CN113736886A - Biomarker for esophageal cancer diagnosis and application thereof - Google Patents
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses a biomarker for esophageal cancer diagnosis and application thereof, wherein the biomarker comprises hsa _ circ _ 0060927. The specific marker of esophageal cancer can be used for auxiliary diagnosis of esophageal cancer, can be conveniently applied to clinic, can quickly and effectively diagnose esophageal cancer, and provides support for clinicians to adopt personalized prevention and treatment schemes in time.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a biomarker for diagnosing esophageal cancer and application thereof.
Background
The incidence of malignant tumors has increased in recent ten years, with over 60% of them being malignant tumors of the digestive system. Esophageal cancer is one of common malignant tumors in China. The occurrence of esophageal cancer is related to the deficiency of nitrosamine-containing substances, the surrounding environment, the regional distribution, genetic factors and vitamins and trace elements in the dietary structure after long-term eating. With the improvement of medical treatment level, new techniques and new methods for diagnosing and treating esophageal cancer are endless, but statistics shows that the death rate of esophageal cancer is not obviously reduced. The reason for this is that more than half of patients are already in the middle and advanced stage at the time of first visit.
With the continuous development of molecular biology technology, people are beginning to focus on the research of biomarkers related to tumors. It is a class of substances that reflect the presence of disease, and their presence or amount may indicate the presence of disease. Biomarkers have been widely used in the clinic since the 60 s of the last century. The detection of the biomarkers is simple, convenient, economical and rapid, and some biomarkers are expressed before morphological changes of tissues and organs occur and are developed to the end of the whole process along with diseases, so the biomarkers are concerned by clinicians. At present, the types of the biomarkers are hundreds of types, but an ideal biomarker which can be independently applied to clinic as diagnosis of esophageal cancer is not found yet. Therefore, the search for specific sensitive biomarkers has become an important direction for clinical and basic research.
Disclosure of Invention
The object of the present invention is to establish a highly sensitive, highly stable method for the diagnosis of esophageal cancer, said method comprising detecting the level of hsa _ circ _0060927 expression in a sample.
In a first aspect, the invention provides the use of a reagent for detecting the expression level of a circRNA in a sample, said circRNA comprising hsa _ circ _0060927, in the manufacture of a means for diagnosing oesophageal cancer.
In the present invention, hsa _ circ _0060927 is located on human chromosome 1 and includes the hsa _ circ _0060927 gene and homologs, mutations, and isoforms thereof. The location on the genome is chr20: 52773707-.
The term "expression level" generally refers to the amount of a biomarker in a biological sample. "expression" generally refers to the process by which information (e.g., gene coding and/or epigenetic information) is converted into structures present and operating in a cell. Thus, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., post-translational modifications of a polypeptide). Transcribed polynucleotides, translated polypeptides, or fragments of a polynucleotide and/or polypeptide modification (e.g., post-translational modification of a polypeptide) should also be considered expressed, whether they are derived from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of a polypeptide (e.g., by proteolysis). "expressed gene" includes genes that are transcribed into a polynucleotide (e.g., mRNA) and then translated into a polypeptide, as well as genes that are transcribed into RNA but not translated into a polypeptide (e.g., transport and ribosomal RNA), and "expressed" refers to a transcribed polynucleotide as a preferred embodiment of the invention.
"increased expression," "increased expression level," "increased level," "elevated expression level," or "elevated level" refers to increased expression or increased level of a biomarker in an individual relative to a control, such as a median expression level of the biomarker in an individual without a disease or disorder (e.g., cancer), an internal control (e.g., a housekeeping biomarker), or a sample from one patient group/population.
"reduced expression", "reduced expression level", "reduced expression level" or "reduced level" refers to reduced expression or reduced level of a biomarker in an individual relative to a control, such as a median expression level of the biomarker in an individual or an internal control (e.g., a housekeeping biomarker) that does not have a disease or disorder (e.g., cancer), or a sample from one patient group/population. In some embodiments, the reduced expression is little or no expression.
In some embodiments, the esophageal cancer comprises esophageal squamous carcinoma, esophageal adenocarcinoma, preferably esophageal squamous carcinoma.
In some embodiments, the reagent for detecting the expression level of the circRNA in the sample comprises a reagent for detecting the expression level of the circRNA in the sample by using a sequencing technology, a nucleic acid hybridization technology or a nucleic acid amplification technology.
In some embodiments, the reagent for detecting the expression level of the circRNA in the sample comprises a reagent for detecting the expression level of the circRNA in the sample by using a high-throughput sequencing technology, a probe hybridization technology, a fluorescent in situ hybridization technology, a gene chip technology or a fluorescent quantitative PCR technology.
