CN109609630A

CN109609630A - Molecular marker and its application for early carcinoma of stomach diagnosis

Info

Publication number: CN109609630A
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: 2019-04-12
Anticipated expiration: 2038-12-03
Also published as: CN109609630B

Abstract

The invention discloses a kind of molecular marker for early carcinoma of stomach diagnosis and its applications.The molecular marker is excretion body miRNA exo-miR-92b-3p and/or exo-let7g-5p, respectively as shown in SEQ ID NO:1 in sequence table and SEQ ID NO:2.Present invention firstly discovers that being compared with normal people, exo-miR-92b-3p the and exo-let7g-5p content in early carcinoma of stomach patient's circulating is significantly increased, and more conventional tumor markers CEA index has higher sensitivity and specificity.And exo-miR-92b-3p and exo-let7g-5p combines or can significantly improve diagnosis after combining with CEA index, therefore, exo-miR-92b-3p and/or exo-let7g-5p can be used as the reliable cycle biomarker of early carcinoma of stomach for early carcinoma of stomach diagnosis.

Description

Molecular marker and its application for early carcinoma of stomach diagnosis

Technical field

The present invention relates to a kind of molecular marker and its application, in particular to a kind of molecule mark for early carcinoma of stomach diagnosis Will object and its application, the invention belongs to pharmaceutical technology fields.

Background technique

It is known that gastric cancer is the common alimentary system malignant tumour for seriously threatening life.According to American Cancer Society Statistics, it is the fifth-largest common malignant tumour in the whole world, the third-largest lethal cancer that cut-off had become to gastric cancer in 2012.2012 There are nearly 1,000,000 gastric cancer new cases, more than 70 ten thousand deaths in the whole world.The incidence gastric cancer rate of developing country male and women and The equal more developed country of the death rate is higher, at present the whole world 70% new cases occur Asia developing country, therein one Partly occur mainly in China, Cancer in China statistical data in 2015 shows that gastric cancer occupies the of China's cancer morbidity and the death rate Two.

Although the disease incidence of gastric cancer is declined in recent years, the death rate is still high, lacks early diagnosis Method and it is easy to produce the main reason for drug resistance is its high lethality rate behind.5 years survival rates of late gastric cancer are only 30%- 50%, and 5 years survival rates of early carcinoma of stomach are up to 90% or more.The early diagnosis of gastric cancer can also be effectively reduced the dead of patient Rate is died, therefore is early diagnosed extremely important to gastric cancer curative effect is improved.Early gastric caacer diagnosis is always Important Problems concerned by people, Gastrocopy traditional at present is still the most reliable means of diagnosing gastric cancer, however gastrocopy belongs to invasive inspection, is not suitable for making For conventional screening test.Mainly there are CEA, CA199, CA724 etc. currently used for clinical stomach cancer marker, but these indexs diagnose Sensitivity and specificity it is lower, for diagnostic value it is limited.Therefore, it is the short slab for evading routine diagnosis, compels to be essential A kind of new, easy-to-use, high sensitive and high specific Noninvasive marker is wanted, Lai Tigao early gastric caacer is even super Early diagnosis or prognosis evaluation.

Exosome is derived from intracellular, merges via multitubular corpusculum with cell serosa and be discharged into extracellular loop The double-deck membranous structure in border, diameter can almost be secreted in 30-100nm by the cell of all kinds, and be present in human body In almost all of body fluid, such as: blood plasma, serum, saliva, urine, ascites etc..Exosome can be used as a kind of " means of transport ", Many intracellular processes are participated in intercellular transport protein and RNA, mediated cancer progress, tumor microenvironment change, blood vessel is raw It is adjusted at, immune response, transfer and drug resistance etc..Blood is important distribution place of the exosome in the circulatory system, serum Exosome is proved to be the main source of miRNA in the circulatory system.Under the protection of the double membrane structure of exosome, in blood A large amount of existing RNA enzyme are fully isolated, so that miRNA keeps stable from being degraded；In addition, there is the guarantor of exosome Shield, physical factor in the blood as caused by freeze thawing the fluctuation of rna content will not influence its level in exosome, even if MiRNA is also still able to maintain its original function when being transferred to the position far from initial cell.Compared to miRNA's total in blood Weak steady state, exosome miRNAs stability is stronger, even can easily also extract in the sample of Long-term Cryopreservation；Separately Outside, there are also selective enrichments and special feature by the miRNAs in exosomes.

Present invention firstly discovers that be compared with normal people, exo-miR-92b-3p in early carcinoma of stomach patient's circulating and Exo-let7g-5p content significantly increases, more conventional tumor markers -- and CEA index has higher diagnosis efficiency.And exo- Diagnosis, exo-miR- can be significantly improved after the joint and its joint CEA index of miR-92b-3p and exo-let7g-5p 92b-3p and exo-let7g-5p is the reliable cycle biomarker of early carcinoma of stomach.

Summary of the invention

It is lower for molecular marker diagnosis positive rate, the sensitivity and specificity for being currently used for early carcinoma of stomach diagnosis Problem, the invention proposes a kind of molecular marker for early carcinoma of stomach diagnosis and its applications.

In order to achieve the above object, present invention employs following technological means:

The invention proposes a kind of molecular marker for early carcinoma of stomach diagnosis, which is excretion body miRNA Exo-miR-92b-3p and/or exo-let7g-5p, wherein SEQ in the nucleotide sequence of exo-miR-92b-3p such as sequence table Shown in ID NO:1, the nucleotide sequence of exo-let7g-5p is as shown in SEQ ID NO:2 in sequence table.

Further, the invention also provides the molecular marker and for detecting excretion body miRNA exo- The reagent of miR-92b-3p and/or exo-let7g-5p expression is used to prepare in the kit for diagnosing early carcinoma of stomach Using.

Wherein, it is preferred that the reagent is primer, it is furthermore preferred that being used for excretion body miRNA exo-miR-92b-3p The upstream primer of expression detection is to express as shown in SEQ ID NO:3 in sequence table for excretion body exo-let7g-5p The upstream primer of level detection is as shown in SEQ ID NO:4 in sequence table, and downstream primer is universal primer.

Wherein, it is preferred that the universal primer is miScript Universal Primer.

Further, the invention also provides a kind of for diagnosing the kit of early carcinoma of stomach, contains in the kit There is the primer for detecting excretion body miRNA exo-miR-92b-3p and/or exo-let7g-5p expression, it is preferred that use In the upstream primer that excretion body miRNA exo-miR-92b-3p expression detects as shown in SEQ ID NO:3 in sequence table, Upstream primer for the detection of excretion body exo-let7g-5p expression as shown in SEQ ID NO:4 in sequence table, draw by downstream Object is universal primer.

