CN114959046A - Kit for detecting lncRNA-NEAT1 in gastric cancer exosomes and application of kit in gastric cancer diagnosis - Google Patents

Kit for detecting lncRNA-NEAT1 in gastric cancer exosomes and application of kit in gastric cancer diagnosis Download PDF

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CN114959046A
CN114959046A CN202210817285.2A CN202210817285A CN114959046A CN 114959046 A CN114959046 A CN 114959046A CN 202210817285 A CN202210817285 A CN 202210817285A CN 114959046 A CN114959046 A CN 114959046A
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gastric cancer
kit
neat1
lncrna
detection
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罗维鹏
于家懿
李悦
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Shandong Baijiawei Biotechnology Co ltd
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Shandong Baijiawei Biotechnology Co ltd
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6851—Quantitative amplification
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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 application of lncRNA-NEAT1 in preparation of a gastric cancer diagnosis marker, and also discloses a kit for detecting lncRNA-NEAT1 in gastric cancer exosomes and application of the kit in diagnostics. The expression quantity of lncRNA NEAT1 in the captured exosomes is detected by using a real-time fluorescent quantitative PCR technology, and the capture method of the gastric cancer cell line exosomes can efficiently capture the gastric cancer exosomes; the in vitro method for early auxiliary diagnosis can provide technical support for early diagnosis and treatment of gastric cancer patients, and has profound significance for development of in vitro diagnosis kits and the like.

Description

Kit for detecting lncRNA-NEAT1 in gastric cancer exosomes and application of kit in gastric cancer diagnosis
Technical Field
The invention relates to tumor cell biology and molecular biology, in particular to a kit for detecting lncRNA-NEAT1 in gastric cancer exosomes and application thereof in gastric cancer serological diagnosis.
Background
Gastric cancer is the fourth largest cancer worldwide, but the onset of gastric cancer is very different geographically, and the incidence rate in asia, especially east asia, is significantly higher than in other regions. China is a big country with gastric cancer, 68 ten thousand gastric cancers are newly developed every year, and the gastric cancer accounts for about 40-50% of gastric cancer patients worldwide. Since there are no obvious clinical symptoms in early stage of gastric cancer, and screening work for early stage gastric cancer is less developed, most of the patients with gastric cancer at present stage are already in advanced stage gastric cancer at the time of diagnosis. The treatment of early gastric cancer mainly comprises surgical resection, and partial patients can be cured by matching radiotherapy and chemotherapy according to the condition of the patient, but the recurrence rate is still 50 percent. The treatment of advanced gastric cancer mainly comprises radiotherapy and chemotherapy. Traditional chemoradiotherapy brings certain survival benefit, but the overall survival rate of the gastric cancer is still not ideal at present, and the 5-year survival rate is about 30%. Early absence of typical symptoms can lead to delayed diagnosis and metastasis. Improving the early diagnosis rate can significantly increase the chances of successful treatment. However, the molecular mechanism of gastric cancer is poorly understood, and it is difficult to find an early effective diagnostic marker. The identification of tumor-associated gene changes can provide a certain idea for the diagnosis and treatment of gastric cancer. High-throughput screening technology makes it possible to identify gene expression profiles, and in 2007, Hutchinson et al obtained by screening through chip technology is named NEAT1 as a long-chain non-coding RNA necessary for maintaining the integrity of subnucleosomes. Approximately 40% of the long non-coding RNAs are transcribed to other genes in an antisense manner, and they regulate the expression of genes encoding adjacent proteins by a cis-acting mechanism and the expression of genes located on other chromosomes by a trans-acting mechanism. Increasing research has shown that long non-coding RNAs play important roles in a variety of tumor biological processes, including tumorigenesis, apoptosis, differentiation, proliferation, angiogenesis, and metastasis.
