CN109295059B - Application of covalent circular closed single-stranded RNA in triple-negative breast cancer kit - Google Patents
Application of covalent circular closed single-stranded RNA in triple-negative breast cancer kit Download PDFInfo
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Abstract
The invention relates to the technical field of genes, and particularly discloses a covalent annular closed single-stranded RNA and application thereof; the method comprises the steps of firstly collecting a suspected triple-negative breast cancer tissue specimen to be detected, extracting total RNA, specifically and reversely transcribing the single-stranded RNA (hsa _ circRNA _007255) with the covalent annular closure into cDNA by taking the total RNA as a template, then carrying out real-time quantitative PCR amplification by using a specific PCR primer, obtaining a relative quantitative delta CT value of the single-stranded RNA with the covalent annular closure by taking beta-actin as an internal reference gene, and prompting the positive expression of the single-stranded RNA with the covalent annular closure when the delta CT is less than or equal to 11.27. The invention is used for the application of prognosis prediction of a three-negative breast cancer patient by relatively quantitatively detecting the expression condition of the bivalent circularly closed single-stranded RNA in a suspected three-negative breast cancer tissue specimen.
Description
Technical Field
The invention relates to the technical field of breast cancer diagnosis, in particular to a covalent circular closed single-stranded RNA and application thereof in triple-negative breast cancer.
Background
Breast cancer is the highest incidence of female malignancies worldwide. As a big population country and a unique aging social form, China has higher growth rate of the incidence rate of breast cancer than other countries and is not optimistic in situation. Breast cancer has not been controlled sufficiently effectively to date, and still exhibits a high mortality rate, mainly due to recurrence and metastasis, and once metastasis occurs, the average survival rate is only about 4-5 years. Triple negative breast cancer refers to breast cancer with negative expression of Estrogen Receptor (ER), Progestogen Receptor (PR) and human epidermal growth factor receptor-2 (HER-2), and accounts for 15% -20% of the whole breast cancer population. The three-negative breast cancer is characterized by strong invasiveness, higher histological grade, poor prognosis, easy occurrence of visceral metastasis, lack of corresponding targets of endocrine therapy and HER-2-directed molecular targeted therapy, and limited selection of treatment means compared with other molecular subtype breast cancers.
Disclosure of Invention
In order to solve the above problems, the present invention aims to provide an application of a covalently circular closed single-stranded RNA in a triple-negative breast cancer kit.
The invention is realized by the following technical scheme:
a covalently closed single-stranded RNA, which is a small non-coding circular RNA derived from an exon region of KIF4A gene and has a sequence shown in SEQ No: 1 is shown.
The single-stranded RNA with covalently closed circular structure (RNA circKIF4A) is one of the endogenous non-coding RNAs. Has the characteristics of stable structure, high abundance, tissue-specific expression and the like. It functions as a competitive endogenous rna (cerna) to regulate gene expression. The circular RNA utilizes a microRNA (miRNA) response element thereof to combine with miRNA so as to block the inhibition effect of miRNA on the target expression, thereby regulating the expression level of other related RNAs. The single-stranded RNA (RNA circKIF4A) with covalent circular closure has the same main biological function as protein coding genes, is a novel tool for judging prognosis markers and targeting therapy of tumors, particularly has high expression in triple negative breast cancer, and provides a novel strategy for overcoming triple negative breast cancer for human beings.
Preferably, the reverse transcription of circKIF4A uses a transcription kit and a reverse transcription specific primer, namely reverse transcription kit a3500 manufactured by Promega and reverse transcription specific primer QPM010 manufactured by jima biotechnology limited, shanghai, respectively.
The system for 20. mu.L reverse transcription reaction was as follows:
reverse transcription procedure: 30 minutes at 16 ℃, 30 minutes at 42 ℃ and 10 minutes at 85 ℃.
(3) CircKIF 4A-specific real-time quantitative PCR
Firstly, diluting reverse transcription products of circKIF4A to 2 times respectively, and then mixing uniformly;
in the circKIF4A specific real-time quantitative PCR, 20 μ L reaction system was as follows:
circKIF4A real-time quantitative PCR reaction program: real-time quantitative PCR amplification was performed using SYBR-Green dye at 95 ℃ for 3 min, 40 cycles, 95 ℃ for 12 sec, 62 ℃ for 35 sec.