Wherein, it is preferred that also contain vacuum blood collection tube-separation gel/coagulant pipe, vacuum blood taking needle in the kit Component, excretion body separation agent, excretion body miRNA extract reagent, Reverse Transcription, real-time fluorescence quantitative PCR reagent and use In detection internal reference hsa-miR-30e-5p expression primer, wherein upstream primer as shown in SEQ ID NO:5 in sequence table, Downstream primer is universal primer.

Wherein, it is preferred that using kit of the present invention for early carcinoma of stomach diagnose when, according to the following steps into Row:

(1) serum exosome separation and its RNA are extracted

Using the exosomes in excretion body separation agent separation serum, then reagent is extracted with excretion body miRNA and is extracted Tiny RNA in exosomes；

(2) excretion body RNA reverse transcription is at cDNA

Using Reverse Transcription by excretion body RNA reverse transcription at cDNA；

(3) PCR amplification cDNA

Using reverse transcription at cDNA as template, any one of in accordance with the following methods using real-time fluorescence quantitative PCR reagent Carry out PCR amplification；1) using for detecting drawing for excretion body miRNA exo-miR-92b-3p and internal reference hsa-miR-30e-5p Object is expanded；2) using for detect the primer of excretion body miRNA hsa-let7g-5p and internal reference hsa-miR-30e-5p into Row amplification；3) using for detecting excretion body miRNA exo-miR-92b-3p, hsa-let7g-5p and internal reference hsa-miR- The primer of 30e-5p carries out joint amplification；

(4) result judgement:

Fluorogenic quantitative detection interpretation of result: using the expression quantity of hsa-miR-30e-5p as reference, miRNA expression quantity is calculated When, the relative quantification of miRNA is shown with Δ Ct value, Δ Ct=Ct (miRNA)-Ct (hsa-miR-30e-5p), wherein in method 1) in, it is judged to the relative expression quantity Δ Ct < 1.690 of hsa-miR-92b-3p to express the positive, on the contrary it is negative for expression；Table Gastric cancer is diagnosed as up to positive；Method 2) in, it is judged to the relative expression quantity Δ Ct < 4.184 of hsa-let7g-5p to express sun Property, otherwise it is negative for expression；Expression positive is diagnosed as gastric cancer；Method 3) in, by the relative expression quantity of hsa-miR-92b-3p Δ Ct < 1.690, hsa-let7g-5p relative expression quantity Δ Ct < 4.184 be judged to expressing the positive, otherwise for feminine gender；When When any index expression of hsa-miR-92b-3p and hsa-let7g-5p is positive, it is diagnosed as early carcinoma of stomach.

Compared to the prior art, beneficial effects of the present invention:

Present invention firstly discovers that be compared with normal people, exo-miR-92b-3p in early carcinoma of stomach patient's circulating and Exo-let7g-5p content significantly increases, more conventional tumor markers -- and CEA index has higher diagnosis efficiency, and exo- Diagnosis, exo-miR- can be significantly improved after the joint and its joint CEA index of miR-92b-3p and exo-let7g-5p 92b-3p and exo-let7g-5p is the reliable cycle biomarker of early carcinoma of stomach.

The diagnosis positive rate of early carcinoma of stomach patients serum CEA, CA199, CA724 be respectively as follows: 9.1%, 11.4% and 2.3%.And the diagnosis positive rate of early carcinoma of stomach patients serum exo-miR-92b-3p is 42% in the present invention；Early carcinoma of stomach patient The diagnosis positive rate of serum exo-let7g-5p is 46%.Early carcinoma of stomach patients serum exo-miR-92b-3p and exo-let7g- Positive rate is 60% after 5p Combining diagnosis.Diagnosis positive rate is significantly higher than existing CEA, CA199, CA724.

Stomach cancer marker research at present is largely confined to circulating RNA detection, however circulating rna stability is poor, pole It is degradable, it is high to blood specimen collection and processing requirement.And the exosomes in serum is because of the protection with duplicature, it is stable to be not easy It is degraded, Conservation environment and detection time is required low.Overcome previous early carcinoma of stomach circulating miRNAs stability difference and inspection The low disadvantage of extracting rate.

The present invention changes the few status of early gastric caacer diagnostic means, provides for early gastric caacer diagnosis a kind of new, simple Easy row, high sensitive and high specific Noninvasive marker, improves early gastric caacer even extreme early diagnosis.Together When exosomes in albumen, nucleic acid etc. be that cell is actively or passively secreted, provided during cancer occurrence and development necessary The functional mass of property, therefore more diagnostic value.And the present invention detects exo-miR- by extracting the exosomes in serum 92b-3p and exo-let7g-5p, it is ensured that the stabilization of result and reliable.

Tumor markers exo-miR-92b-3p and exo-let7g-5p of the invention, in small-scale clinical sample Confirm the diagnosis marker for being suitable as digestive system tumor, more traditional CEA, CA199, CA724 etc. all have better sensitivity Property and specificity, can be used as the tumor markers of mass survey.

The sensibility and specificity of early carcinoma of stomach patients serum CEA diagnosis are respectively as follows: 70%, 42%.And it is early in the present invention The sensibility and specificity of phase Serum Obtained From Advance Gastric Cancer exo-miR-92b-3p diagnosis are respectively as follows: 80%, 58%；Early carcinoma of stomach patient The sensibility and specificity of serum exo-let7g-5p diagnosis are respectively as follows: 88%, 54%.Early carcinoma of stomach patients serum exo-miR- Sensibility and specificity are respectively as follows: 82%, 60% after 92b-3p and exo-let7g-5p Combining diagnosis.

The present invention marks analyte detection early carcinoma of stomach using excretion body miRNA molecule, avoids invasive inspection (such as stomach of tradition Mirror) body bring is injured.

It is the positive detecting any result of gastric cancer CEA, CA199 and CA724 using serum to patient, reuses the present invention and mention The detection kit of confession carries out auxiliary detection, can effectively remove the false positive results of Serologic detection, to avoid to patient Bring huge stress and property loss；The result for serum detection gastric cancer CEA, CA199 and CA724 is yin simultaneously Property, it reuses detection kit provided by the invention and carries out auxiliary detection, can effectively find the false negative knot of Serologic detection Fruit, to save patient vitals in time.

Present invention discover that two kinds of new dynamical specific excretion body miRNA molecule marks for being directed to early carcinoma of stomach Object, to provide new approaches and theoretical foundation as the research and development of early gastric caacer diagnosis marker based on peripheral blood excretion body miRNA.

