CN114438207A - Cyclic RNA biomarker of breast cancer and application thereof - Google Patents

Cyclic RNA biomarker of breast cancer and application thereof Download PDF

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CN114438207A
CN114438207A CN202210030335.2A CN202210030335A CN114438207A CN 114438207 A CN114438207 A CN 114438207A CN 202210030335 A CN202210030335 A CN 202210030335A CN 114438207 A CN114438207 A CN 114438207A
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CN114438207B (en
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王婷
龙方懿
肖洪涛
张晞倩
林红
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Sichuan Cancer Hospital
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Abstract

The invention discloses a circular RNA biomarker circ-GLIS1 of breast cancer and application thereof, wherein the structure of circular RNA circ-GLIS1 is a circular structure formed by reverse splicing of a nucleotide sequence shown in SEQ No.1 and with the 3 'end and the 5' end connected end to end, and the kit comprises a reagent for quantitatively detecting the biomarker. The invention discovers that the expression level of circ-GLIS1 in breast tumor cells is obviously increased, and the proliferation and invasion of breast tumors can be obviously increased after the circ-GLIS1 is over-expressed; therefore, the circ-GLIS1 can be used as a molecular marker for diagnosing the breast cancer, so that the breast cancer can be diagnosed more accurately and quickly, and a new theoretical basis and a new marker are provided for the use of the circ-GLIS1 in the diagnosis and prognosis analysis of the breast cancer.

Description

Cyclic RNA biomarker of breast cancer and application thereof
Technical Field
The invention relates to the technical field of breast cancer diagnosis, in particular to application of a circular RNA circ-GLIS1 biomarker in preparation of a breast cancer diagnostic reagent.
Background
Breast cancer is the most common malignancy in women and is the leading cause of cancer death in women worldwide. At present, the incidence rate of breast cancer rises year by year, and the comprehensive treatment means (including operation, chemotherapy and radiotherapy) has larger individual difference and toxic and side effect, and has great influence on the life quality of patients. Therefore, the search for new breast cancer biomarkers for targeted therapy and personalized therapy is of great clinical significance.
Circular RNA (circular RNA) is novel non-coding RNA, has stable structure and obvious tissue and disease specificity, and is found by research to participate in the occurrence, development, invasion and metastasis of various tumors, so that the circular RNA is an ideal candidate tumor biomarker and an ideal tumor treatment target. In mechanism, the function of circRNA includes regulation of gene expression as a sponge of microrna (mirna) and binding to or translation into proteins with RNA Binding Proteins (RBPs). The CircRNA is rich in miRNA binding sites and plays a role of miRNA sponge in cells, so that the inhibition effect of miRNA on target genes of the CircRNA is relieved, and the expression level of the target genes is increased, and the action mechanism is called a competitive endogenous RNA (cepRNA) mechanism.
With the development of bioinformatics, research on the action mechanism of circRNA in the development of gastric cancer and lung cancer tumors has made great progress, but the action mechanism of circRNA in breast cancer still needs to be further explored. Currently, there is still a lack of specific circRNA biomarkers to efficiently detect breast cancer.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a rapid, sensitive and specific diagnostic indicator for breast cancer and a corresponding diagnostic kit.
In order to achieve the purpose, the technical scheme adopted by the patent comprises the following aspects:
in a first aspect, the present invention provides a circular RNA circ-GLIS1(hsa _ circ _0002079) having a structure in which the nucleotide sequence shown in SEQ No.1 is reverse spliced to form a circular structure with 3 'and 5' ends joined end to end. It should be noted that, the inventors of the present application found for the first time that the circular RNA circ-GLIS1 has the genomic location chr1:54059780-54065951, and the corresponding linear gene is GLIS1 (NM-147193.4); the circular RNA is formed by the circularization of 2 nd and 3 rd exons of the gene GLIS1 and is 1061 bases long; at present, the functional application of circ-GLIS1 in breast cancer is not reported.
In a second aspect, the invention provides a molecular marker of breast cancer, wherein the molecular marker is circular RNA circ-GLIS 1. The inventor of the application finds that the expression level of circ-GLIS1 is obviously up-regulated in breast cancer, and the over-expression of circ-GLIS1 can promote the proliferation and metastasis of the breast cancer, which indicates that circ-GLIS1 can be used as a novel molecular marker for breast cancer diagnosis.