Exosomes are 30-100nm extracellular microvesicles formed by dynamic endocytosis. Research make internal disorder or usurp has shown that almost all cells produce exosomes and are released in extracellular body fluids, including plasma, urine, saliva, amniotic fluid, ascites, cerebrospinal fluid, and the like. After release from the cell surface, the exosomes have the ability to fuse with the plasma membrane of the recipient cell to deliver its contents into the cytoplasm. The content of exosomes is complex, containing various bioactive proteins, lipids, DNA, miRNA, and the like and serving as mediators in cellular communication by transporting these functional substances. Exosomes contain highly enriched specific proteins such as tumor susceptibility gene 101 protein, CD63, heat shock protein 70, heat shock protein 90, CD9, CD81, etc., which are commonly used as markers for identifying exosomes. The exosomes are key factors for mediating the communication of tumor cells and microenvironment cells, and play an important role in promoting the growth of tumors, tumorigenesis, tumor angiogenesis, tumor immune escape, drug resistance, metastasis and the like. Exosomes in peripheral blood may be derived from tumor cells or released by non-tumor cells, so that DNA information carried by the tumor exosomes is diluted by a large amount of non-tumor cell exosome DNA, and the molecular characteristics of tumors cannot be accurately reflected. This is a great problem in clinical application of exosome. Exosomes are double-layer nano vesicles secreted to the outside of cells to mediate signal transduction and substance transportation by human cells secreted by living cells and cancer living cells. The vesicle mainly comprises membrane proteins, microRNAs, exoDNA and the like, is not easy to degrade, and can reflect the health condition information of a patient in real time. Although CD63, CD9, CD81, TSG101 and the like are commonly used for detecting exosomes, they do not have tumor specificity, and thus cause low sensitivity and specificity in tumor gene diagnosis.
Disclosure of Invention
The invention aims to provide a new application of long-chain non-coding lncRNA-NEAT1, in particular to an application of lncRNA-NEAT1 as an exosome diagnosis marker of a gastric cancer cell line and an application in preparation of a liver cancer serological diagnosis reagent or a kit.
The invention also aims to provide a kit for detecting ncRNA-NEAT1 in the exosomes of the serum or the gastric cancer cell line and application of the kit in diagnosis.
The lncRNA-NEAT1 is applied to the preparation of a detection reagent or a detection kit for diagnosing gastric cancer, and the biological sample is selected from: gastric cancer exosomes, fresh tissues or cells, formalin-fixed or paraffin-embedded tissues or cells, blood or body fluids obtained from a subject.
In the prior art, serum is used as a detection object, and the invention also provides a method for using cancer cell exosome as a detection object.
Correspondingly, the invention provides a method for capturing gastric cancer exosomes, wherein the method for capturing gastric cancer cell line MKN-45 exosomes can efficiently capture gastric cancer exosomes; can provide technical support for the development and the expansion of gastric cancer diagnosis technology.
The adopted technical scheme comprises the following steps: s1, screening specific exosome surface protein from a MKN-45 gastric cancer cell line culture system; s2, based on the combination of molecular markers CXCR4, CEA and Claudin 18.2, based on the principle of mutual combination of antigen and antibody, producing monoclonal antibody of CXCR4, CEA and Claudin 18.2 molecules, and specifically binding with exosome from gastric cancer cells; s3 designs an exosome immune-capture system coupled with the molecular combination marker, and efficiently enriches exosomes derived from gastric cancer cells.
Further, the method includes step S4 of binding the antibody to the magnetic bead to make the magnetic bead a carrier for the antibody.
Further, S5 is included for collecting free exosomes by an elution step.
The method for capturing the cancer exosomes further comprises the step S6 of obtaining data of the obtained cancer cell line exosomes through a real-time fluorescence quantitative PCR technology, then analyzing the data through software, and evaluating diagnosis and disease progression of the gastric cancer by comparing PCR data of the exosomes of a normal person and a patient.
The core of the application of lncRNA-NEAT1 in preparing a detection reagent or a detection kit for diagnosing gastric cancer is to detect the expression level of lncRNA-NEAT1 in a biological sample.
