CN109609630B

CN109609630B - Molecular marker for early gastric cancer diagnosis and application thereof

Info

Publication number: CN109609630B
Application number: CN201811467882.7A
Authority: CN
Inventors: 张艳桥; 黄小义; 唐淑丽; 程佳楠; 姚媛菲
Original assignee: Harbin Medical University
Current assignee: Harbin Medical University
Priority date: 2018-12-03
Filing date: 2018-12-03
Publication date: 2023-01-13
Anticipated expiration: 2038-12-03
Also published as: CN109609630A

Abstract

The invention discloses a molecular marker for early gastric cancer diagnosis and application thereof. The molecular marker is exosome miRNA exo-miR-92b-3p and/or exo-let7g-5p, which are respectively shown as SEQ ID NO:1 and SEQ ID NO:2, respectively. Compared with normal people, the content of exo-miR-92b-3p and exo-let7g-5p in the circulating blood of an early gastric cancer patient is obviously increased, and the method has higher sensitivity and specificity compared with the conventional CEA (cancer marker) index. And after the exo-miR-92b-3p and the exo-let7g-5p are combined or combined with a CEA index, the diagnosis rate can be remarkably improved, so that the exo-miR-92b-3p and/or the exo-let7g-5p can be used as a reliable circulating biomarker of early gastric cancer for early gastric cancer diagnosis.

Description

Molecular marker for early gastric cancer diagnosis and application thereof

Technical Field

The invention relates to a molecular marker and application thereof, in particular to a molecular marker for early gastric cancer diagnosis and application thereof, and belongs to the technical field of medicines.

Background

It is well known that gastric cancer is a common serious life-threatening malignancy of the digestive system. According to the statistics of the american cancer society, by 2012, gastric cancer has become the fifth most common malignancy, the third most lethal cancer worldwide. Nearly 100 million new cases of gastric cancer and 70 or more deaths of patients exist in the whole world in 2012. The incidence and mortality of gastric cancer of men and women in developing countries are higher than those of developed countries, 70% of new cases worldwide occur in developing countries of Asia at present, half of cases mainly occur in China, and the statistics of cancer in China in 2015 show that gastric cancer is the second place of cancer incidence and mortality in China.

Despite the recent decline in the incidence of gastric cancer, its mortality rate remains high, and the lack of an early diagnostic method and the ease of developing drug resistance are the main reasons behind its high mortality rate. The 5-year survival rate of the late gastric cancer is only 30% -50%, while the 5-year survival rate of the early gastric cancer can reach more than 90%. Early diagnosis of gastric cancer can also effectively reduce the mortality of patients, so early diagnosis is very important to improve the curative effect of gastric cancer. Early diagnosis of gastric cancer is always a key concern, and at present, the traditional gastroscopy is still the most reliable means for diagnosing gastric cancer, but the gastroscopy belongs to invasive examination and is not suitable for being used as routine screening examination. At present, the clinical gastric cancer markers mainly comprise CEA, CA199, CA724 and the like, but the diagnosis sensitivity and specificity of the markers are low, so that the clinical diagnosis value is limited. Therefore, in order to avoid the short panel of conventional diagnosis, a new, simple, convenient, highly sensitive and highly specific non-invasive marker is urgently needed to improve the early and even ultra-early diagnosis or prognosis evaluation of gastric cancer.

Exosomes are bilayer membranous structures derived from the endosome, released into the extracellular environment via fusion of the multilumenal bodies with the cell plasma membrane, with a diameter of 30-100nm, secreted by almost all types of cells, and present in almost all body fluids in the human body, such as: plasma, serum, saliva, urine, ascites, and the like. Exosomes are capable of acting as a "transporter" to transport proteins and RNA between cells and participate in many intracellular processes, mediating cancer progression, changes in tumor microenvironment, angiogenesis, regulation of immune responses, metastasis, and drug resistance. Blood is an important distribution site for exosomes in the circulatory system, and serum exosomes have been shown to be the major source of miRNA in the circulatory system. Under the protection of the bilayer membrane structure of exosome, RNase enzyme existing in blood in large quantity is sufficiently isolated, so that miRNA is prevented from being degraded and kept stable; in addition, with the protection of exosomes, fluctuations in RNA content in blood due to physical factors such as freeze-thawing do not affect their levels in exosomes, and miRNA can retain its original function even when it is transferred to a location remote from the original cells. Compared with the weak stable state of total miRNA in blood, exosome miRNAs have stronger stability and can be conveniently extracted even in a sample frozen for a long time; in addition, miRNAs in exosomes are selectively enriched and specific.

Compared with normal people, the invention discovers for the first time that the content of exo-miR-92b-3p and exo-let7g-5p in the circulating blood of an early gastric cancer patient is obviously increased, and the diagnosis efficiency is higher than that of a conventional tumor marker-CEA index. And the diagnosis rate can be remarkably improved after the combination of exo-miR-92b-3p and exo-let7g-5p and the combined CEA index thereof, and the exo-miR-92b-3p and exo-let7g-5p are reliable circulating biomarkers of early gastric cancer.

Disclosure of Invention

Aiming at the problem that the existing molecular marker for early gastric cancer diagnosis is low in diagnosis positive rate, sensitivity and specificity, the invention provides a molecular marker for early gastric cancer diagnosis and application thereof.

In order to achieve the purpose, the invention adopts the following technical means:

the invention provides a molecular marker for early gastric cancer diagnosis, which is exosome miRNA exo-miR-92b-3p and/or exo-let7g-5p, wherein the nucleotide sequence of exo-miR-92b-3p is shown in SEQ ID NO:1, and the nucleotide sequence of exo-let7g-5p is shown as SEQ ID NO:2, respectively.

Furthermore, the invention also provides application of the molecular marker and a reagent for detecting the expression level of exosome miRNA exo-miR-92b-3p and/or exo-let7g-5p in preparation of a kit for diagnosing early gastric cancer.

Preferably, the reagent is a primer, and more preferably, the upstream primer for detecting the expression level of the exosome miRNA exo-miR-92b-3p is an upstream primer shown in a sequence table SEQ ID NO:3, the upstream primer for detecting the expression level of the exosome exo-let7g-5p is the primer shown in SEQ ID NO:4, the downstream primers are all universal primers.

Wherein, the Universal Primer is preferably a misScript Universal Primer.

Furthermore, the invention also provides a kit for diagnosing early gastric cancer, which contains a primer for detecting the expression level of exosome miRNA exo-miR-92b-3p and/or exo-let7g-5p, preferably an upstream primer for detecting the expression level of exosome miRNA exo-miR-92b-3p is shown as SEQ ID NO:3, the upstream primer for detecting the expression level of exosome exo-let7g-5p is shown as SEQ ID NO:4, the downstream primers are all universal primers.

Wherein, preferably, the Universal Primer is the MiScript Universal Primer.

