CN113249488A - Early diagnosis kit for gastric cancer cachexia based on exosome miRNA-4433-5p expression level - Google Patents
Early diagnosis kit for gastric cancer cachexia based on exosome miRNA-4433-5p expression level Download PDFInfo
- Publication number
- CN113249488A CN113249488A CN202110732167.7A CN202110732167A CN113249488A CN 113249488 A CN113249488 A CN 113249488A CN 202110732167 A CN202110732167 A CN 202110732167A CN 113249488 A CN113249488 A CN 113249488A
- Authority
- CN
- China
- Prior art keywords
- gastric cancer
- mir
- hsa
- exosome
- plasma
- 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
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
- C12Q2600/158—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
Abstract
The invention discloses an early diagnosis kit for gastric cancer dyscrasia based on an exosome miRNA-4433-5p expression level, which comprises real-time quantitative PCR amplification primers of Hsa-miR-4433-5 p. Collecting peripheral blood of a gastric cancer patient with no diagnosed cachexia, centrifuging to obtain plasma, extracting exosome RNA in the plasma, reversely transcribing miRNA into cDNA serving as a template, and detecting the content of Hsa-miR-4433-5p in the plasma exosome by real-time quantitative PCR (polymerase chain reaction), thereby providing an important reference for predicting gastric cancer cachexia.
Description
Technical Field
The invention belongs to the field of tumor medicine, and relates to a kit for detecting the content of Hsa-miR-4433-5p in plasma exosomes and for early diagnosis of gastric cancer cachexia according to the expression condition of the exosomes miRNA-4433-5 p.
Background
Gastric Cancer (GC) is one of the most common digestive tract malignancies and is characterized by high morbidity and mortality, which are rising year by year. Although the treatment of cancer is being studied with the development of medical technology, the improvement of the survival rate of gastric cancer patients still faces a great challenge due to the high incidence rate of gastric cancer and the characteristic of difficult early diagnosis. Cachexia is a multifactorial metabolic syndrome, and clinically, cachexia is mainly manifested by weight loss and muscle atrophy (with or without fat loss), and is most commonly caused by tumors, called tumor cachexia (Cancer cachexia). Research shows that more than 20 percent of patients with tumor dyscrasia die of dyscrasia directly, the dyscrasia is particularly common in gastrointestinal tumors, and 80 percent of patients with gastric cancer have dyscrasia. After the tumor patients suffer from dyscrasia, the toxicity of the chemotherapeutic drugs is more sensitive, and the life quality is also reduced, so the prognosis of the tumor patients is seriously influenced. Currently, there is no effective means for diagnosing and treating cachexia, and many studies have focused on biomarkers that predict the occurrence of tumor cachexia. However, detection of serum markers is generally not valuable for predicting cachexia, and tumor markers such as CEA, CA19-9, and CA724 only predict the possibility of tumor occurrence, but not the occurrence of cachexia.
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, which is one of the major active substances, is the biological basis on which exosomes exert the above functions. The non-coding RNA mainly includes micro RNA (miRNA), long non-coding RNA (incrna), and circular RNA (crna). Wherein, miRNA is a kind of endogenous single-stranded RNA with a length of 19-25 nt, which plays a fine regulation role on downstream target mRNA through a post-transcriptional gene silencing mechanism and influences malignant transformation, anchorage-independent growth, epithelial-mesenchymal transformation, angiogenesis, drug tolerance and other tumor malignant phenotypes of cells. The exosome can carry miRNA into a circulatory system, and the miRNA contained in the vesicle structure (namely the exosome miRNA) is conveyed to specific target cells in an autocrine, paracrine and endocrine mode, so that the miRNA in the exosome can escape degradation, and the connection between cells and between tissues is promoted.