The circKIF4A specific PCR primers are as follows:
the forward primer is 5'-GAGGTACCCTGCCTGGATCT-3'
The reverse primer is 5'-TGGAATCTCTGTAGGGCACA-3';
the beta-actin specific PCR primer is as follows:
the forward primer is 5'-CATGTACGTTGCTATCCAGGC-3'
The reverse primer was 5'-CTCCTTAATGTCACGCACGAT-3'.
(4) Determination of the Delta CT index
When the delta CT is less than or equal to 11.27, the circKIF4A expresses positively, and the tissue can be diagnosed as triple-negative breast cancer tissue; the Δ CT is the difference between circKIF4A and the mean CT value of β -actin.
The application of the RNA circKIF4A is characterized in that the RNA circKIF4A is applied to a triple-negative breast cancer detection kit. The detection kit comprises:
isopropanol, chloroform, Trizol, DEPC water or enzyme-free water, double distilled water, 5 XTR buffer, 25mM magnesium chloride, 10mM deoxynucleotide triphosphate base, 5U/. mu.l RNase inhibitor, 200U/. mu.l MMLV reverse transcriptase or 25U/. mu.l AMV enzyme, 2 XTR buffer, 5U/. mu.l Taq polymerase, 5. mu.M circKIF4A specific PCR primers, 5. mu.M beta. -actin specific PCR primers, 10. mu.M circular RNA reverse transcription primers, 10. mu.M beta. -actin specific reverse transcription primers.
The covalent circular closed single-stranded RNA (RNA circKIF4A) is used for diagnosing triple-negative breast cancer, and the specific method is as follows:
(1) extraction of tissue RNA
A, putting a tissue specimen into a mortar, adding liquid nitrogen for grinding, grinding into homogenate, adding Trizol and chloroform, shaking uniformly, and placing on ice;
b, centrifuging the mixed solution obtained in the step A at a low temperature, taking the upper-layer water phase, adding pre-cooled isopropanol, mixing uniformly, standing in a refrigerator, and centrifuging at a low temperature; discarding the supernatant, adding ethanol diluted by 75% DEPC water, mixing uniformly, and centrifuging at low temperature; discarding the supernatant, and drying at room temperature to obtain RNA;
c, adding DEPC water into the RNA obtained in the step B for dissolving, measuring the concentration and the quality of the RNA by using a spectrophotometer, detecting the quality of the RNA by using EB gel electrophoresis with the OD260/280 ratio of 1.6-1.8 to obtain the tissue RNA, and storing at-70 ℃;
(2) specific reverse transcription
Carrying out reverse transcription on circKIF4A by using a transcription kit and a reverse transcription specific primer, mixing various reagents except reverse transcriptase into a reverse transcription mixed solution before carrying out reverse transcription reaction, flicking a tube for loading the reagents by fingers, and sucking the reverse transcription mixed solution by a pipettor for several times without using an oscillator;
(3) CircKIF 4A-specific real-time quantitative PCR
Firstly, diluting reverse transcription products of circKIF4A to 2 times respectively, and then mixing uniformly;
(4) determination of the Delta CT index
When the delta CT is less than or equal to 11.27, the circKIF4A is expressed positively, and the tissue can be diagnosed as triple-negative breast cancer tissue; the Δ CT is the difference between circKIF4A and the mean CT value of β -actin.
The application of RNA circKIF4A, wherein the RNA circKIF4A is applied to prognosis prediction of triple negative breast cancer patients.
The single-stranded RNA (RNA circKIF4A) covalently closed in a circular manner in the invention, namely hsa _ circRNA _007255 (chrX: 69549254-. The real-time fluorescent quantitative PCR technology is one of the detection methods for detecting the tissue circular RNA expression authority at present. The applicant firstly detected the expression in 57 cases of triple negative breast cancer tissues and the paired normal breast tissues thereof by a real-time fluorescent quantitative PCR technology, and found that circKIF4A shows high expression in the triple negative breast cancer tissues (FIG. 2). The high expression of circKIF4A in triple negative breast cancer tissues is statistically significant (P < 0.01).