Preferably, the kit further comprises a vacuum blood collection tube-separation gel/coagulant tube, a vacuum blood collection needle assembly, an exosome separation reagent, an exosome miRNA extraction reagent, a reverse transcription reagent, a real-time fluorescent quantitative PCR reagent and a primer for detecting the expression level of internal reference hsa-miR-30e-5p, wherein the upstream primer is shown as SEQ ID NO:5, the downstream primer is a universal primer.

Preferably, the kit of the present invention is used for diagnosing early gastric cancer, and comprises the following steps:

(1) Serum exosome isolation and RNA extraction thereof

Exosomes in serum are separated by an exosome separation reagent, and small RNA in the exosomes is extracted by an exosome miRNA extraction reagent;

(2) Reverse transcription of exosome RNA into cDNA

Reverse transcription reagent is adopted to carry out reverse transcription on the exosome RNA into cDNA;

(3) PCR amplification of cDNA

Performing PCR amplification by using the cDNA which is subjected to reverse transcription as a template and adopting a real-time fluorescent quantitative PCR reagent according to any one of the following methods; 1) Amplifying by using primers for detecting exosome miRNA exo-miR-92b-3p and internal reference hsa-miR-30e-5 p; 2) Amplifying by using primers for detecting exosome miRNA hsa-let7g-5p and internal reference hsa-miR-30e-5 p; 3) Carrying out combined amplification by using primers for detecting exosome miRNA exo-miR-92b-3p, hsa-let7g-5p and internal reference hsa-miR-30e-5 p;

(4) And (4) judging a result:

and (3) analyzing a fluorescence quantitative detection result: when the expression quantity of the miRNA is calculated by taking the expression quantity of the hsa-miR-30e-5p as a reference, the relative quantification of the miRNA is displayed by a delta Ct value, and the delta Ct = Ct (miRNA) -Ct (hsa-miR-30 e-5 p), wherein in the method 1), the relative expression quantity delta Ct of the hsa-miR-92b-3p is judged to be positive in expression, and otherwise, the relative expression quantity delta Ct is judged to be negative in expression; the positive expression is diagnosed as gastric cancer; in the method 2), the relative expression quantity delta Ct of the hsa-let7g-5p is less than 4.184, and the expression is judged to be positive, otherwise, the expression is negative; the positive expression is diagnosed as gastric cancer; in the method 3), the relative expression quantity delta Ct of hsa-miR-92b-3p is less than 1.690, and the relative expression quantity delta Ct of hsa-let7g-5p is less than 4.184, so that the expression is judged to be positive, otherwise, the expression is negative; when any index of the hsa-miR-92b-3p and the hsa-let7g-5p is expressed positively, early gastric cancer is diagnosed.

Compared with the prior art, the invention has the beneficial effects that:

compared with normal people, the invention discovers for the first time that the content of exo-miR-92b-3p and exo-let7g-5p in the circulating blood of a patient with early gastric cancer is obviously increased, the diagnosis efficiency is higher than that of a conventional tumor marker-CEA index, the diagnosis rate can be obviously improved by the combination of exo-miR-92b-3p and exo-let7g-5p and the combined CEA index thereof, and the exo-miR-92b-3p and exo-let7g-5p are reliable circulating biomarkers of the early gastric cancer.

The diagnosis positive rates of CEA, CA199 and CA724 of the serum of the early gastric cancer patient are respectively as follows: 9.1%, 11.4% and 2.3%. The diagnosis positive rate of the serum exo-miR-92b-3p of the early gastric cancer patient is 42 percent; the positive diagnosis rate of serum exo-let7g-5p of early gastric cancer patients is 46%. The positive rate of the early gastric cancer patient after combined diagnosis of serum exo-miR-92b-3p and exo-let7g-5p is 60%. The diagnosis positive rate is obviously higher than that of the existing CEA, CA199 and CA724.

At present, most of research on gastric cancer markers is limited to circulating blood RNA detection, but the circulating blood RNA has poor stability, is easy to degrade and has extremely high requirements on blood sample collection and treatment. And exosomes in serum are stable and not easy to degrade due to the protection of a double-layer membrane, and have low requirements on storage environment and detection time. Overcomes the defects of poor stability and low detection rate of miRNAs in early gastric cancer circulating blood.

The invention changes the current situation that few means for early diagnosis of the gastric cancer are provided, provides a novel, simple, convenient, feasible, high-sensitivity and high-specificity non-invasive marker for early diagnosis of the gastric cancer, and improves the early diagnosis rate of the gastric cancer and even the ultra-early diagnosis rate. Meanwhile, proteins, nucleic acids and the like in the exosomes are actively or passively secreted by cells, and provide necessary functional substances in the process of cancer occurrence and development, so that the exosomes have high diagnostic value. The exosomes in the serum are extracted to detect exo-miR-92b-3p and exo-let7g-5p, so that the stability and reliability of the result can be ensured.

The tumor markers exo-miR-92b-3p and exo-let7g-5p can be proved to be suitable for serving as diagnosis markers of digestive system tumors in small-scale clinical samples, have better sensitivity and specificity compared with the traditional CEA, CA199, CA724 and the like, and can be used as tumor markers for population general survey.

The sensitivity and specificity of serum CEA diagnosis of early gastric cancer patients are respectively as follows: 70 percent and 42 percent. The sensitivity and specificity of the serum exo-miR-92b-3p diagnosis of the early gastric cancer patient are respectively as follows: 80% and 58%; the sensitivity and specificity of serum exo-let7g-5p diagnosis of early gastric cancer patients are respectively as follows: 88 percent and 54 percent. After combined diagnosis of serum exo-miR-92b-3p and exo-let7g-5p of early gastric cancer patients, the sensitivity and specificity are respectively as follows: 82 percent and 60 percent.

The invention uses the exosome miRNA molecular marker to detect early gastric cancer, and avoids the harm to the body caused by traditional invasive examination (such as gastroscopy).

When any result of the gastric cancer CEA, CA199 and CA724 detected by serum of a patient is positive, the detection kit provided by the invention is used for auxiliary detection, so that the false positive result of serological detection can be effectively removed, and huge mental stress and property loss brought to the patient are avoided; meanwhile, the result of detecting gastric cancer CEA, CA199 and CA724 by serum is negative, and the detection kit provided by the invention is used for auxiliary detection, so that the false negative result of serological detection can be effectively found, and the life of a patient can be saved in time.

The invention discovers two novel high-efficiency specific exosome miRNA molecular markers aiming at early gastric cancer, and provides a novel thought and theoretical basis for the research and development of the miRNA based on peripheral blood exosome as the early gastric cancer diagnosis marker.

