CN117398466A - Application of circRNA-FOXK2 gene as target in preparation or screening of breast cancer therapeutic drugs - Google Patents
Application of circRNA-FOXK2 gene as target in preparation or screening of breast cancer therapeutic drugs Download PDFInfo
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Abstract
The invention provides application of a circRNA-FOXK2 gene serving as a target in preparation or screening of a breast cancer therapeutic drug. The invention provides a novel breast cancer marker circRNA-FOXK2 gene, wherein the expression level of the circRNA-FOXK2 gene in breast cancer can be related to the occurrence, development and prognosis of the disease, and the specificity and the sensitivity are higher; based on the breast cancer marker, a novel breast cancer treatment target is provided, a treatment strategy taking the circRNA-FOXK2 gene as the target is developed, and a novel thought and direction are provided for the treatment of breast cancer.
Description
Technical Field
The invention belongs to the field of biotechnology and medicine, and particularly relates to application of a circRNA-FOXK2 gene serving as a target in preparation or screening of a breast cancer therapeutic drug, in particular to a circRNAFOXK2 (hsa_circ_ 0000816) nucleic acid fragment and correlation thereof in growth, proliferation or migration of breast cancer tumors and application thereof in treatment.
Background
According to 2020, global cancer report indicates that the new incidence rate of breast cancer exceeds lung cancer, and the new incidence rate of breast cancer becomes the most common malignant tumor, and the death rate of breast cancer is increased to the 5 th position. At present, the clinic has remarkable improvement on the operation, radiotherapy, chemotherapy and combined treatment methods of breast cancer patients, but the death number of the breast cancer patients is about 230 ten thousand per year, and the death number of the breast cancer patients is in an ascending trend. Despite the tremendous advances made in current research into methods of treating breast cancer, there remains a strong need for a more thorough understanding of the molecular mechanisms of breast cancer occurrence and progression, and for the development of effective and efficacious treatments that effectively reduce the occurrence of death.
Circular RNAs (circRNAs) are a class of covalently closed single-stranded circular RNA molecules formed by reverse splicing without a 5 'terminal cap and a 3' terminal poly (a) tail. The CircRNAs are not degraded by external nucleic acid degrading enzymes, and can be well and stably expressed. Most circular RNAs are under-expressed in cells or tissues, some are expressed higher than their parent linear RNAs, and some are tissue-specific. More and more researches report that a large amount of circular RNA is found in various cancers, and the circular RNA plays an important role in inhibiting tumor progression or promoting tumorigenesis by influencing various cellular malignant processes such as cell proliferation, apoptosis, cell cycle, migration, invasion, drug resistance and the like, and is expected to become a new target of tumor treatment and drugs. Therefore, the research on the function and mechanism of the circular RNA has important scientific significance and clinical value.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide the application of the circRNA-FOXK2 gene serving as a target in the preparation or screening of breast cancer therapeutic drugs. The invention provides a novel breast cancer marker circRNA-FOXK2 gene, wherein the expression level of the circRNA-FOXK2 gene in breast cancer can be related to the occurrence, development and prognosis of the disease, and the specificity and the sensitivity are higher; based on the breast cancer marker, a novel breast cancer treatment target is provided, a treatment strategy taking the circRNA-FOXK2 gene as the target is developed, and a novel thought and direction are provided for the treatment of breast cancer.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the invention provides the use of the circRNA-FOXK2 gene as a target in the preparation or screening of a medicament for the treatment of breast cancer.
The invention also provides application of the circRNA-FOXK2 gene expression inhibitor in preparing or screening medicaments for treating breast cancer.
According to the invention, through annular RNA sequencing analysis, one of annular FOXK2 which is obviously and highly expressed in breast cancer and influences the growth of breast cancer cells is obtained through systematic identification and screening. Early-stage researches show that the circFOXK2 is high in expression in breast cancer cell lines and clinical tissue samples; after knocking down the circFOXK2, the estrogen receptor positive breast cancer cell line MCF-7 and the triple negative breast cancer cell line MDA-MB-231 cells were blocked in the G0/G1 phase, inhibiting cell division, and had a significant effect on their migration, clone formation. Further animal preliminary experiments suggest that knocking down circFOXK2 can significantly promote the anti-tumor effect of death of breast cancer cells. The mechanism finds that the circFOXK2 is used as a molecular sponge of miR-149-3p, regulates and controls the stability of a target gene E2F2, and recruits MCM7 proteins into cell nuclei in cytoplasm to promote the expression of cell cycle genes and regulate the progress of breast cancer. The invention provides scientific basis for annular RNA as a breast cancer treatment strategy and provides a new idea for improving the curative effect of breast cancer treatment by targeting circFOXK2.