The detection reagent or the kit comprises: probes, gene chips, or PCR primers with detection specificity for lncRNA-NEAT 1.
Further, the invention provides a group of PCR primers with detection specificity for lncRNA-NEAT1, and the specific sequences are as follows:
lncRNA NEAT1 upstream primer: 5'-GGCAGGTCTAGTTTGGGCAT-3' the flow of the air in the air conditioner,
a downstream primer: 5'-CCTCATCCCTCCCAGTACC-3';
internal control GAPDH upstream primer: 5'-GCAACTAGGATGGTGTGGCT-3' the flow of the air in the air conditioner,
a downstream primer: 5'-TCCCATTCCCCAGCTCTCATA-3' are provided.
The kit provided by the invention comprises:
A. the reverse transcription system consists of a reverse transcription system buffer solution, a primer, dNTPs, reverse transcriptase and an RNase inhibitor;
B. a primer system, a primer set and a primer set,
lncRNA NEAT1 upstream primer: 5'-GGCAGGTCTAGTTTGGGCAT-3' the flow of the air in the air conditioner,
a downstream primer: 5'-CCTCATCCCTCCCAGTACC-3', respectively;
internal control GAPDH upstream primer: 5'-GCAACTAGGATGGTGTGGCT-3' the flow of the air in the air conditioner,
a downstream primer: 5'-TCCCATTCCCCAGCTCTCATA-3'
C. The amplification system consists of reagents of a SYBR Green Mix kit.
The invention also provides a detection method by using the kit, which comprises the following specific steps:
extracting total RNA from tissue sample with Trizol reagent (Invitrogen, USA), measuring RNA concentration and purity with NanoDrop2000 photometer, performing reverse transcription reaction with kit to obtain cDNA, amplifying cDNA product with SYBR Green Mix kit in 2720 type thermal cycle PCR system,
lncRNA NEAT1 upstream primer: 5'-GGCAGGTCTAGTTTGGGCAT-3' the flow of the air in the air conditioner,
a downstream primer: 5'-CCTCATCCCTCCCAGTACC-3', respectively;
internal control GAPDH upstream primer: 5'-GCAACTAGGATGGTGTGGCT-3', and the adhesive tape is used for adhering the film to a substrate,
a downstream primer: 5'-TCCCATTCCCCAGCTCTCATA-3' are provided.
The PCR amplification conditions include the following three steps: the first step is at 95.0 ℃ for 2 min; the second step is 95.0 ℃ for 15s and 58 ℃ for 40s (40 cycles); third step (separation phase), NEAT1 and GAPDH were automatically recorded at 95.0 ℃ for 15s, 60.0 ℃ for 15s, and 95.0 ℃ for 15s, and the relative expression of lncRNA NEAT1 was expressed as 2-. DELTA.Ct.
Has the advantages that:
the method for capturing the gastric cancer cell line exosomes can efficiently capture the gastric cancer exosomes; the in-vitro method for early auxiliary diagnosis can provide technical support for early diagnosis and treatment of gastric cancer patients, and has far-reaching significance for development of in-vitro diagnosis kits and the like.
Drawings
FIG. 1 shows Ct values of experimental group (MKN-45 gastric cancer cell exosomes) and control group (normal human peripheral blood exosomes).
FIG. 2 is a graph showing the relative expression amounts of lncRNA NEAT1 in the experimental group (MKN-45 gastric cancer cell exosome) and the control group (normal human peripheral blood exosome).
FIG. 3 is a graph comparing the relative expression levels of lncRNA NEAT1 in the experimental group (MKN-45 gastric cancer cell exosomes) and the control group (normal human peripheral blood exosomes).
Detailed Description
Example 1
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only preferred embodiments of the present invention, not all embodiments. The use and purpose of these exemplary embodiments are to illustrate the present invention only, and do not constitute any limitation to the actual scope of the present invention in any form, rather, the scope of the present invention is limited thereto.