Drawings

FIG. 1 is a serum exosome form under a transmission electron microscope;

FIG. 2 is a gel electrophoresis diagram of the RNA size distribution of serum exosomes;

FIG. 3 shows the expression levels and differences of serum exo-miR-92b-3p in early gastric cancer patients and normal persons;

FIG. 4 shows the expression levels and differences of exo-let7g-5p in serum in early gastric cancer patients and normal persons;

FIG. 5 shows the expression level and difference of CEA in serum in early stage gastric cancer patients and normal persons;

FIG. 6 is a ROC curve for serum exo-miR-92b-3 p;

FIG. 7 is a ROC curve for serum exo-let7g-5 p;

FIG. 8 is a ROC curve for serum CEA;

FIG. 9 is a ROC curve for serum exo-miR-92b-3p and exo-let7g-5p combinations;

FIG. 10 is a ROC curve for serum exo-miR-92b-3p and CEA combinations;

FIG. 11 is a ROC curve for serum exo-let7g-5p in combination with CEA;

FIG. 12 is a ROC curve for combinations of serum exo-miR-92b-3p, exo-let7g-5p and CEA;

FIG. 13 shows the relationship between serum exo-miR-92b-3p and gastric cancer neuro-infiltration;

FIG. 14 shows the relationship between serum exo-let7g-5p and gastric cancer neuro-infiltration;

FIG. 15 shows the relationship between serum exo-miR-92b-3p and low adhesion of gastric cancer;

FIG. 16 shows the relationship between serum exo-let7g-5p and low adhesion of gastric cancer;

FIG. 17 shows the relationship between serum exo-miR-92b-3p and gastric cancer differentiation degree;

FIG. 18 shows the relationship between serum exo-let7g-5p and the degree of differentiation of gastric cancer;

FIG. 19 is a graph comparing the accuracy of serological, exosome miRNAs and gastroscopic detection assays using post-operative pathological examination as a standard.

Detailed Description

The following examples are given to illustrate the embodiments of the present invention and the detailed embodiments and specific procedures thereof, but the scope of the present invention is not limited to the following examples.

The room temperatures described in the examples below all refer to 25 ℃.

Example 1: screening of exosome miRNA related to early gastric cancer

1. Patients and samples were included:

in this experiment, a total of 36 early stage gastric cancer patients and 12 normal controls were included.

Inclusion and exclusion criteria for tumor patients included: 1) First diagnosing the patient; 2) During blood collection, the patient does not use any therapeutic drug aiming at gastric cancer, and does not undergo any radiation examination and treatment; 3) No prior or concomitant history of other digestive tract tumors; 4) Early gastric cancer confirmed by postoperative pathology; 5) The clinical and pathological data of the patient are complete; 6) There are no other acute diseases or systemic diseases such as autoimmune diseases, diabetes, coronary heart disease, etc. The blood samples of patients meeting the above conditions were grouped into case groups. The normal control group was derived from a normal physical examination population of a number and age matched to the case group (median age difference < 1%).

2. Sample collection

Collecting 3ml fresh peripheral blood, placing in yellow vacuum blood collection tube-separation gel/coagulant tube, standing at 4 deg.C for 30min, and centrifuging at 4 deg.C 3000 Xg in TOMY MX-307 refrigerated centrifuge for 30min. The upper layer of light yellow serum was aspirated and dispensed into a clean 1.5mL plastic centrifuge tube at 260. Mu.L/tube and stored at-80 ℃ until use. To ensure that the serum does not contain cell debris, the centrifuge tube retains at least 0.5cm of serum; to ensure the homogeneity of the experimental work, it is necessary to ensure that the whole serum preparation process is completed within two hours.

3. Serum Exosomes isolation and RNA extraction thereof

1) Extraction of Exosomes:

the exosome is extracted by using an exotQuickTMexosome Precipitation Solution kit (Cat. # EXOQ 20A-1) of SBI company, and the extraction method specifically comprises the following steps:

mu.L of serum was thawed on ice and centrifuged at 3000 Xg for 30min at 4 ℃ in a TOMY MX-307 refrigerated centrifuge. The supernatant pale yellow serum was aspirated 250. Mu.L/tube and dispensed into a clean 1.5mL plastic centrifuge tube. Adding 25 mu L of Tromboretin D and mixing. Incubating in 37 deg.C water bath for 15min. Followed by centrifugation in a TOMY MX-307 refrigerated centrifuge at 25 ℃ 10000 Xg for 5min and aspiration of the supernatant into a clean 1.5mL plastic centrifuge tube. Add 75. Mu.L of ExoQuick reagent to the centrifuge tube, mix well and place in a refrigerator at 4 ℃ overnight. Centrifuging the overnight treated sample in a TOMY MX-307 refrigerated centrifuge at 4 ℃ and 1500 Xg for 30min, sucking and discarding the supernatant, centrifuging the sample in the TOMY MX-307 refrigerated centrifuge at 4 ℃ and 1500 Xg for 5min again, sucking and discarding the supernatant, and obtaining yellow precipitate below an EP tube as serum exosomes. The yellow precipitate was suspended in 50. Mu.L of 1 XPBS to form a homogeneous pale yellow mixture.

2) Detection under an Exosomes electron microscope:

the Exosomes solution was fixed in 2% paraformaldehyde solution and then serially diluted with distilled water. Transfer 5. Mu.l of the diluted mixture to a clean silicon wafer. After the ultrasonic treatment, the silicon wafer was immersed in ethanol and distilled water for 5 minutes, respectively, in this order. And drying and fixing the microvesicles under the ventilation hood. The silicon wafer was then mounted on an SEM platform. To make the surface conductive, a coating of 2-5nm gold-palladium alloy was applied by sputtering (SPI-Module Sputter, eden Instruments, france) before imaging with a scanning electron microscope (S-4700, japan). Scanning by electron microscope is carried out under the condition of low beam energy (5.0-10.0 kV). To present optimal vesicle morphology under scanning electron microscopy, freshly isolated exosomes were fixed and imaged immediately after isolation. The photos were processed using ImageJ software. The results are shown in FIG. 1. Using ExoQuick ^TM The kit separates and extracts exosomes in serum, particle size distribution of an extract is mainly circular microvesicles with the diameter of about 100nm under the condition of amplification of 80000 x as observed by transmission electron microscopy, and morphological expression of the exosomes is met, so that the extraction method can successfully extract the exosomes in the serum.

3) Exosome RNA extraction

Exosome RNA was extracted with the aid of QIAGEN miRNeasy Micro Kit (Cat. # 217084) by the following method:

(1) add 700. Mu.L of QIAzol to the centrifuge tube, resuspend the pellet completely, and let stand at room temperature for 5 minutes. Then 140. Mu.L of chloroform (Shanghai test chemical) was added and the centrifuge tube cap was closed. Shaking vigorously for 15 seconds, and standing at room temperature for 3 minutes.

(2) TOMY MX-307 refrigerated centrifuge centrifuged at 12,000 Xg for 15min at 4 ℃.

(3) Carefully transfer 350. Mu.L of the upper aqueous phase from the centrifuge tube to a fresh clean 1.5mL plastic centrifuge tube. Add 525. Mu.L of 100% ethanol solution and mix gently.

(4) Extracting 700 μ L of the mixture obtained in (3), adding into RNeasy MinElute spin column, hitachi CT15RE refrigerated centrifuge at 22 deg.C 500 Xg centrifuging 30s → 22 deg.C 800 Xg centrifuging 30s, and discarding the effluent.

