CN109652554B - Application of the circ_SLC7A5 molecular marker in diagnosis esophageal squamous cell carcinoma and prognosis in blood - Google Patents
Application of the circ_SLC7A5 molecular marker in diagnosis esophageal squamous cell carcinoma and prognosis in blood Download PDFInfo
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Abstract
The invention discloses a kind of application of the circ_SLC7A5 molecular marker in blood in diagnosis esophageal squamous cell carcinoma and prognosis, are related to the discovery, detection, application of circ_SLC7A5 molecular marker, design and synthesize out the detection primer for being specifically used for real-time quantitative PCR.Circ_SLC7A5 in blood can be used as the blood plasma marker object of ESCC patient, it is poor to cancer of the esophagus Endoscopic Screening compliance for target group, and the patients with esophageal squamous cell carcinoma overwhelming majority clinically made a definite diagnosis is the status of middle and advanced stage, ideal molecule pre-warning signal is found in esophageal squamous cell carcinoma change process, targetedly carry out endoscopy, definitive pathological diagnosis, to mitigate patient's pain, over-treatment is avoided, medical resource is saved.
Description
Technical field
The present invention relates to oncomolecularbiology field, circ_SLC7A5 molecular marker exists in specially a kind of blood
Diagnose the application in esophageal squamous cell carcinoma and prognosis.
Background technique
The cancer of the esophagus is to seriously threaten one of most common malignant tumour of human health, and there are mainly two types of pathologicals: squamous carcinoma
And gland cancer.90% cancer of the esophagus is squamous cell carcinoma, and distribution has apparent areal variation.China ESCC disease incidence Zhan Quanshi
70% or more boundary, prognosis is poor, and overall survival only has 15%-25% within 5 years.Clinician's Journal of Cancer report in 2016, food
Pipe cancer has become one of highest five kinds of tumours of Chinese disease incidence, and disease incidence ranked third position in male, and is arranged in women
Five.
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 [7], and compared with Microrna in mammalian cell (miRNAs) and long-chain non-coding RNA (lncRNAs), have more
High stability and sequence conservation is 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., such as hypopharyngeal squamous cell carcinoma, squamous carcinoma of larynx, basal-cell carcinoma, ductal adenocarcinoma of pancreas, gastric cancer, liver
Cell cancer, thyroid papillary carcinoma etc..However, circRNAs is rarely reported in esophageal squamous cell carcinoma.
ESCC still assesses the state of an illness without tumor marker at present, and scope is goldstandard, but wound is big, patient is not
It is easily accepted by.
Summary of the invention
To solve the above problems, the invention discloses circ_SLC7A5 molecular markers in a kind of blood in diagnosis oesophagus
Application in squamous carcinoma and prognosis, for target group to cancer of the esophagus Endoscopic Screening compliance oesophagus that is poor, and clinically making a definite diagnosis
The squamous cell carcinoma patients overwhelming majority is that the status of middle and advanced stage proposes that it is pre- to find ideal molecule in esophageal squamous cell carcinoma change process
Alert signal, targetedly carries out endoscopy, and definitive pathological diagnosis avoids over-treatment to mitigate patient's pain, saves medical treatment money
Source.Circ_SLC7A5 in blood plasma can be used as the blood plasma marker object of ESCC patient, this inspection method is easy to be connect by subject
By, more can become ESCC patient early diagnosis, the judgement of disease progression and the effective means of prognosis evaluation.
In order to reach the goals above, the present invention supplies following technical solution:
Application of the circ_SLC7A5 molecular marker in diagnosis esophageal squamous cell carcinoma and prognosis, the circ_ in a kind of blood
The nucleotides sequence of SLC7A5 molecular marker is classified as SEQ ID NO.1.The ID number of circ_SLC7A5 is circ_0040796.
It in the present invention, is real-time quantitative PCR kit for oesophagus squama cancer diagnosis or the reagent of prognosis.
The circ_SLC7A5 molecular marker for being classified as SEQ ID NO.1 according to nucleotides sequence of the invention devises 2 pairs and draws
Object is SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID for detecting the sequence, primer nucleotide sequences
NO.5。
Specific primer difference is as follows:
The invention also discloses a kind of detection method of circ_SLC7A5 molecular marker, including sample Total RNAs extraction,
RNA reverse transcription, real-time quantitative PCR amplification.