With the discovery of mirnas, numerous mirnas have been shown to be involved in inflammatory responses, induction of metastasis, and mediation of cancer invasion, even in pathways of protein synthesis and degradation in skeletal muscle. Tumorigenesis can trigger tumor cell-muscle cell communication, leading to degradation and metabolism of proteins in muscle. Changes in miRNA expression profiles during tumor development can affect the development of muscle-related diseases. Altering the level of miRNA can affect myogenesis, leading to hypertrophy or atrophy of skeletal muscle, and thus to the development of cachexia, perhaps by inducing apoptosis-related signaling pathways or activation of the ubiquitin-protease system within muscle cells, thereby promoting protein degradation in muscle fines.
Hsa-miR-4433-5p is a rare miRNA, and is rarely reported in previous researches. A publication published in Journal of Cancer in 2019 for the first time reports its relationship to chronic myelogenous leukemia, in which Hsa-miR-4433-5p was identified as a miRNA targeting Bcr-Abl, possibly epigenetically regulated by SAHA, a histone deacetylase inhibitor that has been annotated by the U.S. FDA for the treatment of cutaneous T-cell lymphoma. The article suggests that the stable expression of Hsa-miR-4433-5p can inhibit the expression of Bcr-Abl and downstream genes, thereby inhibiting the activity of cancer cells. However, the function and the effect of Hsa-miR-4433-5p in other tumors are not reported.
At present, a kit for predicting gastric cancer dyscrasia by detecting miRNA contained in plasma exosomes in peripheral blood so as to realize convenient, accurate and early diagnosis of gastric cancer dyscrasia is not found.
Disclosure of Invention
The invention aims to provide an early diagnosis kit for gastric cancer dyscrasia based on an exosome miRNA-4433-5p expression level, which can improve the accuracy of early diagnosis for gastric cancer dyscrasia and improve the prognosis of a gastric cancer patient by carrying out real-time quantitative PCR detection on Hsa-miR-4433-5p in a plasma exosome.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting the content of exosome miRNA-4433-5p in plasma comprises the following steps:
1) extracting exosome RNA in plasma of a gastric cancer patient, and carrying out reverse transcription on miRNA in the exosome RNA to obtain a reverse transcription product of exosome miRNA;
2) taking a reverse transcription sample of the exosome miRNA (cDNA obtained by reverse transcription of miRNA in total RNA extracted from the plasma exosome) as a template, amplifying a cDNA sequence corresponding to Hsa-miR-4433-5p (namely exosome miRNA-4433-5p) in the plasma exosome by real-time quantitative PCR, and determining an amplification cycle threshold Ct (referred to as the amplification cycle threshold Ct of Hsa-miR-4433-5 p) of the cDNA sequence corresponding to Hsa-miR-4433-5p in the plasma exosome after the amplification is finished;
3) and calculating the difference delta CT between the amplification cycle threshold Ct of the Hsa-miR-4433-5p and the amplification cycle threshold Ct of the reference gene.
Preferably, in the step 1), the collected peripheral blood of the patient with gastric cancer (gastric cancer confirmed by tissue or cytopathology diagnosis) is centrifuged to obtain plasma, and the exosome RNA is extracted from the plasma.
Preferably, in the step 2), the real-time quantitative PCR amplification primer of the cDNA sequence corresponding to Hsa-miR-4433-5p in the plasma exosome consists of an upstream primer and a downstream primer, wherein the downstream primer is an miRNA universal primer, and the sequence of the upstream primer is as follows:
5`-ATGTCCCACCCCCACTCCTGT-3`。
an early diagnosis kit for gastric cancer dyscrasia, which comprises real-time quantitative PCR amplification primers (such as the upstream primer and the downstream primer) of Hsa-miR-4433-5 p.
Preferably, the Hsa-miR-4433-5p is extracted from peripheral blood of a patient with gastric cancer.
Preferably, the Hsa-miR-4433-5p is extracted from plasma exosomes of gastric cancer patients.
Preferably, when the difference Δ CT between the amplification cycle threshold Ct of Hsa-miR-4433-5p in plasma exosomes of gastric cancer patients and the amplification cycle threshold Ct of the internal reference gene exceeds a limit value (for example, when the difference Δ CT between the amplification cycle threshold Ct of Hsa-miR-4433-5p and the amplification cycle threshold of the internal reference gene U6 satisfies: -2< Δ CT ≦ 0), the closer the difference Δ CT to the limit value (for example, -2) indicates the higher risk of cachexia in the patients.
