CN109371137A - Hsa_circ_0007986 is as new biomarker object space method and application in detection esophagus cancer patient blood serum - Google Patents
Hsa_circ_0007986 is as new biomarker object space method and application in detection esophagus cancer patient blood serum Download PDFInfo
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Abstract
The present invention relates to method of the hsa_circ_0007986 as new biomarker object in a kind of detection esophagus cancer patient blood serum, mainly include that digital pcr detects the biomarker as shown in SEQ ID NO:1.It includes the forward primer as shown in SEQ ID NO:2 and the reverse primer as shown in SEQ ID NO:3 that the digital pcr, which expands used primer,.Hsa_circ_0007986 in the present invention can be used for preparing esophagus cancer diagnosis reagent and/or drug.The method safety that the present invention is used to detect the cancer of the esophagus is noninvasive, high to the detection acceptance Silent cerebral infarction；And accuracy is high, has higher sensitivity and specificity to the cancer of the esophagus, the early screening suitable for the cancer of the esophagus and the biomarker as cancer of the esophagus prognosis, monitors the effect of cancer return and treatment.
Description
Technical field
The invention belongs to oncomolecularbiology fields, and in particular to hsa_circ_ in detection esophagus cancer patient blood serum
0007986 method and purposes as biomarker.
Background technique
The cancer of the esophagus is that the 6th of the 8th most common malignant tumour and cancer related mortality in the world is most common
The cause of disease.The cancer of the esophagus is roughly divided into squamous cell carcinoma and gland cancer from Histopathology, and the above are squamous carcinomas in China about 90%.Although
Cancer of the esophagus correlation diagnosis and treatment means are constantly progressive at present, but the prognosis of patient with esophageal carcinoma is very poor, and 5 years overall survivals are 15%-
34%.Currently, the treatment of the cancer of the esophagus still with operation based on, in conjunction with preoperative neoadjuvant chemicotherapy or postoperative chemoradiation therapy to food
Pipe cancer prognosis raising has certain effect.But the early stage cancer of the esophagus lacks specific symptom, mostly middle and advanced stage when most of patients is made a definite diagnosis,
Surgical indication is lost, mostly can only carry out disease control by treatments such as systemic chemotherapies, but curative effect is still undesirable.So the cancer of the esophagus
Early diagnosis is the key that improve its survival rate and important prerequisite condition.
Eukaryocyte endogenous circular rna is the gene transcript of a kind of annular, without free 5 ' ends or 3 ' ends, nucleic acid
Intermolecular formation is closed 3 ' -5 ' phosphodiester bond (base is at intron lariat RNA cyclization with the combination of 2 ' -5 ' phosphodiester bond),
Constitute single-stranded ring structure.Circular rna is many kinds of, extensively, is largely present in eukaryocyte.So far, global scholar has been
Including people, mouse, zebra fish, drosophila, nematode, saccharomycete, rice animals and plants and fungal cell's interior prediction go out more than 10
Ten thousand kinds of circular rnas.Circular rna biological property is stablized.The stability of circular RNA molecule largely derives from its ring junction
The escape of nuclease caused by structure.More than 1000 kinds circRNA are found in human serum.Circular rna participates in intracellular rna regulation
Network, it is closely related with the occurrence and development of disease, it is likely to become novel biomarker and therapy target.Existing many passes
It is used for the report of tumour early detection and prognosis biomarker in serum/plasma circRNA, is had as tumor markers
Good potential applicability in clinical practice.
Digital pcr, that is, Digital PCR (dPCR), it is a kind of nucleic acid molecules absolute quantitation technology, and reaction and result are sentenced
Reading is influenced to substantially reduce by amplification efficiency.Compared with conventional fluorescent quantitative PCR, have outstanding sensitivity, specificity and
Accuracy, therefore rare mutation detection, expression quantity fine difference are identified and are copied under the detection of denier sample of nucleic acid, complex background
The application of shellfish number variation detection etc. is widely recognized as.Oncology studies, diabetes, in terms of be unfolded
Using.Compared to conventional fluorescent quantitative PCR, digital pcr detection esophagus cancer patient blood serum in circRNA variation side
Mask has a clear superiority.
