the biomarker has the technical advantages of simple operation, low price and short detection period, and can meet the detection requirements of various samples such as blood, tissues and the like.

Description

Technical Field

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

[0002] Esophageal squamous cell carcinoma (ESCC) is a digestive tract malignant tumor originating from human esophageal epithelial cells, with high invasiveness and lethality. Although clinicians and researchers are making every effort to improve the outcome, the incidence of this lethal malignant tumor is still rising rapidly, and the overall 5-year survival rate is still less than 25%. Therefore, early diagnosis and timely treatment are the keys to improving the prognosis of esophageal squamous cell carcinoma. However, due to the lack of obvious symptoms in the early stage of the disease, many patients are diagnosed in the advanced stage. Although non-invasive detection of cancer-related biomarkers (such as alpha-fetoprotein and prostate-specific antigen) has long been widely used as a complementary diagnostic method for specific types of cancer, there is currently no available biomarker with ideal sensitivity and specificity for esophageal squamous cell carcinoma. Although the use of cancer targeted therapy has become an irresistible trend, it is still in its infancy, especially in the field of esophageal squamous cell carcinoma treatment. Therefore, from the perspective of the clinical management of esophageal squamous cell carcinoma, there is an urgent need for sensitive and specific biomarkers, as well as candidate treatment targets.

[0003] Long noncoding RNAs (lncRNAs) are a group of RNA transcripts longer than 200 nucleotides that are involved in a variety of physiological and pathological processes, including carcinogenesis. At the beginning of their discovery, the traditional hypothesis was that most of these types of transcripts were "transcriptional noise" because these elements could not encode and translate proteins. In fact, although most lncRNAs lack open reading frames and do not direct protein synthesis, they still play crucial roles in complex gene regulatory networks, including molecular signals, decoys, guides, and scaffolds. These regulatory modes occur at the epigenetic, transcriptional, and post-transcriptional levels, causing changes in cell state and function. It is estimated that the number of lncRNAs far exceeds that of mRNAs and other non-coding RNAs, such as microRNAs. LncRNAs are not rare molecules; on the contrary, they are the largest class of transcripts in mammals. Unfortunately, most of their regulatory mechanisms have not been fully understood. So far, more and more evidence has shown that the abnormal expression of lncRNAs contributes to the occurrence 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 to prevent the malignant transformation of normal cells. Notably, some lncRNAs not only show cell / tumor-specific expression but also stably exist in the blood and have the potential to serve as key predictive biomarkers for cancer occurrence, metastasis, treatment response, recurrence, and survival.

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

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

[0006] The object of the present invention is to provide the application of lncRNA biomarkers in the diagnosis of esophageal squamous cell carcinoma to solve the defects existing in the application of existing diagnostic biomarkers for esophageal squamous cell carcinoma. The existing technologies for diagnosing esophageal squamous cell carcinoma have the following defects: (1) The imaging signs of esophageal squamous cell carcinoma are limited by the tumor size and do not have high sensitivity and specificity; (2) The specificity and accuracy of serum tumor markers such as squamous cell carcinoma antigen and carcinoembryonic antigen are relatively low; (3) Although histopathology is the "gold standard" for tumor diagnosis, invasive examinations have certain damage to the body, and due to tumor heterogeneity, pathological biopsy still has limitations.

[0007] The present invention is implemented as follows: The present invention provides an application of a reagent for detecting esophageal squamous cell carcinoma markers in the preparation of an esophageal squamous cell carcinoma auxiliary diagnosis or diagnosis product or an esophageal squamous cell carcinoma curative effect monitoring product, and the esophageal squamous cell carcinoma lncRNA markers are 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.