Detailed description of the invention

Fig. 1 is serum excretion volume morphing under transmission electron microscope；

Fig. 2 is serum excretion body RNA size distribution gel electrophoresis figure；

Fig. 3 is expression and difference of the serum exo-miR-92b-3p in early carcinoma of stomach patient and normal person；

Fig. 4 is expression and difference of the serum exo-let7g-5p in early carcinoma of stomach patient and normal person；

Fig. 5 is expression and difference of the change of serum C EA in early carcinoma of stomach patient and normal person；

Fig. 6 is the ROC curve of serum exo-miR-92b-3p；

Fig. 7 is the ROC curve of serum exo-let7g-5p；

Fig. 8 is the ROC curve of change of serum C EA；

Fig. 9 is the ROC curve of serum exo-miR-92b-3p and exo-let7g-5p combination；

Figure 10 is the ROC curve of serum exo-miR-92b-3p and CEA combination；

Figure 11 is the ROC curve of serum exo-let7g-5p and CEA combination；

Figure 12 is the ROC curve of serum exo-miR-92b-3p, exo-let7g-5p and CEA combination；

Figure 13 is the relationship of serum exo-miR-92b-3p and gastric cancer neural invasion；

Figure 14 is the relationship of serum exo-let7g-5p and gastric cancer neural invasion；

Figure 15 is the relationship of serum exo-miR-92b-3p adhesion low with gastric cancer；

Figure 16 is the relationship of serum exo-let7g-5p adhesion low with gastric cancer；

Figure 17 is the relationship of serum exo-miR-92b-3p and gastric cancer differentiation degree；

Figure 18 is the relationship of serum exo-let7g-5p and gastric cancer differentiation degree；

Figure 19 is comparison Serologic detection, excretion body miRNAs detection and gastroscope detection using pathologic examination after operation as standard Etc. detection methods accuracy.

Specific embodiment

Elaborate below to the embodiment of the present invention, following embodiment under the premise of the technical scheme of the present invention into Row is implemented, and gives detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following realities Apply example.

Room temperature described in following embodiment refers both to 25 DEG C.

Embodiment 1: the screening with the higher excretion body miRNA of the early carcinoma of stomach degree of correlation

1, patient and sample are included in:

In this experiment, 36 early carcinoma of stomach patients and 12 normal controls are incorporated altogether.

The selected and exclusion criteria of tumor patient include: 1) head examine patient；2) any needle is previously not used in patient when taking a blood sample To the curative drug of gastric cancer, it is not subjected to any radiativity inspection and treatment yet；3) without previously or with other tumor in digestive tract Medical history；It 4) is early carcinoma of stomach through verified by postoperative pathology；5) patient clinical pathological data is complete；6) without other acute illness or Systemic disease such as autoimmune disease, diabetes, coronary heart disease etc..The blood sample of patient for meeting above-mentioned condition is incorporated into case Group.Normal group matches derived from quantity and age with case group, the normal crowd of physical examination (age median differs < 1%).

2, sample collection

Acquisition 3ml fresh peripheral blood is placed in yellow head vacuum blood collection tube-separation gel/coagulant pipe, is stood at 4 DEG C 30min is centrifuged 30min then at 4 DEG C of 3000 × g of TOMY MX-307 refrigerated centrifuge.The faint yellow serum on upper layer is drawn, with 260 μ L/ pipe is dispensed into clean 1.5mL plastic centrifuge tube, and -80 DEG C save backup.To ensure to be free of cell fragment in serum, from At least retain the serum of 0.5cm high in heart pipe；For the homogeneity for guaranteeing experimental implementation, entire serum preparation process need to be ensured two It is fully completed in hour.

3, serum Exosomes separation and its RNA are extracted

1) Exosomes is extracted:

The extraction of excretion body is by SBI company ExoQuickTMExosome Precipitation Solution kit (Cat.#EXOQ20A-1), extracting method is specific as follows:

260 μ L serum are placed in and are thawed on ice, are centrifuged 30min then at 4 DEG C of 3000 × g of TOMY MX-307 refrigerated centrifuge. The 250 μ L/ pipe of faint yellow serum for drawing upper layer is dispensed into clean 1.5mL plastic centrifuge tube.25 μ L are added Tromboplatin D is mixed.It is placed in 37 DEG C of water-baths and is incubated for 15min.Then in 25 DEG C of TOMY MX-307 refrigerated centrifuge 10000 × g is centrifuged 5min, and Aspirate supernatant is into clean 1.5mL plastic centrifuge tube.75 μ L are added into centrifuge tube ExoQuick reagent mixes, is placed in 4 DEG C of refrigerator overnights.The sample handled overnight is in TOMY MX-307 refrigerated centrifuge 4 DEG C 1500 × g centrifugation 30min centrifugation, inhales and abandons supernatant, 4 DEG C of 1500 × g of TOMY MX-307 refrigerated centrifuge are centrifuged 5min again Centrifugation inhales and abandons supernatant, and the precipitating of yellow is serum exosomes below EP pipe.1 × PBS, 50 μ L is added by yellow mercury oxide Being suspended becomes uniform faint yellow mixed liquor.

2) it is detected under Exosomes Electronic Speculum:

Exosomes solution is fixed in 2% paraformaldehyde solution, then uses distilled water serial dilution.5 μ l dilution is mixed Object is closed to be transferred on clean silicon wafer.After sonicated, silicon wafer impregnates 5 points in ethyl alcohol and distilled water in order respectively Clock.Fixed microcapsule bubble is dried up under ventilator cowling.Then silicon wafer is mounted on SEM platform.In order to make surface conductance, electric with scanning Before sub- microscope (S-4700, Japan) imaging, pass through sputtering (SPI-Module Sputter, Eden Instruments, method State) coating 2-5nm gold-palldium alloy coating.Electron-microscope scanning carries out under the conditions of low beam energies (5.0-10.0kV).To present Best vesica form, the exosomes of fresh separated is fixed, and is imaged immediately after isolation under scanning electron microscope.Use ImageJ Software handles photo.The result is shown in Figure 1.Use ExoQuick^TMKit separates and extracts the excretion body in serum, aobvious in transmitted electron Micro mirror inspection observes that in amplification 80000 × lower extract particle diameter distribution be mainly the round microcapsule bubble of diameter 100nm or so, symbol Close the morphologic features of excretion body, it was demonstrated that the extracting method can successfully extract the excretion body in serum.

3) excretion body RNA is extracted

The extraction of excretion body RNA is mentioned by QIAGEN company miRNeasy Micro Kit kit (Cat.#217084) Take method as follows:

1. 700 μ L QIAzol are added into centrifuge tube, cracking is resuspended completely in precipitating, is stored at room temperature 5 minutes.It adds 140 μ L chloroforms (try chemical industry in Shanghai Shanghai), cover tightly centrifuge tube lid.Acutely concussion 15 seconds, are stored at room temperature 3 minutes.

2. 4 DEG C of 12,000 × g of TOMY MX-307 refrigerated centrifuge are centrifuged 15min.

3. carefully by centrifuge tube, 350 μ L of water phase is transferred in new clean 1.5mL plastic centrifuge tube centrifuge tube at the middle and upper levels.Add Enter 525 μ L, 100% ethanol solution, it is soft to be uniformly mixed.

4. extracting the 700 μ L of mixed liquor 3. obtained to be added in RNeasy MinElute spin column, Hitachi 22 DEG C of 500 × g centrifugation, 800 × g of 30s → 22 DEG C of CT15RE refrigerated centrifuge are centrifuged 30s, abandon efflux.