Preferably, the circRNA-FOXK2 gene is used as a target of RNA interference, and a tumor therapeutic drug or preparation capable of aiming at the circRNA-FOXK2 gene is developed, so that the expression level of the circRNA-FOXK2 gene in tumor cells is reduced.
Preferably, the circRNA-FOXK2 gene is used as a target for RNA interference, and the target sequence is shown as SEQ ID NO. 1.
In a second aspect, the invention provides a nucleic acid molecule medicament for the treatment of breast cancer, said nucleic acid molecule medicament comprising an isolated nucleic acid molecule or a pharmaceutically acceptable salt thereof which reduces the expression level of the circRNA-FOXK2 gene in a tumor cell.
Preferably, the isolated nucleic acid molecule that reduces the expression level of the circRNA-FOXK2 gene in a tumor cell comprises: the circRNA-FOXK2 gene siRNA, circRNA-FOXK2 gene shRNA, circRNA-FOXK2 gene ASO, miRNA or a combination of at least two of them.
Preferably, the sequence of the circRNA-FOXK2 gene siRNA comprises any one of SEQ ID NO: 2-3.
Preferably, the sequence of the circRNA-FOXK2 gene shRNA comprises any one of SEQ ID NOs 4 to 6.
Preferably, the sequence of the circRNA-FOXK2 gene ASO comprises the sequence shown in SEQ ID NO. 7.
Preferably, the circRNA-FOXK2 gene ASO further comprises a nucleotide having a modification selected from the group consisting of: any one or a combination of at least two of thio modification, phosphothio modification, cholesterol modification or morpholino ring modification.
In a third aspect, the present invention provides a lentiviral vector medicament for treating breast cancer, the lentiviral vector medicament comprising a circRNA-FOXK2 gene interfering lentiviral vector or a pharmaceutically acceptable salt thereof, the circRNA-FOXK2 gene interfering lentiviral vector comprising a gene fragment encoding a shRNA of the circRNA-FOXK2 gene in the nucleic acid molecule medicament for treating breast cancer according to the second aspect.
In a fourth aspect, the invention provides a lentivirus medicament for treating breast cancer, the lentivirus medicament contains circRNA-FOXK2 gene interference lentivirus or pharmaceutically acceptable salt thereof, and the circRNA-FOXK2 gene interference lentivirus is formed by virus packaging of a circRNA-FOXK2 gene interference lentivirus vector in the third aspect under the assistance of a lentivirus packaging plasmid and a cell line.
In a fifth aspect, the present invention provides a pharmaceutical composition for treating breast cancer, the pharmaceutical composition comprising any one or a combination of at least two of the nucleic acid molecule drug for treating breast cancer according to the second aspect, the lentiviral vector drug for treating breast cancer according to the third aspect, or the lentiviral drug for treating breast cancer according to the fourth aspect.
Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
In a sixth aspect, the invention provides the use of any one or a combination of at least two of the nucleic acid molecule drug for treating breast cancer according to the second aspect, the lentiviral vector drug for treating breast cancer according to the third aspect, the lentiviral drug for treating breast cancer according to the fourth aspect or the pharmaceutical composition for treating breast cancer according to the fifth aspect in the preparation of a breast cancer treatment product.
Compared with the prior art, the invention has the following beneficial effects:
(1) The present invention provides a novel breast cancer marker, the circRNA-FOXK2 gene, whose expression level in breast cancer may be related to the occurrence, development and prognosis of the disease. The circRNA-FOXK2 gene may have higher specificity and sensitivity than traditional breast cancer markers, such as CEA and CA 15-3.
(2) The invention provides a novel breast cancer treatment target point, and a treatment strategy taking a circRNA-FOXK2 gene as the target point. Interference against the circRNA-FOXK2 gene may help to block the growth, metastasis and invasive capacity of breast cancer cells, thereby providing new ideas and directions for the treatment of breast cancer.
Drawings
FIG. 1 shows the results of expression levels of circFOXK2 in breast cancer cell lines including T47D, HCC1500, MCF7, BT474, SK-BR-3, HCC1806, HCC1937, MDA-MB-231, MDA-MB-468, SUM149PT, BT20 and HS578T, and normal breast epithelial cell line MCF 10A.
Fig. 2 shows the results of expression levels of circFOXK2 in clinical breast tumor tissue (n=27) and adjacent normal tissue (n=27).
FIG. 3 shows the results of detection of the effect of circFOXK2 siRNA knockdown on MCF7 cell growth.
FIG. 4 shows the results of detection of the effect of PCD2.1 plasmid over-expression of circFOXK2 on MCF7 cell growth.