[0008] The present invention has the following beneficial effects: The lncRNA markers screened in the present invention have the technical advantages of high diagnostic sensitivity and strong specificity for esophageal squamous cell carcinoma, have good diagnostic characteristics, and 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 method for auxiliary diagnosis or diagnosis of esophageal squamous cell carcinoma by the biomarker proposed in the present invention has the technical advantages of simple operation, low price, and short detection period, and can be used for the detection requirements of various samples such as blood and tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0010] Figure 1 For the present invention, a diagnostic model for esophageal squamous cell carcinoma was constructed using linear regression analysis, and the optimal λ value was obtained through 10-fold cross-validation; Figure 2 This is a box plot of the expression levels of seven biomarkers, namely 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 people (control) in the present invention; Figure 3 This is a schematic diagram of the ROC curve for the diagnosis of seven biomarkers, namely XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2, and AL445588.1, in esophageal squamous cell carcinoma in the present invention; Figure 4Schematic diagram of ROC curves for the analysis of the diagnostic performance of 7 biomarkers (XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2, and AL445588.1) for esophageal squamous cell carcinoma diagnosis individually; Figure 5 Results graphs for the analysis of the diagnostic performance of pairwise combinations of XIST and LINC00482; XIST and TMLHE-AS1; XIST and AP001437.2; XIST and AL353608.2; XIST and AC087672.2; XIST and AL445588.1 for esophageal squamous cell carcinoma; Figure 6 Results graphs for the analysis of the diagnostic performance of pairwise combinations of LINC00482 and TMLHE-AS1; LINC00482 and AP001437.2; LINC00482 and AL353608.2; LINC00482 and AC087672.2; LINC00482 and AL445588.1; AC087672.2 and AL445588.1 for esophageal squamous cell carcinoma; Figure 7 Results graphs for the analysis of the diagnostic performance 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 for esophageal squamous cell carcinoma; Figure 8 Results graphs for the analysis of the diagnostic performance of triple combinations of 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 for esophageal squamous cell carcinoma; Figure 9For the four - four combinations of the biomarkers 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, the result graph of the diagnostic performance analysis for esophageal squamous cell carcinoma; Figure 10 For the five - five combinations of the biomarkers 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; XIST, LINC00482, TMLHE - AS1, AP001437.2, and AL445588.1, the result graph of the diagnostic performance analysis for esophageal squamous cell carcinoma; Figure 11 For the combination of six biomarkers XIST, LINC00482, TMLHE - AS1, AP001437.2, AL353608.2, and AC087672.2, the result graph of the diagnostic performance analysis for esophageal squamous cell carcinoma. Detailed implementation manners

[0011] Reference to the embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the present invention. For example, features described or illustrated as part of one embodiment can be used in another embodiment to yield a further embodiment.

[0012] The term "biomarker" or "marker" broadly refers to any detectable compound or cell present in or derived from a sample, such as a protein, peptide, proteoglycan, glycoprotein, lipoprotein, cell, or a distinguishing molecule or fragment of any of the foregoing. For example, the detection of a specific antibody or binding to a specific antibody can indicate the presence of a specific antigen (e.g., a 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 upon detection. Biomarkers can be isolated from a sample, measured directly in a sample, or detected or assayed in a sample. Biomarkers can be, for example, functional, partially functional, or non - functional.

[0013] Specifically, a tumor marker refers to a biomarker that appears in samples such as serum, urine, and tissues of tumor patients. The presence of it indicates that there may be tumor cells in the body. Tumor markers are used for applications such as early diagnosis of tumors, monitoring of disease progression, and evaluation of treatment effects by detecting these markers. Since the presence or absence and the amount of tumor markers are related to factors such as the type, location, size, and malignancy of tumors, early diagnosis and disease monitoring of tumors can be achieved by detecting tumor markers, which is beneficial to improving the success rate of tumor treatment and the survival rate of patients. Cell surface markers refer to substances such as proteins and carbohydrates present on the surface of tumor cells. Changes in cell surface markers can reflect the growth status and characteristics of tumor cells and are the most common type of tumor markers. Intracellular markers refer to substances such as proteins and RNA present inside tumor cells.

[0014] The term "sample" refers to a biological specimen obtained from or derived from a subject of interest, and the source of the biological specimen can be a fresh, frozen, and / or preserved organ or tissue sample or a solid tissue generated from a biopsy or primer; blood or any blood component. The term "sample" includes a biological sample that has been manipulated in any way after its acquisition, such as by reagent treatment, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedding in a semi-solid or solid matrix for sectioning purposes.

[0015] In a first aspect, the present invention provides the use of a reagent for detecting esophageal squamous cell carcinoma markers in the preparation of an esophageal squamous cell carcinoma auxiliary diagnosis or diagnosis product or an esophageal squamous cell carcinoma treatment efficacy monitoring product. The esophageal squamous cell carcinoma lncRNA markers are selected from any one of (1)-(2): (1) LINC00482; (2) LINC00482, and including at least one selected from the following markers: XIST, TMLHE-AS1, AP001437.2, AC087672.2, AL353608.2, and AL445588.1.

