CN109295059A - A kind of single stranded RNA of covalent annular closed and its application - Google Patents

A kind of single stranded RNA of covalent annular closed and its application Download PDF

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CN109295059A
CN109295059A CN201811069452.XA CN201811069452A CN109295059A CN 109295059 A CN109295059 A CN 109295059A CN 201811069452 A CN201811069452 A CN 201811069452A CN 109295059 A CN109295059 A CN 109295059A
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single stranded
stranded rna
covalent
annular closed
breast cancer
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谢小明
唐海林
陈涛
刘鹏
黄晓嘉
谢新华
叶锋
肖祥胜
杨露
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Cancer Prevention Center Of Zhongshan University (affiliated Cancer Hospital Of Zhongshan University Zhongshan University Institute Of Oncology)
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Abstract

The present invention relates to gene technology field, single stranded RNA and its application of a kind of covalent annular closed are specifically disclosed；The present invention first collects to be measured doubtful three negative breast cancer tissue's samples, extracted total RNA, using total serum IgE as template by single stranded RNA (hsa_circRNA_007255) specific reverse transcriptase of covalent annular closed at cDNA, real-time quantitative PCR amplification is carried out with Specific PCR primers again, using β-actin as reference gene, the single stranded RNA relative quantification Δ CT value for obtaining covalent annular closed prompts the single stranded RNA of covalent annular closed to express positive as Δ CT≤11.27.Covalently the single stranded RNA of annular closed expresses situation in the negative breast cancer tissue's sample of detection doubtful three that the present invention passes through relative quantification, for the application in three negative breast cancer patients prognosis predictions.

Description

A kind of single stranded RNA of covalent annular closed and its application
Technical field
The present invention relates to breast cancer diagnosis technical field, the single stranded RNA of covalent annular closed and its in three negative breast cancer Application.
Background technique
Breast cancer is the highest female malignant of whole world disease incidence.China is used as populous nation and unique aging Social pattern, breast cancer incidence growth rate are higher than other countries, and situation allows of no optimist.Breast cancer is so far still without being filled Divide effective control, the higher death rate is still presented in breast cancer, and main cause is its recurrence and transfer, and breast cancer once occurs Transfer, average viability probably only have 4-5.Three negative breast cancer refer to estrogen receptor (ER), progesterone receptor (PR) and people The negative breast cancer of skins growth factor receptors -2 (HER-2) expression, accounts for the 15%-20% of entire breast cancer group.Three Negative breast cancer Clinical symptoms is not showed only as that aggressive strong, histological grade is higher, poor prognosis, visceral metastases easily occurs, and lacks The corresponding target spot of weary endocrine therapy and the molecular targeted therapy for HER-2, the selection for the treatment of means is relative to other molecules Hypotype breast cancer is restricted.
Summary of the invention
To solve the above-mentioned problems, the purpose of the present invention is to provide it is a kind of can in triple negative breast cancer highly expressed ring The single stranded RNA of the covalent annular closed of shape.
The second object of the present invention is to provide the application of the single stranded RNA of covalent annular closed.
The present invention is achieved through the following technical solutions:
A kind of single stranded RNA of covalent annular closed, the single stranded RNA of the covalent annular closed are small molecule non-coding ring Shape, from the exon region of KIF4A gene, sequence is as shown in SEQ No: 1.
The single stranded RNA (RNA circKIF4A) of covalent annular closed belongs to one kind of endogenous non-coding RNA.With knot The features such as structure stabilization, abundance height and tissue specific expression.It plays gene expression as competitive endogenous RNA (ceRNA) The effect of regulation.Circular rna utilizes its microRNA (miRNA) response element combination miRNA, to block miRNA to its target The inhibiting effect of expression, to regulate and control the expression of other correlations RNA.Single stranded RNA (the RNA of covalent annular closed CircKIF4A) as protein coding gene, have main biological function, be the new judgement prognostic marker of tumour and The pretty good tool of targeted therapy, the especially high expression in three negative breast cancer, defeat triple negative breast cancer to provide new for the mankind Strategy.