FIG. 5 shows the effect of siRNA knockdown of circFOXK2 on MCF7 cell growth by overexpression of circFOXK2 on PCD2.1 plasmid.
FIG. 6 shows cell cycle analysis of the circFOXK2 postflow in siRNA knockdown MCF7 cells.
FIG. 7 shows cell cycle analysis of PCD2.1 plasmid over-expression of circFOXK2 in MCF7 cells.
FIG. 8 is a graph showing the effect of siRNA knockdown circFOXK2 on scoring of MCF7 cells.
FIG. 9 is a graph showing the effect of siRNA knockdown circFOXK2 on MCF7 cell transwell.
FIG. 10 is the effect of circFOXK2 shRNA knockdown on MCF7 cell growth.
FIG. 11 is the effect of circFOXK2 shRNA knockdown on MCF7 cell colony formation.
FIG. 12 is a graph showing the effect of siRNA knockdown circFOXK2 on MDA-MB-231 cell proliferation.
FIG. 13 is a graph showing the cell cycle effect of siRNA knockdown circFOXK2 on MDA-MB-231 cells.
FIG. 14 is a graph showing the effect of siRNA knockdown circFOXK2 on the ability of MDA-MB-231 cells to migrate.
FIG. 15 is a graph showing the effect of siRNA knockdown circFOXK2 on colony formation in MDA-MB-231 cells.
Fig. 16 shows tumor size and weight results.
FIG. 17 shows the expression of the specifically targeted circFOXK2 ASO (ASO circFOXK 2) and the negative control ASO (ASO NC) transfected in MCF-7 and MDA-MB-231 cells, respectively, and the RT-qPCR analysis.
FIG. 18 is the effect of transfected negative control ASO (ASO NC) or ASO specifically targeting circFOXK2 (ASO circFOXK 2) on MCF-7 and MDA-MB-231 cell proliferation.
FIG. 19 is the effect of transfected negative control ASO (ASO NC) or ASO specifically targeting circFOXK2 (ASO circFOXK 2) on the MCF-7 cell scratch healing assay.
FIG. 20 shows the scratch healing assay of MDA-MB-231 cells transfected with a negative control ASO (ASO NC) or ASO specifically targeting circFOXK2 (ASO circFOXK 2).
FIG. 21 shows the colony formation assay of MCF-7 cells by ASO-CTL and ASO-circFOXK 2.
FIG. 22 shows the test of ASO-CTL and ASO-circFOXK2 against MDA-MB-231 cell colony formation.
FIG. 23 shows ASO-CTL and ASO-circFOXK2 to MCF-7 cell transwell assay.
FIG. 24 shows ASO-CTL and ASO-circFOXK2 vs MDA-MB-231 cell transwell assay.
FIG. 25 is a subcutaneous tumor map of MCF7 cell transfected control ASO or ASO-circFOXK2 transplanted nude mice.
FIG. 26 is a graph showing tumor weight statistics and expression levels of circFOXK2 in transplanted tumors.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1
Screening of circFOXK2
The circular RNA which is remarkably high in the breast cancer and influences the growth of breast cancer cells is obtained through the circular RNA sequencing analysis and systematic identification and screening.
The MCF-7 breast cancer cell line extracts total RNA, sends the total RNA to Ji Ma company for circular RNA sequencing, predicts the top 40 of BSJ reads by three general methods of CIRI2, find_circ and CIRCExplorer2 respectively, and selects 16 circRNAs predicted by the three methods together. Through systematic identification and screening, one of the cyclic RNAs, circFOXK2, which is remarkably high-expressed in breast cancer and affects the growth of breast cancer cells is obtained.
The CircFOXK2 sequence: corresponding to FOXK2 genome EXON2 and EXON3, chr17:82563353-82568201, specifically shown as SEQ ID NO:1, 343nt in total:
gtgcacattcaggttcccgagcacaaacatcaagataacgttcactgccctgtccagcgagaagagagagaagcaggaggcgtctgagtctccagtgaaggccgtacagccacacatctcgcccctgaccatcaacattccagacaccatggcccacctcatcagccctctgccctcccccacgggaaccatcagcgctgcaaactcctgcccctccagcccccggggagcggggtcttcagggtacaaggtgggccgagtgatgccatctgacctcaatttaatggctgacaactcacagcctgaaaatgaaaaggaagcttcaggtggagacagcccgaag。
example 2
This example compares the expression of circFOXK2 in normal breast epithelial cell lines, ER-positive breast cancer cell lines, HER2 positive cell lines and triple negative breast cancer cell lines, and their expression was examined in MCF-7 breast cancer cell lines (breast cancer cell lines include T47D, HCC1500, MCF7, BT474, SK-BR-3, HCC1806, HCC1937, MDA-MB-231, MDA-MB-468, SUM149PT, BT20 and HS 578T).
