CN112779335A - Colorectal cancer early metastasis diagnosis kit based on exosome LncSENP6 expression level - Google Patents

Colorectal cancer early metastasis diagnosis kit based on exosome LncSENP6 expression level Download PDF

Info

Publication number
CN112779335A
CN112779335A CN202110138419.3A CN202110138419A CN112779335A CN 112779335 A CN112779335 A CN 112779335A CN 202110138419 A CN202110138419 A CN 202110138419A CN 112779335 A CN112779335 A CN 112779335A
Authority
CN
China
Prior art keywords
exosome
colorectal cancer
plasma
lncsenp6
metastasis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202110138419.3A
Other languages
Chinese (zh)
Inventor
李纪鹏
张健
朱军
郝俊
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Air Force Medical University of PLA
Original Assignee
Air Force Medical University of PLA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Air Force Medical University of PLA filed Critical Air Force Medical University of PLA
Priority to CN202110138419.3A priority Critical patent/CN112779335A/en
Publication of CN112779335A publication Critical patent/CN112779335A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a colorectal cancer early metastasis diagnosis kit based on an expression level of an exosome LncSENP6, which comprises a real-time quantitative PCR amplification primer of an exosome LncSENP 6. Collecting peripheral blood of a patient with colorectal cancer non-metastasis, centrifuging to obtain plasma, extracting exosome total RNA in the plasma, reversely transcribing the exosome total RNA into cDNA serving as a template, and detecting the content of exosome LncSENP6 in the plasma through real-time quantitative PCR (polymerase chain reaction), thereby providing important reference for predicting the colorectal cancer early-stage metastasis.

