CN113337609A - Early diagnosis kit for gastric cancer cachexia based on exosome miRNA-206 expression level - Google Patents

Early diagnosis kit for gastric cancer cachexia based on exosome miRNA-206 expression level Download PDF

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CN113337609A
CN113337609A CN202110731382.5A CN202110731382A CN113337609A CN 113337609 A CN113337609 A CN 113337609A CN 202110731382 A CN202110731382 A CN 202110731382A CN 113337609 A CN113337609 A CN 113337609A
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gastric cancer
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李纪鹏
张瑞
姜勋亮
王珂
刘俊
丁晓琛
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Air Force Medical University of PLA
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Abstract

The invention discloses an early diagnosis kit for gastric cancer cachexia based on an exosome miRNA-206 expression level, which comprises real-time quantitative PCR amplification primers of Hsa-miR-206. Collecting peripheral blood of a gastric cancer patient with no gastric cancer cachexia diagnosed, centrifuging to obtain plasma, extracting exosome RNA in the plasma, reversely transcribing miRNA in the plasma into cDNA serving as a template, and detecting the content of Hsa-miR-206 in the plasma exosome by real-time quantitative PCR (polymerase chain reaction), thereby providing important reference for predicting gastric cancer cachexia.

Description

Early diagnosis kit for gastric cancer cachexia based on exosome miRNA-206 expression level
Technical Field
The invention belongs to the field of tumor medicine, and relates to a kit for detecting the content of Hsa-miR-206 in plasma exosomes and for early diagnosis of gastric cancer cachexia according to the expression condition of the exosomes miRNA-206.
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-206 is a miRNA specific to vertebrates, so that the miRNA has the commonality of animal miRNAs. Hsa-miR-206 is first transcribed by RNA polymerase II to produce a primary miRNA transcription product (pri-miRNA-206), which is processed in the nucleus by an RNA endonuclease III called Drosha to produce a stem-loop structured miRNA-206 precursor (pre-miRNA-206). Then, pre-miRNA-206 is transported to cytoplasm by Exportin-5 protein, and is further processed by a second RNA endonuclease III Dicer to obtain mature double-stranded RNA molecules, wherein one mature strand is inserted into an RNA Induced Silencing Complex (RISC) and combined with a 3 'end untranslated region (3' UTR) of mRNA of a target gene of the RISC, and the mRNA of the target gene is cracked or protein expression is inhibited, so that the biological characteristics of the RNA induced silencing complex are exerted, and the proliferation, differentiation, apoptosis, migration and other biological behaviors of cells are regulated and controlled. Hsa-miR-206 can inhibit the expression of a plurality of target genes; the same target gene may also be regulated by multiple mirnas. The molecular regulatory network thus formed plays a role in embryonic development, normal physiological function, and pathological processes of various diseases. Hsa-miR-206 is reported to play an important role in a variety of tumors. Hsa-miR-206 is also reported to be closely related to the occurrence of diseases of lung, brain, heart and the like. Takada et al found that the expression level of Hsa-miR-206 steadily increased during embryonic development and tended to decrease after birth. The relation between Hsa-miR-206 and target genes Pax7, Pax3 and the like plays an important role in the development of skeletal muscle. Hsa-miR-206 can also regulate synaptic plasticity and memory by inhibiting the expression of brain-derived neurotrophic factors.
Yan et al prove that Hsa-miR-206 inhibits the growth and transfer of RMS by regulating and controlling target gene Met. Including breast cancer, rhabdomyosarcoma, ovarian cancer, colorectal cancer, lung cancer and the like, Hsa-miR-206 is obviously reduced and plays an important role in the growth process of tumors. 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-206 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-206 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-206 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-206 (namely the exosome miRNA-206) in the plasma exosome by real-time quantitative PCR, and determining an amplification cycle threshold Ct (the amplification cycle threshold Ct of Hsa-miR-206 for short) of the cDNA sequence corresponding to Hsa-miR-206 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-206 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-206 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`-TGGAATGTAAGGAAGTGTGTGG-3`。
an early diagnosis kit for gastric cancer cachexia, which comprises real-time quantitative PCR amplification primers (such as the upstream primer and the downstream primer) of Hsa-miR-206.
Preferably, the Hsa-miR-206 is extracted from peripheral blood of a patient with gastric cancer.
Preferably, the Hsa-miR-206 is extracted from plasma exosomes of gastric cancer patients.
Preferably, when the difference Δ CT between the amplification cycle threshold Ct of Hsa-miR-206 and the amplification cycle threshold Ct of the internal reference gene in plasma exosomes of a gastric cancer patient (e.g., a gastric cancer patient who has not been diagnosed with cachexia) exceeds a limit (e.g., when the difference Δ CT between the amplification cycle threshold of Hsa-miR-206 and the amplification cycle threshold of the internal reference gene U6 satisfies: 2< Δ CT ≦ 4), the closer the difference Δ CT to the limit (e.g., 2) indicates the higher risk of cachexia in the patient.
The Hsa-miR-206 is applied to preparation of a kit for early diagnosis of gastric cancer dyscrasia.
The reverse transcript of Hsa-miR-206 is applied to preparation of a kit for early diagnosis of gastric cancer cachexia.