The expression level of hsa _ circ _0060927 in the sample to be detected can be obtained through high-throughput sequencing, and compared with the result of a sample of a healthy person, the result of the sample to be detected can be used for easily judging whether the sample to be detected has esophageal cancer and the risk of the esophageal cancer. Therefore, the application of obtaining the correlation between the expression level of hsa _ circ _0060927 and esophageal cancer by high-throughput sequencing is also included in the protection scope of the present invention.
In some embodiments, the reagent is a reagent for detecting the expression level of circRNA in a sample by using fluorescence in situ hybridization.
In some embodiments, the detecting the circRNA expression level in the sample by using the fluorescence in situ hybridization technique is realized by counting the fluorescence signal in the cells to judge the circRNA expression level.
In some embodiments, the method for determining the expression level of circRNA by counting the fluorescence signals in cells comprises calculating a Ratio of the total number of the circRNA fluorescence signals in 100 nuclei to the total number of DAPI fluorescence signals in 100 nuclei. In a preferred embodiment, if the Ratio value in the sample is <2, the circRNA expression in the sample is determined to be low, and if the Ratio value in the sample is >2, the circRNA expression in the sample is determined to be high.
In some embodiments, the method for determining the expression level of circRNA by counting the fluorescent signals in the cells comprises counting the fluorescent signals in the cells, and determining that the circRNA is highly expressed in the sample if > 15 circRNA fluorescent signals or clustered circRNA fluorescent signals are present in > 10% of the cells in the sample, or >4 circRNA fluorescent signals are present in > 40% of the cells.
As used herein, a "sample" may be a cell or tissue sample (e.g., a biopsy), a biological fluid, an extract (e.g., a protein or DNA extract obtained from a subject). In particular, the sample may be a tumor sample, e.g. a solid tumor, e.g. esophageal cancer. The sample may be a sample freshly obtained from the subject, or may be a sample that has been processed and/or stored (e.g., frozen, fixed, or subjected to one or more purification, enrichment, or extraction steps) prior to making the determination. Samples of the present invention include, but are not limited to, blood, vitreous humor, lymph, synovial fluid, semen, amniotic fluid, milk, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tissue, stool, and preferably tissue.
In some embodiments, hsa _ circ _0060927 is up-regulated in esophageal cancer samples.
In some embodiments, the reagent comprises a primer, a probe, or a combination thereof.
The term "primer" as used herein refers to a nucleic acid sequence having a short free 3' -hydroxyl group, which is a short nucleic acid that can form a base pair with a complementary template and serves as an origin of replication for the template strand. The primers can prime DNA synthesis in the presence of reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffer solutions and temperatures. The PCR conditions and the lengths of the sense and antisense primers can be appropriately selected according to the techniques known in the art.
The term "probe" as used herein refers to a nucleic acid fragment (e.g., RNA or DNA) corresponding to several bases to several hundred bases that can specifically bind to mRNA, and the presence or absence and expression level of a particular mRNA can be confirmed by a tag. The probe may be prepared in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, or an RNA probe. Suitable probes and hybridization conditions may be appropriately selected according to techniques known in the art.
In some embodiments, the reagent is a probe, and as a preferred embodiment, the sequence of the probe is shown in SEQ ID NO. 1.
In a second aspect, the invention provides a kit for diagnosing oesophageal cancer, said kit comprising reagents for detecting the expression level of circRNA in a sample, said circRNA comprising hsa _ circ _ 0060927.
In some embodiments, the means comprises a kit, chip, or nucleic acid membrane strip.
The kit provided by the invention comprises a reagent for detecting hsa _ circ _0060927, and one or more substances selected from the following group: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent.
The kit of the invention can be also attached with an instruction manual of the kit, wherein the instruction manual describes how to adopt the kit for detection, how to judge the tumor development by using the detection result and how to select a treatment scheme.
In the present invention, the components of the kit may be packaged in the form of an aqueous medium or in a lyophilized form. Suitable containers in the kit generally include at least one vial, test tube, flask, pet bottle, syringe, or other container in which a component may be placed and, preferably, suitably aliquoted. Where more than one component is present in the kit, the kit will also typically comprise a second, third or other additional container in which the additional components are separately disposed. However, different combinations of components may be contained in one vial. The kit of the invention will also typically include a container for holding the reactants, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials may be retained.