5. repeating, i.e. 22 DEG C of 500 × g centrifugation 800 × g of 30s → 22 DEG C centrifugations of Hitachi CT15RE refrigerated centrifuge 30s abandons efflux.

6. 700 μ L Buffer RWT are added in RNeasy MinElute spin column, lid is covered, 22 DEG C of 8000 × g of Hitachi CT15RE refrigerated centrifuge are centrifuged 15s, abandon efflux.

7. 500 μ L Buffer RPE are added in RNeasy MinElute spin column, lid is covered, 22 DEG C of 8000 × g of Hitachi CT15RE refrigerated centrifuge are centrifuged 15s, abandon efflux.

8. 500 μ L, 80% ethanol solution is added in RNeasy MinElute spin column, lid is covered, 22 DEG C of 8000 × g of Hitachi CT15RE refrigerated centrifuge are centrifuged 15s, abandon efflux.Hitachi CT15RE refrigerated centrifuge again 22 DEG C of 21000 × g of machine are centrifuged 2min.

9. RNeasy MinElute spin column is put into the new clean 1.5mL plastic centrifuge that kit provides In pipe centrifuge tube, it is placed in super-clean bench dry.

10. 14 μ L nuclease free deionized water (i.e. kits are added at the center spin column RNeasy MinElute The RNase free water of offer), lid is covered, 5min, 22 DEG C of Hitachi CT15RE refrigerated centrifuge are stored at room temperature 2000 × g is centrifuged 30s → 21000 × g, 22 DEG C of centrifugation 2min, and efflux is exosomes RNA.

Centrifuge tube containing exosomes RNA is placed on ice, dispenses 1.2 μ L into clean 200 μ L plastic centrifuge tubes, And two parts are frozen in -80 DEG C of refrigerators.

4) excretion body rna content detects

Using Life Technologies (ABI) company Agilent Small RNA Kit kit (Cat.#5067- 1548) excretion body rna content is detected.Testing result is shown in Fig. 2, passes through Agilent Small RNA Kit kit measurement excretion The rna content and clip size extracted in body.MiRNA yield is 9 to 30ng in 250 μ L blood plasma, and abundance mainly divides For cloth in the range of length is 18-28nt, 22nt reaches peak value, meets the molecular size of miRNA, it was demonstrated that extracts in excretion body RNA is mainly miRNA.Excretion body RNA extraction method is stable, it is plentiful to extract rna content.

4, Jian Ku, the sequencing of two generations and data analysis

1) library is built

By NEB companyMultiplex Small RNA Library Prep Set for(Set 1) kit (NEB#E7300S) progress excretion body RNA's builds library, then applies QIAGEN company QIAquick PCR Purification Kit kit (Cat.#28104) carries out purification processes, purification to the cDNA of acquisition CDNA afterwards is separated by electrophoresis in 5%PAGE glue 100V 40mins, in the ultraviolet of BIO-RAD colour developing instrument (USA, EN 61010-1) It develops the color under lamp, cutting size is the band in the region 140bp~160bp, further to purpose blob of viscose purification processes to obtain tiny RNA Corresponding purpose cDNA library: all purposes sample standard deviation is diluted to 20ng/ μ L, 5.5 μ L of packing to new clean nuclease free 1.5mL plastic centrifuge tube in, two generation of dry ice inspection company row sequencing.

2) two generation sequencing results and analysis

High through-put sequence identification is carried out using the HiSeq2000Genome Analyzer platform of Illumina company.Sequencing Strategy is set to unidirectional reading, 50 sequencing endless form.Due to of length no more than 23nt of miRNA molecule, 50 bases are read Sequencing strategy the overall length of the miRNA molecule being inserted into library can be completely covered.Come using the perl script optimized by us Handle sequencing data.Abstraction sequence information and data trimming is carried out first, the sequential extraction procedures by Insert Fragment greater than 16nt come out And it integrates.Using the Bowtie software by optimization by after merging sequential file and human mature's miRNA sequence library and gene Group RNA sequence library carries out sequence analysis, reference data from miRNbase latest edition (Rlease 19, update in) and The library file of NCBI recent renewal.MiRNA transcriptional level is got by comparing the miRNA total indicator reading that its measurement reading can compare Value be standardized.The miRNA of standardization reading absolute value >=5 carries out difference analysis.Choose expression up-regulation, AUC >= 8, the variant miRNAs that sensibility and specificity are all larger than 80% is verified, the early stage that the sequencing of two generation of part is just sifted out The miRNAs and its diagnosis situation of gastric cancer serum exosomes expression up-regulation are shown in Table 1.It is total that 6 miRNA molecules is chosen to be tested Card: hsa-miR92b-3p, hsa-miR-21-5p, hsa-let7c-5p, hsa-let7g-5p, hsa-miR-26a-5p, hsa- MiRNA-101-3p (MS00032144, MS00009079, MS00003129, MS00008337, MS00029239, MS00008372, QIAGEN, USA).MiRNA-30e-5p (MS00009401, QIAGEN, USA) is used as endogenous internal reference simultaneously It is expanded for each sample.

The miRNAs and its diagnosis situation for the early carcinoma of stomach serum exosomes expression up-regulation that the sequencing of 1 two generation of table is just sifted out

Embodiment 2: the verifying with the higher excretion body miRNAs of the early carcinoma of stomach degree of correlation

1. patient and sample are included in:

In this experiment, 50 early carcinoma of stomach patients and 50 normal persons are incorporated altogether as control.Tumor patient enters Choosing and exclusion criteria are the same as embodiment 1.

2. the separation of sample collection, serum excretion body and its RNA extraction method are the same as embodiment 1.

3. excretion body RNA reverse transcription is at cDNA

The reverse transcription of excretion body RNA by QIAGEN company miScript II RT Kit kit (Cat.#218161), The specific operation method is as follows:

1. melting excretion body RNA on ice, melt 5x miScript RT Buffer and RNasefree at room temperature water.Flicking each tubule mixes well it, and then of short duration centrifugation is finally placed with collecting the liquid that tube wall and pipe cover On ice.

2. 4ng RNA is added in each PCR pipe, and nuclease free deionized water (the i.e. RNase of kit offer is added Free water) to total volume be 9 μ L.

3. preparing reverse-transcription master mix on ice, a new clean 1.5mL plastics are taken Centrifuge tube, be added 330 μ L 5x miScript HiSpec Buffer, 165 μ L 10x miScript Nucleics Mix and 165 μ L miScript Reverse Transcriptase Mix mix centrifugation, as miScript reverse- transcription master mix.It mixes and is put on ice for after preparing.

4. 6 μ L miScript Reverse Transcriptase Mix is taken to be added in 2. liquid that step obtains, mix Centrifugation.