[0016] The present invention conducts a comprehensive transcriptomic analysis on whole blood samples of 80 esophageal squamous cell carcinoma patients and 40 normal human whole blood samples to characterize the lncRNA expression differences in the whole blood of patients and normal humans, and identifies a group of potential esophageal squamous cell carcinoma biomarkers through linear regression analysis.

[0017] When the above 7 markers are used for combined diagnosis, they have an extremely high AUC value, high diagnostic specificity and sensitivity.

[0018] In other embodiments, those skilled in the art can use at least one of the above eight markers for the auxiliary diagnosis or diagnosis of esophageal squamous cell carcinoma and the efficacy monitoring 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 seven markers.

[0019] In a preferred embodiment of the application of the present invention, the esophageal squamous cell carcinoma lncRNA markers are any combination of the following pairwise markers: XIST and LINC00482; LINC00482 and TMLHE-AS1; LINC00482 and AP001437.2; LINC00482 and AL353608.2; LINC00482 and AC087672.2; LINC00482 and AL445588.1.

[0020] Among them, the marker combination of XIST and LINC00482 relatively has a higher AUC value, and the confidence interval CI is: 0.795 - 0.968. The marker combination of LINC00482 and AC087672.2 relatively has a higher AUC value (0.950), and the confidence interval CI is: 0.893 - 1.000. The marker combination of AL353608.2 and AC087672.2 relatively has a higher AUC value (0.935), and the confidence interval CI is: 0.868 - 1.000.

[0021] In a preferred embodiment of the application of the present invention, the esophageal squamous cell carcinoma lncRNA markers are any combination of the following triple 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.

[0022] Among them, the marker combination of LINC00482, AC087672.2 and AP001437.2 relatively has a higher AUC value (0.955), and the confidence interval CI is: 0.904 - 1.000.

[0023] In a preferred embodiment of the application of the present invention, the esophageal squamous cell carcinoma lncRNA markers are a combination of any one of the following groups of 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, 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.

[0024] Among them, the marker combination of TMLHE-AS1, LINC00482, AP001437.2 and AC087672.2 has a relatively higher AUC value (0.958), and the confidence interval CI is: 0.908 - 1.000. The marker combinations of XIST, LINC00482, TMLHE-AS1, AP001437.2 and AC087672.2; LINC00482, TMLHE-AS1, AP001437.2, AL353608.2 and AC087672.2 have relatively higher AUC values (0.958), and the confidence interval CI is: 0.908 - 1.000. The analysis result graph of the diagnostic performance of the combination of six markers of XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2 shows that the combination of these six markers has a relatively higher AUC value (0.958), and the confidence interval CI is: 0.908 - 1.000.

[0025] In a preferred embodiment of the application of the present invention, the esophageal squamous cell carcinoma lncRNA markers are XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2 and AL445588.1.

[0026] In a preferred embodiment of the application of the present invention, the reagent for detecting esophageal squamous cell carcinoma lncRNA markers is a reagent for detecting the levels of DNA, RNA, protein or cells carrying esophageal squamous cell carcinoma lncRNA markers of esophageal squamous cell carcinoma lncRNA markers.

[0027] In a preferred embodiment of the application 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 method.

[0028] In a preferred embodiment of the application of the present invention, the reagent for detecting esophageal squamous cell carcinoma lncRNA markers is a reagent for detecting the protein level of esophageal squamous cell carcinoma lncRNA markers in a sample by ELISA, Western blotting and / or Elisopt method, or a reagent for detecting the level of cells carrying esophageal squamous cell carcinoma lncRNA markers in a sample by TUNEL detection and / or flow cytometry.

[0029] In a preferred embodiment of the application of the present invention, the product is selected from reagents, reagent kits or chips.

[0030] The chip can also be called a suspension array or a liquid array. It includes a carrier and nucleic acid molecules (such as primers and / or probes) and / or antibodies bound to the surface of the carrier.