In a third aspect, the invention provides the use of a detection reagent for detecting the content of circular RNA circ-GLIS1 in the preparation of a diagnostic kit for breast cancer, wherein the detection reagent is a primer pair capable of amplifying circ-GLIS1, and the base sequences of the primer pair are selected from the sequences shown in any two of SEQ Nos. 2-5.
Preferably, the primer pair comprises an upstream primer and a downstream primer, the base sequence of the upstream primer is selected from a sequence shown as SEQ No. 2-3, and the base sequence of the downstream primer is selected from a sequence shown as SEQ No. 4-5.
In a fourth aspect, the present invention provides a diagnostic kit for breast cancer, which comprises a primer pair for amplifying the circular RNA circ-GLIS1, wherein the structure of the circular RNA circ-GLIS1 is a circular structure with 3 'end and 5' end connected end to end formed by reverse splicing of the nucleotide sequence shown in SEQ No.1, and the base sequences of the primer pair are selected from the sequences shown in any two of SEQ Nos. 2 to 5.
Preferably, the primer pair comprises an upstream primer and a downstream primer, the base sequence of the upstream primer is selected from a sequence shown as SEQ No. 2-3, and the base sequence of the downstream primer is selected from a sequence shown as SEQ No. 4-5.
It should be noted that the diagnostic kit of the present invention includes other reagents required for PCR amplification, such as reverse transcriptase, RNA polymerase, buffer, etc., in addition to the primers capable of amplifying circ-GLIS 1.
In a fifth aspect, the present invention provides the use of circular RNA circ-GLIS1 in the preparation of a pharmaceutical composition against breast cancer. The structure of the circular RNA circ-GLIS1 is a circular structure which is formed by reverse splicing of a nucleotide sequence shown in SEQ No.1 and is connected end to end at the 3 'end and the 5' end. After the cyclic RNA is over-expressed, the proliferation of breast cancer cells can be obviously increased, and the invasion of the breast cancer cells and the growth of breast transplantation tumor can be promoted. The medicament aiming at the circular RNA target of the invention can inhibit the expression of circ-GLIS1 in breast cancer cells.
Preferably, the sequence of the target point aimed by the drug is circular RNA formed by reverse splicing of SEQ No.1, and the drug is oligonucleotide, an oligonucleotide expression vector or a small molecule compound.
In conclusion, the beneficial effects of the invention are as follows:
the invention discovers and confirms the function of circular RNA (circ-GLIS1) as a cancer promoting factor in breast cancer for the first time.
The invention discloses a key role of circ-GLIS1 in the occurrence and development of breast cancer, and lays a foundation for the diagnosis of breast cancer and the research and development of related target drugs. The discovery of the new target point provides powerful means for the accurate treatment of breast cancer patients and the screening and prevention of high risk groups, and has great social significance for improving the life quality of patients.
The circRNA disclosed by the invention has a stable structure and tissue specificity, and the kit for diagnosing and screening the breast cancer developed based on the discovered target point circ-GLIS1 is beneficial to improving the diagnosis efficiency of the breast cancer and establishing an early diagnosis, prevention and intervention system of the breast cancer, so that the family economic burden of a patient can be greatly reduced, and the utilization efficiency of medical resources can be improved.
