CN109234401A - A kind of molecular marker for sdenocarcinoma of stomach diagnosis - Google Patents
A kind of molecular marker for sdenocarcinoma of stomach diagnosis Download PDFInfo
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Abstract
The present invention relates to a kind of molecular marker circ_PSMC3 for sdenocarcinoma of stomach diagnosis, and nucleotide sequence is as shown in SEQ ID NO.1.It is detected by expression quantity of the qRT-PCR to circ_PSMC3 in blood samples of patients sample, the early diagnosis and prognosis evaluation to sdenocarcinoma of stomach can be achieved, for detecting the specific primer of above-mentioned molecular marker, including downstream primer shown in upstream primer shown in SEQ ID NO.2 and SEQ ID NO.3.The verifying discovery of cDNA microarray combination Big Clinical Samples early period, expression of the molecular marker in patients with gastric adenocarcinoma are substantially less than normal person.Clinical detection only needs to acquire micro peripheral blood, traumatic small, is easily received by subject, early diagnosis, the judgement of disease progression and the effective means of prognosis evaluation of patients with gastric adenocarcinoma can be become, the index is used for the high specificity of sdenocarcinoma of stomach diagnosis, and sensibility is high, as a result stable.
Description
Technical field
The present invention relates to a kind of molecular markers for sdenocarcinoma of stomach diagnosis, belong to field of biotechnology.
Background technique
Gastric cancer is to seriously threaten one of most common malignant tumour of human health, there are mainly two types of pathological: gland cancer and
Other types.Most of gastric cancers are gland cancer, and distribution has apparent areal variation.China's incidence gastric cancer rate is in the whole world
High level, prognosis is poor, and overall survival is lower within 5 years.Clinician's Journal of Cancer report in 2016, gastric cancer have become China
One of highest five kinds of tumours of disease incidence, disease incidence ranked second position in male, ranked third position in women.
Currently, the diagnosis of sdenocarcinoma of stomach is very low, clinically used diagnostic techniques such as gastroscope, upper digestive tract radiography, CT etc.,
Due to higher inspection fee and to undertake certain pain and risk is difficult to popularize, tumour mark in conventional Serological testing
Whether Sensitivity Specificity all shows unsatisfactory note object such as CEA etc., and therefore, it is always swollen for finding novel tumor markers
The important topic of tumor research field.
Circular rna (circRNAs) is a kind of new endogenous non-coding RNA, is confirmed as in early stage the 1990s
The transcript of out-of-order exon, structure, function and mechanism are reported in succession, become later 20 years research hotspots.It is different from
The special closed loop of 5' end cap and the end 3' poly (A) tail is the absence of using the end 5' and the end 3' as the conventional linear RNA, circRNAs of end
Structure, and compared with Microrna in mammalian cell (miRNAs) and long-chain non-coding RNA (lncRNAs), have higher
Stability and sequence conservation are because they have nuclease resistant.Recent numerous studies report, in different plant species
CircRNAs has expression.Rapid development based on bioinformatic analysis and high throughput sequencing technologies, researcher have found
Ten hundreds of circRNAs has found that they participate in the disease developments such as vascular lesion, the nervous system disease, tumour.
CircRNAs chip analysis technology has been applied to many tumours, including digestive system tumor, nervous system neoplasm, and urinary system swells
Tumor, head and neck neoplasm etc., as hypopharyngeal squamous cell carcinoma, squamous carcinoma of larynx, basal-cell carcinoma, ductal adenocarcinoma of pancreas, the cancer of the esophagus,
Hepatocellular carcinoma, thyroid papillary carcinoma etc..However, blood plasma circRNAs is rarely reported in gastric cancer.
Summary of the invention
It is an object of the invention to solve the deficiencies in the prior art, a kind of molecular marker for sdenocarcinoma of stomach diagnosis is provided
Object, judges whether patient suffers from sdenocarcinoma of stomach or suffer from the risk of sdenocarcinoma of stomach by the expression of detection molecules marker, from
And clinician is instructed to carry out early intervention and treatment, improve the survival rate and life quality of patient.
Technical solution
A kind of molecular marker for sdenocarcinoma of stomach diagnosis, the molecular marker are circ_PSMC3, nucleotides sequence
Column are as shown in SEQ ID NO.1.
Circular rna has many characteristics, such as that mechanism stable, abundance degree and sample are specific expressed, and the present inventor passes through detection
Circ_PSMC3 expression quantity in patients with gastric adenocarcinoma and normal human blood sample, discovery circ_PSMC3 is in patients with gastric adenocarcinoma and just
Expression in ordinary person's blood sample has differences, and to it is by stages related.