[0031] The chip is, for example, a microfluidic chip. The microfluidic chip is selected from PDMS chips or metal droplet generators of T-type chips, flow focusing type chips or coaxial flow type chips, or PMMA microfluidic chips.

[0032] In a preferred embodiment of the application of the present invention, the product includes probes and / or primers for detecting esophageal squamous cell carcinoma lncRNA markers.

[0033] In a preferred embodiment of the application of the present invention, the product further includes reverse transcriptase. In one embodiment, the reverse transcriptase exists, for example, in the form of a reverse transcriptase mix. In a preferred embodiment of the application of the present invention, the product further includes DNA polymerase, such as selected from hot start DNA polymerases, such as Tth DNA polymerase, Taq DNA polymerase, etc.

[0034] The auxiliary diagnosis or diagnosis product (such as a reagent kit) provided by the present invention may also optionally include any reagents and / or consumables acceptable in the art for PCR reactions or for formulating PCR reaction systems. Specific examples may include, but are not limited to, one or more of dNTP, salts or salt solutions, negative control products, positive control products, blank control products, calibration products and PCR reaction containers.

[0035] Furthermore, the kit includes at least one of a buffer, a detection reagent, a diluent, a washing solution (or a rinsing solution), and is not limited thereto. In one embodiment, the kit further includes a chromogenic solution, a termination solution, an RNA extraction reagent, etc.

[0036] In a preferred embodiment of the application of the present invention, the auxiliary diagnosis or diagnostic use of the esophageal squamous cell carcinoma auxiliary diagnosis or diagnostic product 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 determined that the test sample has a risk of suffering from esophageal squamous cell carcinoma.

[0037] The efficacy monitoring use of the esophageal squamous cell carcinoma efficacy monitoring product includes: if, within one or more esophageal squamous cell carcinoma treatment cycles, the level of the esophageal squamous cell carcinoma lncRNA marker in the later stage of the treatment of the test sample is higher than the level of the esophageal squamous cell carcinoma lncRNA marker in the early stage of the esophageal squamous cell carcinoma treatment, it is determined that the efficacy of the esophageal squamous cell carcinoma is poor, the esophageal squamous cell carcinoma may be resistant to the treatment method, and the treatment plan needs to be adjusted.

[0038] In a preferred embodiment of the application of the present invention, the test sample is selected from esophageal cancer surgical tissue specimens, formalin-fixed esophageal cancer tissues or adjacent tissues, paraffin-embedded esophageal cancer tissues or adjacent tissues, blood, serum or plasma.

[0039] In a preferred embodiment of the application of the present invention, the above-mentioned exosomes are derived from serum, plasma or urine.

[0040] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0041] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0042] The following experiment was conducted to screen and determine lncRNA in 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 of Sichuan University. All subjects voluntarily participated, were orally informed of the purpose of the experiment and signed the informed consent form.

[0043] Experimental subjects: 80 esophageal squamous cell carcinoma patients and 40 healthy normal people collected in West China Hospital were studied. The sociodemographic information of the experimental subjects was recorded in detail, including gender, age, etc. The experimental subjects in each group were matched in terms of age, gender and education level.

[0044] Sample collection: Whole blood samples were collected from each patient and normal individual, subjected to next-generation sequencing, and the down-stream data were subjected to bioinformatics analysis.

[0045] Example 1 Screening lncRNAs as diagnostic markers for esophageal squamous cell carcinoma, which can be used for differential diagnosis of esophageal squamous cell carcinoma. The specific steps are as follows: 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 build the index and calculate the transcript expression levels.

[0046] Construction of the esophageal squamous cell carcinoma diagnostic model: The R package DESeq2 (version 1.33.1) was used to analyze the differentially expressed lncRNAs in the blood of esophageal squamous cell carcinoma patients and normal individuals, with Padj < 0.05 considered statistically significant. The R packages survival (version 3.1-12) and glmnet (version 4.1-3) were used to perform linear regression analysis on the differentially expressed lncRNAs and construct the esophageal squamous cell carcinoma diagnostic model. The Mann-Whitney U test was used to perform statistical analysis on the gene expression levels in the blood of esophageal squamous cell carcinoma patients and normal individuals, with a p-value < 0.05 considered statistically significant, and box plots were made using the R package ggplot2 (version 3.3.0). All analyses were performed in R (version 4.0.2).