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FIG. 1 is a structural map of circ-GLIS1 according to the present invention;
FIG. 2 is a graph of the results of a first generation sequencing; it identified the reverse splice site of circ-GLIS 1;
FIG. 3 is a graph showing the differential expression of circ-GLIS1 of the present invention in normal breast cell MCF-10A and breast tumor cell MCF-7 (n-3;. p < 0.01);
FIG. 4 is a structural map of a pCD5-ciR vector overexpressing circ-GLIS 1;
figure 5 shows verification by qPCR that MCF-7 cells overexpress circ-GLIS1 (n;. 3;. p < 0.01);
FIG. 6 shows the results of detecting overexpression of circ-GLIS1 on MCF-7 cell proliferation by flow cytometry;
FIG. 7 is a statistical plot of the results of the overexpression of circ-GLIS1 on MCF-7 cell proliferation in accordance with the present invention;
FIG. 8 shows the results of cell staining of the invasion assay overexpressing circ-GLIS1 and a control (control);
FIG. 9 is a statistical chart of the results of invasion test on MCF-7 cells by overexpression of circ-GLIS 1;
FIG. 10 is a graph showing the results of examining the expression of circ-GLIS1 in tumor tissues of nude mice overexpressing circ-GLIS1 and a control (control) group by the qPCR method;
FIG. 11 is a photograph of tumor tissues after nude mice overexpressing circ-GLIS1 group and control group (control) were inoculated with transplanted tumors;
FIG. 12 shows the growth of tumor tissue after nude mice were inoculated with transplantation tumor, with circ-GLIS1 levels in the transplantation tumor overexpressing circ-GLIS1 significantly higher than in the control group (control).
Detailed Description
The invention provides the application of circ-GLIS1 in the diagnosis of breast cancer according to the expression of circular RNA circ-GLIS1 in the breast cancer and the research on the regulation effect of the circular RNA circ-GLIS1 on the biological function of human breast cancer cells.
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
Example 1 preparation method and Structure verification of circular RNA circ-GLIS1
Circ-GLIS1(hsa _ Circ _0002079, chr1: 54059780-. The structural features and sequence information of Circ-GLIS1 are shown in FIG. 1 and SEQ No. 1.
Quantitative detection method of circular RNA circ-GLIS 1:
1. RNA extraction: extracting the total RNA of the breast tumor cell MCF-7 by adopting a total RNA extraction kit, and detecting the purity and the concentration of the total RNA by using a NanoDrop3000 micro nucleic acid determinator.
2. Reverse transcription: carrying out reverse transcription to obtain a cDNA template, wherein the reverse transcription reaction comprises the following steps: adding 500ng of template RNA, 1 mu L of random hexamer primer and RNase (RNase) removing water to the total volume of 12 mu L, mixing, and incubating at 70 ℃ for 5 minutes; then, 4. mu.L of 5 Xreaction buffer, 20U of RNase inhibitor, 2. mu.L of 10mM dNTP Mix, and 1. mu.L of reverse transcriptase were sequentially added. After mixing uniformly, reacting at 25 ℃ for 5min, then at 42 ℃ for 60min, and finally at 70 ℃ for 10min to obtain the cDNA template.
3. Recovering circular RNA circ-GLIS 1: specific divergent primers were designed for the reverse splice site of circ-GLIS1, with the following sequences:
Figure BDA0003466205080000061
polymerase Chain Reaction (PCR) was performed using the PCR amplification kit to amplify the reverse spliced fragment of circ-GLIS 1. The experimental steps are as follows: 10 XPCR Buffer 2. mu.L, 25mM MgCl21.2. mu.L, 1.6. mu.L dNTP mix, 500ng cDNA template, Primer-F1. mu. L, Primer-R1. mu.L, 12.1. mu.L enzyme-removed water and 0.1. mu.L amplification enzyme are mixed and amplified, the Primer-F adopted in the embodiment is SEQ No.3, the Primer-R is SEQ No.5, the PCR amplification process: firstly, denaturation is carried out for 5min at 95 ℃; 95 ℃ denaturation for 30s, 60 ℃ annealing for 30s, 72 ℃ extension for 1min, repeating the steps from (m) to (m) for 40 cycles, and finally 72 ℃ treatment for 10 min. And carrying out agarose gel electrophoresis on the amplification product, recovering the amplification product according to the size of the product fragment, and carrying out Sanger sequencing on the amplification product 1 by using a primer pair to verify the circular structure of the circ-GLIS 1. The sequencing results are shown in FIG. 2.
Example 2 detection of expression of circular RNA Circ-GLIS1 in Breast tumor cells and Normal cells of Breast
The expression condition of circ-GLIS1 in a breast tumor cell MCF-7 and a breast normal cell MCF-10A (purchased from cell banks of Chinese academy of sciences, Shanghai) is detected by adopting fluorescent quantitative PCR (qPCR), and the expression of circ-GLIS1 in the breast tumor cell is found to be remarkably higher than that of the normal cell, so that the circ-GLIS1 can be used as a cancer promotion factor and a potential treatment target of breast cancer.