The chip of the invention comprises: a solid support; and oligonucleotide probes immobilized on the solid support in an ordered manner, the oligonucleotide probes specifically corresponding to part or all of the sequence shown in hsa _ circ _ 0060927.
The solid phase carrier of the present invention can be made of various materials commonly used in the field of gene chip, such as but not limited to nylon membrane, glass or silicon slice modified by active group (such as aldehyde group, amino group, etc.), unmodified glass slice, plastic slice, etc.
The hsa _ circ _0060927 chip can be prepared by conventional methods known in the art for biochip fabrication. For example, if a modified glass slide or silicon wafer is used as the solid support and the 5' -end of the probe contains a poly-dT strand modified with an amino group, the oligonucleotide probe may be prepared as a solution, and then spotted on the modified glass slide or silicon wafer using a spotting apparatus, arranged in a predetermined sequence or array, and then fixed by standing overnight, to obtain the circRNA chip of the present invention.
In the present invention, the nucleic acid membrane strip comprises a substrate and an oligonucleotide probe specifically recognizing hsa _ circ _0060927 immobilized on the substrate; the substrate may be any substrate suitable for immobilizing oligonucleotide probes, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass plate, a silica gel wafer, a micro magnetic bead, or the like.
The kit or chip or nucleic acid membrane strip can be used for detecting the expression levels of a plurality of genes (for example, a plurality of genes related to esophageal cancer) including hsa _ circ _0060927 gene, and simultaneously detecting a plurality of markers of esophageal cancer, thereby greatly improving the accuracy of esophageal cancer diagnosis.
In some embodiments, the esophageal cancer comprises esophageal squamous carcinoma, esophageal adenocarcinoma, preferably esophageal squamous carcinoma.
In a third aspect, the present invention provides a device for predicting esophageal cancer, the device comprising:
a processor;
an input module for inputting an expression level of circRNA in a biological sample, said circRNA comprising hsa _ circ _ 0060927.
A computer-readable medium containing instructions that, when executed by the processor, perform an algorithm on the input levels of the biomarkers; and
an output module indicating whether the subject has or is at risk of having esophageal cancer.
An apparatus as applied herein shall at least comprise the above-mentioned modules. The modules of the device are operatively connected to each other. How the modules are operatively linked will depend on the type of module contained in the device.
The processor may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as a memory. Instructions may be directed to a processor that may then program or otherwise configure the processor to implement the present disclosure. Examples of operations performed by a processor may include read, decode, execute, and write-back.
The computer-readable storage medium stores a program for executing the esophageal cancer prediction model constructed by hsa _ circ _ 0060927. The computer readable storage medium, such as computer executable code, may take many forms, including but not limited to tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, any storage device such as in any computer or the like, volatile storage media include dynamic memory, such as the main memory of such computer platforms. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media can take the form of electrical or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency and infrared data communications. Thus, common forms of computer-readable media include, for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards, paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer can read programming code and/or data. Many of these computer readable media may take the form of one or more sequences of one or more instructions that are executable by a processor to perform operations.
In some embodiments, the esophageal cancer comprises esophageal squamous carcinoma, esophageal adenocarcinoma, preferably esophageal squamous carcinoma.
In a fourth aspect, the invention provides the use of circRNA comprising hsa _ circ _0060927 in the construction of a computational model for predicting esophageal cancer.
In some embodiments, the esophageal cancer comprises esophageal squamous carcinoma, esophageal adenocarcinoma, preferably esophageal squamous carcinoma.
Drawings
FIG. 1 is a graph showing the results of an experiment for detecting the expression of hsa _ circ _0060927 using fluorescence in situ hybridization;
FIG. 2 is a statistical plot of hsa _ circ _0060927 expression in 114 esophageal cancer tissues and 66 normal tissues, where P < 0.001;
FIG. 3 is a ROC plot of hsa _ circ _0060927 diagnosis of esophageal cancer.
Detailed Description
The following detailed description of embodiments of the present application will be made with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present application, are given by way of illustration and explanation only, and are not intended to limit the present application.