Preferably, described carry out reverse transcription using transcript reagent box and reverse transcription specific primer, respectively to circKIF4A The reverse produced for the Reverse Transcriptase kit A3500 of Pu Luomaige Promega company and Shanghai Ji Ma Bioisystech Co., Ltd Record specific primer QPM010.
The system of 20 μ L reverse transcription reactions is as follows:
Reverse transcription process: 16 DEG C 30 minutes, 42 DEG C 30 minutes, 85 DEG C 10 minutes.
(3) circKIF4A specificity real-time quantitative PCR
First circKIF4A reverse transcription product is diluted to 2 times respectively, then mixed；
In circKIF4A specificity real-time quantitative PCR, 20 μ L reaction systems are as follows:
CircKIF4A real-time quantitative PCR response procedures: 95 DEG C 3 minutes, 40 circulation, 95 DEG C 12 seconds, 62 DEG C 35 seconds, make Real-time quantitative PCR amplification is carried out with SYBR-Green dyestuff.
The circKIF4A Specific PCR primers are as follows:
Forward primer is 5 '-GAGGTACCCTGCCTGGATCT-3 '
Reverse primer is 5 '-TGGAATCTCTGTAGGGCACA-3 '；
β-actin the Specific PCR primers are as follows:
Forward primer is 5 '-CATGTACGTTGCTATCCAGGC-3 '
Reverse primer is 5 '-CTCCTTAATGTCACGCACGAT-3 '.
(4) measurement of Δ CT index
As Δ CT≤11.27, circKIF4A expression is positive, i.e., the diagnosable tissue is three negative breast cancer tissues；It is described Δ CT is the difference of the mean CT-number of circKIF4A and β-actin.
The RNA circKIF4A is applied to three negative breast cancer detection reagents by the application of the RNA circKIF4A Box.Detection kit includes:
Isopropanol, chloroform, Trizol, DEPC water or without enzyme water, distilled water, 5 × RT Buffer, 25mM chlorine Change magnesium, 10mM triphosphoric acid base deoxynucleotide, 5U/ μ l RNase inhibitor, 200U/ μ l MMLV reverse transcriptase or 25U/ μ l AMV enzyme, 2 × real-time quantitative PCR buffer, 5U/ μ l Taq polymerase, 5 μM of circKIF4A Specific PCR primers, 5 μM of β- Actin Specific PCR primers, 10 μM of circular rna reverse transcriptase primers, 10 μM of β-actin specific reverse transcriptase primers.
The single stranded RNA (RNA circKIF4A) of the covalent annular closed negative breast cancer of diagnosis three, specific method is such as Under:
(1) extracting of tissue RNA
A takes tissue specimen that liquid nitrogen grinding is added in mortar, Trizol and chloroform is added after being ground into homogenate, concussion is uniform It places on ice afterwards；
B is by gained mixed liquor low-temperature centrifugation in step A, and the isopropanol for taking upper strata aqueous phase that pre-cooling is added mixes, and refrigerator is stood Low-temperature centrifugation afterwards；Supernatant is abandoned, the water-reducible ethyl alcohol of 75%DEPC is added and mixes, low-temperature centrifugation；Supernatant is abandoned, is obtained after drying at room temperature RNA；
The dissolution of DEPC water is first added in C into step B in gained RNA, then the concentration and quality of RNA are measured with spectrophotometric, OD260/280 ratio is between 1.6-1.8 and carries out EB gel electrophoresis detection RNA mass, obtains the tissue RNA, -70 DEG C of guarantors It deposits；
(2) specific reverse transcriptase
Reverse transcription is carried out to circKIF4A using transcript reagent box and reverse transcription specific primer, reverse transcription is anti-carrying out The various reagents other than reverse transcriptase must be mixed into reverse transcription mixed liquor before answering, the pipe of installed reagents is flicked with finger, Reverse transcription mixed liquor is inhaled with pipettor and is beaten several times, oscillator cannot be used；
(3) circKIF4A specificity real-time quantitative PCR
First circKIF4A reverse transcription product is diluted to 2 times respectively, then mixed；
(4) measurement of Δ CT index
As Δ CT≤11.27, circKIF4A expression is positive, i.e., the diagnosable tissue is three negative breast cancer tissues；It is described Δ CT is the difference of the mean CT-number of circKIF4A and β-actin.
