CN107083432A - A kind of stomach cancer molecular marker hsa_circ_0000705 and its application - Google Patents
A kind of stomach cancer molecular marker hsa_circ_0000705 and its application Download PDFInfo
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Abstract
The present invention relates to a kind of stomach cancer molecular marker hsa_circ_0000705 and its application, its nucleotide sequence is as shown in SEQ ID NO.1.Molecular marker of the present invention has fabulous stomach organization specificity and stomach cancer correlation, and quantitative detection can be realized by RT PCR methods, discovery and detection for early carcinoma of stomach provide new molecular marker, work as NIH mice treatment level to further improving, with certain meaning.
Description
Technical field
The invention belongs to tumor markers field, more particularly to a kind of stomach cancer molecular marker hsa_circ_0000705 and
It is applied.
Background technology
It is commonly used with high flux genome sequencing technology, it has surprisingly been found that in mammalian cell about
98% transcript is non-coding RNA molecule (noncoding RNA, ncRNA).Have in these non-coding RNA molecules including
Well known at present " special (housekeeping) " non-coding RNA (such as transfer RNAs and ribosomal RNAs
Deng), small non-coding RNA (such as microRNAs and piRNAs), long-chain non-coding RNA (long noncoding RNA,
LncRNA), more is then circular rna (circular RNA, the circRNA) molecule for waiting further investigation.
CircRNA refers to that a class is produced by precursor RNA through alternative splicing and with the reverse covalently bonded in 5' ends and 3' ends
The endogenous circular RNA molecule of conjunction.Domestic and international Preliminary Results show that circRNA is that a class has important biomolecule function
New RNA molecule, it can by with RNA or protein interaction, multiple levels participate in gene expressions and adjusted after transcription, transcription
Control.Simultaneously because circRNA sequences are highly conserved, property is stable, is difficult to be degraded by exonuclease, and with tissue, when
Sequence and disease relative specificity, so circRNA possesses the potential quality that molecular marker is precisely diagnosed as clinical disease.
Stomach cancer is one of most common malignant tumor of digestive tract in the world, and its incidence of disease occupies the 4th in all malignant tumours,
The death rate arranges the 2nd.In China, annual new hair patients with gastric cancer is up to 400,000, occupies first place in the world；Death toll 300,000, accounts for the whole world
Because of mortality of gastric carcinoma number half.The early diagnosis of stomach cancer is global study hotspot, is also the difficult point of research.Long-term clinical is real
Trampling proves that gastric cancer screening is the effective way of early detection and diagnosis of gastric cancer.Unfortunately, even clinic is at first at present
The physical detection means entered are (such as：CT, nuclear magnetic resonance etc.) limit diameter that finds tumour is only 1cm, and conventional tumor markers
Sensitivity and specificity it is also not ideal enough, such as carcinomebryonic antigen (carcinoembryonic antigen, CEA) examination stomach cancer
Sensitivity and specificity are only 36.0% and 86.2% respectively.Therefore, with reference to clinical Cost Benefit Principle, novel tumor mark is developed
It is significant for improving diagnosing gastric cancer rate that will thing precisely diagnoses molecular marker in particular for early carcinoma of stomach.
The content of the invention
The technical problems to be solved by the invention be to provide a kind of stomach cancer molecular marker hsa_circ_0000705 and its
Using the molecular marker has fabulous stomach organization specificity and stomach cancer correlation, and can be real by RT-PCR method
Now quantitatively detect, the discovery and detection for early carcinoma of stomach provide new molecular marker, to further improving when NIH mice is examined
Level is controlled, with certain meaning.
The invention provides a kind of stomach cancer molecular marker hsa_circ_0000705, its nucleotide sequence such as SEQ ID
Shown in NO.1.
The hsa_circ_0000705 is positioned at No. 16 regions of chromosome 58593707~58594266 of people, by CNOT1
Formed after genetic transcription through shearing.Hsa_circ_0000705 is substantially a kind of circular RNA molecule.The present invention is using in digestion
Section's magnifying gastroscope, cell micro-dissections, circRNA chip technologies detect and compare 3 pairs of stomach organizations and cancer beside organism
The difference of circRNA express spectras, wherein circular rna the hsa_circ_0000705 notable low expression in stomach organization.
It is for the hsa_circ_0000705 specific PCR reverse primers detected：
Upstream sequence is 5 '-TAACTGGGGCTCTTCAGCTC-3 '；
Downstream sequence is 5 '-TGGTGGTTGTCTGGCCTTAT-3 '.It is sequenced, is demonstrated by the PCR primer to the primer
The specificity of primer amplification.