5. in PCR instrument (Applied Biosystems,PCR System9700) 37 DEG C be incubated for 1h → 95 DEG C 5mins is incubated for inactivate miScript Reverse Transcriptase Mix, -20 DEG C of refrigerators of reverse transcription product save.

3) PCR amplification cDNA

The PCR process of excretion body cDNA is by QIAGEN companyMiRNA PCR Array kit (Cat.# 218076), the specific operation method is as follows:

1. melting cDNA sample on ice, nuclease free deionized water 1:9 dilution proportion is added.

2. configuring reaction solution by taking 1 plate, 384 orifice plate as an example: the 5mL plastic centrifuge tube of a new clean nuclease free is taken, 1950 μ L 2x QuantiTect SYBR Green PCR Master Mix, 390 μ L 10x miScript are added thereto Universal Primer and 390 μ L 10x miScript Primer Assay mixes centrifugation, takes 7 holes μ L/ that 384 holes are added In plate.

Wherein primer contained in 10 × miScript Primer Assay is primer hsa-miR-92b-3p, hsa- Let7g-5p or hsa-miR-30e-5p.10 × miScript Universal Primer is that Qiagen company designs and synthesizes General reverse primer, the primer be included in Qiagen company miScript SYBR Green PCR Kit kit in.

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. sequentially adding 3 μ L of the cDNA sample after dilution, each cDNA sample repeats 3 holes.

4. after sample-adding, 384 orifice plates are placed in 25 DEG C of 1000xg centrifugation 1min of Labnet C1000 centrifuge.

5. installing into Roche 480PCR instrument operation PCR program, specific procedure is provided that

Table 2

6. saving data after PCR, export, analysis.As a result as follows:

50 couples of early carcinoma of stomach patients and normal control serum exo-miR-92b-3p (SEQ ID are detected using qRT-PCR NO.1), exo-let7g-5p (SEQ ID NO.2) relative expression levels, as a result see Fig. 3-Fig. 5.In relative quantitative assay, The result of qRT-PCR is shown with Δ Ct value:

Δ Ct=Ct (miRNA)-Ct (hsa-miR-30e-5p)

Wherein, Δ Ct value is the difference that fluorescence signal reaches required recurring number when the threshold value of setting in each reaction tube Value, Δ Ct value is bigger, and expression quantity is lower.It is poor using expression of more each index of u-test between early carcinoma of stomach and Normal group It is different.The results show that the Δ Ct value [1.380 (0.234,2.315)] of early stage GC group serum exo-miR-92b-3p is substantially less than just Normal control group [2.480 (1.722,3.122), U=714, P=0.0002]；The Δ Ct of early stage GC group serum exo-let7g-5p Value [4.101 (2.348,5.365)] is substantially less than Normal group [5.617 (4.835,6.327), U=611, P < 0.0001]；Early stage GC group change of serum C EA expression [1.915 (1.083,2.538)] and Normal group no difference of science of statistics [1.780 (1.150,2.445), U=1200, P=0.7329].As a result prompt: change of serum C EA index is in early carcinoma of stomach and normal person In have no significant difference, and be not suitable for the marker diagnosed as early gastric caacer, and serum exo-miR-92b-3p and exo- Let7g-5p can preferably distinguish early carcinoma of stomach patient and normal person compared with change of serum C EA, can be used as GC early diagnosis marker.

In order to determine serum exo-miR-92b-3p, exo-let7g-5p and routine tumor markers CEA to early stage GC's Diagnosis capability draws ROC curve with serum exo-miR-92b-3p, exo-let7g-5p of detection and change of serum C EA data to comment Estimate the value of its auxiliary diagnosis, as Figure 6-Figure 8.The results show that the AUC value of serum exo-miR-92b-3p is 0.714 (P =0.0002,95%CI:0.613~0.815)；Its sensibility and specificity are respectively as follows: 80%, 58%；Serum exo-let7g- The AUC value of 5p is 0.756 (P < 0.0001,95%CI:0.659~0.892), sensibility and specificity be respectively as follows: 88%, 54%；The AUC value of change of serum C EA is 0.520 (95%CI:0.569~0.779), sensibility and specificity be respectively as follows: 70%, 42%, but not statistically significant (P=0.7329)；The result shows that the diagnosis to early stage GC, serum exo-miR-92b-3p, exo- Let7g-5p has higher diagnosis efficiency compared with change of serum C EA, and diagnoses with statistical significance.

The ROC curve of serum exo-miR-92b-3p, exo-let7g-5p and CEA combination is as shown in Fig. 9-Figure 12.Serum Exo-miR-92b-3p combines exo-let7g-5p, serum exo-miR-92b-3p and combines change of serum C EA, serum exo-let7g-5p The diagnosis of early stage GC can be improved in joint change of serum C EA, serum exo-miR-92b-3p joint exo-let7g-5p joint change of serum C EA Efficiency, AUC value are respectively as follows: 0.777,0.722,0.760,0.784, and all have statistical significance (P < 0.05)；Wherein serum Sensibility and specificity are respectively increased to 82% and 60% after exo-miR-92b-3p and exo-let7g-5p joint.

As a result prompt: not only combining for serum exo-miR-92b-3p and exo-let7g-5p can be improved respective diagnosis Efficiency, serum exo-miR-92b-3p, exo-let7g-5p respectively and combination combine with change of serum C EA after can make examining for CEA Disconnected efficiency improves, and has statistical significance, and serum exo-miR-92b-3p and exo-let7g-5p can not only be as GC's Early diagnosis independent tag object also can be used as the auxiliary diagnosis marker of routine serum marker CEA.

Using u-test further to serum exo-miR-92b-3p, exo-let7g-5p and early stage GC clinical pathologic characteristic It is analyzed, as a result as shown in Figure 13-Figure 18.The results show that serum exo-miR-92b-3p expression and early carcinoma of stomach Neural invasion has correlation, and there are the Δ Ct values [4.830 of the serum exo-miR-92b-3p of the patient of neural invasion (2.994,5.923)] it is significantly higher than neural invasion person [3.164 (1.880,4.774), U=185, P=0.0234] does not occur, The expression that the serum exo-miR-92b-3p of the early carcinoma of stomach patient of neural invasion does not occur is significantly higher than in the presence of nerve Infiltration person；Low adhesion is related to early carcinoma of stomach for serum exo-let7g-5p expression, and there are the morning of tumour low-adhesion The Δ Ct value [2.610 (0.952,3.167)] of the serum exo-let7g-5p of phase patients with gastric cancer is significantly higher than without low adhesion Early carcinoma of stomach patient [1.273 (0.047,1.873), U=116.5, P=0.0021], i.e., the early carcinoma of stomach without low adhesion are suffered from The expression of the serum exo-let7g-5p of person is significantly higher than that there are tumour low-adhesion persons；And serum exo-miR-92b-3p The low adhesion and differentiation degree non-correlation (P > 0.05) of expression and early carcinoma of stomach, serum exo-let7g-5p expression Horizontal and neural invasion and the equal non-correlation of differentiation degree (P > 0.05).