(5) Repeating the steps, namely Hitachi CT15RE refrigerated centrifuge centrifuges 30s at the temperature of 22 ℃ and 500 Xg → 30s at the temperature of 22 ℃ and 800 Xg, and discarding effluent.

(6) Add 700. Mu.L Buffer RWT to RNeasy MinElute spin column, cover, hitachi CT15RE refrigerated centrifuge at 22 ℃ 8000 Xg for 15s, and discard the effluent.

(7) Add 500. Mu.L Buffer RPE to RNeasy MinElute spin column, cover, hitachi CT15RE refrigerated centrifuge 22 ℃ 8000 Xg centrifuge 15s, discard effluent.

(8) Add 500. Mu.L of 80% ethanol solution to RNeasy MinElute spin column, cover, hitachi CT15RE refrigerated centrifuge at 22 deg.C 8000 Xg for 15s, and discard the effluent. Then Hitachi CT15RE refrigerated centrifuge 22 ℃ 21000 Xg centrifugal 2min.

(9) RNeasy MinElute spin column was placed in a fresh 1.5mL plastic centrifuge tube provided with the kit and dried on a clean bench.

R.C. in RNeasy MinElute spin column center, 14. Mu.L of nuclease-free deionized water (i.e., RNase free water provided in kit) was added, the lid was closed, and left to stand at room temperature for 5min, and Hitachi CT15RE refrigerated centrifuge was centrifuged at 22 ℃ 2000 Xg for 30s → 21000 Xg and 22 ℃ for 2min, and the effluent was exosomes RNA.

Centrifuge tubes containing exosomes RNA were placed on ice, 1.2 μ L into clean 200 μ L plastic centrifuge tubes and both portions were frozen in a-80 ℃ freezer.

4) Exosome RNA content detection

The exosome RNA content was detected using the Agilent Small RNA Kit (Cat. # 5067-1548) from Life Technologies (ABI). The results are shown in FIG. 2, and the RNA content and fragment size extracted from the exosomes were determined by Agilent Small RNA Kit. The miRNA yield in 250 mu L of plasma is 9-30 ng, the yield is rich, the miRNA is mainly distributed in the length range of 18-28nt, 22nt reaches the peak value and accords with the molecular size of miRNA, and the RNA extracted from exosomes is proved to be mainly miRNA. The method for extracting the exosome RNA is stable, and the extracted RNA content is rich.

4. Library construction, next generation sequencing and data analysis

1) Building warehouse

By NEB Corp

Multiplex Small RNA Library Prep Set for

(Set 1) Kit (NEB # E7300S) was used to perform the library construction of exosome RNA, followed by Purification of the obtained cDNA using QIAGEN QIAquick PCR Purification Kit (Cat. # 28104), electrophoretic separation of the purified cDNA in 5% page gel 100V 40mins, visualization under uv lamp of BIO-RAD visualizer (USA, EN 61010-1), cutting of bands with a size of 140bp to 160bp region, further Purification of target gel mass to obtain target cDNA library corresponding to small RNA: all samples of interest were diluted to 20 ng/. Mu.L and dispensed 5.5. Mu.L into new clean nuclease-free 1.5mL plastic centrifuge tubes for second generation sequencing by dry ice delivery to the laboratory.

2) Second generation sequencing results and analysis

High throughput sequence identification was performed using the HiSeq2000Genome Analyzer platform from Illumina. The sequencing strategy is determined to be a one-way reading and 50 sequencing cycle mode. Since the length of the miRNA molecule does not exceed 23nt, a sequencing strategy that reads 50 bases can completely cover the full length of the miRNA molecules inserted in the library. The sequencing data was processed using Perl scripts that we optimized. Firstly, extracting sequence information, pruning data, and extracting and integrating sequences with inserts larger than 16 nt. The combined sequence file is subjected to homology comparison with a human mature miRNA sequence library and a genome RNA sequence library by using optimized Bowtie software, and reference data are from a latest version of miRNbase (Rlease 19 in the update) and a library file recently updated by NCBI. miRNA transcript levels are normalized by comparing the values from total miRNA reads against which the assay reads can align. And performing differential analysis on miRNA with the normalized absolute reading value of more than or equal to 5. Differential miRNAs with up-regulated expression, AUC (AUC) of more than or equal to 8 and sensitivity and specificity of more than 80% are selected for verification, and part of miRNAs with up-regulated expression of early gastric cancer serum exosomes primarily screened out by second-generation sequencing and diagnosis conditions thereof are shown in Table 1. A total of 6 miRNA molecules were selected for validation: hsa-miR92b-3p, hsa-miR-21-5p, hsa-let7c-5p, hsa-let7g-5p, hsa-miR-26a-5p, hsa-miRNA-101-3p (MS 00032144, MS00009079, MS00003129, MS00008337, MS00029239, MS00008372, QIAGEN, USA). miRNA-30e-5p (MS 00009401, QIAGEN, USA) was used as an endogenous reference and amplified for each sample.

TABLE 1 MiRNAs with up-regulated expression of early gastric cancer serum exosomes primarily screened by second-generation sequencing and diagnosis conditions thereof

Example 2: verification of exosome miRNAs with high correlation with early gastric cancer

1. Patients and samples were included:

in this experiment, a total of 50 early stage gastric cancer patients and 50 normal persons were included as controls. The criteria for inclusion and exclusion of tumor patients were the same as in example 1.

2. The sample collection, serum exosome separation and RNA extraction methods were the same as in example 1.

3. Reverse transcription of exosome RNA into cDNA

The reverse transcription of the exosome RNA was performed by using the QIAGEN misscript II RT Kit (Cat. # 218161), and the specific procedure was as follows:

(1) exosome RNAs were thawed on ice, 5x miScript RT Buffer and rnaefree water were thawed at room temperature. Each vial was flicked to mix well, then centrifuged briefly to collect the liquid on the walls and caps, and finally placed on ice.

(2) 4ng of RNA was added to each PCR tube and nuclease free deionized water (i.e., RNase free water supplied from the kit) was added to a total volume of 9. Mu.L.

(3) Preparing a Reverse-transcription master Mix on ice, taking a new clean 1.5mL plastic centrifuge tube, adding 330 mu L of 5x misscript HiSpec Buffer, 165 mu L of 10x misscript Nucleics Mix and 165 mu L of misscript Reverse transcription Mix, mixing uniformly and centrifuging to obtain the misscript Reverse-transcription master Mix. After compounding, mix and place on ice.

(4) And (3) adding 6 mu L of MiScript Reverse Transcriptase Mix into the liquid obtained in the step (2), mixing uniformly and centrifuging.

(5) In a PCR instrument (Applied Biosystems,

PCR System 9700) incubation at 37 ℃ for 1h → incubation at 95 ℃ for 5mins to inactivate MiScript Reverse Transcriptase Mix, reverse transcription product-20 ℃ refrigerator storage.

3) PCR amplification of cDNA

PCR of exosome cDNA Using QIAGEN

The specific operation method of the miRNA PCR Array kit (Cat. # 218076) is as follows:

(1) the cDNA samples were thawed on ice and diluted in nuclease free deionized water at ratio 1.