Hsa_circ_0000705 low expressions in precancerous lesions and stomach organization.
Present invention also offers a kind of stomach cancer molecular marker hsa_circ_0000705 application, applied to research stomach cancer
The expression of tissue.
The present invention is extracted by sample rna, the quantitative RT-PCR detecting method of RNA Quality Identifications and target, and large sample is tested
Hsa_circ_0000705 is demonstrate,proved in expression in gastric cancer situation, hsa_circ_0000705 is in 79.2% patients with gastric cancer cancerous tissue
Notable low expression.Further large sample detection and lateral comparison hsa_circ_0000705 molecules are in Healthy People gastric tissue, benign
Gastropathy (gastric ulcer, gastritis) tissue, gastric precancerous lesion (dysplasia) tissue and the level in each phase stomach organization and its change
Law, finally identifies the hsa_circ_0000705 only low tables in gastric precancerous lesion (dysplasia) tissue and stomach organization
Reach, with fabulous stomach organization specificity.
The present invention proves that stomach organization hsa_circ_0000705 has by building Receiver Operating Characteristics (ROC) curve
Good sensitivity and specificity.Area is 0.719 (95%CI, 0.648- under hsa_circ_0000705 ROC curve
0.791).When its cutoff value is 9.125, Sensitivity and Specificity is respectively 64.58% and 69.79% (more than existing stomach cancer
Mark CEA, CA19-9 level).With reference to Clinical Pathology of Tumor factor, present invention demonstrates that hsa_circ_ in stomach organization
0000705 expression and tumor stage, Borrmann partings, histological type and tissue CA19-9 expression are closely related.This
Invention proves that hsa_circ_0000705 has fabulous stomach cancer correlation by the studies above.
Beneficial effect
The technologies such as present invention application circRNA chips, quantitative PCR detection, confirm hsa_circ_0000705 only in stomach cancer
Low expression in preceding lesion (dysplasia) tissue and stomach organization, with fabulous stomach organization specificity and stomach cancer correlation,
And quantitative detection can be realized by RT-PCR method, the discovery and detection for early carcinoma of stomach provide new molecular marker,
To further improving when NIH mice treatment level has certain meaning.
Brief description of the drawings
Fig. 1 is hsa_circ_0000705 PCR primer sequencer map；
The amplification curve diagram that Fig. 2 is hsa_circ_0000705 in two tissue specimens；
The melting profile that Fig. 3 is hsa_circ_0000705 in two tissue specimens；
The amplification curve diagram that Fig. 4 is GAPDH in two tissue specimens；
The melting profile that Fig. 5 is GAPDH in two tissue specimens；
Fig. 6 is hsa_circ_0000705 expressions in stomach organization and its pairing cancer beside organism；
Fig. 7 is gastric dysplasia tissue and its expression with hsa_circ_0000705 in normal tissue；
Fig. 8 is hsa_circ_0000705 in Healthy People gastric tissue, Benign Gastric lesion (gastric ulcer, gastritis) tissue, stomach cancer
Expression and its changing rule in preceding lesion (dysplasia) tissue and each phase stomach organization；
Fig. 9 is hsa_circ_0000705 ROC curve.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
Embodiment 1
(1) collection and processing of sample
Surgical operation therapy, removal lesion tissue are carried out to patients with gastric cancer.Outside in stomach cancer focus and away from lesions position 3cm
One piece of soya bean sample size tissue is cut respectively, and the tissue fritter that thickness is no more than 0.5cm is cut into scalpel, 1mL is soaked into
The non-liquid nitrogen pattern product RNA of RNAfixer are preserved in liquid (Tyke of Beijing hundred), are put into standby in -80 DEG C of ultra low temperature freezers.
(2) design of specific PCR reverse primer and synthesis
By means of PrimerPremier primer-design softwares, autonomous Design is used for what hsa_circ_0000705 was detected
Specific PCR reverse primer (divergent primer), and entrust Shanghai Sheng Gong bioengineering limited company to synthesize.Should
Primer upstream primer sequence is 5 '-TAACTGGGGCTCTTCAGCTC-3 ', downstream primer sequence is 5 '-
TGGTGGTTGTCTGGCCTTAT-3’.By the way that the primer PCR product is sequenced, the specificity (Fig. 1) of primer amplification is demonstrated.
(3) Total RNAs extraction and hsa_circ_0000705 RCR detections
1. tissue specimen is taken out from -80 DEG C of ultra low temperature freezers, thaw at RT.