Description

Colorectal cancer early metastasis diagnosis kit based on exosome LncSENP6 expression level
Technical Field
The invention belongs to the field of tumor medicine, and relates to a diagnostic kit for detecting the content of exosome LncSENP6 in a plasma sample and predicting early colorectal cancer metastasis according to the expression condition of exosome LncSENP 6.
Background
Colorectal cancer (CRC) is one of the most common digestive tract malignancies and is characterized by high morbidity and mortality, which increase year by year. The data show that the main reasons for failure of colorectal cancer therapy are local recurrence and distant metastasis after surgery. Of these, approximately 23% of patients have metastatic disease at the time of visit, approximately 40% of patients develop metastasis during treatment, and metastasis of colorectal cancer is responsible for more than half of patients' deaths, and the 5-year overall survival rate for metastatic colorectal cancer patients is less than 10%. 15% of patients benefit from early diagnosis and early treatment of liver metastasis, and the 5-year survival rate can be increased to 40% -74%, so that detection of early colorectal cancer metastasis is crucial to improving the survival rate of patients. Early diagnosis and prediction of the occurrence of metastasis in colorectal cancer patients has become one of the hot spots of clinical research in recent years.
Many studies are currently focused on biomarkers that can predict the metastatic sites of primary tumors. However, since the detection of serum tumor markers is not valuable for predicting metastasis, tumor markers such as CEA and CA19-9 can only predict the probability of tumor occurrence, but cannot predict whether tumor metastasis occurs. One of the exosomes functions as a mediator of intercellular communication, closely related to the proliferation, apoptosis, drug resistance and metastasis of tumors. Non-coding RNA, one of the major active substances, is the biological basis on which tumor exosomes exert the above functions. The non-coding RNA mainly includes micro RNA (miRNA), long non-coding RNA (lncRNA), circular RNA (ciRNA) and the like. The functional studies of LncRNA and miRNA are more complete and thorough. Long non-coding RNAs are a class of transcripts that are greater than 200 nucleotides in length. With the intensive research on LncRNAs, a great deal of research has confirmed that LncRNAs are differentially expressed in tumor tissues and play an important role in the development of tumors. LncRNAs in tumor cells can be encapsulated into exosomes and enter blood circulation through cell secretion, so that the exosomes LncRNAs become a potential tumor diagnosis and prognosis biomarker.
At present, no research report about the differential expression and related functions of LncSENP6 is found. NONC ODE TRANSCRIPT ID of LncSENP6 is NONHSAT 251227.1; NONCODE Gene ID NONHSAG044151.3, Chromosome chr6, Start Site 75285013, End Site 75296757.
Disclosure of Invention
The invention aims to provide a colorectal cancer early metastasis diagnosis kit based on the expression level of an exosome LncSENP6, which can improve the accuracy of colorectal cancer early metastasis diagnosis and improve the prognosis condition of a colorectal cancer patient by analyzing the content of the exosome LncSENP6 in plasma.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting the content of exosome LncSENP6 in plasma comprises the following steps:
1) extracting exosome total RNA in plasma of a colorectal cancer patient;
2) amplifying a cDNA sequence corresponding to an exosome LncSENP6 in plasma by real-time quantitative PCR by using a reverse transcript of the exosome total RNA (the reverse transcript is cDNA obtained by reverse transcription of the exosome total RNA) as a template;
3) after the amplification is finished, the amplification cycle threshold Ct of the cDNA sequence corresponding to the exosome LncSENP6 (referred to as the amplification cycle threshold Ct of the exosome LncSENP 6) is determined.
Preferably, the step 1) specifically comprises the following steps: peripheral blood of a collected colorectal cancer non-metastatic patient (for example, a metastatic focus is not found by imaging detection) is centrifuged to obtain plasma, and exosome total RNA is extracted from the plasma.
Preferably, in the step 2), the amplification primer pair for real-time quantitative PCR is:
upstream primer LncSENP 6F: 5' -CTCTGTTGGCCGTGGCTTTA-3 ″
Downstream primer LncSENP 6R: 5 '-TACTGCTAATCCTCCCCCGA-3'.
Preferably, in the step 2), the amplification reaction system of the real-time quantitative PCR comprises 40-400 nmol/L of upstream primer and 40-400 nmol/L of downstream primer.
Preferably, in the step 2), the amplification reaction procedure of the real-time quantitative PCR is as follows: 1) 30s at 95 ℃; 2) 5s at 95 ℃ and 30-34 s at 60 ℃ for 40 cycles.
A kit for diagnosing early colorectal cancer metastasis, which comprises a detection reagent for analyzing the content of exosome lncsnp 6 in the plasma of a colorectal cancer patient.