The specific method is as follows: the first step, Total RNAs extraction
A. blood plasma Total RNAs extraction
Blood plasma, serum Total RNAs extraction use mir Vana PARIS Kit (Ambion1566, USA) kit, ultraviolet point
Light photometric determination concentration of specimens and purity.
B. total tissue RNA extracts Trizol method and extracts tissue and cell total rna, and ultraviolet specrophotometer measures concentration of specimens
And purity.
Second step, RNA reverse transcription and real-time quantitative PCR
Using Prime-Script by RNA reverse transcription at cDNA TM One step RT-PCR kit, glyceraldehyde 3 phosphate is de-
Hydrogen enzyme (GAPDH) is used as internal contrast.Owned using ABI7500 system and SYBR Green PCR Master Mix
QRT-PCR reaction.In order in accurate validation ESCC circ_SLC7A5 express multiple variation, by the Ct value of calculating relative to from
GAPDH (Δ Ct=Cttested-CtGAPDH) standardization of same sample amplification, and use-Δ Ct method is estimated to change
Value.In triplicate, all reactions are independent in triplicate to ensure the repeatabilities of all data for each sample.circ_
The Cutoff value of SLC7A5 is -3.503, when detect-Δ Ct be greater than this value when for ESCC, specificity is 81.13%,
Sensibility is 67.82%.
The invention also discloses a kind of for oesophagus squama cancer diagnosis and the real time quantitative PCR detecting reagent kit of prognosis, including
It is designed and synthesized out and is specifically used in real time according to the circ_SLC7A5 molecular marker that nucleotides sequence is classified as SEQ ID NO.1
The detection primer of quantitative PCR；SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ in specific primer such as sequence table
ID NO.5。
The purpose of the present invention is to provide the circ_SLC7A5 molecular markers that a kind of nucleotides sequence is classified as SEQ ID NO.1
Object.
The present invention has the advantage that
(1) purposes of circ_SLC7A5 is proposed.(2) circular rna has mechanism stable, abundance degree and sample specificity
The features such as expression, and circ_SLC7A5 expression quantity in ESCC patient and normal human blood sample has differences, and with by stages
Correlation, therefore circ_SLC7A5 has the application prospect as esophageal squamous cell carcinoma biomarker.
The present invention be directed to target groups to cancer of the esophagus Endoscopic Screening compliance esophageal squamous cell carcinoma that is poor, and clinically making a definite diagnosis
The patient overwhelming majority is that the status of middle and advanced stage proposes, ideal molecule early warning letter is found in esophageal squamous cell carcinoma change process
Number, endoscopy is targetedly carried out, definitive pathological diagnosis avoids over-treatment, save medical resource to mitigate patient's pain.
Circ_SLC7A5 in blood plasma can be used as the blood plasma marker object of ESCC patient, this inspection method is easy to be received by subject, more
Early diagnosis, the judgement of disease progression and the effective means of prognosis evaluation of ESCC patient can be become.
Detailed description of the invention
Fig. 1, display circ_SLC7A5 derive from SLC7A5exons 7-10；Amplified production insertion is used to Sanger to survey
Their overall length is determined in the T- carrier of sequence；
Fig. 2, with the abundance of circ_SLC7A5 and SLC7A5 mRNA in the GC cell of RNase R processing；
Fig. 3, with the abundance of circ_SLC7A5 and SLC7A5 mRNA in the GC cell of D actinomycin D processing；
The significant adjacent non-cancer tissue higher than ESCC patient of expression of Fig. 4, circ_SLC7A5 expression；
The expression of the expression of Fig. 5, circ_SLC7A5 in ESCC patients blood plasma is significant to be higher than normal person；
Fig. 6, ROC curve have been used for the assessment potential diagnostic value of circ_SLC7A5, and area (AUC) is under ROC curve
0.7717；
The total survivorship curve of Fig. 7, Kaplan-Meier shows that the patient with high circ_SLC7A5 expression in blood shows and deposits
Live time shortens；
Fig. 8, circ_SLC7A5 and patient with esophageal carcinoma Overall survival are analyzed.
Specific embodiment
With reference to the accompanying drawings and detailed description, the present invention is furture elucidated, it should be understood that following specific embodiments are only
For illustrating the present invention rather than limiting the scope of the invention.