The Hsa-miR-4433-5p is applied to preparation of a kit for early diagnosis of gastric cancer dyscrasia.
The reverse transcription product of Hsa-miR-4433-5p is applied to preparation of a kit for early diagnosis of gastric cancer cachexia.
The application of the Hsa-miR-4433-5p amplification primer in preparing the kit for early diagnosis of gastric cancer dyscrasia.
The invention has the beneficial effects that:
according to the miRNA development diagnosis kit which is screened out according to high-throughput sequencing and is closely related to gastric cancer dyscrasia, the occurrence of the gastric cancer dyscrasia can be predicted by detecting the expression of the specific miRNA in the plasma exosomes of the gastric cancer patients, and the kit is not easily influenced by other basic diseases, so that a more accurate and reliable reference standard is provided for clinical medication and nutritional support of the gastric cancer patients. The diagnosis kit can be developed based on mature technologies such as exosome RNA extraction and miRNA detection, only needs to collect a small amount of blood, is suitable for most of gastric cancer patients, and can quickly and conveniently obtain a diagnosis result.
Furthermore, the invention can rapidly, accurately and reliably detect the expression of Hsa-miR-4433-5p in the plasma exosome of the gastric 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. 2A is the results of high throughput sequencing analysis of plasma exosomes (differentially expressed miRNA volcano) for 3 patients with gastric cancer cachexia and 2 patients with gastric cancer non-cachexia.
Fig. 2B is the high throughput sequencing analysis results (differential expression miRNA cluster maps) of plasma exosomes from 3 patients with gastric cancer cachexia and 2 patients with gastric cancer non-cachexia.
FIG. 3 shows the expression of the screened Hsa-miR-4433-5p in plasma exosomes of gastric cancer patients; wherein: normal represents the gastric cancer non-dyscrasia group; cachexia represents gastric cancer Cachexia group; p is less than or equal to 0.05, with statistical differences.
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 the detailed information of the patient, collecting peripheral blood of the gastric cancer patient by using a 5mL anticoagulation blood collection tube, centrifuging the peripheral blood sample at 4 ℃ and 3000r for 15min within 30min after collection, taking supernatant, namely blood plasma, respectively marking, and freezing and storing the blood plasma to a refrigerator at minus 80 ℃. Peripheral blood collection site: the first subsidiary hospital of the university of military medical and air force for digestive diseases hospital; collecting time: 7 months in 2020 to 12 months in 2020; peripheral blood was collected during surgery.
2. Screening of miRNAs related to gastric cancer cachexia in plasma exosomes of gastric cancer patients
2.1 plasma exosome extraction and whole transcriptome sequencing
Plasma exosomes of 3 gastric cancer cachexia patients and 2 gastric cancer non-cachexia patients were randomly extracted, and an exosome sample cryopreserved at-80 ℃ was sent to Beijing Enzekangtai Biotechnology Co., Ltd to perform whole transcriptome sequencing on the sample.
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 a gastric cancer patient by using a 5mL anticoagulation blood collection tube, centrifuging the sample at 4 ℃ and 3000r for 15min 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 differentially expressed miRNAs related to gastric cancer cachexia
According to the results of high-throughput sequencing on the complete transcriptome of plasma exosomes of 3 patients with gastric cancer dyscrasia and 2 patients with gastric cancer non-dyscrasia, the change of the expression profiles of miRNAs in the plasma exosomes of the patients with gastric cancer dyscrasia and the patients with gastric cancer non-dyscrasia is analyzed, so that 24 miRNAs which are differentially expressed in the exosomes of the patients with gastric cancer dyscrasia are screened, wherein the up-regulation expression is 12, and the down-regulation expression is 12 (see fig. 2A and 2B). 3 miRNAs with the largest differential expression fold are selected as candidate molecules, and are verified by clinical samples to be consistent with a sequencing result.