For detecting the specific primer of above-mentioned molecular marker, including upstream primer and SEQ shown in SEQ ID NO.2
Downstream primer shown in ID NO.3.
Upstream primer sequence are as follows: 5 '-GTTTAGGGTCCCTGCCCTTTG-3 '；
Downstream primer sequence are as follows: 5 '-GTGTTGGGCTGGAAGCCATC-3 '.It is sequenced by the PCR product to the primer,
Demonstrate the specificity of primer amplification.
Above-mentioned specific primer is preparing or is screening the application in sdenocarcinoma of stomach diagnostic medicine.
A kind of diagnostic kit of sdenocarcinoma of stomach, including above-mentioned specific primer.
The utility model has the advantages that passing through the present invention provides a kind of molecular marker circ_PSMC3 for sdenocarcinoma of stomach diagnosis
QRT-PCR the expression quantity of circ_PSMC3 in blood samples of patients sample is detected, it can be achieved that early diagnosis to sdenocarcinoma of stomach and
Prognosis evaluation, the verifying discovery of cDNA microarray combination Big Clinical Samples early period, the circ_PSMC3 molecular marker are suffered from sdenocarcinoma of stomach
Expression in person is substantially less than normal person, and its expression and clinical stages and patient's life cycle are highly relevant.It is clinical
Detection only needs to acquire micro peripheral blood (5ml), traumatic small, is easier to be received by subject, can more become the morning of patients with gastric adenocarcinoma
It examines, the effective means of the judgement of disease progression and prognosis evaluation, is analyzed by ROC curve, AUC value 0.9326, P <
0.0001, high specificity of the index for sdenocarcinoma of stomach diagnosis is reflected, sensibility is high.As a result stable, have extensive clinical
Application prospect.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of circ_PSMC3；
Fig. 2 is ROC curve of the circ_PSMC3 to patients with gastric adenocarcinoma and healthy population；
Fig. 3 is that qRT-PCR detects expression of the circ_PSMC3 in patients with gastric adenocarcinoma blood plasma and healthy population blood plasma；
Fig. 4 is expression of the circ_PSMC3 in patients with gastric adenocarcinoma cancerous tissue and cancer beside organism；
The correlation of Fig. 5 tracing analysis circ_PSMC3 expression and survival for survival.
Specific embodiment
The present invention will be further explained below with reference to the attached drawings and specific examples.
Embodiment 1
1. case selection
Patient tissue and blood sources are studied to cure during 2013 in Augusts, 2016 in the attached Nanjing of Nanjing Medical University
Institute receives the patients with gastric adenocarcinoma of radical cure or palliative operation excision.Normal control blood sample derives from Nanjing No.1 Hospital
People taking physical examination, normal healthy controls crowd exclude malignant tumour medical history and gastrointestinal tract related disease medical history.All marks of this experiment
This obtains patient or its trustee signs informed consent, and the research of related to human body specimen obtains Nanjing No.1 Hospital
Ethics Committee's approval.
It is as follows that research patient is included in standard:
A. sdenocarcinoma of stomach is turned out to be through postoperative pathological (to carry out according to the 7th edition (2009) sdenocarcinoma of stomach TNM stage standard clinical
By stages)；
B. receive the patient of radical cure or palliative resction, while the person that excludes visiting before operation between hospital stay；
C. present illness history, family history, personal history and various inspections of data are perfect.
GC cancer patient's blood sample 106 in this research, normal human blood sample 21.
The collection of clinical data is broadly divided into patients with gastric adenocarcinoma and normal person's two parts, is both needed to comprising general information: including
Name, gender, age, occupation, medical history, family history, feminine menstrual history and obsterical history of patient etc..Patients with gastric adenocarcinoma master
It will being hospitalized from data to arrange by patients with gastric adenocarcinoma.Mainly include outside above-mentioned general information, should also include Clinical symptoms, including
The histological type of tumour, position, size, infiltration degree, lymph node are whether there is or not transfer, tumor cell differentiation degree, and tumour TNM points
Phase, operating time, postoperative whether there is or not the data such as chemicotherapy and conventional auxiliary examination.It obtains proprietary follow-up simultaneously to agree to, inspection
The fruit data that comes to an end, which is improved, to be saved.