As shown in Figures 1 to 8, the invention discloses circ_SLC7A5 molecular markers in a kind of blood in diagnosis oesophagus
Application in squamous carcinoma and prognosis, the nucleotides sequence of circ_SLC7A5 molecular marker are classified as SEQ ID NO.1.
The sequencing of 1.RNA mulberry lattice
(1) amplified production insertion is used in the T- carrier of mulberry lattice (Sanger) sequencing determine their overall length.Design
Different primers is to confirm that circ_SLC7A5's returns splice junction.Primer is synthesized by Invitrogen company (Chinese Shanghai), Sang Ge
Sequencing is completed by the Realgene company of Nanjing of China.Testing result is as shown in Figure 1.
2.RNase R digestion and actinomycin D digestion
2mg total serum IgE is incubated 20 minutes at 37 DEG C, is added or is added without 3U/mg RNase R, then uses RNeasy
MinElute kit.For actinomycin D treatment, by 2mg total serum IgE at 37 DEG C with or be added without 1mg D actinomycin D (unwrapping wire
Rhzomorph D, Sigma) it incubates together, and the detection gained RNA respectively at 0,6,12,18 hour.Testing result such as Fig. 2, Fig. 3 institute
Show.
3. experimental subjects
Using the attached Nanjing hospital Oncological Surgery of Nanjing Medical University as study base, Oncological Surgery accepts food for medical treatment throughout the year for this research
Pipe squamous cell carcinoma patients, and sample storehouse is established, case-data abundant can be provided for this research.Study patient tissue and blood sources in
Receive the patients with esophageal squamous cell carcinoma of radical cure or palliative operation excision during 2013 in Augusts, 2017 in our hospital.Normal control blood
Liquid sample standard deviation derives from Nanjing No.1 Hospital's people taking physical examination, and normal healthy controls crowd excludes malignant tumour medical history and oesophagus
Related disease medical history.All samples of this experiment obtain patient or its trustee signs informed consent, related to human body specimen
Research obtain Ethics Committee, Nanjing No.1 Hospital approval.
It is as follows that research patient is included in standard:
A. esophageal squamous cell carcinoma is turned out to be (according to the 7th edition (2009) cancer of the esophagus TNM stage standard through postoperative pathological
Carry out clinical 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.
ESCC cancer patient's blood sample 87 in this research, normal human blood sample 53, ESCC tissue and cancer beside organism 23 are right.
4. clinical data acquires
The collection of clinical data is broadly divided into patients with esophageal squamous cell carcinoma and normal person's two parts, is both needed to comprising general information: packet
Include name, gender, age, occupation, medical history, family history, feminine menstrual history and the obsterical history etc. of patient.Esophageal squamous cell carcinoma is suffered from
Person is mainly from the data to arrange in hospital of patients with esophageal squamous cell carcinoma.Mainly include outside above-mentioned general information, should also include clinical special
Sign, whether there is or not transfer, tumor cell differentiation degree to swell for histological type, position, size, infiltration degree, lymph node including tumour
Tumor TNM stage, operating time, postoperative whether there is or not the data such as chemicotherapy and conventional auxiliary examination.Proprietary follow-up is obtained simultaneously
Agree to, inspection result data, which is improved, to be saved.
5. sample is collected
It is divided into two parts, a part is the collection of blood, preoperative primary (5ml/ pipe) use of taking a blood sample of every patients with esophageal squamous cell carcinoma
In subsequent experimental research.Generation haemolysis should be avoided in whole blood sample.It is ethylenediamine tetrem that wherein plasma sample, which collects heparin tube,
Acid disodium (Ethylenediaminetetraacetic acid, EDTA) anticoagulant tube, whole blood sample should complete blood plasma in 0.5h
Be collected by centrifugation and freeze；It is that routine biochemistry promotees solidifying pipe that serum sample, which collects heparin tube, and sample should complete the precipitation of serum in 2h
Centrifugation freezes.Whole blood sample removes cell component influence by two step centrifugal process, first in 2000g, 4 DEG C, is centrifuged 10min,
Then careful transfer supernatant is into new EP pipe, and then 12000g, is centrifuged 10min, finally completes blood plasma and serum sample by 4 DEG C
It dispenses (400 μ l/ pipe), is put into -80 DEG C of refrigerator long-term preservations.