Preferably, the detection reagent comprises a real-time quantitative PCR amplification primer pair (namely the upstream primer LncSENP6F and the downstream primer LncSENP6R) of a cDNA sequence corresponding to the exosome LncSENP 6.
Preferably, when the amplification cycle threshold Ct or the relative expression quantity of the exosome LncSENP6 in the plasma of the colorectal cancer non-metastatic patient is within a limit value (for example, the amplification cycle threshold Ct of LncSENP6 is less than or equal to 27, and the amplification cycle threshold Ct of the internal reference gene is 18-20), the expression level of the exosome LncSENP6 in the plasma of the colorectal cancer non-metastatic patient is high, and the transfer risk is avoided, and when the amplification cycle threshold Ct or the relative expression quantity of the exosome LncSENP6 in the plasma of the colorectal cancer non-metastatic patient exceeds the limit value (for example, the amplification cycle threshold Ct of LncSENP6 is greater than 27, and the amplification cycle threshold Ct of the internal reference gene is 18-20), the expression level of the exosome LncSENP6 in the plasma of the patient is low, and the transfer risk is avoided.
The invention has the beneficial effects that:
according to the invention, a diagnostic kit is developed according to exosomes LncSENP6 (expression of exosomes LncSENP6 in plasma of a colorectal cancer metastasis patient is down-regulated by analysis and found out that the exosomes LncSENP6 in the plasma of the colorectal cancer metastasis patient is verified to be consistent with a sequencing result) screened by high-throughput sequencing, the occurrence of early colorectal cancer metastasis can be predicted by detecting the expression of exosome specificity LncSENP6 in the plasma of a colorectal cancer non-metastasis patient, and the early colorectal cancer metastasis is not easily influenced by other basic diseases, so that an accurate and reliable reference standard is provided for clinical medication and nutrition support of the colorectal cancer patient. The diagnosis kit is developed based on mature technologies such as exosome RNA extraction, LncRNA detection and the like, only a small amount of blood needs to be collected, the diagnosis kit is suitable for most colorectal cancer patients, can quickly and conveniently obtain a diagnosis result, and provides a new target for molecular mechanism research and diagnosis technology development of colorectal cancer metastasis.
Furthermore, the invention can rapidly, accurately and reliably detect the expression change of the exosome-specific LncSENP6 in the plasma of the colorectal cancer patient by designing the sequence of the real-time quantitative PCR amplification primer.
Drawings
Figure 1A is electron microscopy (TEM) identification of extracted exosomes.
Fig. 1B is Nanoparticle Tracking Analysis (NTA) of extracted exosomes.
FIG. 1C is a Western Blotting identification of the extracted exosomes.
FIG. 2 shows the results of high throughput sequencing of plasma exosomes from 5 patients with colorectal carcinoma in situ and 5 patients with colorectal carcinoma lymph node metastasis.
FIG. 3 shows the expression of LncSENP6 in plasma of colorectal cancer patients: wherein: n0 represents colorectal carcinoma in situ group; n + represents colorectal cancer lymph node metastasis group; p < 0.05 with statistical differences.
FIG. 4 shows the expression of LncSENP6 in the tissues of colorectal cancer patients; wherein: p < 0.001, with a very significant statistical difference in LncSENP6 expression in colorectal cancer tissue (Tumor) and para-cancer tissue (Normal).
Detailed Description
The present invention will be described in further detail with reference to the following drawings and examples, which are illustrative of the present invention and are not intended to limit the scope of the present invention.
1. Collection of samples
Registering detailed information of a patient, collecting peripheral blood of a colorectal cancer patient by using a 5mL anticoagulation blood collection tube, centrifuging a peripheral blood sample at 4 ℃ for 15min at 1000g within 30min after collection, and taking supernatant, namely plasma; collecting cancer tissue specimens and tissue specimens beside cancer of patients with colorectal cancer at the same time; respectively marking and freezing to-80 ℃ in a refrigerator. The sample for each patient was divided into three parts: plasma, tissue adjacent to cancer, cancer tissue. The collection place comprises: the first subsidiary hospital of the university of military medical and air force for digestive diseases hospital; collecting time: 8 months in 2019-2020 and 8 months; peripheral blood and tissues are collected on the same day, peripheral blood is collected in the morning of the operation day, and tissues are collected during the operation.
2. Screening LncRNAs related to colorectal cancer lymph node metastasis in plasma exosomes of colorectal cancer patients
2.1 plasma exosome extraction and whole transcriptome sequencing
Plasma exosomes were randomly extracted from 5 colorectal carcinoma-in-situ patients and 5 colorectal carcinoma lymph node metastasis patients, and the exosome samples cryopreserved at-70 ℃ were sent to Beijing Enzekangtai Biotechnology Ltd for whole transcriptome sequencing of the samples.