The RNA circKIF4A is applied to three negative breast cancer patients prognosis predictions by the application of RNA circKIF4A.
Single stranded RNA (RNA circKIF4A) i.e. hsa_circRNA_007255 of covalent annular closed in the present invention (chrX:69549254-69553539), the exon region (Fig. 1) of KIF4A gene (Xq13.1) is derived from.Real-time fluorescence Quantitative PCR technique is one of the detection method of current detection tissue circular rna expression authority.Applicant passes through real-time fluorescence first Quantitative PCR technique detects 57 triple negative breast cancer tissues and it matches the expression in normal galactophore tissue, as a result sends out Existing, high expression (Fig. 2) is presented in circKIF4A in triple negative breast cancer tissue.CircKIF4A is in triple negative breast cancer tissue Height expresses statistically significant (P < 0.01).
In order to preferably determine expression of the circKIF4A in triple negative breast cancer tissue, further analyze The influence of circKIF4A patient with breast cancer's prognosis negative for three, the applicant have detected again by way of in situ hybridization The expression of circKIF4A in 240 three negative patients with breast cancer.Follow-up Data shows that circKIF4A is highly expressed There are 27 death in 100 triple negative breast cancer patients, and in 140 triple negative breast cancer patients of circKIF4A low expression Only 11 death；It always survives through the highly expressed triple negative breast cancer patient of Kaplan-Meier statistical analysis circKIF4A Rate is lower than the patient of circKIF4A low expression, and difference statistically significant (P < 0.01) (Fig. 3) prompts circKIF4A three High expression in negative breast cancer, is the molecular marker for predicting triple negative breast cancer prognosis.The present invention is triple negative breast cancer prognosis Prediction provides strong technical support and molecular biology mechanism, has far-reaching clinical meaning and generalization.
In previous research work, the applicant utilizes circular rna high throughput chip (Arraystar Human CircRNA Array V2) the screening triple negative breast cancer phase in the Carcinoma side normal tissue of 3 pair of three negative breast cancer tissue and pairing Close circular rna differential expression spectrum.It is greater than 2 times according to fold differences, the standard of P < 0.05 finds that triple negative breast cancer is related cyclic annular RNA has 258, wherein up-regulation circular rna has 84, lowering circular rna has 174.The circular rna of subsequent 5 up-regulations of picking QRT-PCR verifying is carried out in 51 pairs of triple negative breast cancer tissues and the Carcinoma side normal tissue of pairing.The results show that RNA hsa_ CircRNA_007255 (circKIF4A) is significantly higher than corresponding cancer beside organism in three negative expression in breast highests. It is consistent with chip results.
By further fabric study, inventor's discovery: high expression is presented in circKIF4A in triple negative breast cancer tissue (Fig. 2).Patient (figure of the highly expressed triple negative breast cancer patient overall survival of circKIF4A lower than circKIF4A low expression 3).This prompt circKIF4A is the molecular marker for predicting triple negative breast cancer prognosis.
Of the invention confirms circKIF4A high expression in triple negative breast cancer by research, can be used for three negative breasts Application in carninomatosis people's prognosis prediction is applied to field of medicaments, provides that a kind of cost performance is high, three negative cream of application easy to spread Gland cancer diagnostic kit.
Figure of description
Fig. 1 circKIF4A schematic diagram
Fig. 2 real-time fluorescence quantitative PCR is surveyed expression of the circKIF4A in triple negative breast cancer tissue and is changed
The relationship of Fig. 3 circKIF4A and triple negative breast cancer patient prognosis
The relationship of Fig. 4 circKIF4A and triple negative breast cancer patient disease-free survival rate.