(2) Taking a 1-plate 384-well plate as an example, a reaction solution was prepared: a fresh clean nuclease-free 5mL plastic centrifuge tube was taken, added to 1950. Mu.L of 2 xQuantiTect SYBR Green PCR Master Mix, 390. Mu.L of 10 xMiScript Universal Primer and 390. Mu.L of 10 xMiScript Primer Assay, mixed and centrifuged, and 7. Mu.L/well was added to a 384 well plate.

Wherein the Primer contained in the 10 XmiScript Primer Assay is Primer hsa-miR-92b-3p, hsa-let7g-5p or hsa-miR-30e-5p.10 XmiScript Universal Primer is a Universal downstream Primer designed and synthesized by Qiagen, which is included in the Qiagen's miScript SYBR Green PCR Kit.

hsa-miR-92b-3p:5'TATTGCACTCGTCCCGGC3’(SEQ ID NO.3)

hsa-let7g-5p:5'TGAGGTAGTAGTTTGTAC3’(SEQ ID NO.4)

hsa-miR-30e-5p:5'TGTAAACATCCTTGACTG3’(SEQ ID NO.5)

(3) mu.L of the diluted cDNA samples were added sequentially, and 3 wells were repeated for each cDNA sample.

(4) After the sample application, the 384-well plate is placed in a Labnet C1000 centrifuge and centrifuged at 25 ℃ and 1000Xg for 1min.

(5) Installing the PCR program into a Roche 480PCR instrument to run, wherein the specific program setting is as follows:

TABLE 2

(6) And after the PCR is finished, storing the data, exporting and analyzing. The results were as follows:

relative expression levels of 50 pairs of early gastric cancer patients and normal control serum exo-miR-92b-3p (SEQ ID NO. 1) and exo-let7g-5p (SEQ ID NO. 2) are detected by applying qRT-PCR, and the results are shown in a graph 3-5. In relative quantitative analysis, the results of qRT-PCR are shown as Δ Ct values:

ΔCt＝Ct(miRNA)-Ct(hsa-miR-30e-5p)

wherein, the delta Ct value is the difference of the cycle number required when the fluorescence signal reaches a set threshold value in each reaction tube, and the larger the delta Ct value is, the lower the expression amount is. The expression difference of each index between early gastric cancer and a normal control group is compared by using a u test. The results show that the Δ Ct value [1.380 (0.234, 2.315) ] of early GC group serum exo-miR-92b-3p is significantly lower than that of the normal control group [2.480 (1.722, 3.122), U =714, p =0.0002]; the delta Ct value [4.101 (2.348, 5.365) ] of early GC group serum exo-let7g-5p was significantly lower than that of normal control group [5.617 (4.835, 6.327), U =611, p < 0.0001]; the early GC group serum CEA expression level [1.915 (1.083, 2.538) ] was not statistically different from the normal control group [1.780 (1.150, 2.445), U =1200, p =0.7329]. And (4) prompting by a result: the serum CEA index has no obvious difference in early gastric cancer and normal people, and is not suitable to be used as a marker for early diagnosis of gastric cancer, and the serum exo-miR-92b-3p and exo-let7g-5p can better distinguish early gastric cancer patients from normal people than the serum CEA and can be used as a GC early diagnosis marker.

In order to determine the diagnostic capability of serum exo-miR-92b-3p, exo-let7g-5p and a conventional tumor marker CEA on early GC, an ROC curve is drawn by using detected serum exo-miR-92b-3p, exo-let7g-5p and serum CEA data to evaluate the value of auxiliary diagnosis, and the value is shown in FIGS. 6-8. The results showed that the AUC value for serum exo-miR-92b-3P was 0.714 (P =0.0002, 95% CI; the sensitivity and specificity are respectively as follows: 80% and 58%; the AUC value for serum exo-let7g-5P was 0.756 (P < 0.0001, 95% CI: 88 percent and 54 percent; the AUC value of serum CEA was 0.520 (95% ci: 70%,42%, but not statistically significant (P = 0.7329); the result shows that the serum exo-miR-92b-3p and exo-let7g-5p have higher diagnosis efficiency than serum CEA in the early GC diagnosis, and the diagnosis has statistical significance.

ROC curves for combinations of serum exo-miR-92b-3p, exo-let7g-5p and CEA are shown in FIGS. 9-12. The serum exo-miR-92b-3p combined exo-let7g-5p, the serum exo-miR-92b-3p combined serum CEA, the serum exo-let7g-5p combined serum CEA, the serum exo-miR-92b-3p combined exo-let7g-5p combined serum CEA can improve the diagnosis efficiency of early GC, and the AUC values are respectively as follows: 0.777, 0.722, 0.760 and 0.784, and all have statistical significance (P is less than 0.05); wherein the sensitivity and specificity of the serum exo-miR-92b-3p and exo-let7g-5p after combination are respectively improved to 82% and 60%.

And (4) prompting by a result: the serum exo-miR-92b-3p and exo-let7g-5p can be combined to improve the diagnostic efficiency of each serum exo-miR-92b-3p and exo-let7g-5p, the serum exo-miR-92b-3p and exo-let7g-5p and the combination of the serum exo-miR-92b-3p and the serum CEA can improve the diagnostic efficiency of the CEA and have statistical significance, and the serum exo-miR-92b-3p and the exo-let7g-5p can be used as an independent marker for early diagnosis of GC and can also be used as an auxiliary diagnostic marker for a conventional serum marker CEA.

Serum exo-miR-92b-3p, exo-let7g-5p and early GC clinical pathological characteristics are further analyzed by applying a u test, and the results are shown in FIGS. 13-18. The results show that the expression level of serum exo-miR-92b-3p is related to the neural infiltration of early gastric cancer, the delta Ct value [4.830 (2.994, 5.923) ] of the serum exo-miR-92b-3p of a patient with the neural infiltration is remarkably higher than that of a patient without the neural infiltration [3.164 (1.880, 4.774), U =185, P =0.0234], namely the expression level of the serum exo-miR-92b-3p of an early gastric cancer patient without the neural infiltration is remarkably higher than that of a patient with the neural infiltration; the expression level of serum exo-let7g-5p is related to low adhesiveness of early gastric cancer, the delta Ct value [2.610 (0.952, 3.167) ] of the serum exo-let7g-5p of an early gastric cancer patient with low adhesiveness of a tumor is obviously higher than that of an early gastric cancer patient without low adhesiveness [1.273 (0.047, 1.873), U =116.5, P =0.0021], namely the expression level of the serum exo-let7g-5p of the early gastric cancer patient without low adhesiveness is obviously higher than that of the early gastric cancer patient with low adhesiveness of the tumor; the expression level of serum exo-miR-92b-3P is not related to the low adhesion and differentiation degree of early gastric cancer (P & gt 0.05), and the expression level of serum exo-let7g-5P is not related to the nerve infiltration and differentiation degree (P & gt 0.05).