Wherein, the extraction of plasma exosome adopts exosome to draw the kit fast (the use of kit is divided into two stages, and exosome is drawn in first stage), includes the following step:
1) collecting peripheral blood of patients with colorectal cancer by using a 5mL anticoagulation blood collection tube, centrifuging the sample at 4 ℃ for 15min at 1000g within 30min after collection, and taking supernatant, namely plasma;
2) the resulting plasma (1-2 mL) was filtered into 5mL EP tubes using a 0.8 μm filter to remove cell debris and larger cell vesicles;
3) adding an equal volume of XBP Buffer (the ratio of the plasma and the XBP Buffer in the previous step is 1:1) into an EP tube, immediately and gently reversing and uniformly mixing for 5 times;
4) transferring the mixed solution to exoEasy spin column, centrifuging for 1min at 500g, discarding the liquid, and returning the column to the collection tube (if liquid is still on the membrane of the column, centrifuging for 1min again at 5000 g);
5) adding 3.5mL of XWP Buffer into a centrifugal column, centrifuging for 5min at 5000g, and discarding the liquid and a collecting pipe;
6) placing the centrifugal column in a new collecting tube, eluting with 400 μ L XE Buffer, and centrifuging for 5min at 5000g to obtain eluate, i.e. exosome solution.
Referring to fig. 1A, 1B and 1C, the identification results of the extracted exosomes are specifically as follows:
1) and (3) displaying by an electron microscope: the exosome has complete morphological structure and uniform distribution;
2) nanoparticle Tracking Analysis (NTA) results show: the grain diameter (diameter) of the exosome is about 125 nm;
3) western Blotting showed: exosomes express the membrane marker molecules CD9, CD81, and the intrabursal marker molecule TSG-101.
2.2 screening of differentially expressed LncRNAs associated with colorectal cancer lymph node metastasis
Based on the results of high throughput sequencing of the whole transcriptome of plasma exosomes of 5 colorectal carcinoma in situ patients and 5 colorectal carcinoma lymph node metastasis patients, changes in expression profiles of LncRNAs in plasma exosomes of colorectal carcinoma in situ patients and colorectal carcinoma lymph node metastasis patients were analyzed, and 5 LncRNAs with the largest differential expression fold in exosomes of colorectal carcinoma lymph node metastasis patients were selected as candidate molecules, wherein lncSENP6 (FIG. 2) and another lncRNA were confirmed from clinical samples and were consistent with the sequencing results.
3. Verifying the relationship between LncSENP6 and colorectal cancer metastasis
3.1, screening candidate molecules LncSENP6 with large differential expression fold related to colorectal cancer lymph node metastasis according to a high-throughput sequencing result, and verifying the expression condition of the candidate molecules LncSENP6 in plasma exosomes of colorectal cancer metastasis patients through real-time quantitative PCR.
3.1.1LncSENP6 and design of primers for amplifying reference genes
LncSENP6F：5`-CTCTGTTGGCCGTGGCTTTA-3`
LncSENP6R：5`-TACTGCTAATCCTCCCCCGA-3`
The upstream primer GAPDHF: 5' -CTCCTCCACCTTTGACGCTG-3 ″
The downstream primer GAPDHR: 5' -TCCTCTTGTGCTCTTGCTGG-3 ″
3.1.2 Total RNA extraction from plasma exosomes of patients with colorectal cancer
The method for extracting the total RNA of the exosome adopts an exosome rapid extraction kit (the use of the kit is divided into two stages, and the total RNA of the exosome is extracted in the second stage), and comprises the following steps:
1) collecting peripheral blood of patients with colorectal cancer by using a 5mL anticoagulation blood collection tube, centrifuging the sample at 4 ℃ for 15min at 1000g within 30min after collection, and taking supernatant, namely plasma;
2) the resulting plasma (1-2 mL) was filtered into 5mL EP tubes using a 0.8 μm filter to remove cell debris and larger cell vesicles;
3) adding an equal volume of XBP Buffer (the ratio of the plasma and the XBP Buffer in the previous step is 1:1) into an EP tube, immediately and gently reversing and uniformly mixing for 5 times;
4) transferring the mixed solution to exoEasy spin column, centrifuging for 1min at 500g, discarding the liquid, and returning the column to the collection tube (if liquid is still on the membrane of the column, centrifuging for 1min again at 5000 g);
5) adding 3.5mL of XWP Buffer into a centrifugal column, centrifuging for 5min at 5000g, and discarding the liquid and a collecting pipe;
6) placing the spin column in a new collection tube;
7) adding 700. mu.L of QIAzol to the membrane of the centrifugal column, centrifuging for 5min at 5000g, obtaining lysate and transferring to a 2mL EP tube;
8) vortex briefly and shake, and incubate for 5min at room temperature (15-25 ℃);
9) adding 90 mu L of chloroform into the EP tube, tightly covering the EP tube, and violently shaking for 15 s;
10) incubating for 2-3 min at room temperature (15-25 ℃);
11) centrifuging at 12000g and 4 ℃ for 15 min;