2. sample is collected
It is the collection of blood, every patients with gastric adenocarcinoma is preoperative to take a blood sample primary (5ml/ pipe) for subsequent experimental research.It is all
Generation haemolysis should be avoided in blood sample.It is disodium ethylene diamine tetraacetate that wherein plasma sample, which collects heparin tube,
(Ethylenediaminetetraacetic acid, EDTA) anticoagulant tube, whole blood sample should complete the centrifugation of blood plasma in 0.5h
Collection freezes；It is that routine biochemistry promotees solidifying pipe that serum sample, which collects heparin tube, and the precipitation centrifugation that sample should complete serum in 2h is frozen
It deposits.Whole blood sample removes cell component influence by two step centrifugal process, first in 2000g, 4 DEG C, is centrifuged 10min, then small
The heart shifts supernatant into new EP pipe, then 12000g, 4 DEG C, is centrifuged 10min, finally completes blood plasma and serum sample packing
(400 μ l/ pipe), is put into -80 DEG C of refrigerator long-term preservations.
3. Total RNAs extraction
Blood plasma, serum Total RNAs extraction use mirVana PARIS Kit (Ambion1566, USA) kit, specific to walk
It is rapid as follows:
1) all reagents are both needed to be restored to and use at room temperature, take out and melt spare, eluent in plasma/serum sample ice face
95 DEG C of pre- stand-by heats；
2) the EP pipe of added isometric 2X Denaturing Solution is added in 400 μ l plasma/serum samples
In, mixing fullys shake；
3) isometric Acid-Phenol:Chloroform is added, concussion mixes 1min, then at room temperature 10000g from
Heart 5min；
4) carefully transfer upper strata aqueous phase is managed to new EP, and records transfer water phase volume, then adds 1.25 times of volumes
100% ethyl alcohol shifts lysate/alcohol mixture into EP pipe, after mixing well and passes through filter (10000g is centrifuged 30s)；
5) add 700 1 cleaning filter of μ l mi RNA Wash Solution 1 time (10000g is centrifuged 15s), be then added
500 2/3 cleaning filters of μ l Wash Solution 2 times (10000g is centrifuged 15s)；
6) RNA (10000g is centrifuged 30s) on the deoxyribonuclease washing filter apparatus of 95 DEG C of 50 μ l preheatings, it is -80 DEG C long
Phase saves；
7) ultraviolet specrophotometer measurement concentration of specimens and purity.
4.RNA reverse transcription and qPCR amplification
(1) configuration of reverse transcription system
1. taking out Reverse Transcriptase kit from -20 DEG C of refrigerators, by 2 kinds of enzyme insertion ice chest ice, other reagents melt on ice,
RNase Free H2O room temperature melts spare.
2. according to surveyed RNA concentration, RNA volume needed for calculating the every pipe of reverse transcription, and DEPC water volume is calculated simultaneously.
RNA and DEPC water total volume are no more than 7ul.Note: RNA multigelation is easily degraded, once it is determined that the RNA matter of sample extraction
Amount preferably, should according to pairing standard by cancer and cancer as far as possible by RNA disposably whole reverse transcriptions at cDNA.Such as paired sample
RNA amount is big, more than the amount of 10 parts of reverse transcriptions, it is contemplated that the cost of reverse transcription only can do reverse transcription by 10 parts of amount.
3. taking PCR pipe to be placed on ice, being put into sterile no enzyme 200ul PCR pipe as needed and marking by number.Pipettor
After drawing appropriate RNA, pipette tips are inserted perpendicularly into PCR pipe bottom, at the uniform velocity softly get liquid, can reduce or even does not generate bubble, it is complete
Confirmation pipette tips are without obvious residual afterwards.
4. pipettor draws appropriate DEPC water, pipette tips against rear wall and squeeze into PCR pipe.Because sample rna before is vertical
Straight that tube bottom is added, then pipe surrounding side wall can be considered uncontaminated area, any angle that oneself can be accustomed to againsts eight unions
Front/rear/left/right side wall slowly at the uniform velocity gets liquid, can reduce or even not generate bubble.
5. reverse transcription Step1: gDNAEraser 1ul and 5x gDNA Eraser Buffer 2ul is added in every pipe.By step
Rapid 4 operation is pasted different directions side wall respectively and is added.
6. being placed on eight union centrifuges, gently bullet falls tube bottom or tube wall bubble, is centrifuged in short-term.
7. 42 DEG C 2 minutes on RT-PCR instrument, product is placed in spare on ice after reaction.
8. reverse transcription Step2: taking 1.5ml EP to manage, according to formula as below, configure every tube reaction
5x PrimerScript Buffer2 4ul
Rnase Free dH2O 4ul
PrimerScript RT Enzyme Mix I 1ul
RTPrimer Mix 1ul
Set reaction condition: 37 DEG C of 15min, 85 DEG C of 5sec, 4 DEG C of maintenances.