[0047] By observing the abnormal lncRNA molecular characteristics of esophageal squamous cell carcinoma and constructing the diagnostic model of esophageal squamous cell carcinoma, it was found that the esophageal squamous cell carcinoma diagnostic model composed of 7 lncRNAs, namely XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AL589863.2, AC245052.7, and AL357497.1, had good diagnostic value and could be used as biomarkers for the diagnosis of esophageal squamous cell carcinoma ( Figure 1 )

[0048] Compared with normal individuals, the expression levels of XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2, and AL445588.1 were increased in the blood of esophageal squamous cell carcinoma patients ( Figure 2 )

[0049] Example 2 Based on the results of Example 1, in this example, an individual disease risk detection kit was constructed for the assessment of the risk of esophageal squamous cell carcinoma. The kit consists of RNA extraction reagents and reverse transcription reagents, and can be used to construct a detection kit for the diagnosis and monitoring of esophageal squamous cell carcinoma.

[0050] In this example, 40 samples of esophageal squamous cell carcinoma patients and 20 samples of healthy normal individuals different from the above samples were collected for transcriptomic sequencing analysis, and the diagnostic performance analysis of esophageal squamous cell carcinoma was carried out using 7 biomarkers (XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2, and AL445588.1).

[0051] The ROC curve for the diagnosis of esophageal squamous cell carcinoma showed that the diagnostic AUC value of the 7 biomarkers was 0.960, with high specificity and sensitivity, and had high accuracy in predicting esophageal squamous cell carcinoma ( Figure 3 )

[0052] 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 for the differential diagnosis of esophageal squamous cell carcinoma, and for the auxiliary differential diagnosis of esophageal squamous cell carcinoma patients, the operation is simple, the price is low, the detection time is short, and the sensitivity and specificity are strong, which has important clinical significance.

[0053] Example 3 In this example, for the samples of Example 2, the diagnostic performance analysis of esophageal squamous cell carcinoma was carried out separately using 7 biomarkers (XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2, and AL445588.1).

[0054] The results are referred to Figure 4 As shown, the results showed that the AUC of the 7 biomarkers for the individual diagnosis of esophageal squamous cell carcinoma was greater than 0.85.

[0055] Example 4 Compared with Example 3, in this example, the diagnostic performance analysis of esophageal squamous cell carcinoma was carried out using a combination of two biomarkers, and the samples evaluated were the samples of Example 2.

[0056] Figure 5The graphs of the analysis results of the diagnostic performance of pairwise combinations of the biomarkers A, B, C, D, E, and F in [[]] being XIST and LINC00482; XIST and TMLHE-AS1; XIST and AP001437.2; XIST and AL353608.2; XIST and AC087672.2; XIST and AL445588.1 for esophageal squamous cell carcinoma. Figure 5 The results showed that the biomarker combination of XIST and LINC00482 relatively had a higher AUC value, and the confidence interval CI was: 0.795 - 0.968.

[0057] Figure 6 The graphs of the analysis results of the diagnostic performance of pairwise combinations of the biomarkers A, B, C, D, E, and F in [[]] being LINC00482 and TMLHE-AS1; LINC00482 and AP001437.2; LINC00482 and AL353608.2; LINC00482 and AC087672.2; LINC00482 and AL445588.1; AC087672.2 and AL445588.1 for esophageal squamous cell carcinoma. Figure 6 The results showed that the biomarker combination of LINC00482 and AC087672.2 relatively had a higher AUC value (0.950), and the confidence interval CI was: 0.893 - 1.000. Slightly lower than the combination of the 7 biomarkers in this application.

[0058] Figure 7 The graphs of the analysis results of the diagnostic performance of pairwise combinations of the biomarkers A, B, C, D, E, F, G, H, and I in [[]] being 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 for esophageal squamous cell carcinoma. Figure 7 The results showed that the biomarker combination of AL353608.2 and AC087672.2 relatively had a higher AUC value (0.935), and the confidence interval CI was: 0.868 - 1.000. Slightly lower than the combination of the 7 biomarkers in this application.

[0059] Example 5 Compared with Example 3, in this example, a combination of multiple biomarkers was used for the analysis of the diagnostic performance of esophageal squamous cell carcinoma, and the samples evaluated were the samples of Example 2.