Detection method of circular RNA circ-GLIS 1: total RNA of the breast tumor cell MCF-7 and normal breast cell MCF-10A is extracted by adopting a total RNA extraction kit, and the purity and concentration of the total RNA are detected by a NanoDrop3000 trace nucleic acid determinator. Then, reverse transcription is carried out by adopting the method to obtain a cDNA template for qPCR detection, and the experimental steps are as follows: 2 xqPCR Mix 10 uL, 500ng cDNA template, Primer-F1 u L, Primer-R1 uL and 7.4 uL of enzyme-removed water are mixed evenly and then qPCR reaction is carried out, the amplification process is as follows: firstly, pretreating for 2min at 50 ℃; 95 ℃ enzyme activation for 2min, 95 ℃ denaturation for 15s, 60 ℃ annealing/extension for 20s, and 40 cycles of third to fourth steps, and a dissolution curve. The detection results are shown in fig. 3, and the results show that: the expression of circular RNA circ-GLIS1 circ-GLIS1 in MCF-7 was approximately 3.11-fold higher than in MCF-10A, indicating that circ-GLIS1 was significantly upregulated in tumor cells.
Example 3 construction of the Circ-GLIS1 overexpression vector and its role in promoting proliferation and invasion of Breast cancer cells
The pCD5-ciR vector (Giscomb, Guangzhou, Biotechnology GmbH) overexpressing circ-GLIS1 was constructed to amplify a full-length 1061bp sequence of hsa _ circ _0002079 by PCR, and EcoRI and BamHI were ligated into pCD5-ciR by double digestion. The pCD5-ciR vector structure is shown in FIG. 4.
The effect of overexpression of circ-GLIS1 on the proliferation and invasion capacity of breast tumor cells was observed by transfecting the breast tumor cells with pCD5-ciR vector overexpressing circ-GLIS 1. The test steps are as follows: inoculating MCF-7 cells into a 12-hole plate, and transfecting when the MCF-7 cells grow to 70% of fusion; ② adding 1 μ g of circ-GLIS1 pCD5-ciR carrier and 2 μ L P3000 reagent into 50 μ L serum-free culture medium, and mixing uniformly; ③ adding 2 uL Lipofectamine 3000 into 50 uL serum-free culture medium; mixing the diluted overexpression vector with a Lipofectamine reagent, and standing at room temperature for 20min to form a complex; fifthly, 100 mu L of over-expression vector-Lipofectamine compound is added into a cell hole containing 900 mu L of serum-free culture medium, cells are cultured in an incubator containing 5% CO2 at 37 ℃ for 6h, then the transfection culture medium is sucked off, and the complete culture medium is replaced. After transfection for 48h, the overexpression condition of circ-GLIS1 was detected by the qPCR method, and the test results showed that compared with the pCD5-ciR blank vector control group, the overexpression fold of circ-GLIS1 pCD5-ciR group was about 1608 times, while the expression of the corresponding linear GLIS1 mRNA was not affected, which proved that the overexpression vector was successfully constructed and had better specificity, and the results are shown in FIG. 5.
MCF-7 cells were transfected with circ-GLIS1 pCD5-ciR overexpression vector using the above method, and a pCD5-ciR blank vector control group was used, followed by EdU cell proliferation assay, as follows: preparing a cell culture medium containing 10 mu M EdU, replacing the transfected cell culture medium with an EdU culture medium, digesting, centrifuging and collecting cells after incubating for 2h, fixing the cells for 30min by 1mL of 4% paraformaldehyde, neutralizing the cells for 5min by using 3mL of 2mg/mL glycine, washing the cells by PBS, adding 1mL of 0.5% Triton X-100 penetrant, incubating for 10min at room temperature, washing the cells by PBS, staining the cells for 10min by 200 mu L of Apollo staining reaction solution, centrifuging, collecting the cells, washing the cells for 3 times by using 0.5% Triton X-100 penetrant, and performing flow detection after resuspending the cells by 0.5mL of PBS. The results of the tests are shown in fig. 6 and 7, and show that: the number of proliferation cells of the EdU marker after overexpression of circ-GLIS1 was about 1.9 times that of the control group (control), suggesting that overexpression of circ-GLIS1 significantly increased the proliferation of MCF-7 cells.