To better determine the expression of circKIF4A in triple negative breast cancer tissues, further analysis of the effect of circKIF4A on the prognosis of triple negative breast cancer patients, the applicant tested the expression level of circKIF4A in the tumors of 240 triple negative breast cancer patients again by in situ hybridization. Follow-up data showed that 27 out of 100 triple negative breast cancer patients with high expression of circKIF4A died, while only 11 out of 140 triple negative breast cancer patients with low expression of circKIF4A died; the Kaplan-Meier statistical analysis shows that the overall survival rate and disease-free survival rate of the circKIF4A high-expression triple-negative breast cancer patient are lower than those of the circKIF4A low-expression patient, the difference is statistically significant (P is less than 0.01) (fig. 3 and fig. 4), and the fact that the circKIF4A is high-expression in triple-negative breast cancer is suggested to be a molecular marker for predicting the prognosis of triple-negative breast cancer. The invention provides powerful technical support and molecular biology basis for prognosis prediction of triple negative breast cancer, and has profound clinical significance and popularization.
In previous work, the applicant screened triple negative breast cancer-associated circular RNA differential expression profiles in 3 pairs of triple negative breast cancer tissues and paired paracancerous normal tissues using a circular RNA high throughput chip (Array star Human circRNA Array V2). 258 triple negative breast cancer-associated circular RNAs were found according to the criteria of fold difference greater than 2 fold and P < 0.05, with 84 upregulated circular RNAs and 174 downregulated circular RNAs. Subsequently, 5 upregulated circular RNAs were picked for qRT-PCR validation in 51 pairs of triple negative breast cancer tissues and paired paracancerous normal tissues. The results show that RNA hsa _ circRNA _007255(circKIF4A) is most highly expressed in triple negative breast cancer tissue and significantly higher than corresponding paracarcinoma tissue. Consistent with the chip results.
Through further organization studies, the inventors found that: circKIF4A showed high expression in triple negative breast cancer tissues (fig. 2). The overall survival of circKIF 4A-highly expressed triple negative breast cancer patients was lower than that of circKIF 4A-less expressed patients (fig. 3). This suggests that circKIF4A is a molecular marker for predicting prognosis of triple negative breast cancer.
Research proves that circKIF4A is highly expressed in triple negative breast cancer, can be applied to prognosis prediction of triple negative breast cancer patients, is applied to the field of medicine, and provides a triple negative breast cancer diagnosis kit which is high in cost performance and easy to popularize and apply.
Drawings
FIG. 1 schematic representation of circKIF4A
FIG. 2 real-time fluorescent quantitative PCR assay for changes in circKIF4A expression in triple negative breast cancer tissue
FIG. 3 correlation of circKIF4A with prognosis of triple negative breast cancer patients
FIG. 4 relationship between circKIF4A and disease-free survival rate in triple negative breast cancer patients.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments to assist those skilled in the art in understanding the present invention.
Composition of triple negative breast cancer tissue diagnosis kit (50 times reaction)
100ml of isopropanol, 100ml of chloroform, 50ml of Trizol, 10ml of DEPC water or enzyme-free water, 10ml of double distilled water, 1ml of 5 Xreverse transcription buffer, 1ml of 25mM magnesium chloride, 1ml of 10mM deoxynucleotide triphosphate, 500. mu.l of 5U/. mu.l RNase inhibitor, 50. mu.l or 50. mu.l of 25U/. mu.l AMV enzyme of 200U/. mu.l MMLV reverse transcriptase, 2ml of 2 Xreal-time PCR buffer, 50. mu.l of 5U/. mu.l Taq polymerase,
mu.l of 5. mu.M circKIF 4A-specific PCR primers
The forward primer is 5'-GAGGTACCCTGCCTGGATCT-3'
The reverse primer is 5'-TGGAATCTCTGTAGGGCACA-3'
5 μ M beta-actin specific PCR primers 30 μ l
The forward primer is 5'-CATGTACGTTGCTATCCAGGC-3'
The reverse primer is 5'-CTCCTTAATGTCACGCACGAT-3'
Mu.l of 10. mu.M circular RNA reverse transcription primer (Shanghai Jima Biotechnology Co., Ltd., QPM 010).