Example 3: preparation and application of early gastric cancer serum exo-miR-92b-3p and exo-let7g-5p detection and identification kit

1. Reagent kit

The kit provided by the embodiment comprises a primer, a vacuum blood collection tube, a separation gel/coagulant tube, a vacuum blood collection needle assembly, an exosome separation reagent, an exosome miRNA extraction reagent, a reverse transcription reagent and a real-time fluorescent quantitative PCR reagent.

Upstream primers for real-time quantitative PCR amplification of hsa-miR-92b-3p (SEQ ID NO. 1), hsa-let7g-5p (SEQ ID NO. 2), and hsa-miR-30e-5p (SEQ ID NO.6, internal reference) were synthesized by QIAGEN. The primers are shown in the following table 3:

TABLE 3

Primer and method for producing the same	Sequence (5 '-3')	SEQ ID NO.
			hsa-miR-92b-3p	TATTGCACTCGTCCCGGC	3
hsa-let7g-5p	TGAGGTAGTAGTTTGTAC	4
			hsa-miR-30e-5p	TGTAAACATCCTTGACTG	5

The downstream primer is a universal downstream primer designed and synthesized by Qiagen, and the primer is contained in a MiScript SYBR Green PCR Kit of Qiagen.

The exosome-separating agent was obtained from the exotic biosensor Precipitation Solution kit (Cat. # EXOQ 20A-1) from SBI. Exosome miRNA extraction reagent was obtained from QIAGEN miRNeasy Micro Kit (Cat. # 217084). The RNA reverse transcription reagent was obtained from the MiScript II RT Kit (Cat. # 218161) from QIAGEN. Real-time fluorescent quantitative PCR reagents were obtained from the miScript SYBR Green PCR Kit from Qiagen (Cat. # 218076).

2. The results of the serological tests were verified using the kit provided in this example:

after the patient uses serum to detect whether any one of the gastric cancer serum tumor markers CEA, CA199 and CA724 is positive or the detection result is completely negative, the kit is used for further detection, and the result of serological detection is judged. The operation method of the kit comprises the following steps:

1) Serum exosome isolation and RNA extraction thereof

(1) 3ml of fresh peripheral blood was collected in a vacuum blood collection tube-separation gel/coagulant tube, left to stand at 4 ℃ for 30min, and centrifuged at 3000 Xg at 4 ℃ for 30min in a Thermo ST16R refrigerated centrifuge to collect serum.

(2) mu.L of serum was centrifuged for 30min at 3000 Xg in Thermo ST16R refrigerated centrifuge at 4 ℃. Absorbing 250 mu L of upper serum, adding 25 mu L of Tromboretin D, mixing uniformly, incubating in a water bath at 37 ℃ for 15min, centrifuging at 25 ℃ 10000 Xg by a thermo ST16R refrigerated centrifuge for 5min, and absorbing the supernatant into a clean 1.5mL plastic centrifuge tube. Add 75. Mu.L of ExoQuick reagent to the centrifuge tube, mix well and place in a refrigerator at 4 ℃ overnight. The overnight treated sample was centrifuged at 1500 Xg for 30min at 4 ℃ in a Thermo ST16R refrigerated centrifuge, the supernatant was discarded, and the supernatant was again centrifuged at 1500 Xg for 5min at 4 ℃ in a Thermo ST16R refrigerated centrifuge, and the yellow precipitate below the EP tube was serum exosomes. The yellow precipitate was suspended in 50. Mu.L of 1 XPBS to form a homogeneous pale yellow mixture.

(3) Adding 700. Mu.L QIAzol into the sample, resuspending and lysing the precipitate, standing at room temperature for 5mins, adding 140. Mu.L chloroform (Shanghai test chemical), covering the centrifugal tube, shaking vigorously for 15s, and standing at room temperature for 3mins.

(4) Thermo ST16R Freeze centrifuge centrifuged 15mins at 12,000 Xg at 4 ℃.

(5) The upper aqueous phase was carefully separated from the 350. Mu.L centrifuge tube and transferred to a fresh clean 1.5mL plastic centrifuge tube. Add 525. Mu.L of 100% ethanol solution and mix gently.

(6) Extracting 700 μ L of the mixed solution obtained in (5) and adding into RNeasy MinElute spin column (column for short), and removing effluent → Termo micro21R refrigerated centrifuge 22 deg.C 500 × g centrifugation 30s → 22 deg.C 800 × g centrifugation 30s, and discarding effluent.

(7) 700. Mu.L of Buffer RWT, and a Termo micro21R refrigerated centrifuge at 22 ℃ 8000 Xg were added to the column and centrifuged for 15s, and the effluent was discarded.

(8) 500 μ L Buffer RPE, termo micro21R refrigerated centrifuge, 8000 Xg at 22 deg.C was added to the column and centrifuged for 15s, and the effluent was discarded.

(9) Add 500. Mu.L of 80% ethanol solution to the column, cover the lid, centrifuge with a Termo micro21R refrigerated centrifuge at 22 ℃ 8000 Xg for 15s, discard the effluent. Then, the mixture is centrifuged for 2mins at a temperature of 22 ℃ and 21000 Xg by a Termo micro21R refrigerated centrifuge.

The column was placed in a fresh, clean 1.5mL plastic centrifuge tube provided with the kit and dried on a clean bench.

Adding 14 mu L of nuclease-free deionized water (namely RNase free water provided by the kit) into the center of the column, covering a cover, standing for 5min at room temperature, and centrifuging for 30s → 21000 Xg at the temperature of 22 ℃ by using a Termo micro21R refrigerated centrifuge and for 2min at the temperature of 22 ℃, wherein the effluent is exosomes RNA, subpackaging a small amount of RNA for detecting the concentration of the RNA, and placing the rest on ice.

2) Reverse transcription of exosome RNA into cDNA

(1) 4ng of RNA was added to each PCR tube and nuclease free deionized water (i.e., RNase free water supplied from the kit) was added to a total volume of 9. Mu.L.

(2) Preparing a Reverse-transcription master Mix on ice, taking a new clean 1.5mL plastic centrifuge tube, adding 330 mu L of 5x misscript HiSpec Buffer, 165 mu L of 10x misscript Nucleics Mix and 165 mu L of misscript Reverse transcription Mix, mixing and centrifuging to obtain the misscript Reverse-transcription master Mix. After compounding, mix and place on ice.

(3) Adding 6 mu.L of MiScript Reverse Transcriptase Mix into the liquid obtained in the step (1), mixing and centrifuging.

(4) In a PCR instrument (Applied Biosystems,

PCR System 9700) incubation at 37 ℃ for 1h → incubation at 95 ℃ for 5mins to inactivate the MiScript Reverse Transcriptase Mix, subpackaging a portion of the Reverse transcription product on ice, and storing the remaining product at-20 ℃ in a refrigerator for review.