3. The relation between Hsa-miR-4433-5p and gastric cancer dyscrasia is verified
3.1 according to the candidate molecule Hsa-miR-4433-5p which is screened out by high-throughput sequencing and is related to gastric cancer cachexia and has differential expression multiple of 10.61 times, the expression condition of the candidate molecule Hsa-miR-4433-5p in the plasma exosomes of the gastric cancer patients is verified by real-time quantitative PCR.
3.1.1Hsa-miR-4433-5p and reference gene U6 amplification primer design
Hsa-miR-4433-5p amplification primer:
the upstream primer F1: 5' -ATGTCCCACCCCCACTCCTGT-3 ″
The downstream primer R1: QIAGEN MiScript SYBR Green PCR Kit Universal primer
Amplification primers of an internal reference gene U6:
the upstream primer F2: 5' -AAAGCAAATCATCGGACGACC-3 ″
The downstream primer R2: QIAGEN MiScript SYBR Green PCR Kit Universal primer
3.1.2 plasma exosome RNA extraction for gastric cancer patients
The exosome RNA extraction adopts an exosome rapid extraction kit (the use of the kit is divided into two stages, and the total exosome RNA is extracted in the second stage), and the exosome RNA extraction method comprises the following steps:
1) collecting peripheral blood of a gastric cancer patient by using a 5mL anticoagulation blood collection tube, centrifuging the sample at 4 ℃ and 3000g for 15min 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 2mLEP 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 RNA of exosomes was taken and reverse-transcribed using a reverse transcription Kit (MiScript II RT Kit (50)) from QIAGEN, 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: 90min at 37 ℃; 5min at 95 ℃ and 10min at 4 ℃.
The cDNA in the reaction system was diluted (1:2) with ultrapure water.
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 20 μ L: 2 XTB Green Premix Ex Taq II 10. mu.L, 10. mu.M forward primer 0.5. mu.L, 10. mu.M reverse primer 0.5. mu. L, cDNA template 2. mu.L, and deionized water to 20. mu.L.
The method is carried out on a Bio-Rad CFX Manager real-time quantitative PCR instrument according to a real-time quantitative PCR three-step amplification standard program:
1) pre-denaturation: 180s at 95 ℃;
2) and (3) PCR reaction: 95 ℃ for 10s, 56 ℃ for 15s, 72 ℃ for 15s, for a total of 54 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 relative expression quantity of Hsa-miR-4433-5p and the reference gene U6 is calculated by the method.
3.2 results
The gene expression of the plasma samples of 66 gastric cancer patients was detected by Real-time PCR. Among them, 36 patients with gastric cancer are non-cachexia, and 30 patients with gastric cancer are cachexia. The result shows (figure 3), compared with the gastric cancer non-dyscrasia group, the expression level of Hsa-miR-4433-5p in the plasma exosome of the gastric cancer dyscrasia group patient is obviously increased, and the statistical difference is realized.
4. Early diagnosis of gastric cancer dyscrasia
According to the invention, the expression of Hsa-miR-4433-5p in the plasma exosome of the gastric cancer cachexia patient is found to be up-regulated through high-throughput sequencing. The real-time quantitative PCR result shows that Hsa-miR-4433-5p is highly expressed in the plasma exosome of the gastric cancer cachexia patient, which is consistent with the high-throughput sequencing result. Based on the above findings, Hsa-miR-4433-5p is presumed to play a key role in the occurrence and development of gastric cancer cachexia. Therefore, Hsa-miR-4433-5p in the plasma exosome is a Biomarker (BIOMARK) for gastric cancer cachexia, and provides a new target for prediction and early diagnosis of gastric cancer cachexia. The following is a detailed illustration of the design and development of methods for predicting gastric cancer cachexia and for early diagnosis directed to this target.