12) transferring the uppermost layer water sample liquid to a new 2mL EP tube, adding anhydrous ethanol with the volume twice that of the EP tube, and blowing and beating the mixture for a plurality of times by using a liquid transfer gun to uniformly mix the mixture;
13) placing RNeasy MinElute spin column centrifugal column in 2mL collecting tube, sucking 700 μ L of the mixed solution obtained in step 12), transferring to centrifugal column, covering with cover, centrifuging at 12000g room temperature for 15s, and discarding liquid;
14) sucking the residual mixed liquid in the step 12), repeating the step 13), transferring and centrifuging, and discarding the liquid;
15) adding 700 μ L of RWT Buffer to the centrifugal column, covering the column with a cover, centrifuging at 12000g for 15s, and discarding the liquid;
16) adding 500 μ L of RPE Buffer into the centrifugal column, covering the centrifugal column, centrifuging at 12000g for 15s, and discarding the liquid;
17) adding 500 μ L of RWT Buffer into the centrifugal column, covering the centrifugal column with a cover, centrifuging at 12000g for 2min, and discarding the liquid;
18) placing the centrifugal column in a new 2mL collecting tube, opening a cover, centrifuging for 5min at the maximum rotating speed of a centrifuge to air-dry the membrane of the centrifugal column, and discarding the collecting tube;
19) placing the centrifugal column in a new collecting tube of 1.5mL, adding 14 μ L of RNase-free water on the central membrane of the centrifugal column, covering the cover tightly, standing for 1min, then centrifuging at full speed for 1min to elute RNA, thus obtaining an exosome total RNA solution, and carrying out RNA concentration determination.
3.1.3 reverse transcription
Mu.g of total exosome RNA was collected and subjected to reverse transcription using a reverse transcription kit (Prime Script RT reagent kit) of TaKaRa, and the reaction system (20. mu.L) was: 5 XPrime Script RT Master Mix 4. mu.L, supplementary RNase Free dH2O to 20. mu.L. The reaction conditions are as follows: 15min at 37 ℃; 5s at 85 ℃ and 10min at 4 ℃.
Diluting cDNA in the reaction system (1:2) with ultrapure water, and further adjusting the cDNA content according to the quantitative result of the internal reference gene, wherein the Ct value of the internal reference gene is about 18 generally.
3.1.4 Real-time quantitative PCR (Real-time PCR)
The Real-time PCR reaction was performed using TB Green Premix Ex Taq II reagent from TaKaRa, three replicates per sample.
The reaction system is 10 μ L: 2 XTB Green Premix Ex Taq II 5. mu.L, 10. mu.M upstream primer 0.4. mu.L, 10. mu.M downstream primer 0.4. mu. L, cDNA template 1. mu. L, ROX 0.2.2. mu.L, and deionized water to 10. mu.L.
On a Roche LightCycler 96 real-time quantitative PCR instrument, the method is carried out according to a real-time quantitative PCR two-step amplification standard program:
stage 1 pre-denaturation: 30s at 95 ℃;
stage 2PCR reaction: 5s at 95 ℃ and 30s at 60 ℃ for 40 cycles.
After the reaction was completed, data analysis was performed. After adjusting the baseline cycle and calculating the threshold, adopting 2 according to the Ct value automatically obtained by the instrument-ΔΔCtThe expression level of LncSENP6 relative to the reference Gene (GAPDH) was calculated by the method.
Gene expression was detected in plasma samples from 94 colorectal cancer patients by Real-time PCR. Among them, 46 patients with colorectal cancer without metastasis and 48 patients with colorectal cancer with metastasis. The results show (fig. 3) that lncsnp 6 expression levels were lower in plasma exosomes of colorectal cancer metastasis group patients compared to the non-metastasis group, and were statistically different.
3.2 according to the high-throughput sequencing result, screening candidate molecules LncSENP6 with large differential expression fold related to colorectal cancer lymph node metastasis, and verifying the expression condition of the candidate molecules LncSENP6 in colorectal cancer metastasis patient tissues through Real-time PCR.
3.2.1LncSENP6 and design of primers for amplifying reference genes
LncSENP6F：5`-CTCTGTTGGCCGTGGCTTTA-3`
LncSENP6R：5`-TACTGCTAATCCTCCCCCGA-3`
The upstream primer GAPDHF: 5' -CTCCTCCACCTTTGACGCTG-3 ″
The downstream primer GAPDHR: 5' -TCCTCTTGTGCTCTTGCTGG-3 ″
3.2.2 extraction of RNA from cancer tissue and tissue adjacent to cancer of patient with colorectal cancer
1) Firstly, removing fibers and fat of tumor tissues and tissues beside cancer, adding the cleaned fibers and fat into an EP (EP) tube, and simultaneously adding two grinding beads with the diameter of 2mm and 2mL of RNase-free into the EP tube;
2) adding 1mL of precooled Trizol into each tube of the sample after the first-step treatment, placing an EP tube into a high-speed tissue grinder, and grinding the tissue for 60s (60 HZ);
3) adding 200 μ L chloroform into EP tube, shaking, mixing, standing for 5min for layering;
4) centrifuging at 4 deg.C and 12000rpm for 15min, and sucking about 400 μ L of upper layer water sample liquid into a new 1.5mL EP tube with 200 μ L pipette;
5) adding isopropanol with the same volume, mixing well, standing at room temperature for 10 min;
6) centrifuging at 4 deg.C and 12000rpm for 15min, and removing supernatant;