3) PCR amplification of cDNA

(1) Taking the PCR tube for each sample as an example, 5. Mu.L of 2 × QuantiTect SYBR Green PCR Master Mix, 1. Mu.L of 10 × MiScript Universal Primer, 1. Mu.L of 10 × MiScript Primer Assay, 1. Mu.L of cDNA sample, and 2. Mu.L of nuclease free deionized water (i.e., RNase free water provided by the kit) were added to a clean PCR tube, and 3 wells were repeated for each Primer Assay sample. Wherein the 10 XmiScript Primer Assay contains primers hsa-miR-92b-3p, hsa-let7g-5p or hsa-miR-30e-5p.10 XmiScript Universal Primer is a Universal downstream Primer designed and synthesized by Qiagen, which is included in the Qiagen's miScript SYBR Green PCR Kit.

hsa-miR-92b-3p:5'TATTGCACTCGTCCCGGC3’(SEQ ID NO.3)

hsa-let7g-5p:5'TGAGGTAGTAGTTTGTAC3’(SEQ ID NO.4)

hsa-miR-30e-5p:5'TGTAAACATCCTTGACTG3’(SEQ ID NO.5)

(2) The 384 well plates were placed in a Labnet C1000 centrifuge and centrifuged at 1000Xg for 1min at 25 ℃.

(3) The PCR program is installed in a Roche 480PCR instrument to run, and the specific program setting method comprises the following steps:

TABLE 4

(4) And after the PCR is finished, storing the data, exporting and analyzing.

4) Analysis of fluorescent quantitative detection results

When calculating the miRNA expression amount with reference to the expression amount of hsa-miR-30e-5p, the relative quantification of miRNA is shown as Δ Ct value, Δ Ct = Ct (miRNA) -Ct (hsa-miR-30 e-5 p).

The detection method of the kit for detecting and identifying gastric cancer serum exo-miR-92b-3p and exo-let7g-5p comprises the following steps: (1) serological detection: extracting blood to be detected, and detecting by using serological tumor markers (CEA, CA199 and CA 724); (2) detection by a kit: extracting exosome, exosome miRNA extraction reagent and reverse transcription reagent in blood to be detected by using exosome separation reagent to prepare exosome cDNA (complementary deoxyribonucleic acid) of the blood to be detected as a real-time fluorescence quantitative PCR template, adding primers and the real-time fluorescence quantitative PCR reagent for reaction, and obtaining the expression quantity of miRNAs by using 95 ℃ 15mins → 94 ℃ 15s,55 ℃ 30s,70 ℃ 30s and 40 cycles → 4 ℃ infinity, wherein the expression quantity is compared with the expression quantity of miR-30e-5p in the serum exosome; and (3) judging the result: (1) when the serological detection of blood to be detected is positive, if the kit detects that the expression quantity of hsa-miR-92b-3p or hsa-let7g-5p in a patient serum exosome is up-regulated, and after removing the internal reference primer of hsa-miR-30e-5p, the relative expression quantity delta Ct of hsa-miR-92b-3p is less than 1.690 or the relative expression quantity delta Ct of hsa-let7g-5p is less than 4.184, or both of the two are met, the serological detection is judged to be positive; (2) when the blood serological detection to be detected is negative, if the kit detects that the expression quantity of hsa-miR-92b-3p and hsa-let7g-5p is reduced, the relative expression quantity delta Ct of the hsa-miR-92b-3p is more than or equal to 1.690, and the relative expression quantity delta Ct of the hsa-let7g-5p is more than or equal to 4.184, judging that the serological detection is negative; if the kit detects hsa-miR-92b-3p and the expression quantity of hsa-let7g-5p is up-regulated, after removing the internal reference primer of hsa-miR-30e-5p, the relative expression quantity delta Ct of hsa-miR-92b-3p is less than 1.690, and the relative expression quantity delta Ct of hsa-let7g-5p is less than 4.184, judging that the serological detection negative is false negative; and if other results appear, the serology judgment is consistent, and the detection result does not need to be further judged.

3. The kit provided in this example was used for early diagnosis of gastric cancer:

(1) Selection of a sample:

in this experiment, a total of 50 early stage gastric cancer patients were included without any treatment.

Inclusion and exclusion criteria for tumor patients included: 1) First diagnosing the patient; 2) During blood collection, the patient does not use any therapeutic drug aiming at gastric cancer, and does not undergo any radiation examination and treatment; 3) No prior or concomitant history of other digestive tract tumors; 4) The patient has undergone gastroscopy biopsy before operation; 5) Early gastric cancer as confirmed by postoperative pathology; 6) The clinical pathological data of the patient is complete; 7) There is no history of other acute or systemic diseases.

(2) Serum exosome isolation and RNA extraction thereof

Peripheral blood of a patient is collected by a vacuum blood collection needle assembly, fresh peripheral blood is collected in a vacuum blood collection tube-separation gel/coagulant tube, and serum is collected. Serum exosomes are separated by adopting an exosome separation kit, and then small RNA in the exosomes is extracted by adopting an exosome miRNA extraction kit. The specific operation method is the same as above.

(3) Reverse transcription of exosome RNA into cDNA

The exosome RNA was reverse transcribed into cDNA using a reverse transcription kit. The specific operation method is the same as above.

(4) PCR amplification of cDNA

And carrying out PCR amplification by using the reverse transcribed cDNA as a template and adopting a real-time fluorescent quantitative PCR reagent. The method is divided into 3 experimental groups, wherein the experimental group 1 adopts primer hsa-miR-92b-3p and internal reference primer hsa-miR-30e-5p for amplification, the experimental group 2 adopts primer hsa-let7g-5p and internal reference primer hsa-miR-30e-5p for amplification, and the experimental group 3 adopts primer hsa-miR-92b-3p and hsa-let7g-5p combination and internal reference primer hsa-miR-30e-5p for amplification. The specific operation method is the same as above.

In practice, we have found that the following problems can occur if absolute quantitative PCR is performed: 1. the operation steps are more complicated, so that the accuracy of the result is correspondingly reduced; 2. the Agilent Small RNA Kit (Cat. # 5067-1548) which is used for absolute quantification detects the content of the exosome RNA and then carries out the following reverse transcription and PCR steps, and the Agilent Small RNA Kit has the defects of high cost, short storage period and high operation difficulty. In view of the above problems, we improved the operation, and changed the absolute quantitative PCR into the relative quantitative PCR to test the expression level of exo-miR-92b-3p and exo-let7g-5 p. Internal control exo-miR30e-5p is used for relative quantification.

(5) And (4) judging a result:

and (3) analyzing a fluorescence quantitative detection result: when the expression quantity of the miRNA is calculated by taking the expression quantity of the hsa-miR-30e-5p as a reference, the relative quantification of the miRNA is shown by a delta Ct value, wherein the delta Ct = Ct (miRNA) -Ct (hsa-miR-30 e-5 p), wherein in the experimental group 1, the relative expression quantity delta Ct of the hsa-miR-92b-3p is judged to be positive in expression, and otherwise, the relative expression quantity delta Ct is judged to be negative in expression; the positive expression of the protein is diagnosed as gastric cancer. In the experimental group 2, the relative expression quantity delta Ct of hsa-let7g-5p is less than 4.184, and the result is judged to be positive expression, otherwise, the result is negative expression; the positive expression of the gene is diagnosed as gastric cancer. In the experimental group 3, the relative expression quantity delta Ct of hsa-miR-92b-3p is less than 1.690, and the relative expression quantity delta Ct of hsa-let7g-5p is less than 4.184, so that the expression is judged to be positive, otherwise, the expression is negative; when any index of the hsa-miR-92b-3p and the hsa-let7g-5p is expressed positively, early gastric cancer is diagnosed.