The effect of over-expression of circ-GLIS1 on MCF-7 cell invasion was further examined. The test procedure was as follows: precooling the Transwell plate, spreading the gel by adopting a thin gel gelling method, and using a precooled liquid-transfer sucker to transfer the Matrigel matrix to 50 mu L/cm2Adding into the upper side of the membrane, standing at 37 deg.C for 30min to solidify; after MCF-7 cells are digested and centrifuged, the cells are blown off by DMEM medium containing 1% serum, the cells are inoculated on the upper side of the membrane, and the medium containing 10% serum is added on the lower side of the membrane; placing the Transwell plate into a cell culture box for further culture for 12h, taking out the chamber, wiping off residual cells and matrix glue on the upper side of the membrane by using a cotton ball, soaking the lower surface of the Transwell in 4% paraformaldehyde solution after washing by using PBS, and fixing for 30min; stained with a crystal violet solution, photographed using microscopic observation, and the number of cells invading the lower side of the membrane was counted. The test results show that the number of the cells which are invaded after the circ-GLIS1 is over-expressed is about 5.3 times of that of a control group (control), and the over-expression of the circ-GLIS1 can promote the invasion of MCF-7 cells, and the test results are shown in figures 8 and 9.
Example 4 Circ-GLIS1 promotes growth of nude mouse breast cancer transplantable tumors
The test was carried out using BALB/c female nude mice of 4-6 weeks of age. Pancreatin digestion of MCF-7 cells transfected with circ-GLIS1 overexpression or blank vector, preparation of cell suspension with culture medium, and adjustment of cell concentration to 5 × 106Each cell was injected at 100. mu.L/mL under the second breast pad of the right chest wall of nude mice, and 6 nude mice were inoculated with each cell, and the growth of tumor was measured. Measuring the longest diameter and the shortest diameter of the transplanted tumor by using a vernier caliper according to the formula: volume is longest diameter x shortest diameter2The tumor volume was calculated 2. Nude mice were sacrificed at week 3 of the experiment, tumor size was measured and volume was calculated, and total RNA extracted from tumor tissue to verify circ-GLIS1 expression. As shown in FIGS. 10 to 12, it was found that the level of circ-GLIS1 in the transplanted tumor overexpressing circ-GLIS1 was significantly higher than that of the control group (control), and the expression amount was about 40.8 times that of the control group; and overexpression of circ-GLIS1 promoted the growth of transplanted tumors, with a tumor formation volume of about 4.1 times that of control after three weeks.
The above-mentioned embodiments only express the specific embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
SEQ ID NO.1
TGAATGGGAGCTACGGACACCGTACCCCGGGCTCAGAGAAGAGCCTGCTGGACCTGGACCTTGCTGAGGGCCCTGGCCCCACCTGCTGCCAGGGCCTGTTTCTCCCTGCAGGAAGCCCACCGCCCCGGGCTCACCCCCAAGCTTGTGAGAGGCTGCTGCATTTCCCCCACCCTGACAGGTCACCTAGACCCCAGGCCACGTATGTGAACGGCAGCCTCCCAACCACACAACACATCAAACAGGAGTCCTTGCCCGACTACCAAGCCATGGCAGAGGCCCGCACATCCCTGTCTGCCCACTGTCGGGGCCCGCTGGCCACTGGCCTGCACCCAGACCTGGACCTCCCGGGCCGAAGCCTCGCCACCCCTGCGCCTTCCTGCTACCTTCTGGGCAGCGAACCCAGCTCTGGCCTGGGCCTCCAGCCCGAGACCCACCTCCCCGAGGGCAGCCTGAAGCGGTGCTGCGTCTTGGGCCTACCCCCCACCTCCCCAGCCTCCTCCTCACCCTGTGCCTCCTCCGACGTCACCTCCATCATCCGCTCCTCCCAGACGTCTCTGGTCACCTGTGTAAATGGACTCCGGAGCCCCCCTCTGACGGGAGATCTGGGGGGCCCTTCCAAGCGGGCCCGGCCTGGCCCTGC