4.1Hsa-miR-4433-5p and reference Gene Real-Time PCR primer design
The upstream primer F1: 5' -ATGTCCCACCCCCACTCCTGT-3 ″
The downstream primer R1: the QIAGEN MiScript SYBR Green PCR Kit universal primer is adopted
The upstream primer F2: 5' -AAAGCAAATCATCGGACGACC-3 ″
The downstream primer R2: the QIAGEN MiScript SYBR Green PCR Kit universal primer is adopted
4.2 sample pretreatment
Collecting peripheral blood of gastric cancer (cachexia and non-cachexia) patient with 5mL anticoagulation blood collection tube (EDTA), centrifuging the sample at 4 deg.C and 3000r for 15min within 30min after collection, collecting supernatant as blood plasma, and storing at-80 deg.C.
4.3 Rapid, simple and convenient detection of gene expression level of Hsa-miR-4433-5p in plasma exosome by using Real-Time PCR method
1) Primary reagent
2) Main instrument
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 5-fold to a concentration of 10. mu. mol/L.
4.4 adopt 2-ΔΔCtCalculating the relative expression quantity of Hsa-miR-4433-5p in the plasma exosome
After the Real-Time PCR amplification reaction program, the instrument automatically calculates the value representing the relative expression quantity according to the Ct value.
In quantification, the difference delta CT between the amplification cycle threshold Ct of Hsa-miR-4433-5p in the plasma exosome of the gastric cancer cachexia group patient and the amplification cycle threshold Ct of the internal reference gene U6 is not higher than-2 (the delta CT is less than or equal to-2, the expression level of Hsa-miR-4433-5p in the plasma exosome of the patient is high), while the difference delta CT between the amplification cycle threshold Ct of Hsa-miR-4433-5p in the plasma exosome of the gastric cancer non-cachexia group patient and the amplification cycle threshold Ct of the internal reference gene U6 is higher than-2 (the delta CT > -2, the expression level of Hsa-miR-4433-5p in the plasma exosome of the patient is low). According to the quantitative results of a large number of clinical samples, the occurrence of cachexia in a gastric cancer patient is predicted by using the Δ CT value of-2 as a limit, the risk of cachexia increases as Δ CT (> -2) approaches-2, and when the Δ CT value of a gastric cancer patient who has not diagnosed cachexia decreases to 0, the gastric cancer patient is judged to have cachexia.
In a word, the invention proves that specific exosome miRNA (Hsa-miR-4433-5 p) is closely related to the generation and development of gastric cancer dyscrasia through experiments, so that Hsa-miR-4433-5p is taken as an important target in gastric cancer genomics, and important reference can be provided for early diagnosis and later nutritional support of gastric cancer dyscrasia by collecting a small amount of peripheral blood of a gastric cancer patient and detecting and analyzing the expression of Hsa-miR-4433-5p in plasma exosomes.
<110> China people liberation military and military medical university
<120> early diagnosis kit for gastric cancer cachexia based on exosome miRNA-4433-5p expression level
<160> 2
<210>1
<211>21
<212> DNA
<213> Artificial Synthesis
<400> 1
atgtcccacc cccactcctg t 21
<210>2
<211>21
<212> DNA
<213> Artificial Synthesis
<400> 2
aaagcaaatc atcggacgac c 21
Claims (10)
1. A method for detecting the content of exosome miRNA-4433-5p in blood plasma is characterized by comprising the following steps: the method comprises the following steps:
1) extracting exosome RNA in plasma of a gastric cancer patient, and carrying out reverse transcription on miRNA in the exosome RNA to obtain a reverse transcription product of exosome miRNA;
2) amplifying Hsa-miR-4433-5p in the plasma exosome by real-time quantitative PCR (polymerase chain reaction) by taking the reverse transcription copy of the exosome miRNA as a template, and determining an amplification cycle threshold of the Hsa-miR-4433-5p in the plasma exosome;
3) and calculating the difference delta CT between the amplification cycle threshold of the Hsa-miR-4433-5p and the amplification cycle threshold of the reference gene.
