CN109280707A - A kind of circular rna hsa_circPDE4D_040 and its specificity amplification primer and application - Google Patents
A kind of circular rna hsa_circPDE4D_040 and its specificity amplification primer and application Download PDFInfo
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Abstract
The invention discloses a kind of circular rna hsa_circPDE4D_040 and its specificity amplification primer and applications, wherein, for the nucleotide sequence of the circular rna as shown in SEQ ID NO:1, the low expression in Colorectal Carcinoma cell can be used as the molecular marker of screening colorectal cancer；The present invention also provides a kind of for expanding the specificity amplification primer of the circular rna, the specificity amplification primer includes upstream primer and downstream primer, wherein, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO:2, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO:3；The invention also discloses application of the specificity amplification primer in the kit that preparation is used for colorectal cancer screening and the kits including the specificity amplification primer.
Description
Technical field
The invention belongs to oncomolecularbiology field more particularly to a kind of circular rnas for diagnosis of colorectal carcinoma, use
Specificity amplification primer and application in the circular rna.
Background technique
Colorectal cancer is the malignant tumour occurred in lower digestive tract, has become global public health problem.With
The raising of living standards of the people, the disease incidence of colorectal carcinoma are in significantly raised trend, disease incidence and lethality in recent years
It ranks among the best, seriously affects people's lives quality.Early diagnosis, early treatment can greatly reduce colorectal cancer deterioration
Further occurrence occurs with the development of molecular level and to colorectal cancer, development, treats further grinding for related mechanism
Study carefully, screening to targeting diagnosis and targeted therapy molecular marker and research become colorectal cancer study field of greatest concern it
One.
Circular rna (Circular RNA, circRNA) is a kind of without the end 5' cap and the end 3' poly (A) tail
Bar and with covalent bond formed ring structure non-coding RNA molecule, variable sheer is mainly passed through by precursor RNA (pre-mRNA)
Processing generates.Most of circRNA is positioned in cytoplasm, and minority is positioned in nucleus, most of to be formed by exon.
CircRNA is not easy to be degraded by exonuclease, can more stably be present in organism compared with linear rna, different
There is conservative in species, while there is expression specificity in different tissues and stage of development, most of circRNA are different
Between species rich content and all have tissue specificity, have stable expression in tissue, saliva, blood and excretion body,
This makes circRNA become the ideal biological marker that tumor patient diagnosis, treatment and prognosis are tracked.
In recent years, about the existing many reports of the relationship research between circRNA and tumour, clear circRNA has
Have and adjusts the functions such as tumor cell proliferation, apoptosis, vascularization, metabolism and drug resistance.Zhang etc. passed through to different lactation periods
Two groups of mouse breast tissue carry out the detection of circRNA chip, find circRNA in different lactation mouse breast tissue
Expression have very big difference, two periods detect the most of differences of circRNA type.Therefore, circRNA may be Colon and rectum
The novel targets of the accurate diagnosis and treatment of cancer, discovery circRNA marker relevant with screening colorectal cancer, have early prevention and treatment colorectal cancer
There are important meaning and value.
Summary of the invention
The application's is designed to provide the following aspects:
In a first aspect, the application provides a kind of circular rna, the nucleotide sequence of the circular rna such as SEQ ID NO:1 institute
Show, wherein two nucleotide of starting are cyclic binding site with two nucleotide of most end.
Full name of the circular rna in circ-RNA database circBank is hsa_circPDE4D_040, referred to as
CircPDE4D_040, the number in circBase database are hsa_circ_0072567.
Fig. 1 is the gene structure figure of the circular rna, as shown in Figure 1, the circular rna hsa_circPDE4D_040
Sequence 161bp, between 58476420-58489362 on No. 5 chromosome antisense strands of the mankind, by PDE4D gene
The shearing of 5-7 exon is cyclized, and CDS indicates the circular rna from code area.F and R respectively represents the gene-specific primer
Position, arrow represent primer initiation direction.
According to a second aspect of the present application, circular rna hsa_circPDE4D_040 described in first aspect present invention is provided
The purposes of molecular marker as screening colorectal cancer.
The research of the invention finds that relative to colorectal cancer patients Carcinoma side normal tissue, circular rna hsa_circPDE4D_
040 significantly lowers in Colorectal Carcinoma.