ATCGACGGACAGCCATGAGGGCAGCTTGCAACTTGAAGCCTGCCGGAAGGCGAGCTTCCTGAAGCAGGAACCCGCGGATGAGTTTTCAGAGCTCTTTGGGCCTCACCAGCAGGGCCTGCCGCCCCCCTATCCCCTGTCTCAGTTGCCGCCTGGCCCAAGCCTTGGAGGCCTGGGGCTGGGCCTGGCAGGCAGGGTGGTGGCCGGGCGGCAGGCGTGCCGCTGGGTGGACTGCTGTGCAGCCTATGAGCAGCAGGAGGAGCTGGTGCGGCACATCGAGAAGAGCCACATCGACCAGCGCAAGGGCGAGGACTTCACCTGCTTCTGGGCTGGCTGCGTGCGCCGCTACAAGCCCTTCAACGCCCGCTACAAGCTGCTCATCCACATGCGAGTGCACTCGGGCGAGAAGCCCAACAAGTGCATG
SEQ ID NO.2
GAGGACTTCACCTGCTTCTG
SEQ ID NO.3
GCCCAACAAGTGCATGTGAA
SEQ ID NO.4
TACGGTGTCCGTAGCTCCCA
SEQ ID NO.5
ATGCAGCAGCCTCTCACAAG

Claims (5)

1. The application of the detection reagent for the content of circular RNA circ-GLIS1 in the preparation of a diagnostic kit for breast cancer, wherein the structure of circular RNA circ-GLIS1 is a circular structure formed by reverse splicing of a nucleotide sequence shown in SEQ No.1 and connected end to end at the 3 'end and the 5' end; the detection reagent comprises a primer pair capable of amplifying the circular RNA circ-GLIS1, wherein the base sequences of the primer pair are selected from the sequences shown as any two of SEQ Nos. 2-5.
2. A diagnostic kit for breast cancer, which comprises a primer pair capable of amplifying the circular RNA circ-GLIS1, wherein the structure of the circular RNA circ-GLIS1 is a circular structure formed by reverse splicing of a nucleotide sequence shown in SEQ No.1 and the 3 'end and the 5' end are connected end to end, and the base sequences of the primer pair are selected from any two sequences shown in SEQ Nos. 2 to 5.
3. The use of the reagent for detecting the content of circular RNA circ-GLIS1 according to claim 1 in the preparation of a diagnostic kit for breast cancer or the diagnostic kit for breast cancer according to claim 2, wherein the primer pair comprises an upstream primer and a downstream primer, the base sequence of the upstream primer is selected from the sequences shown in any one of SEQ Nos. 2-3, and the base sequence of the downstream primer is selected from the sequences shown in any one of SEQ Nos. 4-5.
4. Use of cyclic RNA circ-GLIS1 in the manufacture of a pharmaceutical composition against breast cancer, wherein the use of cyclic RNA circ-GLIS1 in the manufacture of a medicament for inhibiting proliferation and migration of breast cancer cells or treating breast cancer; the structure of the circular RNA circ-GLIS1 is a circular structure which is formed by reverse splicing of a nucleotide sequence shown in SEQ No.1 and is connected end to end at the 3 'end and the 5' end.
5. Use of the circular RNA circ-GLIS1 in the preparation of a pharmaceutical composition against breast cancer according to claim 4, wherein the target of the drug is circular RNA circ-GLIS1, and the drug is an oligonucleotide, an oligonucleotide expression vector or a small molecule compound.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110229900A (en) * 2019-06-21 2019-09-13 苏州吉玛基因股份有限公司 Gene hsa_circ_0103520 relevant to breast cancer diagnosis and treatment and its application
CN110257522A (en) * 2019-07-17 2019-09-20 苏州吉玛基因股份有限公司 Gene hsa_circ_0045881 relevant to breast cancer diagnosis and treatment and its application

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110229900A (en) * 2019-06-21 2019-09-13 苏州吉玛基因股份有限公司 Gene hsa_circ_0103520 relevant to breast cancer diagnosis and treatment and its application
CN110257522A (en) * 2019-07-17 2019-09-20 苏州吉玛基因股份有限公司 Gene hsa_circ_0045881 relevant to breast cancer diagnosis and treatment and its application

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