7) adding 1mL of 75% ethanol, fully washing, centrifuging at 7500rpm for 5min at 4 ℃, and removing supernatant;
8) placing the EP tube in a super clean bench for air drying for 4min, and obtaining total RNA if white precipitate is visible at the bottom;
9) after air drying, dissolving with 20 μ L DEPC water;
10) RNA quantification: taking 2 mu L of RNA obtained in the step 9, measuring the A260/A280 value of the RNA by using an ultraviolet spectrophotometer, calculating the purity and the concentration of the extracted total RNA, and diluting the total RNA according to the experiment requirement.
3.2.3 sample Gene expression detection
The tissue samples were analyzed for the relative expression of lncsnp 6 with reference to 3.1.3 and 3.1.4.
By carrying out gene expression detection on 36 colorectal cancer tissue samples and corresponding paracarcinoma tissue samples, the results show that: referring to fig. 4, lncsnp 6 expression was lower in cancer tissues and was very statistically different compared to paracancerous tissues.
4. Early metastasis diagnosis of colorectal cancer
The expression of LncSENP6 in plasma exosomes of colorectal cancer lymph node metastasis patients is found to be down-regulated through high-throughput sequencing. Real-time quantitative PCR results showed that lncsnp 6 is under-expressed in both plasma exosomes and tissues of colorectal cancer metastasis patients, consistent with high-throughput sequencing results. Based on the above findings, it is presumed that exosome lncsnp 6 plays a certain role in the occurrence and development of colorectal cancer, and is closely related to the metastasis of the patient's postoperative pathology. Therefore, the exosome LncRNA is a Biomarker (BIOMARK) of colorectal cancer metastasis, and can provide a new idea and means for prediction and diagnosis of early colorectal cancer metastasis.
LncSENP6 provides a new target for early colorectal cancer metastasis prediction and diagnosis, and can be used for designing and developing a new method for diagnosing early colorectal cancer metastasis directly aiming at the target. The following are specific examples.
4.1LncSENP6 and reference Gene Real Time PCR primer design
LncSENP6F：5`-CTCTGTTGGCCGTGGCTTTA-3`
LncSENP6R：5`-TACTGCTAATCCTCCCCCGA-3`
The upstream primer GAPDHF: 5' -CTCCTCCACCTTTGACGCTG-3 ″
The downstream primer GAPDHR: 5' -TCCTCTTGTGCTCTTGCTGG-3 ″
4.2 sample pretreatment
Collecting peripheral blood of a patient with unknown colorectal cancer metastasis (metastasis and non-metastasis) by using a 5mL anticoagulation blood collection tube (EDTA), centrifuging the sample at 1000g for 15min at 4 ℃ within 30min after collection, taking supernatant, namely blood plasma, and marking the blood plasma for later use at-80 ℃.
4.3 Rapid and simple detection of Gene expression level of LncSENP6 in plasma exosomes by Real-Time PCR method
1) Primary reagent
Figure BDA0002927906320000082
2) Main instrument
Figure BDA0002927906320000081
Figure BDA0002927906320000091
3) Preparation of the solution
Preparing a primer storage solution: the primer freeze-dried powder is centrifuged at 12000rpm for 1min, and the corresponding ddH is added according to the primer tube wall2O/TE was dissolved at a concentration of 100. mu. mol/L.
Preparing a primer working solution: primer stock solution with concentration of 100. mu. mol/L was added to ddH2O/TE was diluted 10-fold to a concentration of 10. mu. mol/L.
4.4 adopt 2-ΔΔCtCalculating the relative expression quantity of LncSENP6
After the Real-Time PCR amplification reaction program, the instrument automatically calculates the value representing the relative expression quantity according to the Ct value.
According to the quantitative results of a large number of clinical samples, the Ct values corresponding to the plasma exosomes LncSENP6 of the colorectal cancer metastasis group patients are all higher than 27, and the Ct values corresponding to the plasma exosomes LncSENP6 of the colorectal cancer non-metastasis group patients are all lower than 27, so the Ct value 27 is used as a limit value for predicting the metastasis risk of colorectal cancer in situ patients.
In conclusion, the invention proves that the exosome LncSENP6 is closely related to the occurrence and development (metastasis) of colorectal cancer tumors through experiments, so that the exosome LncSENP6 is used as an important target in colorectal cancer genomics, and important references can be provided for the prediction of early colorectal cancer metastasis and the later nutritional support by collecting a small amount of peripheral blood of a colorectal cancer patient and detecting and analyzing the expression of the exosome LncSENP6 in plasma.
<110> China people liberation military and military medical university
<120> colorectal cancer early metastasis diagnosis kit based on expression level of exosome lncSENP6
<160> 4
<210>1
<211>20
<212> DNA
<213> Artificial Synthesis
<400> 1
ctctgttggc cgtggcttta 20
<210>2
<211>20
<212> DNA
<213> Artificial Synthesis
<400> 2
tactgctaat cctcccccga 20
<210>3
<211>20
<212> DNA
<213> Artificial Synthesis
<400> 3
ctcctccacc tttgacgctg 20
<210>4
<211>20
<212> DNA
<213> Artificial Synthesis
<400> 4
tcctcttgtg ctcttgctgg 20