2. The method for detecting the content of exosome miRNA-4433-5p in plasma according to claim 1, wherein the method comprises the following steps: in the step 2), the real-time quantitative PCR amplification primer of Hsa-miR-4433-5p in the plasma exosome consists of an upstream primer and a downstream primer, wherein the downstream primer is a miRNA universal primer, and the sequence of the upstream primer is as follows:
5`-ATGTCCCACCCCCACTCCTGT-3`。
3. an early diagnosis kit for gastric cancer dyscrasia, which is characterized in that: the kit comprises a real-time quantitative PCR amplification primer pair of Hsa-miR-4433-5 p.
4. The kit for the early diagnosis of gastric cancer cachexia according to claim 3, wherein: the Hsa-miR-4433-5p is extracted from peripheral blood of a gastric cancer patient.
5. The kit for the early diagnosis of gastric cancer cachexia according to claim 3, wherein: the Hsa-miR-4433-5p is extracted from plasma exosomes of gastric cancer patients.
6. The kit for the early diagnosis of gastric cancer cachexia according to claim 5, wherein: the real-time quantitative PCR amplification primer of Hsa-miR-4433-5p in the plasma exosome consists of an upstream primer and a downstream primer, wherein the downstream primer is a miRNA universal primer, and the sequence of the upstream primer is as follows:
5`-ATGTCCCACCCCCACTCCTGT-3`。
7. the kit for the early diagnosis of gastric cancer cachexia according to claim 5, wherein: when the difference delta CT between the amplification cycle threshold of Hsa-miR-4433-5p in the plasma exosome of the gastric cancer patient and the amplification cycle threshold of the internal reference gene exceeds a limit value, the closer the difference delta CT is to the limit value, the higher the risk of the patient suffering from dyscrasia is.
Application of Hsa-miR-4433-5p in preparation of early diagnosis kit for gastric cancer dyscrasia.
The application of the reverse transcript of Hsa-miR-4433-5p in the preparation of the kit for early diagnosis of gastric cancer cachexia.
Application of an amplification primer of Hsa-miR-4433-5p in preparation of an early diagnosis kit for gastric cancer dyscrasia.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110732167.7A CN113249488A (en) | 2021-06-29 | 2021-06-29 | Early diagnosis kit for gastric cancer cachexia based on exosome miRNA-4433-5p expression level |
| CN202210753426.9A CN115287355A (en) | 2021-06-29 | 2022-06-29 | Early diagnosis kit for gastric cancer dyscrasia based on exosome miRNA-4433-5p expression level |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110732167.7A CN113249488A (en) | 2021-06-29 | 2021-06-29 | Early diagnosis kit for gastric cancer cachexia based on exosome miRNA-4433-5p expression level |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113249488A true CN113249488A (en) | 2021-08-13 |
Family
ID=77190173
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110732167.7A Pending CN113249488A (en) | 2021-06-29 | 2021-06-29 | Early diagnosis kit for gastric cancer cachexia based on exosome miRNA-4433-5p expression level |
| CN202210753426.9A Pending CN115287355A (en) | 2021-06-29 | 2022-06-29 | Early diagnosis kit for gastric cancer dyscrasia based on exosome miRNA-4433-5p expression level |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210753426.9A Pending CN115287355A (en) | 2021-06-29 | 2022-06-29 | Early diagnosis kit for gastric cancer dyscrasia based on exosome miRNA-4433-5p expression level |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN113249488A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106460068A (en) * | 2014-06-16 | 2017-02-22 | 东丽株式会社 | Stomach cancer detection kit or device, and detection method |
| US20170114124A1 (en) * | 2015-10-22 | 2017-04-27 | Northwestern University | SLIT-ROBO-Myo9-RHOA PATHWAY AND CANCER |
| CN106701964A (en) * | 2017-01-17 | 2017-05-24 | 中国人民解放军第三军医大学第附属医院 | Serum exosome miRNA biological marker and kit for early diagnosis of gastric cancer |