9. fastening pipe lid, it is taken off from pipe support, is placed on centrifuge tube shelf, having bubble, person can flick tube bottom, in short-term
Centrifugation resets after confirming that the liquid of every pipe equals the interior bubble-free of consistent and pipe on ice.
10. operating the computer, setting reaction condition, cDNA is placed in -20 DEG C of preservations after 15-20min.
(2) QPCR is expanded
For the specific primer of detection molecules marker circ_PSMC3, including upstream primer and downstream primer, upstream
Primer sequence are as follows: 5 '-GTTTAGGGTCCCTGCCCTTTG-3 ' (SEQ ID NO.2), downstream primer sequence are as follows: 5 '-
GTGTTGGGCTGGAAGCCATC-3′(SEQ ID NO.3)。
It is used as internal contrast, GAPDH_F:5 '-with glyceraldehyde 3 phosphate dehydrogenase (GAPDH)
TGCACCACCAACTGCTTAGC-3 ', GAPDH_R:5 '-GGCATGGACTGTGGTCATGAG-3 '.Each sample, which is arranged 3, puts down
Row pipe, all amplified reactions are repeated three times the above reliability to guarantee result.
1. getting out ice chest, cDNA, primer, PCR reagent (the SYBR Premix of sample to be tested are taken out from -20 DEG C of refrigerators
Ex TaqTM II, ROC Reference Dye II), it thaws on ice.
2. getting the liquid relief of eight unions and Guan Gai, eight union framves, tweezers, sterile 1.5ml EP pipe, EP pipe support, suitable range ready
Device (2ul, 20ul are that must use range), aseptic thin-film gloves.
3. the cDNA to have thawed, primer, PCR reagent to be first successively placed on oscillator and vibrate in short-term, then be put into centrifuge
Of short duration centrifugation is gone up, on ice stand for standby use.
4. 1.5ml EP is taken to manage, eight union PCR Mix (matching according to 19ul total volume) are prepared by every hole formula below:
SYBR Premix Ex TaqTM II 10ul
Forward Primer
1ul*3 is to simplify operation to be configured to mix primer
Reverse Primer
ROC Reference Dye/II 0.4ul*4
cDNA 1ul
dH2O 7.6ul
Reaction condition:
ABI 7300/7500 Real-Time PCR System, StepOnePlusTM
Stage1:95℃30sec Reps 1
Stage2:95℃5sec,60℃30-34sec,Reps 40
Stage3:95℃15sec,60℃1min,95℃5sec,Reps 1
Note: 60 DEG C of time settings in Stage2: 7500 Fast Real-Time PCR System/
StepOnePlusTM:30 sec, 7300:31 sec, 7500:34 sec.
5. 2ul pipettor range is adjusted to 1ul, successively by each sample and multiple holes are preset in the position of eight unions
The cDNA of sample is added.
6. turning off fluorescent lamp, all bright sources are shielded, take corresponding amount to be added to ROC Reference Dye/II
In the EP pipe of the installation PCR Mix stood before, rear concussion is mixed, then is centrifuged in short-term, to ensure that the Mix dispensed is to mix.
7. 20ul pipettor range is adjusted to 19ul, absorption Mix successively againsts eight union rear walls again and gets liquid, every time
Confirmation pipette tips are without obvious residual after having beaten.
8. clamping eight union lid one side edges with tweezers, it is gently placed in nozzle.New aseptic thin-film gloves are worn, gently by eight
Union is taken out from ice chest together with eight union framves and is placed in desktop, light that a side pipe lid is pressed to fix, then firmly fastens pipe lid rapidly.
Marking pen is marked in the both ends Guan Gai, then gently detains eight union both ends bottoms from the bottom to top, it is taken off from pipe support, is placed in
On eight centrifugation framves, having bubble, person can flick tube bottom, be centrifuged in short-term, after confirming that the liquid of every pipe equals the interior bubble-free of consistent and pipe, set
In on ice.
9. operating the computer, reaction condition is set, race solubility curve is added, purpose item is determined by melt curve analysis analysis and electrophoresis
Band, Δ Δ CT method carry out relative quantification.
5. data analysis and result
It is examined using t and the method circ_PSMC3 of Chi-square Test and survival analysis and GC patient clinical data and pre- by stages
Correlation afterwards.Receiver Operating Characteristics' (ROC) curve is carried out to assess its diagnostic value.All statistical analysis use SPSS
V.17.0, for Windows is carried out.For all as a result, P < 0.05 is considered statistically significant.