Some is the collection of tissue specimen.Clinic, which is collected, receives radical cure or palliative operation excision because of esophageal squamous cell carcinoma
Patient tumor tissues and cancer by normal esophageal tissue specimen.Control group sample from same patient cancer beside organism (away from
From borderline tumor at least 5cm), pathologic examination after operation does not find that incisxal edge cancer cell remains.Normal group by fresh tumor tissue and cancer
It is cleaned twice with pre- cold saline as early as possible first after knitting in vitro, subsequent clip about soya bean size tissue has been put into RNA protection liquid
Cryopreservation tube in mix well.All samples after obtaining it is first quick-frozen in liquid nitrogen container for 24 hours, be then put into -80 DEG C of refrigerators in time
Medium-term and long-term preservation, to be used to subsequent experimental.
6. main primer
7. Total RNAs extraction
A. blood plasma Total RNAs extraction
Blood plasma, serum Total RNAs extraction use mir Vana PARIS Kit (Ambion1566, USA) kit, specific to walk
It is rapid as follows:
All reagents are both needed to be restored to and use at room temperature, take out and melt spare, eluent 95 in plasma/serum sample ice face
DEG C pre- stand-by heat；
400 μ l plasma/serum samples are added in the EP pipe of added isometric 2X Denaturing Solution,
Mixing fullys shake；
Isometric Acid-Phenol:Chloroform is added, concussion mixes 1min, and then 10000g is centrifuged at room temperature
5min；
Careful 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)；
Add 700 1 cleaning filter of μ l mi RNA Wash Solution 1 time (10000g is centrifuged 15s), 500 μ are then added
2/3 cleaning filter of l Wash Solution 2 times (10000g is centrifuged 15s)；
RNA (10000g is centrifuged 30s) on the deoxyribonuclease washing filter apparatus of 50 95 DEG C of μ l preheating, it is -80 DEG C long-term
It saves；
Ultraviolet specrophotometer measures concentration of specimens and purity.
B. total tissue RNA extracts Trizol method and extracts tissue and cell total rna, and specific step is as follows:
After cleaning porcelain grinding body, with DEPC liquid soaked overnight (at least 12h), ultralow temperature then is put into after high pressure steam sterilization
Refrigerator cold-storage；
Prepare sterile no enzyme pipette tips, EP pipe, 4 DEG C of centrifuge it is spare to the cold
Cryopreserved tissue (about 50mg) is taken out out of ultra low temperature freezer, surface frozen stock solution is wiped, is filled in mortar (- 40 DEG C of pre-coolings)
Divide grinding, adds liquid nitrogen in grinding to prevent tissue RNA from degrading.Powdery is ground into tissue samples；
Every part of sample adds 1ml Trizol lysate, blows even rear transfer lysate with pipette tips and manages to EP, stands in ice face
5min decomposes it completely；
Chlorination imitates 0.2ml, acutely shakes 15s, stands 2min at room temperature, 4 DEG C, 12000g is centrifuged 15min；
Careful transfer upper strata aqueous phase avoids drawing cotton-shaped middle layer as far as possible into new EP pipe.Then add 0.5ml isopropyl
Alcohol, concussion mix, stand 10min at room temperature, and subsequent 4 DEG C, 12000g is centrifuged 10min.It is now spare with 75% ethyl alcohol；
It carefully discards supernatant, 75% ethyl alcohol 1ml concussion cleaning precipitating, subsequent 7500g, 4 DEG C of centrifugation 5min is added；
It finally discards supernatant, sucks residual liquid as far as possible with pipette tips, be air-dried at room temperature, according to precipitating size, 30-60 is added
μ l DEPC water dissolves RNA precipitate；
Ultraviolet specrophotometer measures concentration of specimens and purity.