Embodiment 3: the preparation of early carcinoma of stomach serum exo-miR-92b-3p and exo-let7g-5p Testing and appraisal kit With use

1, kit

Kit provided by the present embodiment, by primer, vacuum blood collection tube-separation gel/coagulant pipe and vacuum blood taking needle Component, excretion body separation agent, excretion body miRNA extract reagent, Reverse Transcription and real-time fluorescence quantitative PCR reagent composition.

For expanding hsa-miR-92b-3p (SEQ ID NO.1), hsa-let7g-5p (SEQ ID NO.2), hsa- The upstream primer of the real-time quantitative PCR of miR-30e-5p (SEQ ID NO.6, internal reference) is synthesized by QIAGEN company.Primer is as follows Table 3:

Table 3

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

Downstream primer is the general reverse primer that Qiagen company designs and synthesizes, which is included in Qiagen company In miScript SYBR Green PCR Kit kit.

Excretion body separation agent comes from SBI company ExoQuickTMExosome Precipitation Solution reagent Box (Cat.#EXOQ20A-1).Excretion body miRNA extracts reagent and comes from QIAGEN company miRNeasy Micro Kit kit (Cat.#217084).MiScript II RT Kit kit (Cat.# of the RNA reverse transcription reagents from QIAGEN company 218161).MiScript SYBR Green PCR Kit kit of the real-time fluorescence quantitative PCR reagent from Qiagen company (Cat.#218076)。

2, the verifying of results of serological detection is carried out using the provided kit of the present embodiment:

Any result of gastric cancer serum tumor markers CEA, CA199 and CA724 are surveyed for sun using serum blood examination to patient Property or after testing result is complete negative, detected using this kit further progress, judge the result of Serologic detection.Reagent Box operating method is as follows:

1) serum exosome separation and its RNA are extracted

1. 3ml fresh peripheral blood is collected in vacuum blood collection tube-separation gel/coagulant pipe, in 4 DEG C of standing 30min, then It is centrifuged 30min in 4 DEG C of 3000 × g of Thermo ST16R refrigerated centrifuge, collects serum.

2. taking 260 μ L serum, 30min is centrifuged in 4 DEG C of 3000 × g of Thermo ST16R refrigerated centrifuge.Draw upper layer 250 25 μ L Tromboplatin D are added in μ L serum, mix, and 37 DEG C of water-baths are incubated for 15min, Thermo ST16R refrigerated centrifuge 25 DEG C of 10000 × g of machine are centrifuged 5min, and Aspirate supernatant is into clean 1.5mL plastic centrifuge tube.75 μ L are added into centrifuge tube ExoQuick reagent mixes, is placed in 4 DEG C of refrigerator overnights.The sample handled overnight is in Thermo ST16R refrigerated centrifuge 4 DEG C 1500 × g centrifugation 30min centrifugation, inhales and abandons supernatant, again the 4 DEG C of 1500 × g centrifugations of Thermo ST16R refrigerated centrifuge 5min centrifugation inhales and abandons supernatant, and the precipitating of yellow is serum exosomes below EP pipe.1 × PBS, 50 μ L is added by yellow Precipitating, which is suspended, becomes uniform faint yellow mixed liquor.

3. 700 μ L QIAzol are added into sample, cracking is resuspended in precipitating, and 140 μ L chloroforms are added after being stored at room temperature 5mins (trying chemical industry in Shanghai Shanghai), covers tightly centrifuge tube lid, and acutely concussion 15 seconds, are stored at room temperature 3mins.

4. 4 DEG C of 12,000 × g of Thermo ST16R refrigerated centrifuge are centrifuged 15mins.

5. 350 μ L centrifuge tube of careful separation water phase at the middle and upper levels, is transferred to new clean 1.5mL plastic centrifuge tube centrifuge tube In.525 μ L, 100% ethanol solution is added, it is soft to be uniformly mixed.

6. the 700 μ L of mixed liquor 5. obtained is extracted to be added in RNeasy MinElute spin column (abbreviation pillar), 22 DEG C of 500 × g centrifugation, 800 × g of 30s → 22 DEG C of Termo micro21R refrigerated centrifuge are centrifuged 30s, abandon efflux → Termo 22 DEG C of 500 × g centrifugation, 800 × g of 30s → 22 DEG C of micro21R refrigerated centrifuge are centrifuged 30s, abandon efflux.

7. be added in pillar 700 μ 22 DEG C of 8000 × g of L Buffer RWT, Termo micro21R refrigerated centrifuge from Heart 15s abandons efflux.

8. be added in pillar 500 μ 22 DEG C of 8000 × g of L Buffer RPE, Termo micro21R refrigerated centrifuge from Heart 15s abandons efflux.

9. 500 μ L, 80% ethanol solution is added in pillar, lid, Termo micro21R refrigerated centrifuge 22 are covered DEG C 8000 × g is centrifuged 15s, abandons efflux.22 DEG C of 21000 × g of Termo micro21R refrigerated centrifuge are centrifuged 2mins again.

10. pillar is put into the new clean 1.5mL plastic centrifuge tube centrifuge tube that kit provides, it is placed in super-clean bench Middle drying.

14 μ L nuclease free deionized waters (i.e. the RNase free water of kit offer), lid is added at pillar center Good lid, is stored at room temperature 5min, and 22 DEG C of 2000 × g of Termo micro21R refrigerated centrifuge are centrifuged 30s → 21000 × g, 22 DEG C It is centrifuged 2min, efflux is exosomes RNA, dispenses a small amount of RNA for detecting RNA concentration, residue is placed on ice.

2) excretion body RNA reverse transcription is at cDNA

1. 4ng RNA is added in each PCR pipe, and nuclease free deionized water (the i.e. RNase of kit offer is added Free water) to total volume be 9 μ L.

2. preparing reverse-transcription master mix on ice, a new clean 1.5mL plastics are taken 330 μ L 5x miScript HiSpec Buffer, 165 μ L 10x miScript Nucleics are added in centrifuge tube centrifuge tube Mix and 165 μ L miScript Reverse Transcriptase Mix mixes centrifugation, as miScript reverse- transcription master mix.It mixes and is put on ice for after preparing.

3. 6 μ L miScript Reverse Transcriptase Mix is taken to be added in 1. liquid that step obtains, mix Centrifugation.

4. in PCR instrument (Applied Biosystems,PCR System9700) 37 DEG C be incubated for 1h → 95 DEG C be incubated for 5mins to inactivate miScript Reverse Transcriptase Mix, portioning section reverse transcription product is placed in On ice, -20 DEG C of refrigerator preservations of resultant product can be used for rechecking.