The application of the amplification primer of Hsa-miR-206 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-206 in the plasma exosomes of gastric cancer patients 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-206 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 (namely 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 plasma 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. Verify the relationship between Hsa-miR-206 and gastric cancer dyscrasia
3.1 screening out candidate molecules Hsa-miR-206 with 18.03 times of differential expression fold related to gastric cancer cachexia according to high-throughput sequencing, and verifying the expression condition of the candidate molecules Hsa-miR-206 in plasma exosomes of gastric cancer patients through real-time quantitative PCR.
3.1.1Hsa-miR-206 and reference gene U6 amplification primer design
Hsa-miR-206 amplification primer:
the upstream primer F1: 5' -TGGAATGTAAGGAAGTGTGTGG-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 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 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 upstream primer 0.5. mu.L, 10. mu.M downstream 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, wherein:
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 the Hsa-miR-206 and the reference gene U6 is calculated by the method.
3.2 results
Gene expression was detected in plasma samples from 69 patients with gastric cancer by Real-time PCR. Among them, 37 patients with gastric cancer are non-cachexia, and 32 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-206 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-206 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-206 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-206 is presumed to play a key role in the occurrence and development of gastric cancer cachexia. Therefore, Hsa-miR-206 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-206 and reference Gene Real-Time PCR primer design
The upstream primer F1: 5' -TGGAATGTAAGGAAGTGTGTGG-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-206 in plasma exosome by using Real-Time PCR method
1) Primary reagent
Figure BDA0003139359190000081
2) Main instrument
Figure BDA0003139359190000082
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-206 in the plasma exosome by the method
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 the Hsa-miR-206 in the plasma exosomes of the gastric cancer dyscrasia patients 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 the Hsa-miR-206 in the plasma exosomes of the patients is high), while the difference delta CT between the amplification cycle threshold Ct of the Hsa-miR-206 in the plasma exosomes of the gastric cancer dyscrasia patients and the amplification cycle threshold Ct of the internal reference gene U6 is higher than 2 (the delta CT is greater than 2, and the expression level of the Hsa-miR-206 in the plasma exosomes of the patients is low). According to the quantitative results of a large number of clinical samples, the Δ CT value is 2 as a limit value to predict the occurrence of cachexia of a gastric cancer patient, the closer Δ CT (>2) is to 2, the higher the risk of cachexia, and when the Δ CT value of a gastric cancer patient who has not diagnosed cachexia is reduced to 4, the gastric cancer patient is judged to have cachexia.
In a word, the invention proves that the specific exosome miRNA, namely Hsa-miR-206 is closely related to the generation and development of gastric cancer dyscrasia through experiments, so that Hsa-miR-206 is taken as an important target in gastric cancer genomics, and important references 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-206 in plasma exosomes.
<110> China people liberation military and military medical university
<120> early diagnosis kit for gastric cancer cachexia based on exosome miRNA-206 expression level
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<213> Artificial Synthesis
<400> 1
tggaatgtaa ggaagtgtgt gg 22
<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-206 in 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-206 in the plasma exosome by real-time quantitative PCR (polymerase chain reaction) by taking the reverse transcript of the exosome miRNA as a template, and determining an amplification cycle threshold of the Hsa-miR-206 in the plasma exosome;
3) and calculating the difference delta CT between the amplification cycle threshold of the Hsa-miR-206 and the amplification cycle threshold of the reference gene.
2. The method for detecting the content of exosome miRNA-206 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-206 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`-TGGAATGTAAGGAAGTGTGTGG-3`。
3. an early diagnosis kit for gastric cancer dyscrasia, which is characterized in that: the kit comprises real-time quantitative PCR amplification primers of Hsa-miR-206.
4. The kit for the early diagnosis of gastric cancer cachexia according to claim 3, wherein: the Hsa-miR-206 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-206 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-206 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`-TGGAATGTAAGGAAGTGTGTGG-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-206 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-206 in preparation of early diagnosis kit for gastric cancer cachexia.
The application of the reverse transcript of Hsa-miR-206 in preparing the kit for early diagnosis of gastric cancer cachexia.
Application of an amplification primer of Hsa-miR-206 in preparation of an early diagnosis kit for gastric cancer cachexia.
CN202110731382.5A 2021-06-29 2021-06-29 Early diagnosis kit for gastric cancer cachexia based on exosome miRNA-206 expression level Pending CN113337609A (en)

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