8.RNA reverse transcription and real-time quantitative PCR
Using Prime-Script by RNA reverse transcription at cDNA TM One step RT-PCR kit, glyceraldehyde 3 phosphate is de-
Hydrogen enzyme (GAPDH) is used as internal contrast.Owned using ABI7500 system and SYBR Green PCR Master Mix
QRT-PCR reaction.In order in accurate validation ESCC circ_SLC7A5 express multiple variation, by the Ct value of calculating relative to from
GAPDH (Δ Ct=Cttested-CtGAPDH) standardization of same sample amplification, and use-Δ Ct method is estimated to change
Value.In triplicate, all reactions are independent in triplicate to ensure the repeatabilities of all data for each sample.circ_
The Cutoff value of SLC7A5 is -3.503, when detect-Δ Ct be greater than this value when for ESCC, specificity is 81.13%,
Sensibility is 67.82%.
9, data are analyzed:
The as shown in Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8 is using t inspection and Chi-square Test and the method circ_ of survival analysis
SLC7A5 and ESCC patient clinical data and the correlation of prognosis by stages.Receiver Operating Characteristics' (ROC) curve is carried out to assess
Its diagnostic value.V.17.0, all statistical analysis are carried out using SPSS for Windows.For all as a result, P < 0.05
It is considered statistically significant.
Therefore, the circ_SLC7A5 in blood plasma can be used as the blood plasma marker object of ESCC patient, and circ_SLC7A5 exists
Expression quantity has differences in ESCC patient and normal human blood sample, and to it is by stages related, therefore circ_SLC7A5 have at
For the application prospect of esophageal squamous cell carcinoma biomarker.
Data are as follows:
Note:*P < 0.05.
The technical means disclosed in the embodiments of the present invention is not limited only to technological means disclosed in above embodiment, further includes
Technical solution consisting of any combination of the above technical features.It should be pointed out that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.
Sequence table
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Claims (8)
1. circ_SLC7A5 molecular marker reagent is in preparation diagnosis esophageal squamous cell carcinoma and prognosis product in a kind of detection blood
Using the nucleotides sequence of the circ_SLC7A5 molecular marker is classified as SEQ ID NO.1.
2. circ_SLC7A5 molecular marker reagent diagnoses oesophagus in preparation in a kind of detection blood as described in claim 1
Application in squamous carcinoma and prognosis product, it is characterised in that: for oesophagus squama cancer diagnosis or the reagent of prognosis be real-time quantitative PCR
Kit.
3. circ_SLC7A5 molecular marker reagent is in preparation diagnosis food in a kind of detection blood as claimed in claim 1 or 2
Application in pipe squamous carcinoma and prognosis product, it is characterised in that: the circ_SLC7A5 of SEQ ID NO.1 is classified as according to nucleotides sequence
It is SEQ ID NO.4, SEQ ID that molecular marker, which devises 1 pair of primer for detecting the sequence, primer nucleotide sequences,
NO.5。
4. circ_SLC7A5 molecular marker reagent diagnoses oesophagus in preparation in a kind of detection blood as described in claim 1
Application in squamous carcinoma and prognosis product, it is characterised in that: the detection method of circ_SLC7A5 molecular marker, including sample are total
RNA extraction, RNA reverse transcription, real-time quantitative PCR amplification.
5. circ_SLC7A5 molecular marker reagent diagnoses oesophagus in preparation in a kind of detection blood as claimed in claim 4
Application in squamous carcinoma and prognosis product, it is characterised in that: the detection method specific steps packet of circ_SLC7A5 molecular marker
It includes, the first step, extracts blood plasma total serum IgE using mir Vana PARIS Kit kit, extract total tissue RNA using Trizol method
And cell total rna, second step are sweet using Prime-Script TM One step RT-PCR kit by RNA reverse transcription at cDNA
Oily aldehyde 3- phosphate dehydrogenase is used as internal contrast, carries out institute using ABI7500 system and SYBR Green PCR Master Mix
There is qRT-PCR reaction.
6. a kind of for oesophagus squama cancer diagnosis or the real time quantitative PCR detecting reagent kit of prognosis, it is characterised in that: including basis
The circ_SLC7A5 molecular marker that nucleotides sequence is classified as SEQ ID NO.1, which designs and synthesizes out, is specifically used for real-time quantitative
The detection primer of PCR.
7. as claimed in claim 6 for oesophagus squama cancer diagnosis or the real time quantitative PCR detecting reagent kit of prognosis, feature
It is: SEQ ID NO.4, SEQ ID NO.5 in specific primer such as sequence table.
8. the circ_SLC7A5 molecular marker that a kind of nucleotides sequence is classified as SEQ ID NO.1.
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