Specific embodiment
The present invention is described in further detail With reference to embodiment, to help those skilled in the art's reason The solution present invention.
Triple negative breast cancer organizational diagnosis kit forms (50 secondary response)
Isopropanol 100ml, chloroform 100ml, Trizol 50ml, DEPC water or without enzyme water 10ml, distilled water 10ml, 5 × RT Buffer 1ml, 25mM magnesium chloride 1ml, 10mM triphosphoric acid base deoxynucleotide 1ml, 5U/ μ l RNase inhibitor 500 50 μ l or 25U/ μ lAMV enzyme of μ l, 200U/ μ l MMLV reverse transcriptase 50 μ l, 2 × real-time quantitative PCR buffer 2ml, 5U/ μ 50 μ l of l Taq polymerase,
5 μM of 50 μ l of circKIF4A Specific PCR primers
Its forward primer is 5 '-GAGGTACCCTGCCTGGATCT-3 '
Its reverse primer is 5 '-TGGAATCTCTGTAGGGCACA-3 '
5 μM of 30 μ l of β-actin Specific PCR primers
Its forward primer is 5 '-CATGTACGTTGCTATCCAGGC-3 '
Its reverse primer is 5 '-CTCCTTAATGTCACGCACGAT-3 '
10 μM of 20 μ l of circular rna reverse transcriptase primer (Shanghai Ji Ma Bioisystech Co., Ltd, QPM010).
10 μM of 0 μ l of β-actin specific reverse transcriptase primer 2 (Shanghai Ji Ma Bioisystech Co., Ltd, QPM010).
The detection of circKIF4A in tissue samples
1, the extracting of RNA is organized
It takes and liquid nitrogen grinding sample is added in tissue specimen and mortar；0.6ml Trizol mortar sample is added in mortar, Medication spoon is added in tube pipe after being ground into homogenate；0.4ml Trizol is added in tube pipe；Chlorination imitates 200 μ l/mlTrizol In Tube, 15-30s is shaken with hand, places 5min on ice, 4 DEG C of 12000g are centrifuged 15min；Carefully upper strata aqueous phase is taken to enter newly In tube, the isopropanol 0.5ml/mlTrizol that pre-cooling is added is mixed, and -20 DEG C of refrigerators stand 20min, 4 DEG C of 12000g centrifugations 10min；Supernatant is abandoned, the water-reducible ethyl alcohol 1-2ml of 75%DEPC is added and mixes, 4 DEG C of 7500g are centrifuged 5min, as far as possible abandoning supernatant, room The dry 5-10min of temperature, is added DEPC water 10-20 μ l and dissolves RNA.Spectrophotometric measures the concentration and quality of RNA, OD260/280 Ratio is between 1.6-1.8 and carries out EB gel electrophoresis detection RNA mass, -70 DEG C of preservations.
2, the Reverse Transcriptase kit (A3500) and Shang Haiji of Pu Luomaige Promega company specific reverse transcriptase: are used The reverse transcription specific primer (QPM010) of Ma Bioisystech Co., Ltd production carries out reverse transcription to circKIF4A.20 μ L are inverse The system of responsive transcription is as follows:
Various reagents other than reverse transcriptase must be mixed into reverse transcription mixed liquor before carrying out reverse transcription reaction, used Finger flicks the pipe of installed reagents, and reverse transcription mixed liquor is inhaled with pipettor and is beaten several times, oscillator cannot be used.
Reverse transcription process: 16 DEG C 30 minutes, 42 DEG C 30 minutes, 85 DEG C 10 minutes.
3, circKIF4A specificity real-time quantitative PCR: circKIF4A reverse transcription product is first diluted to 2 times respectively, so After mix.20 μ L reaction systems are as follows:
CircKIF4A real-time quantitative PCR response procedures: 95 DEG C 3 minutes, 40 circulation, 95 DEG C 12 seconds, 62 DEG C 35 seconds.
Real-time quantitative PCR amplification is carried out using SYBR-Green dyestuff.