Mu.l of 10. mu.M beta-actin specific reverse transcription primer (Kshima Biotechnology Co., Ltd., QPM 010).
Detection of circKIF4A in tissue samples
1. Extraction of tissue RNA
Taking a tissue specimen and adding liquid nitrogen into a mortar to grind the specimen; adding 0.6ml of Trizol mortar specimen into a mortar, grinding into homogenate, and adding the homogenate into a tube by using a spoon; 0.4ml Trizol was added to the tube; adding chloroform 200 μ l/ml Trizol into Tube, shaking by hand for 15-30s, standing on ice for 5min, and centrifuging at 4 deg.C 12000g for 15 min; carefully taking the upper water phase into a new tube, adding 0.5ml/ml of precooled isopropanol into the new tube, uniformly mixing, standing the mixture in a refrigerator at the temperature of-20 ℃ for 20min, and centrifuging the mixture at the temperature of 4 ℃ for 10min at 12000 g; discarding the supernatant, adding 1-2ml ethanol diluted with 75% DEPC water, mixing, centrifuging at 4 deg.C 7500g for 5min, discarding the supernatant, drying at room temperature for 5-10min, and adding 10-20 μ l DEPC water to dissolve RNA. The concentration and the quality of RNA are measured by spectrophotometry, the OD260/280 ratio is between 1.6 and 1.8, and the RNA quality is detected by EB gel electrophoresis and stored at the temperature of-70 ℃.
2. Specific reverse transcription: reverse transcription of circKIF4A was performed using a reverse transcription kit (a3500) from Promega, and a reverse transcription specific primer (QPM010) produced by jima biotechnology, shanghai. The system for 20. mu.L reverse transcription reaction was as follows:
before the reverse transcription reaction is performed, various reagents other than the reverse transcriptase are mixed into a reverse transcription mixture, a tube containing the reagents is flicked with a finger, and the reverse transcription mixture is pipetted several times, but an oscillator cannot be used.
Reverse transcription procedure: 30 minutes at 16 ℃, 30 minutes at 42 ℃ and 10 minutes at 85 ℃.
3. circKIF 4A-specific real-time quantitative PCR: the reverse transcription products of circKIF4A were diluted to 2 times, respectively, and then mixed well. The 20 μ L reaction was as follows:
circKIF4A real-time quantitative PCR reaction program: 95 ℃ for 3 minutes, 40 cycles, 95 ℃ for 12 seconds, 62 ℃ for 35 seconds.
Real-time quantitative PCR amplification was performed using SYBR-Green dye.
PCR specific primers for circKIF4A were:
the forward primer is 5'-GAGGTACCCTGCCTGGATCT-3'
The reverse primer is 5'-TGGAATCTCTGTAGGGCACA-3'
Beta-actin is used as an internal reference gene, and the PCR primer sequence is as follows:
the forward primer is 5'-CATGTACGTTGCTATCCAGGC-3'
The reverse primer is 5'-CTCCTTAATGTCACGCACGAT-3'
(4)ΔCTMeasurement of the index: delta CTThe difference value of the average Ct value of circKIF4A to be detected and the internal reference beta-actin in the same sample is referred to. Namely circKIF4A Δ CT＝circKIF4A MeanCT-Control MeanCTIn the present invention,. DELTA.CTIs cAverage C of ircKIF4A with beta-actinTThe difference of the values is used to obtain a relative quantitative Delta CT value, and the judgment is carried out, the result shows that the Delta C is the difference between the tested 57 cases of triple negative breast cancer tissuesTWhen the expression is less than or equal to 11.27, 35 cases of circKIF4A positive expression exist, and the positive rate is 61.40%.