| US20180000910A1 (en) * | 2012-11-26 | 2018-01-04 | Modernatx, Inc. | Terminally modified rna |
-
2021
- 2021-06-29 CN CN202110732167.7A patent/CN113249488A/en active Pending
-
2022
- 2022-06-29 CN CN202210753426.9A patent/CN115287355A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180000910A1 (en) * | 2012-11-26 | 2018-01-04 | Modernatx, Inc. | Terminally modified rna |
| CN106460068A (en) * | 2014-06-16 | 2017-02-22 | 东丽株式会社 | Stomach cancer detection kit or device, and detection method |
| US20170114124A1 (en) * | 2015-10-22 | 2017-04-27 | Northwestern University | SLIT-ROBO-Myo9-RHOA PATHWAY AND CANCER |
| CN106701964A (en) * | 2017-01-17 | 2017-05-24 | 中国人民解放军第三军医大学第附属医院 | Serum exosome miRNA biological marker and kit for early diagnosis of gastric cancer |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115287355A (en) | 2022-11-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bonome et al. | A gene signature predicting for survival in suboptimally debulked patients with ovarian cancer | |
| Hanke et al. | A robust methodology to study urine microRNA as tumor marker: microRNA-126 and microRNA-182 are related to urinary bladder cancer | |
| Liu et al. | Identification of genes differentially expressed in human primary lung squamous cell carcinoma | |
| Ramachandran | Functional association of oestrogen receptors with HPV infection in cervical carcinogenesis | |
| Schwertheim et al. | Analysis of deregulated miRNAs is helpful to distinguish poorly differentiated thyroid carcinoma from papillary thyroid carcinoma | |
| Tian et al. | Up-regulation of miR-21 expression predicate advanced clinicopathological features and poor prognosis in patients with non-small cell lung cancer | |
| CN108949969B (en) | Application of long-chain non-coding RNA in colorectal cancer | |
| Pang et al. | Cytogenetic and expression profiles associated with transformation to androgen‐resistant prostate cancer | |
| Ghanbari et al. | Expression Analysis of Previously Verified Fecal and Plasma Down-regulated MicroRNAs (miR-4478, 1295-3p, 142-3p and 26a-5p), in FFPE Tissue Samples of CRC Patients. | |
| CN106755293B (en) | Lung cancer auxiliary diagnosis related lncRNA marker and application thereof | |
| CN115287354A (en) | Early diagnosis kit for gastric cancer cachexia based on exosome miRNA-206 expression level | |
| EP4144859A1 (en) | Tumor detection reagent and kit | |
| WO2010004562A2 (en) | Methods and compositions for detecting colorectal cancer | |
| CN113249488A (en) | Early diagnosis kit for gastric cancer cachexia based on exosome miRNA-4433-5p expression level | |
| He et al. | Aberrant alternative polyadenylation is responsible for survivin up-regulation in ovarian cancer | |
| US20160258028A1 (en) | METHODS AND COMPOSITIONS FOR DETECTING COLORECTAL CANCER USING MICRO RNAs | |
| CN111118146A (en) | Application of LncRNA in diagnosis of breast cancer chemotherapy-related myocardial injury patient | |
| CN113322322A (en) | Early diagnosis kit for gastric cancer cachexia based on exosome miRNA-432-5p expression level | |
| CN112553341B (en) | Renal clear cell carcinoma SCNN1 primer, diagnostic kit and application thereof | |
| CN106148511B (en) | Predictive marker and kit for recurrence risk of liver cancer patient after resection | |
| Papaconstantinou et al. | The role of variations within microRNA in inflammatory bowel disease | |
| CN111575374B (en) | Molecular marker for early pancreatic tumor detection, detection method and application thereof | |
| CN112779336B (en) | Colorectal cancer early metastasis diagnosis kit based on exosome LncCLDN23 expression level | |
| CN111334577A (en) | Laryngeal squamous carcinoma molecular marker hsa _ circ _0004547, detection method and application | |
| CN112779335A (en) | Colorectal cancer early metastasis diagnosis kit based on exosome LncSENP6 expression level |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20210813 |