Therefore, the circular rna hsa_circPDE4D_040 can be used for colorectal cancer screening.
Second aspect, the application also provide the purposes of molecular marker of the circular rna as screening colorectal cancer.
The third aspect, the application also provide it is a kind of for expanding the specificity amplification primer of circular rna described in first aspect,
The specificity amplification primer includes upstream primer and downstream primer, wherein
The nucleotide sequence of the upstream primer is as shown in SEQ ID NO:2；
The nucleotide sequence of the downstream primer is as shown in SEQ ID NO:3.
Specifically, the nucleotide sequence of the upstream primer are as follows: 5 '-GCCACCATAACAGACACGGA-3 '；
The nucleotide sequence of the downstream primer are as follows: 5 '-TGGCCAAGACCTGAGCAAAT-3 '.
In a kind of achievable mode, the G/C content of the upstream primer is 55.0%, and the GC of the downstream primer contains
Amount is 50.0%, wherein the G/C content refers in 4 kinds of bases of DNA, ratio shared by guanine and cytimidine.
Further, the TM value of the upstream primer is 60.04, the TM value 59.89 of the downstream primer, wherein described
TM value refers to the melting temperature of upstream primer or downstream primer.
The inventors discovered that cancerous tissue and patient using specificity amplification primer provided by the present application to Patients With Rectal Carcinoma
RNA described in first aspect in itself cancer beside organism is expanded, and as shown in Examples 1 and 22, amplification is analyzed,
ROC curve is obtained, as a result as shown in figure 3, area is 0.960 under ROC curve, this shows to expand using specificity provided by the present application
Increase the amplified production that primer obtains as single band, no non-specific amplification, the specificity amplification primer can be used as this kind
The Specific marker of detection.
Fourth aspect, the application also provide a kind of method for expanding circular rna described in first aspect, which comprises
Step 1, synthesize the first chain cDNA: mixed raw material, the raw material include circular rna described in first aspect, with power traction
Object, ddH2O, dNTP mixed liquor, reverse transcription buffer, RNase inhibitor, reverse transcriptase are reacted according to the first temperature programmed control；
Step 2, PCR amplification is carried out to the first chain cDNA made from step 1: prepares amplification system, the amplification system packet
Include downstream primer, ddH shown in the cDNA of step 1 preparation, upstream primer, the third aspect shown in the third aspect2O, PCR amplification
Mix is reacted according to the second temperature programmed control.
In a kind of achievable mode, in step 1,
The mixing according to the following ratio of the raw material: circular rna described in 1 μ l first aspect, 1 μ l random primer, 10 μ l
ddH2O, 2 μ l dNTP mixed liquors, 4 μ l reverse transcription buffers, 1 μ l RNase inhibitor, 1 μ l reverse transcriptase, wherein described
DNTP mixed liquor includes dATP, dGTP, dCTP and dTTP；
First program are as follows: keep the temperature 5min under the conditions of 25 DEG C, then keep the temperature 60min under the conditions of 42 DEG C, finally 70
5min is kept the temperature under the conditions of DEG C.Further, reverse transcription is carried out using ABI PCR amplification instrument, it is standby that the cDNA is placed in -80 DEG C of storages
With.
Further, in step 2,
The amplification system is prepared according to the following ratio: the cDNA of 2 μ l steps 1 preparation, 0.8 μ l is as described in the third aspect
Upstream primer, downstream primer described in the 0.8 μ l third aspect, 6.4 μ l ddH2O, 10 μ l PCR amplification Mix；
Second program is the initial denaturation 30s at 95 DEG C, then to keep the temperature 5s at 95 DEG C, keep the temperature 15s at 60 DEG C
40 circulations are carried out, then keep the temperature 5s at 95 DEG C, 60s are kept the temperature at 60 DEG C, finally in 72 DEG C of reaction 5min.
5th aspect, the application also provide the kit that aforementioned specificity amplification primer is used for colorectal cancer screening in preparation
In application.
6th aspect, the application also provide a kind of kit for colorectal cancer screening, and the kit includes third
Specificity amplification primer described in aspect.
In a kind of achievable mode, the kit further includes reverse transcriptase, buffer, ddH2O, archaeal dna polymerase
At least one of with dNTP mixed liquor.
The hsa_ in the method detection Colorectal Carcinoma of real-time fluorescence quantitative PCR can be used in the kit
CircPDE4D_040 is used for screening colorectal cancer.