The PCR specific primer of circKIF4A are as follows:
Its forward primer is 5 '-GAGGTACCCTGCCTGGATCT-3 '
Its reverse primer is 5 '-TGGAATCTCTGTAGGGCACA-3 '
β-actin is used as reference gene, PCR primer sequence are as follows:
Its forward primer is 5 '-CATGTACGTTGCTATCCAGGC-3 '
Its reverse primer is 5 '-CTCCTTAATGTCACGCACGAT-3 '
(4)ΔCTThe measurement of index: Δ CTRefer in same sample, circKIF4A to be checked and internal reference β-actin Average Ct values Difference.That is circKIF4A Δ CT=circKIF4A MeanCT-control MeanCT, the present invention in, Δ CTFor The average C of circKIF4A and β-actinTThe difference of value obtains relative quantification Δ CT value, and is judged, the results show that In 57 triple negative breast cancer tissues of detection, as Δ CTWhen≤11.27, the circKIF4A expression positive has 35, and positive rate is 61.40%.
The Prognostic significance of single stranded RNA (the RNA circKIF4A) and triple negative breast cancer patient of covalent annular closed
With hybridization in situ technique, applicant has detected the expression feelings of circKIF4A in 240 triple negative breast cancer tissues Then condition carries out prognosis follow-up to the triple negative breast cancer patient of every an example detection, circKIF4A is highly expressed as the result is shown Triple negative breast cancer patient's overall survival is lower than the patient (Fig. 3) of circKIF4A low expression.This prompt circKIF4A is prediction The molecular marker of triple negative breast cancer prognosis.
Above studies have shown that detecting the expression of circKIF4A in triple negative breast cancer tissue, there is good stability. CircKIF4A can be used as the specificity molecular marker of triple negative breast cancer patient's prognosis prediction, the yin of circKIF4A and three The property significant correlation of Prognosis in Breast Cancer, can be used for the application in triple negative breast cancer patient's prognosis prediction.
Above-described embodiment, only presently preferred embodiments of the present invention, is not intended to limit the invention practical range, therefore all with this The equivalent change or modification that feature described in invention claim and principle are done should all be included in scope of the invention as claimed Within.
Sequence number
Denomination of invention: a kind of single stranded RNA of covalent annular closed and its application
Applicant: Zhongshan Univ. Cancer Cure Center
The single stranded RNA sequence of covalent annular closed
GTATTAATATTAACCGAGGCCTCCTATGCTTGGGAAATGTAATCAGTGCTCTTGGAGATGACAAAAAGG GTGGCTTTGTGCCCTACAGAGATTCCAAGTTGACTCGACTGCTTCAAGATTCTCTAGGAGGTAATAGC CATACTC TTATGATAGCCTGTGTGAGTCCTGCTGACTCCAATCTAGAGGAAACATTAAATACCCTTCGC TATGCTGACAGAG CAAGAAAAATCAAGAACAAACCTATTGTTAATATTGATCCCCAGACAGCTGAACT TAATCATCTAAAGCAACAGG TACAACAGCTACAAGTCTTGTTGCTACAGGCCCATGGAGGTACCCTGC CTGGATCTATAAC。
Sequence table
<110>Zhongshan Univ. Cancer Cure Center (Tumor Hospital Attached to Zhongshan Univ., institute of oncology of Zhongshan University)
<120>a kind of single stranded RNA of covalent annular closed and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 262
<212> RNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
gaaaaaaccg aggccccagc gggaaagaac aggccggaga gacaaaaagg gggcggccca 60
cagagaccaa ggaccgacgc caagaccagg aggaaagcca accagaagcc gggagccgcg 120
acccaacaga ggaaacaaaa accccgcagc gacagagcaa gaaaaacaag aacaaaccag 180
aaagacccca gacagcgaac aacacaaagc aacaggacaa cagcacaagc ggcacaggcc 240
caggaggacc cgccggacaa ac 262

Claims (9)

1. a kind of single stranded RNA of covalent annular closed, it is characterised in that；The single stranded RNA of the covalent annular closed is small molecule Non-coding is cyclic annular, and from the exon region of KIF4A gene, sequence is as shown in SEQ No: 1.