2. preserving liquid from the non-liquid nitrogen pattern product RNA of RNA fixer takes out tissue block, after being blotted with filter paper, many site clips
50mg~100mg is organized, and is placed in 2mL without in RNase centrifuge tubes, is added Trizol reagents (U.S. Invitrogen) 1mL, use hand
The formula homogenizer of holding fully is homogenized to after without visible solid, is stored at room temperature 10min.
3. chloroform is extracted：Addition 0.2mL chloroforms, vortex oscillation 10 seconds, after standing 5 minutes at room temperature, 4 DEG C
Under, 12000rpm is centrifuged 15 minutes, is drawn upper strata aqueous phase (rather few not indiscriminate principle, can typically get 400 μ L) and is moved into a new nothing
In RNase centrifuge tubes.
4. isopropanol precipitating：Added and the isometric isopropanol of the upper strata aqueous phase in previous step is without RNase centrifuge tubes,
Mix, after standing 20 minutes under the conditions of 4 DEG C, 12000rpm is centrifuged 10 minutes at 4 DEG C, and abandoning supernatant must precipitate.
5.75% ethanol is washed：It is 75% ethanol 1mL (- 20 degree precooling), top that mass fraction is added in above-mentioned precipitation
Reciprocal time, 12000rpm is centrifuged 5 minutes at 4 DEG C, abandons supernatant, then with 75% ethanol repeated washing once, at 4 DEG C 12000rpm from
The heart 5 minutes, abandons supernatant.
6. dry RNA：Previous step is abandoned after supernatant, and 12000rpm is centrifuged 1 minute at 4 DEG C again, it is seen that tube wall residual liquid
Ttom of pipe is come together in, is slowly absorbed with 100 μ L pipettors after residual liquid, the translucent white RNA precipitate of a needle point size, room temperature is seen
Under the conditions of dry 1 minute, plus 10 μ L without RNase water dissolve, blow and beat repeatedly, obtain RNA extract solutions, -80 DEG C save backup.
7.RNA reverse transcriptions：(U.S. is purchased from GoScript reverse transcriptions (reverse transcription, RT) kit
Promega companies) to the RNA extract solutions progress reverse transcription reaction of previous step, cDNA solution is obtained, -20 DEG C is put and saves backup, is had
Gymnastics is made to carry out according to the kit specification：9.5 μ L RNA extract solutions, 1 μ L Random are added in 0.5mL centrifuge tubes
Primers、1μL PCR Nucleotide Mix、2μL MgCl2、1μL Reverse Transcriptase、4μL
Reaction Buffer and 0.5 μ L Ribonuclease Inhibitor；Then 25 DEG C be incubated 5 minutes, 42 DEG C 1 hour,
15 minutes are incubated at 70 DEG C again, 80 μ L is added without RNase water, just obtains cDNA solution.
8.PCR is detected：With qPCR Master MixDetection kit (being purchased from Promega companies of the U.S.) is to upper
The cDNA solution of step carries out quantitative fluorescent PCR (instrument is purchased from Stratagene companies of the U.S.), and the primer of amplification is hsa_circ_
(upstream sequence is 5 '-TAACTGGGGCTCTTCAGCTC-3 ' to 0000705 specific amplification upstream and downstream primer, and downstream sequence is
5 '-TGGTGGTTGTCTGGCCTTAT-3 '), concrete operations are carried out according to detection kit and quantitative real time PCR Instrument specification：
Each 1 μ L of hsa_circ_0000705 specific amplification upstream and downstream primers are separately added into thin-walled transparent fluorescent quantitative PCR reaction tube,
12.5μL qPCR Master Mix, 5.5 μ L are without RNase water and 5 μ L cDNA；PCR reaction conditions are：95 DEG C of pre- changes
Property 5 minutes；Then 94 DEG C are denatured 15 seconds, and 55 DEG C are annealed 30 seconds, and 70 DEG C extend 30 seconds, totally 45 circulations；Curve analysis：95
DEG C 1 minute, 55 DEG C 30 seconds；Then 95 DEG C are to slowly warm up to, heating rate is 0.2 DEG C/sec.Finally obtain as shown in Figure 2
Hsa_circ_0000705 amplification curves and hsa_circ_0000705 melting curves as shown in Figure 3, can be with from amplification curve
Obtain two detected samples Ct values be respectively 24.24 and 33.18, from melting curve it is recognized that the curve be compared with
Narrow simple spike, it can be seen that the hsa_circ_0000705 of each sample by specific amplification, without primer dimer and
The interference of miscellaneous band.It is sequenced by hsa_circ_0000705 PCR primer, demonstrates the specificity (Fig. 1) of primer amplification.