Claims (10)

1. A method for detecting the content of exosome LncSENP6 in plasma, which is characterized by comprising the following steps: the method comprises the following steps:
1) extracting exosome RNA in plasma of a colorectal cancer patient;
2) amplifying an exosome LncSENP6 in plasma by real-time quantitative PCR by using a reverse transcript of the exosome RNA as a template;
3) the amplification cycle threshold Ct of exosome lncsnp 6 was determined.
2. The method for detecting the content of exosome LncSENP6 in plasma according to claim 1, wherein the method comprises the following steps: the step 1) specifically comprises the following steps: the collected peripheral blood of a patient with colorectal cancer non-metastasis is centrifuged to obtain plasma, and exosome RNA is extracted from the plasma.
3. The method for detecting the content of exosome LncSENP6 in plasma according to claim 1, wherein the method comprises the following steps: in the step 2), the amplification primer pair of the real-time quantitative PCR is as follows:
upstream primer LncSENP 6F: 5' -CTCTGTTGGCCGTGGCTTTA-3 ″
Downstream primer LncSENP 6R: 5 '-TACTGCTAATCCTCCCCCGA-3'.
4. The method for detecting the content of exosome LncSENP6 in plasma according to claim 1, wherein the method comprises the following steps: in the step 2), an amplification reaction system of the real-time quantitative PCR comprises 40-400 nmol/L of upstream primers and 40-400 nmol/L of downstream primers.
5. The method for detecting the content of exosome LncSENP6 in plasma according to claim 1, wherein the method comprises the following steps: in the step 2), the amplification reaction procedure of the real-time quantitative PCR is as follows: 1) 30s at 95 ℃; 2) 5s at 95 ℃ and 30-34 s at 60 ℃ for 40 cycles.
6. A colorectal cancer early metastasis diagnosis kit, characterized in that: the kit comprises a detection reagent for analyzing the content of an exosome LncSENP6 in the plasma of a colorectal cancer patient.
7. The kit for diagnosing early metastasis of colorectal cancer according to claim 6, wherein: the detection reagent comprises a real-time quantitative PCR amplification primer pair of an exosome LncSENP 6.
8. The kit for diagnosing early metastasis of colorectal cancer according to claim 7, wherein: the real-time quantitative PCR amplification primer pair comprises:
upstream primer LncSENP 6F: 5' -CTCTGTTGGCCGTGGCTTTA-3 ″
Downstream primer LncSENP 6R: 5 '-TACTGCTAATCCTCCCCCGA-3'.
9. The kit for diagnosing early metastasis of colorectal cancer according to claim 7, wherein: when the amplification cycle threshold Ct or the relative expression quantity of the exosome LncSENP6 in the plasma of the colorectal cancer non-metastatic patient is within the limit value, the patient is indicated to have no metastasis risk, and when the amplification cycle threshold Ct or the relative expression quantity of the exosome LncSENP6 in the plasma of the colorectal cancer non-metastatic patient exceeds the limit value, the patient is indicated to have the metastasis risk.
10. The kit for diagnosing early metastasis of colorectal cancer according to claim 9, wherein: the limit of the amplification cycle threshold Ct is 27.
CN202110138419.3A 2021-02-01 2021-02-01 Colorectal cancer early metastasis diagnosis kit based on exosome LncSENP6 expression level Pending CN112779335A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110138419.3A CN112779335A (en) 2021-02-01 2021-02-01 Colorectal cancer early metastasis diagnosis kit based on exosome LncSENP6 expression level