2. the application of the single stranded RNA of covalent annular closed described in claim 1, it is characterised in that；The covalent ring-type is closed The single stranded RNA of conjunction is applied to three negative breast cancer detection kits.
3. the covalently application of the single stranded RNA of annular closed as claimed in claim 2, it is characterised in that: described three negative breast cancer inspections Test agent box includes:
Trizol, chloroform, isopropanol, water, RT Buffer, magnesium chloride, triphosphoric acid base deoxynucleotide, RNA enzyme Inhibitor, enzyme, circKIF4A Specific PCR primers, β-actin Specific PCR primers, real-time quantitative PCR buffer, Taq are poly- Synthase, circular rna reverse transcriptase primer, β-actin specific reverse transcriptase primer；
The water is one or more of no enzyme water, DEPC water or distilled water composition, the enzyme be MMLV reverse transcriptase or AMV enzyme.
4. the covalently application of the single stranded RNA of annular closed as claimed in claim 3, which is characterized in that described three negative breast cancer 50 The detection kit of secondary response includes:
Isopropanol 100ml, chloroform 100ml, Trizol 50ml, DEPC water or without enzyme water 10ml, distilled water 10ml, 5 × it is inverse Transcription buffer 1ml, 25mM magnesium chloride 1ml, 10mM triphosphoric acid base deoxynucleotide 1ml, 500 μ of 5U/ μ l RNase inhibitor L, 50 μ l or 25U/ μ l AMV enzyme of 200U/ μ l MMLV reverse transcriptase, 50 μ l, 0.2 × real-time quantitative PCR buffer 2ml, 5U/ μ l 50 μ l of Taq polymerase, 5 μM of 50 μ l of circKIF4A Specific PCR primers, 5 μM of 30 μ l of β-actin Specific PCR primers, 10 μM 20 μ l of circular rna reverse transcriptase primer, 10 μM of 0 μ l of β-actin specific reverse transcriptase primer 2.
5. the application of the single stranded RNA of covalent annular closed described in claim 1, it is characterised in that；By the RNA CircKIF4A is applied to three negative breast cancer patients prognosis predictions.
6. the application of the single stranded RNA of covalent annular closed as described in any one of claim 3 or 4, it is characterised in that: described CircKIF4A Specific PCR primers are as follows:
Forward primer is 5 '-GAGGTACCCTGCCTGGATCT-3 '
Reverse primer is 5 '-TGGAATCTCTGTAGGGCACA-3 '；
β-actin the Specific PCR primers are as follows:
Forward primer is 5 '-CATGTACGTTGCTATCCAGGC-3 '
Reverse primer is 5 '-CTCCTTAATGTCACGCACGAT-3 '.
7. the application of the single stranded RNA of covalent annular closed as described in any one of claim 3 or 4, it is characterised in that: described Reverse transcription is carried out to circKIF4A and uses transcript reagent box and reverse transcription specific primer, respectively Pu Luomaige Promega is public The reverse transcription specific primer QPM010 of the Reverse Transcriptase kit A3500 of department and the production of Shanghai Ji Ma Bioisystech Co., Ltd.
8. the application of the single stranded RNA of covalent annular closed as described in claim 3 or 4, it is characterised in that: the 20 μ L reverse transcription The system of reaction is as follows:
Reverse transcription process: 16 DEG C 30 minutes, 42 DEG C 30 minutes, 85 DEG C 10 minutes.
9. the application of the single stranded RNA of covalent annular closed as described in claim 3 or 4, it is characterised in that: the covalent ring-type In the single stranded RNA specificity real-time quantitative PCR of closure, 20 μ L reaction systems are as follows:
The single stranded RNA real-time quantitative PCR response procedures of covalent annular closed: 95 DEG C 3 minutes, 40 circulations, 95 DEG C 12 seconds, 62 DEG C 35 seconds, real-time quantitative PCR amplification was carried out using SYBR-Green dyestuff.
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