Example 1 Gene differential expression
First, experimental materials and methods
1. Main experimental equipment and instrument
TABLE 1 Experimental Equipment, Instrument
| Apparatus, instrument | Source |
| Tissue microtome (RM2135 type) | Leica, Germany |
| Spreading and baking machine (CS-VI type) | Xianggan Hongye medical instruments Co Ltd |
| Digital display electric heating insulation can (303-4A type) | Shanghai sunshine laboratory instruments Co Ltd |
| SCILOGEX palm centrifuge (D1008 type) | Beijing Ke Boser Biotech Co |
| Laser confocal microscope (LSM900) | Zeiss Co, Germany |
| Image acquisition system (Qcapture) | QIMAGING, Canada |
| Rapid mixer (XK96-A type) | Jiangsu Xinkang medical instruments Co., Ltd |
| EppendorfResearch pipettor | Eppendorf, Germany |
| Electronic balance (PB3002-S) | METTLER, Switzerland |
2. Tissue chip preparation
The tissue chip (HESOS180Su08) was prepared by the national center for engineering research on biochips Shanghai. 114 patients with esophageal cancer have the first onset, and do not undergo radiotherapy, chemotherapy and endocrine treatment before operation. The pathological types are esophageal squamous cell carcinoma, which comprises 114 esophageal cancer tissues and 66 normal esophageal tissues. Of 114 esophageal cancer specimens, 84 men (73.68%) and 30 women (26.32%) were divided by sex; divided by age, 33 people < 60 years old (28.95%), > 60 years old 81 people (71.05%); according to the United statesThe Joint Committee for cancer (AJCC) 7 th edition clinical staging, 58 cases at stages I-II (56.67%), 56 cases at stages III-IV (43.33%); by histological grouping, 47 cases of grade I (41.23%), 67 cases of grade II-III (58.77%); grouped according to the presence or absence of lymph metastasis, 61 cases of lymph node metastasis (53.51%), and 53 cases of lymph node metastasis (46.49%); grouping by T stages, T1-227 cases (23.68%), T3-487 cases (76.32%). All tissue wax blocks were routinely pathologically sectioned followed by HE staining and marked on the section according to typical pathological sites. A tissue chip making instrument is used for punching a receptor wax block (the diameter is 1.5mm), then a corresponding tissue chip is obtained according to the marked typical pathological part and is placed in a receptor wax block array hole, and the tissue number is recorded. Serial sections were made at a thickness of 4um using a microtome (Leica germany) to prepare histological chip sections, and each spot was again HE-stained for pathological diagnosis.
3. Detection of expression of hsa _ circ _0060927 Using Fluorescence In Situ Hybridization (FISH)
4um paraffin sections were placed on pre-treated slides. The sections were baked overnight at 50 ℃ and then subjected to xylene deparaffinization and hydration, and gradient alcohol. Deionized water for 5min, incubating the slices with proteinase K for 10min, adding hybridization solution, and sealing for 60 min. Then hybridized with hsa _ circ _0060927 probe (the probe sequence is shown in SEQ ID NO.1, specifically 5'-TCTTCCCCTTCCCTGAGGCGTATTA-3') at 37 ℃ overnight. Washing the section with 4 XSSC, 2 XSSC, 1 XSSC and PBS, dripping Cy5 fluorescent dye, incubating for 60min in dark, washing with PBS, performing DAPI counterstaining, and finally reading under a laser confocal microscope. Interpretation criteria of results: at least 100 cells were counted and Ratio values were counted. Ratio value is the total number of red signals in 100 nuclei/the total number of blue signals in 100 nuclei. If Ratio <2 is a negative result, indicating that the sample hsa _ circ _0060927 is low in expression; if the Ratio >2 is a positive result, prompting that the hsa _ circ _0060927 of the sample is highly expressed; furthermore, positive results were obtained if > 15 red signals or clustered red signals were present in > 10% of the cells, or >4 red signals were present in > 40% of the cells, indicating high expression of hsa _ circ _0060927 in the sample. The above red signal (hsa _ circ _0060927) and blue signal (DAPI) were counted manually by two pathologists and technicians.
Second, experimental results
As shown in figure 1, figure 2, table 2, hsa _ circ _0060927 was up-regulated in esophageal cancer compared to normal controls.
TABLE 2 expression of hsa _ circ _0060927 in esophageal carcinoma
| Gene | C_readcount | N_readcount | log2FoldChange | pval | padj |
| hsa_circ_0060927 | 300.719 | 5.212 | 5.845 | 0.000 | 0.000 |
Example 2 diagnostic Performance analysis
First, experiment method
The results of FISH detection of expression of hsa _ circ _0060927 were analyzed and scored. The operator working characteristic ROC (receiver operating characteristic) curve and the area under the curve (AUC value) are used for measuring the discrimination of the model, and the closer the AUC value is to 1, the better the discrimination of the model is. Open SPSS, import grouping and score result, click "Analyze-ROC curve", pop up interface, import A2 column data (score result), and appear ROC curve after confirmation. The diagnostic efficacy of individual applications of hsa _ circ _0060927 was calculated and analyzed using the expression level of hsa _ circ _0060927 in the patient as an independent variable and the maximum Youden finger [ (specificity + sensitivity) -1] available as the cut off value.