Summary of the invention
Pass through digital pcr technology it is an object of the invention to express for serum circRNA for the above-mentioned prior art
In esophagus cancer patient blood serum and the differential expression of normal control serum detection hsa_circ_0007986, suffer from this, as the cancer of the esophagus
Person's serum biomarkers provide support for the early detection and early treatment of clinically patient with esophageal carcinoma.
The present invention is achieved by the following technical solutions: hsa_circ_ in a kind of detection esophagus cancer patient blood serum
0007986 method as new biomarker object mainly includes the digital pcr detection mark of the biology as shown in SEQ ID NO:1
Remember object.
Further, the digital pcr expand used in primer include the forward primer such as SEQ ID NO:2 shown in
The reverse primer as shown in SEQ ID NO:3.
Further, it further includes internal reference U6 primer, forward primer such as SEQ that the digital pcr, which expands used primer,
Shown in ID NO:4, reverse primer is as shown in SEQ ID NO:5.
Further, the detection method comprises the following steps:
(1) serum sample is collected, serum RNA is extracted；
(2) by step (1) total serum IgE reverse transcription at cDNA；
(3) cDNA of step (2) is used into such as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
Primer shown in NO:5 is expanded, and the table of hsa_circ_0007986 in serum sample described in digital pcr quantitative analysis is passed through
Up to level, related data is obtained；
(4) data of step (3) are subjected to the analysis of Crystal Reader array of droplets.
The present invention also provides hsa_circ_0007986 in a kind of esophagus cancer patient blood serum to exist as new biomarker object
It is used to prepare the application of cancer of the esophagus auxiliary diagnosis or curative effect predication reagent and/or drug.
Further, the reagent and/or drug include the nucleotide sequence as shown in SEQ ID NO:1.
Further, the reagent of the cancer of the esophagus auxiliary diagnosis or outcome prediction is real-time quantitative PCR detection reagent.
Further, the PCR detection reagent includes the forward primer as shown in SEQ ID NO:2 and such as SEQ ID NO:
Reverse primer shown in 3；And internal reference U6 primer, the forward primer of the internal reference U6 primer is as shown in SEQ ID NO:4, reversely
Primer is as shown in SEQ ID NO:5.
Further, the real-time quantitative PCR is detected as digital pcr.
Further, the cancer of the esophagus is esophageal squamous cell carcinoma.
Beneficial effects of the present invention: the present invention relates to the gene markers and application thereof for detecting the cancer of the esophagus, further relate to
The method that the cancer of the esophagus is detected using the gene marker.Compared with prior art, the present invention is for detecting the cancer of the esophagus
Method is the content based on hsa_circ_0007986 on gene marker, and the method that the present invention is used to detect the cancer of the esophagus also has
Following advantage: (1) safety is noninvasive, high to the detection acceptance Silent cerebral infarction；(2) accuracy is high, to oesophagus
Cancer has higher sensitivity and specificity, the early screening suitable for the cancer of the esophagus and the biological marker as cancer of the esophagus prognosis
Object monitors the effect of cancer return and treatment；(3) easy to operate, user experience is good, is easy to carry out recurrence of Esophageal Carcinoma and transfer
Dynamic monitoring.Gene marker in the present invention can be combined with other clinical indices, be the screening of the cancer of the esophagus, diagnosed, control
It treats and provides more accurately judgement with prognosis.
Detailed description of the invention
Fig. 1 is that digital pcr detection discovery hsa_circ_0007986 expresses up-regulation (P < 0.0001) in oesophagus cancer-serum
Schematic diagram.
Fig. 2 is the sensibility and specificity schematic diagram for evaluating hsa_circ_0007986 as cancer of the esophagus biomarker.
Specific embodiment
Embodiment 1 verifies differential expression of the hsa_circ_0007986 in esophagus squameous patient and healthy person
1. the collection of serum sample: every patient with esophageal carcinoma and control group provide 5mL new blood, and upper layer is collected after centrifugation
Serum, -80 DEG C of preservations.Control is healthy person, and laboratory sample information is shown in Table 1, table 2.
The essential information of 1:12, table offer serum patient with esophageal carcinoma
The essential information of 2:12 normal controls of table
2. the extraction of serum RNA, concrete operation step is referring to the operating process in TRIzol LS Reagent specification.
3. reverse transcription: by serum RNA reverse transcription at cDNA, reverse transcription reaction system is as follows, is shown in Table 3:
Table 3: reverse transcription reaction system
Reaction condition are as follows: 37 DEG C of reaction 15min, 85 DEG C of reaction 5s, 4 DEG C.