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110138419.3A CN112779335A (en) 2021-02-01 2021-02-01 Colorectal cancer early metastasis diagnosis kit based on exosome LncSENP6 expression level

Publications (1)

Publication Number Publication Date
CN112779335A true CN112779335A (en) 2021-05-11

Family

ID=75760325

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110138419.3A Pending CN112779335A (en) 2021-02-01 2021-02-01 Colorectal cancer early metastasis diagnosis kit based on exosome LncSENP6 expression level

Country Status (1)

Country Link
CN (1) CN112779335A (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101010434A (en) * 2004-05-06 2007-08-01 霍夫曼-拉罗奇有限公司 SENP1 as a marker for cancer
CN105018594A (en) * 2015-04-27 2015-11-04 广州医科大学附属第三医院 Early-diagnosis marker for colorectal cancer and related kit
CN111518907A (en) * 2020-05-13 2020-08-11 广西医科大学附属肿瘤医院 Marker for diagnosing liver metastasis of colorectal cancer

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101010434A (en) * 2004-05-06 2007-08-01 霍夫曼-拉罗奇有限公司 SENP1 as a marker for cancer
CN105018594A (en) * 2015-04-27 2015-11-04 广州医科大学附属第三医院 Early-diagnosis marker for colorectal cancer and related kit
CN111518907A (en) * 2020-05-13 2020-08-11 广西医科大学附属肿瘤医院 Marker for diagnosing liver metastasis of colorectal cancer

Similar Documents

Publication Publication Date Title
Mugoni et al. Circulating RNAs in prostate cancer patients
CN108179190B (en) Plasma exosome circRNA marker of non-small cell lung cancer and detection primer and kit thereof
CN107447033B (en) Colorectal cancer diagnosis biomarker and application thereof
CN104152452B (en) A kind of blood miRNA marker relevant to hepatocarcinoma and application thereof
CN105177174A (en) miRNA (microribonucleic acid) biomarkers and detection kit for colon cancer diagnosis
CN105219867A (en) For miRNA biomarker and the detection kit of diagnosing gastric cancer
CN114150066B (en) Application of exosomes CDA, HMGN1 and the like in lung cancer diagnosis
CN110229899B (en) Plasma marker combinations for early diagnosis or prognosis prediction of colorectal cancer
CN109593852B (en) Serum miRNA marker related to nasopharyngeal carcinoma auxiliary diagnosis and application thereof
CN108103199B (en) Circulating miRNA marker related to ovarian cancer auxiliary diagnosis and application thereof
CN112779336B (en) Colorectal cancer early metastasis diagnosis kit based on exosome LncCLDN23 expression level
CN115287354A (en) Early diagnosis kit for gastric cancer cachexia based on exosome miRNA-206 expression level
CN112779335A (en) Colorectal cancer early metastasis diagnosis kit based on exosome LncSENP6 expression level
CN112553341B (en) Renal clear cell carcinoma SCNN1 primer, diagnostic kit and application thereof
CN106086178B (en) Serum miRNA marker related to gastric cancer auxiliary diagnosis and application thereof
CN109266750B (en) Biomarker for nasopharyngeal carcinoma diagnosis and application
CN109266751B (en) Biomarker combination for nasopharyngeal carcinoma diagnosis and application
CN109536612B (en) Plasma miRNA marker related to nasopharyngeal carcinoma auxiliary diagnosis and application thereof
CN111575374A (en) Molecular marker for early pancreatic tumor detection, and detection method and application thereof
CN112176060B (en) Plasma non-coding RNA and primer set for detecting expression level thereof and colorectal cancer detection kit
CN114752676B (en) Application of reagent for detecting hsa_circ_0099132 in preparation of auxiliary diagnosis kit for colorectal cancer of human
CN115948546B (en) Exosome miRNA biomarker for breast cancer and application thereof
CN111518914B (en) MiRNA marker combination, kit and method for detecting breast cancer
CN112159845B (en) Application of circular RNA as pancreatic cancer diagnosis marker
CN116121245B (en) Small molecule RNA and application thereof in early diagnosis of endometriosis

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20210511