Applicants have discovered that low expression is presented in Colorectal Carcinoma cell in circular rna described herein, that is, knot
The content of circular rna described in rectum cancer tissue is far below the rna content in cancer beside organism, therefore, the circular rna hsa_
CircPDE4D_040 may be used as the molecular marker of colorectal cancer screening, moreover, provided by the present application for expanding the ring
The specificity amplification primer of shape RNA has high stability, specificity and sensibility to the circular rna, wherein in the tissue
Detection sensitivity up to 90%, specificity is up to 90%.
Detailed description of the invention
Fig. 1 shows hsa_circPDE4D_040 gene structure figure；
Fig. 2 shows real-time fluorescence quantitative PCR detection hsa_circPDE4D_040 in Colorectal Carcinoma and cancer beside organism
Differential expression figure；
Fig. 3 shows the ROC curve of hsa_circPDE4D_040 testing result in Colorectal Carcinoma.
Specific embodiment
The following describes the present invention in detail with reference to examples, the features and advantages of the invention will with these explanation and
It becomes more apparent from, is clear.But these examples are only exemplary, do not constitute any limit to protection scope of the present invention
System.
Embodiment
In embodiment, hsa_circPDE4D_040 screening the primer is limited by giving birth to work bioengineering (Shanghai) share
Company's synthesis；Trizol Reagent (article No. 15596018) is purchased from Invitrogen company；Chloroform (article No. 20100927) purchase
From Beijing chemical industry, reverse transcription reagent box is purchased from Thermo company (article No. K1622)；Real time fluorescent quantitative SYBR Premix Ex
Taq II (article No. RR820) is purchased from Takara.
Clinical sample: the cancerous tissues of 10 Patients with Colorectal Cancer, cancer beside organism's sample are by institute of hospital general of Ningxia Medical University
It provides.
Expression of the 1 PCR reaction detection hsa_circPDE4D_040 of embodiment in Colorectal Carcinoma
1, specificity amplification primer is designed:
Specificity amplification primer sequence and relevant information designed by the applicant are as shown in table 1:
1 specificity amplification primer sequence of table and relevant information
2, the extraction of total serum IgE:
(1) Trizol method extract Colorectal Carcinoma in total serum IgE: take 1g Colorectal Carcinoma, under the conditions of liquid nitrogen into
1mL Trizol is added in liquid nitrogen and continues to grind, to which Trizol to be ground to until the tissue is in pulverulence for row grinding
It after dry powder, is stored at room temperature, the centrifugation that all liq is transferred to 1.5ml RNase-free is collected after dry powder turns to liquid
Guan Zhong；
(2) fluid sample made from step 1 cracks after five minutes at room temperature, adds according to the Trizol grinding homogenate of every 1mL
Chloroform is added in the ratio for entering 0.2mL chloroform, covers tightly pipe lid, is vortexed after concussion 15s, stands 5min, under the conditions of 4 DEG C 12000rpm from
The heart 15 minutes, mixing liquid was layered as lower layer's chloroform phase after centrifugation, and middle layer albumin layer, upper layer colourless aqueous phase, RNA is whole to be assigned
In water phase；
(3) water phase is transferred in new centrifuge tube, 0.5mL isopropanol is added according to the Trizol grinding homogenate of every 1mL
Ratio isopropanol is added into system, mix, after -20 DEG C of precipitating 30min, 12000rpm centrifugation 15min, is obtained under the conditions of 4 DEG C
Precipitating, RNA are all present in precipitating；
(4) supernatant is removed, pre-cooling is added into system according to the ratio that 1mL is added in every 1mL Trizol into system
75% ethyl alcohol cleans RNA precipitate 2 times, mildly vibrates centrifuge tube, and suspend precipitating, and 7500rpm is centrifuged 5 minutes under the conditions of 4 DEG C；
(5) ethanol solution is removed, is dried 5-10 minutes at room temperature, until ethyl alcohol volatilizees, but is completely dried RNA not, Xiang Ti
The ddH without RNA enzyme is added in system2Centrifuge tube is added in O water (DEPC water), and 60 DEG C are incubated for 5 minutes, obtains total serum IgE；
(6) total serum IgE extracted determines the concentration of total serum IgE using the concentration and purity of NanoDrop ND-2000 measurement RNA
It can satisfy the requirement of subsequent experimental with purity, total rna solution packing is placed in -80 DEG C of preservations.