[0060] Figure 8 Among them, A, B, C, D, E, F, G, H, and I are XIST, LINC00482, and TMLHE-AS1 respectively; 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. The figure showing the analysis results of the diagnostic performance of the triple combinations of the markers. Figure 8 The results showed that the marker combination of LINC00482, AC087672.2, and AP001437.2 relatively had a higher AUC value (0.955), and the confidence interval CI was: 0.904 - 1.000. Slightly lower than the combination of the 7 markers in this application.

[0061] Figure 9 Among them, A, B, C, and D are XIST, LINC00482, AP001437.2, and AC087672.2 respectively; TMLHE-AS1, LINC00482, AP001437.2, and AC087672.2; XIST, LINC00482, TMLHE-AS1, and AP001437.2; XIST, LINC00482, TMLHE-AS1, and AL353608.2. The figure showing the analysis results of the diagnostic performance of the quadruple combinations of the markers for esophageal squamous cell carcinoma. Figure 9 The results showed that the marker combination of TMLHE-AS1, LINC00482, AP001437.2, and AC087672.2 relatively had a higher AUC value (0.958), and the confidence interval CI was: 0.908 - 1.000. Slightly lower than the combination of the 7 markers in this application.

[0062] Figure 10A, B, C, and D in it are 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; XIST, LINC00482, TMLHE-AS1, AP001437.2, and AL445588.1. It is a graph showing the analysis results of the diagnostic performance of the five-by-five combinations of the markers for esophageal squamous cell carcinoma. Figure 10 The results show that the marker combinations of XIST, LINC00482, TMLHE-AS1, AP001437.2, and AC087672.2; LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, and AC087672.2 have relatively higher AUC values (0.958), and the confidence interval CI is: 0.908 - 1.000. It is slightly lower than the combination of the 7 markers in this application.

[0063] Figure 11 It is a graph showing the analysis results of the diagnostic performance of the combination of six markers, namely XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, and AC087672.2, for esophageal squamous cell carcinoma. Figure 11 The results show that the combination of these six markers has relatively higher AUC values (0.958), and the confidence interval CI is: 0.908 - 1.000.

[0064] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting esophageal squamous cell carcinoma markers in the preparation of an esophageal squamous cell carcinoma auxiliary diagnosis or diagnosis product or an esophageal squamous cell carcinoma curative effect monitoring product, characterized in that, The lncRNA markers for esophageal squamous cell carcinoma are 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.

2. The application according to claim 1, characterized in that, The lncRNA markers for esophageal squamous cell carcinoma are any combination of the following pairwise 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 application according to claim 1, characterized in that, The lncRNA markers for esophageal squamous cell carcinoma are any combination of the following three-way 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 application according to claim 1, characterized in that, The lncRNA markers for esophageal squamous cell carcinoma are any combination 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, 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 application according to claim 1, characterized in that, The esophageal squamous cell carcinoma lncRNA markers are XIST, LINC00482, TMLHE-AS1, AP001437.2, AL353608.2, AC087672.2, and AL445588.

1.

6. The application according to claim 1, wherein The reagent for detecting esophageal squamous cell carcinoma markers is a reagent for detecting the levels of DNA, RNA, protein, or cells carrying the esophageal squamous cell carcinoma lncRNA markers of the esophageal squamous cell carcinoma lncRNA markers, and the product is selected from reagents, reagent kits, or chips.

7. The application according to claim 6, wherein The reagent for detecting esophageal squamous cell carcinoma markers is a reagent for detecting the RNA levels of the 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 methods.

8. The application according to claim 1, wherein The auxiliary diagnosis or diagnostic use of the esophageal squamous cell carcinoma auxiliary diagnosis or diagnostic product 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 determined that the test sample has a risk of suffering from esophageal squamous cell carcinoma.

9. The application according to claim 1, wherein The efficacy monitoring use of the esophageal squamous cell carcinoma efficacy monitoring product includes: if, within one or more esophageal squamous cell carcinoma treatment cycles, the level of the esophageal squamous cell carcinoma lncRNA marker in the later stage of the 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 determined that the efficacy of esophageal squamous cell carcinoma is poor and the treatment plan needs to be adjusted.

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

Images