3) PCR amplification cDNA

1. 5 μ L 2x QuantiTect SYBR are added in clean PCR pipe by taking the PCR pipe of each sample as an example Green PCR Master Mix、1μL 10×miScript Universal Primer、1μL 10×miScript Primer Assay, 1 μ L cDNA sample and 2 μ L nuclease free deionized waters (the i.e. RNase free of kit offer Water), each Primer Assay sample repeats 3 holes.Wherein contain primer in 10 × miScript Primer Assay Hsa-miR-92b-3p, hsa-let7g-5p or hsa-miR-30e-5p.10 × miScript Universal Primer is The general reverse primer that Qiagen company designs and synthesizes, the primer are included in the miScript SYBR of Qiagen company In Green PCR Kit 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. 384 orifice plates are placed in 25 DEG C of 1000xg centrifugation 1min on Labnet C1000 centrifuge.

3. installing into Roche 480PCR instrument operation PCR program, specific procedure setting method is as follows:

Table 4

4. saving data after PCR, export, analysis.

4) fluorogenic quantitative detection interpretation of result

Using the expression quantity of hsa-miR-30e-5p as reference, when calculating miRNA expression quantity, the relative quantification of miRNA is with Δ Ct value shows, Δ Ct=Ct (miRNA)-Ct (hsa-miR-30e-5p).

The detection method of gastric cancer serum exo-miR-92b-3p and exo-let7g-5p Testing and appraisal kit, the method Are as follows: (1) Serologic detection: extracting blood to be measured, utilizes serology tumor-marker analyte detection (CEA, CA199 and CA724)；(2) Kit detection: the excretion body in blood to be measured is extracted using excretion body separation agent, excretion body miRNA extracts reagent and reversion Record reagent prepares blood excretion body cDNA to be measured as real-time fluorescence quantitative PCR template, and primer and real-time fluorescence quantitative PCR is added Reagent is reacted, 95 DEG C of 15mins → 94 DEG C 15s, 55 DEG C of 30s, 70 DEG C of 30s, and → 4 DEG C of ∞ of 40 circulations obtain miRNAs's Expression quantity, compared with the expression quantity of miR-30e-5p in autoserum excretion body；(3) result judges: 1. when blood serum to be measured When learning test positive, if the intracorporal hsa-miR-92b-3p or hsa-let7g-5p expression of kit detection patients serum's excretion Amount up-regulation, remove hsa-miR-30e-5p internal control primer after, the relative expression quantity Δ Ct < 1.690 of hsa-miR-92b-3p or The relative expression quantity Δ Ct < 4.184 of hsa-let7g-5p, or both be all satisfied, then judge that the Serologic detection positive is correct； 2. when blood serum to be measured detects feminine gender, if under kit detection hsa-miR-92b-3p and hsa-let7g-5p expression quantity It adjusts, the relative expression quantity Δ Ct of relative expression quantity Δ Ct >=1.690 of hsa-miR-92b-3p and hsa-let7g-5p >= 4.184, then judge that Serologic detection feminine gender is correct；If kit detects hsa-miR-92b-3p and hsa-let7g-5p is expressed Amount up-regulation, remove hsa-miR-30e-5p internal control primer after hsa-miR-92b-3p relative expression quantity Δ Ct < 1.690 and The relative expression quantity Δ Ct < 4.184 of hsa-let7g-5p, then judge Serologic detection feminine gender for false negative；If there are other knots Fruit, it is believed that serology judgement is consistent, without further judging testing result.

3, early carcinoma of stomach diagnosis is carried out using the provided kit of the present embodiment:

(1) selection of sample:

In this experiment, it is included in 50 early carcinoma of stomach patients without any treatment altogether.

The selected and exclusion criteria of tumor patient include: 1) head examine patient；2) any needle is previously not used in patient when taking a blood sample To the curative drug of gastric cancer, it is not subjected to any radiativity inspection and treatment yet；3) without previously or with other tumor in digestive tract Medical history；4) biopsy inspection under the preoperative once row gastroscope of patient；It 5) is early carcinoma of stomach through verified by postoperative pathology；6) patient clinical pathology provides Material is complete；7) without other acute illness or systemic disease medical history.

(2) serum exosome separation and its RNA are extracted

Patient peripheral's blood is acquired using vacuum blood collection needle assemblies, is collected in vacuum blood collection tube-separation gel/coagulant pipe Fresh peripheral blood, and collect serum.Serum exosomes is separated using excretion body separating kit, then with excretion body miRNA Extracts kit extracts the tiny RNA in exosomes.Concrete operation method is same as above.

(3) excretion body RNA reverse transcription is at cDNA

Using reverse transcription reagent box by excretion body RNA reverse transcription at cDNA.Concrete operation method is same as above.

(4) PCR amplification cDNA

Using reverse transcription at cDNA as template, using real-time fluorescence quantitative PCR reagent carry out PCR amplification.It is divided into 3 experiments Group, experimental group 1 are expanded using primer hsa-miR-92b-3p and internal control primer hsa-miR-30e-5p, and experimental group 2 uses primer Hsa-let7g-5p and internal control primer hsa-miR-30e-5p amplification, experimental group 3 use primer hsa-miR-92b-3p and hsa- Let7g-5p joint and internal control primer hsa-miR-30e-5p amplification.Concrete operation method is same as above.

In practical operation we have found that if carrying out absolute quantitation PCR has following problems: 1. operating procedures can be more numerous It is trivial, cause the accuracy of result that can accordingly reduce；2. the Agilent Small RNA Kit kit that need to be applied when absolute quantitation (Cat.#5067-1548) next reverse transcription and PCR step are carried out again after detecting excretion body rna content, and Agilent Small RNA Kit kit has that costly, Storage period is short, and the disadvantage that operation difficulty is high.In view of problem above, Wo Mengai Into operation, absolute quantitation PCR is changed to the expression water that relative quantification PCR examines exo-miR-92b-3p, exo-let7g-5p It is flat.Internal reference exo-miR30e-5p is used for relative quantification.

(5) result judgement:

Fluorogenic quantitative detection interpretation of result: using the expression quantity of hsa-miR-30e-5p as reference, miRNA expression quantity is calculated When, the relative quantification of miRNA is shown with Δ Ct value, Δ Ct=Ct (miRNA)-Ct (hsa-miR-30e-5p), wherein testing In group 1, it is judged to the relative expression quantity Δ Ct < 1.690 of hsa-miR-92b-3p to express the positive, on the contrary it is negative for expression；Table Gastric cancer is diagnosed as up to positive.In experimental group 2, the relative expression quantity Δ Ct < 4.184 of hsa-let7g-5p is judged to expressing The positive, on the contrary it is negative for expression；Expression positive is diagnosed as gastric cancer.In experimental group 3, by the relative expression of hsa-miR-92b-3p Amount Δ Ct < 1.690, hsa-let7g-5p relative expression quantity Δ Ct < 4.184 be judged to expressing the positive, otherwise for feminine gender；When When any index expression of hsa-miR-92b-3p and hsa-let7g-5p is positive, it is diagnosed as early carcinoma of stomach.