And (3) analyzing detection results of serum tumor markers CEA, CA19-9 and CA 724: the expression is positive if CEA is more than 5ng/ml, CA199 is more than 37U/ml and CA724 is more than 6.9U/ml, otherwise, the expression is negative; when any serum tumor marker is positive, early gastric cancer is diagnosed.

(6) The accuracy of detection methods such as serological detection, exosome miRNAs detection, gastroscope detection and the like is compared by taking postoperative pathological examination as a standard.

As a result: the specific results are shown in FIG. 19. The diagnosis positive rates of the serum tumor markers CEA, CA199 and CA724 in early gastric cancer are respectively 10%, 0% and 14%; the diagnosis positive rate of serum exo-miR-92b-3p of an early gastric cancer patient is 42%; the positive diagnosis rate of serum exo-let7g-5p of early gastric cancer patients is 46 percent; after combined diagnosis of serum exo-miR-92b-3p and exo-let7g-5p of a patient with early gastric cancer, the positive rate is 60%; the diagnosis positive rate of the traditional invasive gastroscopy in early gastric cancer is 88%.

The results show that:

through detection, the diagnosis positive rate of the traditional invasive gastroscopy in early gastric cancer can reach 88 percent, and although the gastroscopy has higher positive diagnosis rate, the characteristics of large invasiveness, high cost and the like limit wide clinical application value. The diagnosis positive rates of CEA, CA199 and CA724, which are traditional non-invasive marker detection methods, in early gastric cancer are only 10%, 0% and 14%, respectively, and these indexes have very limited clinical diagnosis value for early gastric cancer. The serum exo-miR-92b-3p and exo-let7g-5p are used as a novel, simple, convenient, feasible, high-sensitivity and high-specificity non-invasive marker discovered by the invention, and the marker has better diagnosis advantages compared with the traditional blood tumor marker, wherein the diagnosis positive rate of the serum exo-miR-92b-3p of an early gastric cancer patient is 42%; the positive diagnosis rate of serum exo-let7g-5p of early gastric cancer patients is 46 percent; is significantly superior to the traditional serum marker examination. Meanwhile, the positive rate of the early gastric cancer patient after combined diagnosis of the serum exo-miR-92b-3p and exo-let7g-5p can reach 60%, and is obviously improved compared with the independent detection of the serum exo-miR-92b-3p and exo-let7g-5 p.

Sequence listing

<110> Harbin university of medicine

<120> molecular marker for early diagnosis of gastric cancer and application thereof

<130> KLPI180879

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 22

<212> RNA

<213> hsa-miR-92b-3p

<400> 1

uauugcacuc gucccggccu cc 22

<210> 2

<211> 22

<212> RNA

<213> hsa-let7g-5p

<400> 2

ugagguagua guuuguacag uu 22

<210> 3

<211> 18

<212> DNA

<213> artificial sequence

<400> 3

tattgcactc gtcccggc 18

<210> 4

<211> 18

<212> DNA

<213> artificial sequence

<400> 4

tgaggtagta gtttgtac 18

<210> 5

<211> 18

<212> DNA

<213> artificial sequence

<400> 5

tgtaaacatc cttgactg 18

<210> 6

<211> 22

<212> RNA

<213> hsa-miR-30e-5p

<400> 6

uguaaacauc cuugacugga ag 22

Claims

1. Application of exosome miRNA exo-miR-92b-3p and exo-let7g-5p combination in preparation of kit for diagnosing early gastric cancer, wherein the nucleotide sequence of exo-miR-92b-3p is shown in a sequence table in SEQ ID NO:1, and the nucleotide sequence of exo-let7g-5p is shown as SEQ ID NO:2, respectively.

2. The application of the reagent for detecting the expression levels of exosome miRNA exo-miR-92b-3p and exo-let7g-5p in the preparation of a kit for diagnosing early gastric cancer.

3. The use of claim 2, wherein the agent is a primer.

4. The use of claim 3, wherein the upstream primer for detecting the expression level of the exosome miRNA exo-miR-92b-3p is the primer shown in SEQ ID NO:3, the upstream primer for detecting the expression level of the exosome exo-let7g-5p is the primer shown in SEQ ID NO:4, the downstream primers are all universal primers.

5. The use of claim 4, wherein the Universal Primer is a mixcript Universal Primer.

6. A kit for diagnosing early gastric cancer is characterized by comprising primers for detecting expression levels of exosome miRNA exo-miR-92b-3p and exo-let7g-5p, a vacuum blood collection tube-separation gel/coagulant tube, a vacuum blood collection needle assembly, an exosome separation reagent, an exosome miRNA extraction reagent, a reverse transcription reagent, a real-time fluorescence quantitative PCR reagent and primers for detecting expression levels of an internal parameter hsa-miR-30e-5p, wherein the upstream primers for detecting expression levels of exosome miRNA exo-miR-92b-3p are shown as SEQ ID NO:3, the upstream primer for detecting the expression level of exosome exo-let7g-5p is shown as SEQ ID NO:4, the downstream primers are all Universal primers, and the Universal primers are miScript Universal primers; the upstream primer for detecting the expression level of internal reference hsa-miR-30e-5p is shown as SEQ ID NO:5, the downstream primer is a universal primer.

7. The kit according to claim 6, wherein the diagnosis of early gastric cancer is performed according to the following steps:

(1) Serum exosome isolation and RNA extraction thereof

(2) Reverse transcription of exosome RNA into cDNA

Reverse transcription of exosome RNA into cDNA by using a reverse transcription reagent;

(3) PCR amplification of cDNA

Carrying out PCR amplification by using the reverse transcribed cDNA as a template and adopting a real-time fluorescent quantitative PCR reagent according to the following method; carrying out combined amplification by using primers for detecting exosome miRNA exo-miR-92b-3p, hsa-let7g-5p and internal reference hsa-miR-30e-5 p;

(4) And (4) judging a result:

and (3) analyzing a fluorescence quantitative detection result: when the expression quantity of the miRNA is calculated by taking the expression quantity of the hsa-miR-30e-5p as a reference, the relative quantification of the miRNA is shown by a delta Ct value, wherein the delta Ct = Ct (miRNA) -Ct (hsa-miR-30 e-5 p), the relative expression quantity delta Ct of the hsa-miR-92b-3p is less than 1.690, the relative expression quantity delta Ct of the hsa-let7g-5p is less than 4.184, the expression is judged to be positive, and the expression quantity is negative otherwise; when any index of the hsa-miR-92b-3p and the hsa-let7g-5p is expressed positively, early gastric cancer is diagnosed.