In 24 parts of digital pcr quantitative analysis 4. (12 offer serum patient with esophageal carcinoma and 12 normal controls) serum sample
The expression of hsa_circ_0007986, digital pcr Evagreen reaction system such as the following table 5:
Table 5: digital pcr Evagreen reaction system
Wherein upstream primer F includes as shown in SEQ ID NO:2 and the nucleotide sequence as shown in SEQ ID NO:4；Under
Swimming primer R includes the purpose hsa_circ_ as shown in SEQ ID NO:3 and the nucleotide sequence as shown in SEQ ID NO:5
0007986 carries out simultaneously with internal reference amplification.
Crystal digital pcr loop parameter such as the following table 6:
Table 6:Crystal digital pcr loop parameter
The expression of hsa_circ_0007986 in the cancer of the esophagus and control group serum is detected through the above steps, is used
Crystal Reader software is acquired and is analyzed to data by array of droplets, due to purpose circRNA starting copy number
More, then the droplet number generated is more, therefore, can be direct in purpose circRNA situation identical with internal reference amplification efficiency
The quantitative copy number that purpose circRNA is obtained relative to internal reference changes: then CP purpose/CP internal reference uses SPSS22.0 software
It is further to be statisticallyd analyze, as a result as shown in Figure 1, detecting discovery hsa_circRNA-0007986 in oesophagus by digital pcr
Expression up-regulation (P < 0.0001) in cancer-serum illustrates that hsa_circRNA-0007986 is expressed in esophagus cancer patient blood serum height.
By drawing ROC (receiver operating characteristic curve, abbreviation ROC curve) curve
And under calculated curve area (AUC) to evaluate the sensibility and specificity of hsa_circ_0007986 diagnosis of esophageal cancer.Judgement mark
It is quasi- are as follows: as AUC < 0.5, to indicate that diagnosis is meaningless；When AUC=0.5-0.7, indicate that diagnostic accuracy is lower；AUC=0.7-
When 0.9, indicate that diagnostic accuracy is medium；When AUC > 0.9, indicate that diagnostic accuracy is high.Hsa_circRNA- of the invention
0007986 AUC is 0.9796 (95%CI=0.9170-1.042), and P < 0.01 (as shown in Figure 2) illustrates with patient with esophageal carcinoma
Hsa_circRNA-0007986 is higher as the accuracy of the cancer of the esophagus and the biomarker of prognosis in serum.
Embodiment 2 verifies hsa_circ_0007986 as the cancer of the esophagus and predicts reagent
1, digital pcr kit forms
(1) forward primer: such as SEQ ID NO:2；Reverse primer: such as SEQ ID NO:3；
(2) internal reference U6 primer: forward primer is as shown in SEQ ID NO:4；Reverse primer such as SEQ ID NO:5；
(3) other reagents are referring to SYBR Premix Ex TaqTM II (Tli RNaseH Plus) quantification kit
(Code No.RR820A)。
2, hsa_circRNA-0007986 reagent is subjected to digital pcr detection
(1) preparation of serum RNA
The blood serum sample of other 20 esophageal squamous cell carcinomas carninomatosis people is chosen, the reference of RNA concrete operation step is extracted
TRIzol LS Reagent specification operating process.(NanoDrop is quantified with NanoDrop ND-1000 nucleic acid quantification instrument again
Technologies, Wilmington, Delaware) quantitative extracted RNA purity and concentration, denaturing formaldehyde gel electrophoresis matter
The integrality for the RNA that inspection ensures to extract.
(2) cDNA synthesis and detection
Using TaKaRa kit PrimeScriptTM RT reagent Kit with gDNA Eraser
(PerfectReal Time) (article No. RR047A) will test qualified total serum IgE and carry out reverse transcription reaction.
(3) digital pcr detects
Digital pcr response procedures are using digital pcr Evagreen reaction system and process in embodiment 1.
(4) testing result
As the result is shown: other 20 of selection test patients with esophageal squamous cell cancer-serum and control group enlarged sample amount
Card detects 17 in up-regulation using digital pcr, and Positive rate reaches 85%.Result above demonstrates again that the index is being eaten
Universal high expression in pipe cancer-serum.We carry out above-mentioned sample to be repeated 3 times inspection, and as a result repeatability shows this hair up to 100%
The repeatability and stability of bright detection oesophagus cancer-serum hsa_circ_0007986 kit are preferable.