Collecting peripheral blood of a normal person, standing the peripheral blood by using a heparin anticoagulation tube or an anticoagulation blood collection tube for 5-10 minutes, centrifuging at 3000 rpm for 15 minutes at a low speed, taking supernatant for later use, transferring the supernatant into a new EP tube without RNA enzyme for later use, and storing the supernatant in liquid nitrogen at-80 ℃ for later use if long-term storage is required.
II, MKN-45 human gastric cancer cell culture operation: (1) and (3) cell recovery: the tube containing 1mL of cell suspension was thawed quickly by shaking in a 37 ℃ water bath, and 4mL of medium was added and mixed well. Centrifuging at 1000RPM for 4 min, discarding supernatant, adding 1-2mL culture medium, and blowing. All cell suspensions were then added to the flask and incubated overnight (or the cell suspensions were added to a 10cm dish and approximately 6ml of medium was added and incubated overnight). The next day the fluid was changed and cell density was checked. (2) Cell passage: if the cell density reaches 80-90%, subculture can be carried out. Firstly, discarding culture supernatant, and rinsing cells for 1-2 times by PBS (phosphate buffer solution) without calcium and magnesium ions; secondly, adding 1ml of digestive juice (0.25% Trypsin-0.53mM EDTA) into a culture bottle, placing the culture bottle in an incubator at 37 ℃ for digesting for 1-2 minutes, observing the digestion condition of cells under a microscope, if most of the cells become round and fall off, quickly taking back to an operation table, and adding a small amount of culture medium after tapping a few culture bottles to stop digesting; supplementing culture medium according to 6-8 mL/bottle, gently beating uniformly, sucking out, centrifuging for 4 minutes under the condition of 1000RPM, discarding supernatant, supplementing 1-2mL culture solution, and blowing uniformly; finally, the cell suspension was mixed according to 1: 2 portions were dispensed into new dishes or flasks containing 6ml of medium.
Thirdly, separating and purifying normal human peripheral blood exosomes: centrifuge at 300 Xg for 10 min and collect the supernatant. Centrifuge at 2000 Xg for 10 min and take the supernatant. Centrifuge at 10,000 Xg for 30 minutes and collect the supernatant. 100,000 Xg, at 4 ℃ for 90 minutes, removing the supernatant, the remaining precipitation PBS heavy suspension, again 100,000 Xg centrifugation for 90 minutes. FIG. 1 is a differential ultracentrifugation profile of exosomes. Differential ultracentrifugation is the "gold standard" for the isolation of exosomes, achieved using multiple cycles of centrifugation at centrifugal force from 300 × g to 100,000 × g. After each centrifugation step, the pellets, such as cells, cell debris, apoptotic bodies, etc., were removed, and the supernatant was collected and centrifuged. After final centrifugation, the supernatant was removed and the exosomes were collected containing pellet and contaminating proteins and the centrifugation was performed at 4 ℃.
The specificity enrichment of the exosomes derived from the gastric cancer cell line MKN-45, although CD63, CD9, CD81, TSG101 and the like are commonly used for detecting the exosomes, the exosomes do not have tumor specificity, and therefore the sensitivity and specificity of tumor gene diagnosis are low. Besides high-speed centrifugal separation of exosomes, immunomagnetic bead screening is also a main method for separating exosomes, and exosomes from tumor sources and exosomes from non-tumor sources can be distinguished by specific molecular markers on the surface of the tumor exosomes. The immunocapture of the exosome from the gastric cancer cell line mainly depends on a specific molecular marker on the surface of the exosome, so that the exosome from the gastric cancer is ensured not to be diluted by the exosome from the peripheral blood, and the sensitivity and specificity of early screening and diagnosis of the gastric cancer are improved. The molecular marker combination CXCR4, CEA, Claudin 18.2 are taken as the basis to combine pannel, and monoclonal antibodies of CXCR4, CEA, Claudin 18.2 molecules are produced by the principle of combining antigen and antibody with each other, so as to specifically bind with exosomes from pancreatic cancer. On the basis, an exosome immune capture system coupled with the molecular combination marker is designed to enrich exosomes from gastric cancer sources. After a certain treatment, the antibody can be combined on the magnetic beads, so that the magnetic beads become carriers of the antibody. The antibody on the magnetic bead is combined with the specific antigen substance to form an antigen-antibody-magnetic bead immune complex, and the complex moves under the action of a magnetic field, so that the complex is complex, the purpose of separating the specific antigen is achieved, and the method is a key technology for separating exosomes from gastric cancer.