Fig. 1 is the structural schematic diagram of circ_PSMC3, it can be seen that circ_PSMC3 comes from PSMC gene, and length is
502bp。
Fig. 2 is ROC curve of the circ_PSMC3 to patients with gastric adenocarcinoma and healthy population, it can be seen that AUC 0.9326
(95%CI=0.8862-0.9791, specificity are 95.24%, sensibility 85.85%, P < 0.0001), this illustrates circ_
PSMC3 is applied to the diagnosis accuracy with higher of sdenocarcinoma of stomach, can be used as the molecular marker of patients with gastric adenocarcinoma diagnosis.
Fig. 3 is that qRT-PCR detects expression of the circ_PSMC3 in patients with gastric adenocarcinoma blood plasma and healthy population blood plasma
(106 patients with gastric adenocarcinoma blood plasma and 21 healthy population blood plasma), it can be seen that Circ_PSMC3 table in Plasma of Patient With Gastric Cancer
It is lower than healthy population up to level, the expression of Lymph Node Metastasis patients with gastric cancer, which is lower than, does not shift patients with gastric cancer.
Fig. 4 is that expression of the circ_PSMC3 in patients with gastric adenocarcinoma cancerous tissue and cancer beside organism (suffer from by 106 sdenocarcinomas of stomach
Person's cancerous tissue and cancer beside organism), it can be seen that circ_PSMC3 is substantially less than group by corresponding cancer in the expression of patients with gastric cancer cancerous tissue
It knits.
106 patients with gastric adenocarcinoma prognosis survivorship curves are analyzed, tracing analysis circ_PSMC3 is expressed Fig. 5 for survival
The horizontal correlation with survival, it can be seen that in patients with gastric adenocarcinoma, Circ_PSMC3 expression and prognosis existence
Rate is negatively correlated, illustrates that circ_PSMC3 can be used as the marker of patients with gastric adenocarcinoma diagnosis and prognosis evaluation.
Sequence table
<110>Cao Hongyong
<120>a kind of molecular marker for sdenocarcinoma of stomach diagnosis
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 502
<212> DNA
<213>ring-type RNa (circ_PSMC3)
<400> 1
ctttgacagt gagaaggctg gggaccggga ggtgcagagg acaatgctgg agcttctgaa 60
ccagctggat ggcttccagc ccaacaccca agttaaggta attgcagcca caaacagggt 120
ggacatcctg gaccccgccc tcctccgctc gggccgcctt gaccgcaaga tagagttccc 180
gatgcccaat gaggaggccc gggccagaat catgcagatc cactcccgaa agatgaatgt 240
cagtcctgac gtgaactacg aggagctggc ccgctgcaca gatgacttca atggggccca 300
gtgcaaggct gtgtgtgtgg aggcgggcat gatcgcactg cgcaggggtg ccacggagct 360
cacccacgag gactacatgg aaggcatcct ggaggtgcag gccaagaaga aagccaacct 420
acaatactac gcctagggca cacaggccag ccccagtctc acggctgaag tgcgcaataa 480
aagatggttt agggtccctg cc 502
<210> 2
<211> 21
<212> DNA
<213>ring-type RNa (circ_PSMC3)
<400> 2
gtttagggtc cctgcccttt g 21
<210> 3
<211> 20
<212> DNA
<213>ring-type RNa (circ_PSMC3)
<400> 3
gtgttgggct ggaagccatc 20
Claims (4)
1. a kind of molecular marker for sdenocarcinoma of stomach diagnosis, which is characterized in that the molecular marker is circ_PSMC3,
Nucleotide sequence is as shown in SEQ ID NO.1.
2. the specific primer for detecting molecular marker described in claim 1, which is characterized in that the specific primer packet
Include downstream primer shown in upstream primer shown in SEQ ID NO.2 and SEQ ID NO.3.
3. specific primer described in claim 2 is preparing or is screening the application in sdenocarcinoma of stomach diagnostic medicine.
4. a kind of diagnostic kit of sdenocarcinoma of stomach, which is characterized in that including specific primer described in claim 2.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110894528A (en) * | 2019-09-22 | 2020-03-20 | 潘文胜 | CircRNA marker for diagnosing gastric poorly differentiated adenocarcinoma and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110894528A (en) * | 2019-09-22 | 2020-03-20 | 潘文胜 | CircRNA marker for diagnosing gastric poorly differentiated adenocarcinoma and application thereof |
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