Embodiment described above only expresses one of embodiment of the invention, and description is more specific and detailed
Carefully, but it cannot be understood as the limitations to patent of invention range.It should be pointed out that for the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the present invention
Protection scope.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Sun Yat-sen Memorial Hospital
<120>detect esophagus cancer patient blood serum in hsa_circ_0007986 as new biomarker object space method and application
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Claims (10)
1. detecting method of the hsa_circ_0007986 as new biomarker object in esophagus cancer patient blood serum, feature exists
In mainly including digital pcr detection biomarker as shown in SEQ ID NO:1.
2. detection method as described in claim 1, which is characterized in that it includes such as that the digital pcr, which expands used primer,
Forward primer shown in SEQ ID NO:2 and the reverse primer as shown in SEQ ID NO:3.
3. detection method as claimed in claim 2, which is characterized in that the digital pcr expands used primer and further includes
Internal reference U6 primer, forward primer is as shown in SEQ ID NO:4, and reverse primer is as shown in SEQ ID NO:5.
4. detection method a method according to any one of claims 1-3, which comprises the following steps:
(1) serum sample is collected, serum RNA is extracted；
(2) by step (1) total serum IgE reverse transcription at cDNA；
(3) cDNA of step (2) is used such as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 institute
The primer shown is expanded, by the expression of hsa_circ_0007986 in serum sample described in digital pcr quantitative analysis,
Obtain related data；
(4) data of step (3) are subjected to the analysis of Crystal Reader array of droplets.
5. hsa_circ_0007986 is being used to prepare the cancer of the esophagus as new biomarker object in a kind of esophagus cancer patient blood serum
The application of auxiliary diagnosis or curative effect predication reagent and/or drug.
6. application as claimed in claim 5, which is characterized in that the reagent and/or drug include such as SEQ ID NO:1
Shown in nucleotide sequence.
7. application as claimed in claim 5, which is characterized in that the reagent of the cancer of the esophagus auxiliary diagnosis or outcome prediction
For real-time quantitative PCR detection reagent.
8. the use as claimed in claim 7, which is characterized in that the PCR detection reagent includes as shown in SEQ ID NO:2
Forward primer and the reverse primer as shown in SEQ ID NO:3；And internal reference U6 primer, the forward primer of the internal reference U6 primer
As shown in SEQ ID NO:4, reverse primer is as shown in SEQ ID NO:5.
9. application as claimed in claim 7 or 8, which is characterized in that the real-time quantitative PCR is detected as digital pcr.
10. application as claimed in claim 5, which is characterized in that the cancer of the esophagus is esophageal squamous cell carcinoma.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825596A (en) * | 2019-04-19 | 2019-05-31 | 中国医学科学院肿瘤医院 | The internal reference miRNA of cancer of the esophagus sample and its application |
| CN111304322A (en) * | 2019-12-19 | 2020-06-19 | 徐州市肿瘤医院 | Preparation method of kit for joint detection of esophageal cancer by four novel circRNAs |
| CN111778338A (en) * | 2020-08-06 | 2020-10-16 | 南京医科大学 | Application of circular RNA biomarker |
| CN114751961A (en) * | 2022-06-14 | 2022-07-15 | 中山大学孙逸仙纪念医院 | circ0005199-173aa protein and application thereof in preparation of esophageal cancer diagnosis product |
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| CN105524924A (en) * | 2015-12-29 | 2016-04-27 | 广州永诺生物科技有限公司 | Cyclic RNA circ-ZKSCAN1 use |
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| CN111304322B (en) * | 2019-12-19 | 2022-04-08 | 王强 | Preparation method of kit for joint detection of esophageal cancer by four novel circRNAs |
| CN111778338A (en) * | 2020-08-06 | 2020-10-16 | 南京医科大学 | Application of circular RNA biomarker |
| CN111778338B (en) * | 2020-08-06 | 2021-08-03 | 南京医科大学 | Application of circular RNA biomarker |
| CN114751961A (en) * | 2022-06-14 | 2022-07-15 | 中山大学孙逸仙纪念医院 | circ0005199-173aa protein and application thereof in preparation of esophageal cancer diagnosis product |
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