As shown in fig. 2, exosomes can be enriched on solid matrices after first immobilizing antibodies recognizing exosome-specific markers on such solid matrices and incubating the exosome-containing liquids with antibody-conjugated solid matrices. Free exosomes may be collected by additional elution steps.
And fifthly, analyzing the expression of the lncRNA NEAT1 in the exosome of the gastric cancer cell line by a real-time fluorescence quantitative PCR technology.
Total RNA was extracted from the tissue sample using Trizol reagent (Invitrogen, usa) using the exosomes obtained by the above method. RNA concentration and purity were determined using a NanoDrop2000 luminometer. And carrying out reverse transcription reaction by using the kit to obtain cDNA. The CDNA product was amplified using the SYBR GREEN MIX kit in a 2720 thermal cycling PCR system. LNCRNA NEAT1 upstream primer: 5'-GGCAGGTCTAGTTTGGGCAT-3', downstream primer: 5'-CCTCATCCCTCCCAGTACC-3', respectively; internal control GAPDH upstream primer: 5'-GCAACTAGGATGGTGTGGCT-3', downstream primer: 5'-TCCCATTCCCCAGCTCTCATA-3' are provided. The PCR amplification conditions include the following three steps: the first step is at 95.0 ℃ for 2 min; the second step is 95.0 ℃ for 15s and 58 ℃ for 40s (40 cycles); in the third step (separation phase), NEAT1 and GAPDH were automatically recorded at 95.0 ℃ for 15s, 60.0 ℃ for 15s, and 95.0 ℃ for 15 s. The relative expression level of IncRNA NEAT1 was expressed as 2-. DELTA.Ct. The experiment was repeated 3 times.
The raw data of the Q-PCR experiment are shown in FIGS. 1 and 2. FIG. 3 shows the relative expression levels of IncRNA NEAT1 in the experimental group (MKN-45 gastric cancer cell exosome) and the control group (normal human peripheral blood exosome)
The invention designs an exosome immune capture system coupled with the molecular combination markers by combining the molecular markers CXCR4, CEA and Claudin 18.2 as the basis, and can efficiently obtain exosomes enriched with gastric cancer sources. The lncRNA NEAT1 is detected by real-time fluorescent quantitative PCR, and auxiliary diagnosis and disease progress prediction are carried out on early gastric cancer patients by analyzing the expression quantity of the gene, so that the kit has great significance for subsequent treatment of gastric cancer.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention are included in the scope of the present invention.

Claims (10)

  1. Application of lncRNA-NEAT1 in preparation of gastric cancer diagnostic markers.
  2. Application of lncRNA-NEAT1 in preparation of detection reagents or detection kits for diagnosing gastric cancer.
  3. 3. The use of incRNA-NEAT 1 in the preparation of a detection reagent or a detection kit for the diagnosis of gastric cancer according to claim 2, wherein the detection reagent or the detection kit is a serological detection reagent or kit.
  4. 4. The use of incRNA-NEAT 1 in the preparation of a detection reagent or a detection kit for the diagnosis of gastric cancer according to claim 2 or 3, wherein the detection reagent or the kit is used for detecting the expression level of incRNA-NEAT 1 in a biological sample.