Prognostic relation between covalently circular closed single-stranded RNA (RNA circKIF4A) and triple negative breast cancer patient
The applicant tested the expression of circKIF4A in 240 triple negative breast cancer tissues by in situ hybridization, followed by prognostic follow-up of each of the tested triple negative breast cancer patients, which revealed that the overall survival rate of the circKIF4A highly expressed triple negative breast cancer patients was lower than that of the circKIF 4A-less expressed patients (fig. 3), and the disease-free survival rate of the circKIF4A highly expressed triple negative breast cancer patients was lower than that of the circKIF 4A-less expressed patients (fig. 4). This suggests that circKIF4A is a molecular marker for predicting prognosis of triple negative breast cancer.
The research shows that the test of the expression of circKIF4A in the triple negative breast cancer tissue has good stability. The circKIF4A can be used as a specific molecular marker for prognosis prediction of triple-negative breast cancer patients, and the circKIF4A is obviously related to the prognosis of triple-negative breast cancer and can be used for application in prognosis prediction of triple-negative breast cancer patients.
The above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, so that equivalent changes or modifications made by the features and principles of the present invention as described in the claims should be included in the scope of the present invention.
Serial number
The invention name is as follows: application of covalent circular closed single-stranded RNA in triple-negative breast cancer kit
The applicant: center for preventing and treating tumors of Zhongshan university
Covalently circular closed single-stranded RNA sequences
Sequence listing
<110> Zhongshan university tumor prevention and treatment center (Zhongshan university affiliated tumor hospital, Zhongshan university tumor research institute)
<120> single-stranded RNA with covalent circular closure and application thereof
<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 (7)
1. The application of a reagent for detecting the expression level of covalent circular closed single-stranded RNA in preparing a kit for predicting prognosis of triple negative breast cancer is characterized in that; the covalent circular closed single-stranded RNA is a micromolecule non-coding circular shape and is derived from an exon region of a KIF4A gene, and the covalent circular closed single-stranded RNA is highly expressed in triple-negative breast cancer tissues;
the covalently circularly closed single-stranded RNA sequence is as follows:
GTATTAATATTAACCGAGGCCTCCTATGCTTGGGAAATGTAATCAGTGCTCTTGGAGATGACAAAAAGGGTGGCTTTGTGCCCTACAGAGATTCCAAGTTGACTCGACTGCTTCAAGATTCTCTAGGAGGTAATAGCCATACTCTTATGATAGCCTGTGTGAGTCCTGCTGACTCCAATCTAGAGGAAACATTAAATACCCTTCGCTATGCTGACAGAGCAAGAAAAATCAAGAACAAACCTATTGTTAATATTGATCCCCAGACAGCTGAACTTAATCATCTAAAGCAACAGGTACAACAGCTACAAGTCTTGTTGCTACAGGCCCATGGAGGTACCCTGCCTGGATCTATAAC。
2. use of the reagent for detecting the expression level of the covalent circular closed single-stranded RNA according to claim 1 in the preparation of a kit for prognosis of triple negative breast cancer, wherein: the kit comprises:
trizol, trichloromethane, isopropanol, water, a reverse transcription buffer solution, magnesium chloride, base triphosphate deoxynucleotide, an RNase inhibitor, an enzyme, a circKIF4A specific PCR primer, a beta-actin specific PCR primer, a real-time quantitative PCR buffer solution, Taq polymerase, a circular RNA reverse transcription primer and a beta-actin specific reverse transcription primer;
the water is one or a combination of several of enzyme-free water, DEPC water or double distilled water, and the enzyme is MMLV reverse transcriptase or AMV enzyme.
3. The use of the reagent for detecting the expression level of the covalent circular closed single-stranded RNA according to claim 2 in the preparation of a kit for prognosis prediction of triple negative breast cancer, wherein the kit comprises:
100ml of isopropanol, 100ml of chloroform, 50ml of Trizol, 10ml of DEPC water or enzyme-free water, 10ml of double distilled water, 1ml of 5 Xreverse transcription buffer, 1ml of 25mM magnesium chloride, 1ml of 10mM deoxynucleotide triphosphate, 500. mu.l of 5U/. mu.l RNase inhibitor, 50. mu.l or 25U/. mu.l AMV enzyme of 200U/. mu.l MMLV reverse transcriptase, 2ml of 0.2 Xreal-time quantitative PCR buffer, 50. mu.l of 5U/. mu.l Taq polymerase, 50. mu.l of 5. mu.M circKIF 4A-specific PCR primer, 30. mu.l of 5. mu.M beta. -action-specific PCR primer, 20. mu.l of 10. mu.M circular RNA reverse transcription primer, and 20. mu.l of 10. mu.M beta. -action-specific reverse transcription primer.