3, the synthesis of the first chain cDNA sequence:
PCR pipe is taken to configure reverse transcription system, reverse transcription system are as follows: 1 μ L total serum IgE (about 500ng), 1 μ L random primer
(Thermo company), 10 μ L ddH2O (Tiangeng biotech firm), 2 μ L dNTP mixed liquors (dATP, dGTP, dCTP and dTTP,
Invitrogen company), 4 μ L reverse transcription buffers (Thermo company), 1 μ L RNase inhibitor (Thermo company), 1 μ L is anti-
Transcriptase (Thermo company), 20 μ L of total volume.
Reaction condition are as follows: keep the temperature 5min at 25 DEG C；60min is kept the temperature at 42 DEG C, keeps the temperature 5min at 70 DEG C.Use ABI PCR
Amplification instrument carries out reverse transcription.The cDNA that reverse transcription obtains is placed in -80 DEG C of long term storages or saves backup in -20 DEG C.
4, Hsa_circPDE4D_040 gene cDNA amplification verifying:
The cDNA for taking 2 μ L step 3 reverse transcriptions to obtain prepares system and carries out PCR amplification；
PCR amplification system are as follows: 2 μ L cDNA, upstream primer shown in 0.8 μ L table 1, downstream primer shown in 0.8 μ L table 1,
10 μ L PCR amplification Mix, 6.4 μ L ddH2O, 20 μ L of total volume；
Reaction condition is initial denaturation 30s at 95 DEG C, then carries out 40 to keep the temperature 5s at 95 DEG C, keep the temperature 15s at 60 DEG C
A circulation, then 5s is kept the temperature at 95 DEG C, 60s is kept the temperature at 60 DEG C, finally in 72 DEG C of reaction 5min.
Take 2 μ L reaction products in 1.5g agarose/100ml 1 × TAE buffer, 120V voltage is verified under the conditions of 25min
Whether PCR product is single specificity amplified band.
5, pcr amplification reaction:
The cDNA sample obtained using reverse transcription is detected.
Reaction system are as follows: 2 μ L cDNA, 0.8 μ L upstream primer, 0.8 μ L downstream primer, 6.4 μ L ddH2O (Tiangeng biology
Company), 10 μ L fluorescent quantitative PCR Mix (SYBGREEN dyestuff), 20 μ L of total volume；
Real-time PCR reactions condition: 95 DEG C of initial denaturation 5min；Then to keep the temperature 10s at 98 DEG C, heat preservation 30s is carried out at 62 DEG C
40 circulations.
Amplified reaction carries out on real-time fluorescence quantitative PCR instrument LightCyler480, expands while amplifying target genes
Increase the control of GAPDH internal reference, the primer gene order table such as SEQ ID NO:4 (upstream primer) and SEQ ID of the internal reference control
Shown in NO:5 (downstream primer), and pass through 2(-△△CT)The relative expression quantity of method calculating gene.
For embodiment 1, different samples are taken respectively, are repeated 10 times.
Wherein, as can be seen that amplified peak in hsa_circPDE4D_040 gene real-time fluorescence quantitative PCR solubility curve figure
It is single, no miscellaneous peak, it was demonstrated that primer specificity is outstanding, and amplification experiment is normal.
Expression of the 2 PCR reaction detection hsa_circPDE4D_040 of embodiment in colorectal cancer cancer beside organism
The process of embodiment 1 is repeated, difference is: in step 2, extracting the total serum IgE in colorectal cancer cancer beside organism.
For embodiment 2, different samples are taken respectively, are repeated 10 times.
The PCR interpretation of result of Examples 1 to 2
1, gene expression analysis
Wherein, the expression of results of hsa_circPDE4D_040 is as shown in Figure 2:
In 10 pairs of tissues of Fig. 2, relative to colorectal cancer cancer beside organism, circular rna hsa_circPDE4D_040 exists
Expression in Colorectal Cancer is significantly lowered, and lowers 10 times or more.
The above result shows that circular rna hsa_circPDE4D_040 has in Colorectal Carcinoma and cancer beside organism
Differential expression, specifically, the circular rna hsa_circPDE4D_040 are low expression in Colorectal Carcinoma, therefore, described
Circular rna hsa_circPDE4D_040 can be used for the screening of colorectal cancer.