Blood serum tumor markers CEA, CA19-9 and CA724 Analysis of test results: with CEA > 5ng/ml, CA199 > 37U/ Ml, CA724 > 6.9U/ml are judged to expressing the positive, otherwise negative for expression；Any blood serum tumor markers are that expression is positive When, it is diagnosed as early carcinoma of stomach.

(6) using pathologic examination after operation as standard, the inspection such as comparison Serologic detection, excretion body miRNAs detection and gastroscope detection The accuracy of survey method.

As a result: concrete outcome such as Figure 19.The diagnosis of blood serum tumor markers CEA, CA199 and CA724 in early carcinoma of stomach Positive rate is respectively 10%, 0% and 14%；The diagnosis positive rate of early carcinoma of stomach patients serum exo-miR-92b-3p is 42%； It is 46% that early carcinoma of stomach patients serum exo-let7g-5p, which diagnoses positive rate,；Early carcinoma of stomach patients serum exo-miR-92b-3p and Positive rate is 60% after exo-let7g-5p Combining diagnosis；Diagnosis positive rate of the invasive gastrocopy of tradition in early carcinoma of stomach It is 88%.

As a result illustrate:

Through detecting, diagnosis positive rate of the invasive gastrocopy of tradition in early carcinoma of stomach is up to 88%, although gastroscope is examined Looked into higher positive diagnosing rate, but its it is traumatic it is big, spend the features such as high to limit extensive clinical value.And conduct Traditional Noninvasive marker test mode -- CEA, CA199 and CA724, the diagnosis positive rate in early carcinoma of stomach only divide Not Wei 10%, 0% and 14%, these indexs are very limited to the diagnostic value of early carcinoma of stomach.Serum exo-miR-92b- 3p and exo-let7g-5p as present invention discover that one kind is new, easy-to-use, high sensitive and high specific non-intruding Property marker, showing has compared with the better Diagnostic Superiority of conventional blood tumor markers, wherein early carcinoma of stomach patients serum The diagnosis positive rate of exo-miR-92b-3p is 42%；Early carcinoma of stomach patients serum exo-let7g-5p diagnoses positive rate 46%；It is significantly better than traditional blood serum designated object inspection.Meanwhile early carcinoma of stomach patients serum exo-miR-92b-3p and exo- Positive rate individually detects bright up to 60% compared with serum exo-miR-92b-3p, exo-let7g-5p after let7g-5p Combining diagnosis It is aobvious to improve.

Sequence table

<110>Harbin Medical University

<120>molecular marker for early carcinoma of stomach diagnosis and its application

<130> KLPI180879

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 22

<212> RNA

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

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<210> 2

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<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

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<212> RNA

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

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Claims

1. the molecular marker for early carcinoma of stomach diagnosis, it is characterised in that the sub- marker is excretion body miRNA exo-miR- 92b-3p and/or exo-let7g-5p, the wherein nucleotide sequence of exo-miR-92b-3p such as SEQ ID NO:1 institute in sequence table Show, the nucleotide sequence of exo-let7g-5p is as shown in SEQ ID NO:2 in sequence table.

2. molecular marker described in claim 1 is used to prepare the application in the kit for diagnosing early carcinoma of stomach.

3. prepared by the reagent for detecting excretion body miRNAexo-miR-92b-3p and/or exo-let7g-5p expression The application in kit for diagnosing early carcinoma of stomach.

4. application as claimed in claim 3, which is characterized in that the reagent is primer, it is preferred that is used for excretion body The upstream primer of miRNAexo-miR-92b-3p expression detection is to be used for excretion as shown in SEQ ID NO:3 in sequence table The upstream primer of body exo-let7g-5p expression detection is as shown in SEQ ID NO:4 in sequence table, and downstream primer is Universal primer.

5. application as claimed in claim 4, which is characterized in that the universal primer is miScript Universal Primer。

6. a kind of for diagnosing the kit of early carcinoma of stomach, which is characterized in that containing for detecting excretion body in the kit The primer of miRNA exo-miR-92b-3p and/or exo-let7g-5p expression, it is preferred that be used for excretion body miRNA The upstream primer of exo-miR-92b-3p expression detection is used for excretion body exo- as shown in SEQ ID NO:3 in sequence table For the upstream primer of let7g-5p expression detection as shown in SEQ ID NO:4 in sequence table, downstream primer is universal primer.

7. kit as claimed in claim 6, which is characterized in that the universal primer is miScript Universal Primer。

8. kit as claimed in claim 6, which is characterized in that also contain vacuum blood collection tube-separation in the kit Glue/coagulant pipe, vacuum blood collection needle assemblies, excretion body separation agent, excretion body miRNA extract reagent, Reverse Transcription, in real time Quantitative fluorescent PCR reagent and primer for detecting internal reference hsa-miR-30e-5p expression, wherein upstream primer such as sequence In list shown in SEQ ID NO:5, downstream primer is universal primer.

9. such as the described in any item kits of claim 6-8, which is characterized in that when for early carcinoma of stomach diagnosis, according to following Step carries out:

(1) serum exosome separation and its RNA are extracted

(2) excretion body RNA reverse transcription is at cDNA

(3) PCR amplification cDNA

Using reverse transcription at cDNA as template, carried out any one of in accordance with the following methods using real-time fluorescence quantitative PCR reagent PCR amplification；1) using for detect the primer of excretion body miRNA exo-miR-92b-3p and internal reference hsa-miR-30e-5p into Row amplification；2) it uses and is expanded for detecting the primer of excretion body miRNA hsa-let7g-5p and internal reference hsa-miR-30e-5p Increase；3) using for detecting excretion body miRNA exo-miR-92b-3p, hsa-let7g-5p and internal reference hsa-miR-30e-5p Primer carry out joint amplification；

(4) result judgement:

Fluorogenic quantitative detection interpretation of result: using the expression quantity of hsa-miR-30e-5p as reference, when calculating miRNA expression quantity, The relative quantification of miRNA shows with Δ Ct value, Δ Ct=Ct (miRNA)-Ct (hsa-miR-30e-5p), wherein in method 1) In, it is judged to the relative expression quantity Δ Ct < 1.690 of hsa-miR-92b-3p to express the positive, on the contrary it is negative for expression；Expression Positive is diagnosed as gastric cancer；Method 2) in, it is judged to the relative expression quantity Δ Ct < 4.184 of hsa-let7g-5p to express sun Property, otherwise it is negative for expression；Expression positive is diagnosed as gastric cancer；Method 3) in, by the relative expression quantity of hsa-miR-92b-3p Δ Ct < 1.690, hsa-let7g-5p relative expression quantity Δ Ct < 4.184 be judged to expressing the positive, otherwise for feminine gender；When When any index expression of hsa-miR-92b-3p and hsa-let7g-5p is positive, it is diagnosed as early carcinoma of stomach.