(4) calculating of hsa_circ_0000705 expressions
It is essentially identical with hsa_circ_0000705 detection methods, it is different simply by the upstream and downstream primer of amplification by
GAPDH specific amplification upstream and downstream primers replace hsa_circ_0000705, and detection obtains the expansion of GAPDH expressions in sample
Increase curve (Fig. 4) and melting curve (Fig. 5), the Ct values that two detected samples can be obtained from amplification curve are respectively
29.87 with 26.25；It is recognized that the curve is narrower simple spike from melting curve, it can be seen that each sample
GAPDH is by specific amplification, the interference without primer dimer and miscellaneous band.The present embodiment sample cDNA CtGAPDHValue≤30,
RNA (i.e. cDNA) is up-to-standard.GAPDH specific amplification upstream and downstream primer sequences used are：Upstream sequence is 5 '-
ACCCACTCCTCCACCTTTGAC-3’；Downstream sequence is 5 '-TGTTGCTGTAGCCAAATTCGTT-3 '.
Utilize the Ct values of hsa_circ_0000705 and GAPDH in same sample, so that it may according to formula Δ Ct=
CtcircRNA-CtGAPDHThe Δ Ct values of the hsa_circ_0000705 are calculated, the hsa_ just can determine whether according to the size of Δ Ct values
Circ_0000705 relative expression levels；The small person of Δ Ct values, its correspondence circRNA expression is high；And the big person of Δ Ct values,
Its correspondence circRNA expression is low.
(5) stomach organization hsa_circ_0000705 expressions checking analysis
By above-mentioned qRT-PCR technologies, large sample verifies hsa_circ_0000705 by stomach organization and its pairing cancer
Expression in tissue, as a result shows hsa_circ_0000705 notable low expression (figures in 79.2% patients with gastric cancer cancerous tissue
6).Gastric dysplasia (gastric dysplasia, GD) is gastric precancerous lesion generally acknowledged at present, with obvious canceration
Tendency.It is whether related to gastric precancerous lesion in order to disclose tissue hsa_circ_0000705, detected and compared by qRT-PCR
Gastric dysplasia tissue and its expression with hsa_circ_0000705 in normal tissue.As a result show, hsa_
Circ_0000705 expressions in stomach organization in addition to significantly reducing, and in the gastric precancerous lesion stage, its expression just has significantly
Reduce (Fig. 7).With reference to clinical and pathological data, stomach organization hsa_circ_0000705 expressions and stomach cancer are further analyzed
The potential relation of clinical pathological factors.As shown in Table 1, the hsa_circ_0000705 expressions and tumor stage of stomach organization
(P=0.024), Borrmann partings (P=0.005), histological type (P=0.045) and tissue CA19-9 expression (P=
0.010) it is etc. closely related.
Stomach organization hsa_circ_0000705 expressions (the Δ C of table 1t) and patient clinical pathological factor correlation analysis
(6) difference group patient hsa_circ_0000705 expressions are analyzed
Stomach cancer is a multistage, multi-step and the process of evolution step by step.In order to disclose hsa_circ_
Whether 0000705 take part in the evolution process step by step of stomach cancer, using qRT-PCR technology for detection and compare hsa_circ_0000705
Molecule is in Healthy People gastric tissue, Benign Gastric lesion (gastric ulcer, gastritis) tissue, gastric precancerous lesion (dysplasia) tissue and each phase
Level and its changing rule (Fig. 8) in stomach organization.As a result show, hsa_circ_0000705 expressions are except in stomach cancer
Outside being significantly reduced in tissue and Precancerous Lesion (atypical hyperplasia tissue), in Gastric benign lesion (gastric ulcer and gastritis) tissue
In substantially do not change (Fig. 8), point out hsa_circ_0000705 as gastric cancer screening mark may have good spy
The opposite sex.By building Receiver Operating Characteristics (ROC) curve, further to prove that stomach organization hsa_circ_0000705 has good
Good sensitivity and specificity (Fig. 9).Area is 0.719 (95%CI, 0.648- under hsa_circ_0000705 ROC curve
0.791) (Fig. 9).When its cutoff value is 9.125, Sensitivity and Specificity is respectively 64.58% and 69.79% (more than existing
Stomach cancer marker CEA, CA19-9 level).
The present embodiment proves hsa_circ_0000705 only in gastric precancerous lesion (dysplasia) tissue by the studies above
And low expression in stomach organization, with fabulous stomach organization specificity and stomach cancer correlation, and qRT-PCR methods can be passed through
Realize quantitative detection.