Second, experimental results
As shown in FIG. 3 and Table 3, hsa _ circ _0060927 has better diagnostic performance.
TABLE 3 hsa _ circ _0060927 diagnostic Performance
| Gene | AUC | Degree of specificity | Sensitivity of the device |
| hsa_circ_0060927 | 0.858 | 0.991 | 0.697 |
The preferred embodiments of the present application have been described in detail with reference to the accompanying drawings, however, the present application is not limited to the details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the technical idea of the present application, and these simple modifications are all within the protection scope of the present application.
Sequence listing
<110> Hospital of Hebei medical university
<120> biomarker for esophageal cancer diagnosis and application thereof
<141> 2021-09-14
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tcttcccctt ccctgaggcg tatta 25
Claims (10)
1. The application of the reagent for detecting the expression level of the circRNA in the sample in preparing the tool for diagnosing the esophageal cancer is characterized in that the circRNA comprises hsa _ circ _0060927, preferably, the esophageal cancer comprises esophageal squamous cell carcinoma and esophageal adenocarcinoma, and preferably, the esophageal cancer is esophageal squamous cell carcinoma.
2. The use according to claim 1, wherein the reagents comprise reagents for detecting the expression level of circRNA in the sample by sequencing, nucleic acid hybridization or nucleic acid amplification techniques.
3. The use according to claim 1, wherein the reagent comprises a reagent for detecting the expression level of the circRNA in the sample by using a high throughput sequencing technology, a probe hybridization technology, a fluorescence in situ hybridization technology, a gene chip technology or a fluorescence quantitative PCR technology, preferably the reagent is a reagent for detecting the expression level of the circRNA in the sample by using a fluorescence in situ hybridization technology, preferably the detection of the expression level of the circRNA in the sample by using a fluorescence in situ hybridization technology is realized by counting fluorescence signals in cells to judge the expression level of the circRNA.
4. The use according to claim 3, wherein said method for determining the expression level of circRNA by counting the fluorescence signals in cells comprises calculating a Ratio of the total number of circRNA fluorescence signals in 100 nuclei to the total number of DAPI fluorescence signals in 100 nuclei, preferably determining that the circRNA is under-expressed in a sample if the Ratio in the sample is <2, and determining that the circRNA is under-expressed in a sample if the Ratio in the sample is > 2.
5. The use according to claim 3, wherein said method of counting the fluorescent signals in cells to determine the level of expression of circRNA comprises counting the fluorescent signals in cells and determining high expression of said circRNA in a sample if > 15 or more fluorescent signals of circRNA are present in 10% of the cells or in clusters, or >4 fluorescent signals of circRNA are present in 40% of the cells.
6. The use of claim 1, wherein the sample comprises blood, vitreous humor, lymph fluid, synovial fluid, semen, amniotic fluid, milk, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tissue, stool, and preferably the sample is tissue.
7. The use according to any one of claims 1 to 6, wherein the reagent comprises a primer, a probe or a combination thereof, preferably wherein the reagent is a probe, preferably wherein the probe has the sequence shown in SEQ ID No. 1.
8. A tool for diagnosing esophageal cancer, wherein the tool comprises a reagent for detecting the expression level of circRNA in a test sample, wherein the circRNA comprises hsa _ circ _0060927, preferably the tool comprises a kit, a chip or a nucleic acid membrane strip, preferably the esophageal cancer comprises esophageal squamous cell carcinoma and esophageal adenocarcinoma, preferably the esophageal cancer is esophageal squamous cell carcinoma.
9. A device for predicting esophageal cancer, the device comprising:
a processor;
an input module for inputting an expression level of circRNA in a biological sample, said circRNA comprising hsa _ circ _ 0060927.
A computer-readable medium containing instructions that, when executed by the processor, perform an algorithm on the input levels of the biomarkers; and
an output module indicating whether the subject has or is at risk of having esophageal cancer;
preferably, the esophageal cancer comprises esophageal squamous carcinoma and esophageal adenocarcinoma, and preferably, the esophageal cancer is esophageal squamous carcinoma.
The application of circRNA in constructing a computational model for predicting esophageal cancer, wherein the circRNA comprises hsa _ circ _0060927, preferably, the esophageal cancer comprises esophageal squamous carcinoma and esophageal adenocarcinoma, and preferably, the esophageal cancer is esophageal squamous carcinoma.
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