  5. 5. Use of lncRNA-NEAT1 according to claim 2 or 3 in preparing a detection reagent or a detection reagent for diagnosing gastric cancer
    Use in a kit, wherein said detection reagent or kit comprises: probes, gene chips, or PCR primers with detection specificity for lncRNA-NEAT 1.
  6. 6. The use of incRNA-NEAT 1 in the preparation of a detection reagent or a detection kit for the diagnosis of gastric cancer according to claim 4, wherein the biological sample is selected from the group consisting of: gastric cancer exosomes, fresh tissues or cells, formalin-fixed or paraffin-embedded tissues or cells, blood or body fluids obtained from a subject.
  7. 7. The use of incRNA-NEAT 1 in the preparation of a detection reagent or a detection kit for the diagnosis of gastric cancer according to claim 4, wherein the biological sample is gastric cancer exosomes.
  8. 8. The use of incRNA-NEAT 1 in the preparation of a detection reagent or a detection kit for the diagnosis of gastric cancer according to claim 5, wherein the PCR primers with detection specificity for incRNA-NEAT 1 are as follows:
    lncRNA NEAT1 upstream primer: 5'-GGCAGGTCTAGTTTGGGCAT-3' the flow of the air in the air conditioner,
    a downstream primer: 5'-CCTCATCCCTCCCAGTACC-3', respectively;
    internal control GAPDH upstream primer: 5'-GCAACTAGGATGGTGTGGCT-3' the flow of the air in the air conditioner,
    a downstream primer: 5'-TCCCATTCCCCAGCTCTCATA-3' are provided.
  9. 9. A kit for detecting lncRNA-NEAT1 in gastric cancer exosomes, which is characterized by comprising:
    A. the reverse transcription system consists of a reverse transcription system buffer solution, a primer, dNTPs, reverse transcriptase and an RNase inhibitor;
    B. a primer system, a primer set and a primer set,
    lncRNA NEAT1 upstream primer: 5'-GGCAGGTCTAGTTTGGGCAT-3' the flow of the air in the air conditioner,
    a downstream primer: 5'-CCTCATCCCTCCCAGTACC-3', respectively;
    internal control GAPDH upstream primer: 5'-GCAACTAGGATGGTGTGGCT-3' the flow of the air in the air conditioner,
    a downstream primer: 5'-TCCCATTCCCCAGCTCTCATA-3'
    C. The amplification system consists of reagents of a SYBR Green Mix kit.
  10. 10. The detection method by using the kit for detecting lncRNA-NEAT1 in gastric cancer exosomes as shown in claim 9 comprises the following specific steps:
    extracting total RNA from tissue sample with Trizol reagent (Invitrogen, USA), measuring RNA concentration and purity with NanoDrop2000 photometer, performing reverse transcription reaction with kit to obtain cDNA, amplifying cDNA product with SYBR Green Mix kit in 2720 type thermal cycle PCR system,
    lncRNA NEAT1 upstream primer: 5'-GGCAGGTCTAGTTTGGGCAT-3' the flow of the air in the air conditioner,
    a downstream primer: 5'-CCTCATCCCTCCCAGTACC-3', respectively;
    internal control GAPDH upstream primer: 5'-GCAACTAGGATGGTGTGGCT-3' the flow of the air in the air conditioner,
    a downstream primer: 5'-TCCCATTCCCCAGCTCTCATA-3', respectively;
    the PCR amplification conditions include the following three steps: the first step is at 95.0 ℃ for 2 min; the second step is 95.0 ℃ for 15s and 58 ℃ for 40s (40 cycles); third step (separation phase), NEAT1 and GAPDH were automatically recorded at 95.0 ℃ for 15s, 60.0 ℃ for 15s, and 95.0 ℃ for 15s, and the relative expression of lncRNA NEAT1 was expressed as 2-. DELTA.Ct.
CN202210817285.2A 2022-07-12 2022-07-12 Kit for detecting lncRNA-NEAT1 in gastric cancer exosomes and application of kit in gastric cancer diagnosis Pending CN114959046A (en)

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