SEQUENCE LISTING
<110>Attached Hospital of Medical College, Ningbo Univ.
<120>A kind of stomach cancer molecular marker hsa_circ_0000705 and its application
<130> 1
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 559
<212> DNA
<213>Artificial sequence
<400> 1
gagtgtttct caggagctat cagaaactat cctcaccatg gtagccaatt gcagtaatgt 60
tatgaataag gccagacaac caccacctgg agttatgcca aaaggacgtc ctcctagtgc 120
tagcagctta gatgccattt ctcctgttca ggtaaatgag tgctacattt gatacatctt 180
catttgccat agatttggaa tagaataagc tcctatgtca ttaataaatt gtctgaattt 240
attacatttt agataactgc atgtctagca tccacttatt ttaaaggaga tatgtaaata 300
ggcattgtag ttaacaatag attttatcat caaatagaac gtgactctaa gaggaaatat 360
acagacaatt tatttaggta aatgaagagg gtttcttttt aaaatatgaa ttacgtagac 420
tcttgagaca taagcactgc ctttgaacct gatgtgtctt gtttgtagct tcacgggcca 480
agcaacagtg ctagagcata acgacttgtt ataactgggg ctcttcagct ctcaactgaa 540
ctgctctttt aaaaacaag 559
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
taactggggc tcttcagctc 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
tggtggttgt ctggccttat 20
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
acccactcct ccacctttga c 21
<210> 5
<211> 22
<212> DNA
<213>Artificial sequence
<400> 5
tgttgctgta gccaaattcg tt 22
Claims (5)
1. a kind of stomach cancer molecular marker hsa_circ_0000705, it is characterised in that：Its nucleotide sequence such as SEQ ID NO.1
It is shown.
2. a kind of stomach cancer molecular marker hsa_circ_0000705 according to claim 1, it is characterised in that：It is described
Hsa_circ_0000705 is positioned at No. 16 regions of chromosome 58593707~58594266 of people, is passed through after CNOT1 genetic transcriptions
Shearing is formed.
3. a kind of stomach cancer molecular marker hsa_circ_0000705 according to claim 1, it is characterised in that：For institute
State hsa_circ_0000705 detection specific PCR reverse primer be：
Upstream sequence is 5 '-TAACTGGGGCTCTTCAGCTC-3 '；
Downstream sequence is 5 '-TGGTGGTTGTCTGGCCTTAT-3 '.
4. a kind of stomach cancer molecular marker hsa_circ_0000705 according to claim 1, it is characterised in that：It is described
Hsa_circ_0000705 low expressions in precancerous lesions and stomach organization.
5. a kind of stomach cancer molecular marker hsa_circ_0000705 as claimed in claim 1 application, it is characterised in that：Should
Expression for studying stomach organization.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109666741A (en) * | 2019-01-30 | 2019-04-23 | 江苏万成生物医学研究院有限公司 | A kind of application of new gastric cancer marker gene circPTPDC1 |
| CN110894528A (en) * | 2019-09-22 | 2020-03-20 | 潘文胜 | CircRNA marker for diagnosing gastric poorly differentiated adenocarcinoma and application thereof |
| CN110982906A (en) * | 2019-12-31 | 2020-04-10 | 广西医科大学 | Primer pair for detecting hsa _ circ _0032969, application thereof and kit |
| CN111876487A (en) * | 2020-08-17 | 2020-11-03 | 深圳大学 | Tumor marker based on circular RNA, specific detection primer and molecular detection method and application in gastric cancer diagnosis |
-
2017
- 2017-05-27 CN CN201710390996.5A patent/CN107083432A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109666741A (en) * | 2019-01-30 | 2019-04-23 | 江苏万成生物医学研究院有限公司 | A kind of application of new gastric cancer marker gene circPTPDC1 |
| CN109666741B (en) * | 2019-01-30 | 2022-06-10 | 江苏万成生物医学研究院有限公司 | Application of novel gastric cancer marker gene circPTPDC1 |
| CN110894528A (en) * | 2019-09-22 | 2020-03-20 | 潘文胜 | CircRNA marker for diagnosing gastric poorly differentiated adenocarcinoma and application thereof |
| CN110982906A (en) * | 2019-12-31 | 2020-04-10 | 广西医科大学 | Primer pair for detecting hsa _ circ _0032969, application thereof and kit |
| CN111876487A (en) * | 2020-08-17 | 2020-11-03 | 深圳大学 | Tumor marker based on circular RNA, specific detection primer and molecular detection method and application in gastric cancer diagnosis |
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