2, ROC curve is analyzed
The test data obtained to embodiment 1 and embodiment 2 is analyzed, and obtains ROC curve, as shown in Figure 3, wherein
Area under the curve AUC is 0.960, indicates that detection target spot can be as the Specific marker for detecting the circular rna, also, examine
Sensitivity is surveyed up to 90%, specificity is up to 90%.
One skilled in the art will appreciate that area is between 1.0 and 0.5 under ROC curve, and in the case where AUC > 0.5, AUC
Closer to 1, illustrate that diagnosis effect is better.AUC has lower accuracy in 0.5-0.7, and AUC is fixed in 0.7-0.9
True property, AUC have high accuracy at 0.9 or more, which, which is greater than 0.7, indicates the spy that detection target spot can be detected as this kind
Anisotropic marker.
Combine detailed description and exemplary example that the application is described in detail above, but these explanations are simultaneously
It should not be understood as the limitation to the application.It will be appreciated by those skilled in the art that without departing from the application spirit and scope,
A variety of equivalent substitution, modification or improvements can be carried out to technical scheme and embodiments thereof, these each fall within the application
In the range of.The protection scope of the application is determined by the appended claims.
Sequence table
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Claims (9)
1. a kind of circular rna, which is characterized in that full name of the circular rna in circ-RNA database circBank be
Hsa_circPDE4D_040, nucleotide sequence is as shown in SEQ ID NO:1.
2. the purposes that circular rna described in claim 1 is used as the molecular marker of screening colorectal cancer.
3. the specificity amplification primer for expanding circular rna described in claim 1, which is characterized in that the specific amplification
Primer includes upstream primer and downstream primer, wherein
The nucleotide sequence of the upstream primer is as shown in SEQ ID NO:2；
The nucleotide sequence of the downstream primer is as shown in SEQ ID NO:3.
4. a kind of method of circular rna described in amplification claim 1, which is characterized in that the described method includes:
Step 1, synthesize the first chain cDNA: mixed raw material, the raw material include circular rna, random primer described in claim 1,
ddH2O, dNTP mixed liquor, reverse transcription buffer, RNase inhibitor, reverse transcriptase are reacted according to the first temperature programmed control；
Step 2, PCR amplification is carried out to the first chain cDNA made from step 1: prepares amplification system, the amplification system includes step
The cDNA of rapid 1 preparation, upstream primer as stated in claim 3, downstream primer as stated in claim 3, ddH2O, PCR expands
Increase mix, is reacted according to the second temperature programmed control.
5. according to the method described in claim 4, it is characterized in that, in step 1,
The mixing according to the following ratio of the raw material: circular rna described in 1 μ l claim 1,1 μ l random primer, 10 μ l ddH2O,
2 μ l dNTP mixed liquors, 4 μ l reverse transcription buffers, 1 μ l RNase inhibitor, 1 μ l reverse transcriptase, wherein the dNTP mixing
Liquid includes dATP, dGTP, dCTP and dTTP；
First program are as follows: keep the temperature 5min under the conditions of 25 DEG C, then keep the temperature 60min under the conditions of 42 DEG C, finally in 70 DEG C of items
5min is kept the temperature under part.
6. according to the method described in claim 4, it is characterized in that, in step 2,
The amplification system is prepared according to the following ratio: the cDNA of 2 μ l steps 1 preparation, on 0.8 μ l is as claimed in claim 3
Swim primer, 0.8 μ l downstream primer as claimed in claim 3,6.4 μ l ddH2O, 10 μ l PCR amplification Mix；
Second program is the initial denaturation 30s at 95 DEG C, is then carried out with keeping the temperature 5s at 95 DEG C, keeping the temperature 15s at 60 DEG C
40 circulations, then 5s is kept the temperature at 95 DEG C, 60s is kept the temperature at 60 DEG C, finally in 72 DEG C of reaction 5min.
7. application of the specificity amplification primer as claimed in claim 3 in the kit that preparation is used for colorectal cancer screening.
8. a kind of kit for colorectal cancer screening, which is characterized in that the kit includes spy as claimed in claim 3
Specific amplification primers.
9. kit according to claim 8, which is characterized in that the kit further include reverse transcriptase, buffer,
ddH2O, at least one of archaeal dna polymerase and dNTP mixed liquor.
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