4. Use of a reagent for detecting the expression level of a covalently closed circular single-stranded RNA according to any one of claims 2 or 3 in the preparation of a kit for prognosis of triple negative breast cancer, wherein: the circKIF4A specific PCR primers are as follows:
the forward primer is 5'-GAGGTACCCTGCCTGGATCT-3'
The reverse primer is 5'-TGGAATCTCTGTAGGGCACA-3';
the beta-actin specific PCR primer is as follows:
the forward primer is 5'-CATGTACGTTGCTATCCAGGC-3'
The reverse primer was 5'-CTCCTTAATGTCACGCACGAT-3'.
5. Use of a reagent for detecting the expression level of a covalently closed circular single-stranded RNA as claimed in claim 2 or 3 for the preparation of a kit for prognosis of triple negative breast cancer, wherein: reverse transcription was performed with circKIF4A using a transcription kit and reverse transcription specific primers.
6. Use of the reagent for detecting the expression level of the covalent circular closed single-stranded RNA according to claim 2 or 3 in the preparation of a kit for predicting prognosis of triple negative breast cancer, wherein: the system for 20. mu.L reverse transcription reaction was as follows:
reverse transcription procedure: 30 minutes at 16 ℃, 30 minutes at 42 ℃ and 10 minutes at 85 ℃.
7. Use of a reagent for detecting the expression level of a covalently closed circular single-stranded RNA as claimed in claim 2 or 3 for the preparation of a kit for prognosis of triple negative breast cancer, wherein: in the covalent circular closed single-stranded RNA specific real-time quantitative PCR, a 20 mu L reaction system is as follows:
covalent circular closed single-stranded RNA real-time quantitative PCR reaction program: real-time quantitative PCR amplification was performed using SYBR-Green dye at 95 ℃ for 3 min, 40 cycles, 95 ℃ for 12 sec, 62 ℃ for 35 sec.
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| CN107254519A (en) * | 2017-06-06 | 2017-10-17 | 重庆市肿瘤研究所 | The screening technique of triple negative breast cancer specificity circular rna |
| CN107475258A (en) * | 2017-08-22 | 2017-12-15 | 中山大学肿瘤防治中心 | A kind of RNAcircEPSTI1 and its application in three cloudy breast cancer |
| CN108220433A (en) * | 2017-05-16 | 2018-06-29 | 深圳市晋百慧生物有限公司 | Markers for breast cancer and its application |
| CN108251424A (en) * | 2017-12-19 | 2018-07-06 | 天利康（天津）科技有限公司 | A kind of single stranded circle RNA and DNA and its preparation method and application |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108220433A (en) * | 2017-05-16 | 2018-06-29 | 深圳市晋百慧生物有限公司 | Markers for breast cancer and its application |
| CN107254519A (en) * | 2017-06-06 | 2017-10-17 | 重庆市肿瘤研究所 | The screening technique of triple negative breast cancer specificity circular rna |
| CN107475258A (en) * | 2017-08-22 | 2017-12-15 | 中山大学肿瘤防治中心 | A kind of RNAcircEPSTI1 and its application in three cloudy breast cancer |
| CN108251424A (en) * | 2017-12-19 | 2018-07-06 | 天利康（天津）科技有限公司 | A kind of single stranded circle RNA and DNA and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| Cell-Type Specific Features of Circular RNA Expression;Julia Salzman et al.;《PLOS GENETICS》;20130905;第9卷(第9期);第1页右栏第2-3段，第2页左栏第1段，第4页左栏第3段，图1，表S2、表S3 * |
| 乳腺癌中KIF4A基因表达的临床意义及基因富集分析;王亚男 等;《医学研究杂志》;20180831;第47卷(第8期);第74页右栏第2段，表2、图2 * |
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