FIG. 1 shows the results of expression levels of circFOXK2 in breast cancer cell lines including T47D, HCC1500, MCF7, BT474, SK-BR-3, HCC1806, HCC1937, MDA-MB-231, MDA-MB-468, SUM149PT, BT20 and HS578T, and normal breast epithelial cell line MCF 10A.
Fig. 2 shows the results of expression levels of circFOXK2 in clinical breast tumor tissue (n=27) and adjacent normal tissue (n=27).
The results show that the circFOXK2 is found to exhibit high expression in breast cancer cell lines and clinical tissue samples.
Example 3
In vitro cell experiment to knock down the effect of circRNA-FOXK2 on breast cancer cell lines
(1) In this example, two independent specific siRNAs targeting the sequence of circFOXK2 BJTS and one control siRNA were designed, and MCF-7 cells and MDA-MB-231 cells were transfected respectively to detect the mobility, colony formation and transfer capacity of circFOXK2 to MCF-7 cells.
si-circFOXK2-1：5’-GAAGGUGCACAUUCAGGUUTT-3’(SEQ ID NO:2)；
si-circFOXK2-2：5’-AGACAGCCCGAAGGUGCACAU-3’(SEQ ID NO:3)；
Control siRNA (si-CTL): 5'-UUTTGCUGAAAGUCGAGCAAG-3' (SEQ ID NO: 7).
(2) This example also constructs a control shRNA plasmid (pLKO.1 cloning vector) and a specific shRNA of two-three independent targeting circFOXK2 BJS sequences designed for another system, lentiviruses infected MCF-7 cells and MDA-MB-231 cells, and evaluated the proliferation, cell mobility, colony formation, and infiltration capacity of circFOXK2 on MCF-7 and MDA-MB-231 cells.
The step of evaluating the ability of cells to infiltrate includes:
(1) The cell culture chamber (Corning) was placed in a 24-well plate and 800. Mu.L of serum medium was added to the outer chamber.
(2) Cells were digested and counted, resuspended in 200. Mu.L of serum-free medium, and 2-5 ten thousand cells were inoculated in each cell and incubated at 37℃for 24-36 hours.
(3) The cells were washed 2 times with cold PBS and then fixed with 4% paraformaldehyde solution at 4℃for 15min.
(4) The chamber was washed 2 times with cold PBS and stained with crystal violet stain for 10 minutes at room temperature.
(5) Washing with cold PBS for 5-10 times, finally erasing cells in the chamber with a cotton swab, reversely buckling the chamber, putting the chamber into an ultra-clean workbench, and air-drying for 10 minutes.
(6) Photographs were taken with an inverted microscope and analyzed for cell migration with Image J.
The steps of cell proliferation assessment include:
(1) siRNA was transfected into cells requiring knockdown treatment, 48 hours post-transfection was designated as day 1, while cells not requiring knockdown treatment were designated as day 1 after plating, and so on every day.
(2) The initial inoculum size was 1000-2000 cells/well by inoculating MCF-7 cells and MDA-MB-231 cells into 96-well plates.
(3) When measuring cell activity, adding 20 μl MTS solution into 100 μl DMED culture medium, mixing, sucking out the culture medium in the well plate with a pipette, adding mixed solution of culture medium and MTS solution, and mixing at 37deg.C and 5% CO 2 The reaction was stopped by adding 25. Mu.L of 10% SDS solution to the reaction mixture for 1 hour in the incubator.
(4) The absorbance of the well plate was measured at 490nm using an microplate reader at different time points.
The step of evaluating the cell mobility includes:
(1) Cells were seeded on 6 plates, preferably at the initial seeding rate of the next day. 37 ℃,5% CO 2 Culturing for 3-4 days, and changing fresh culture medium every day.
(2) After the next day of cell confluence, each well was scored with a 200 μl gun head, the floating cells were washed out with PBS and fresh medium was added.
(3) And placing the 6-hole plate under an inverted microscope, selecting a proper scratch area for photographing, recording the photographing position, and photographing at the same scratch position every day 2-3 days later.
(4) The scratch area changes were analyzed with ImageJ and the wound healing rate was calculated.
The step of evaluating cell colony formation includes:
(1) Cells were seeded in 6-well plates at an initial seeding rate of 500-1000 cells per well. 37 ℃,5% CO 2 The culture was performed for 7-14 days, and fresh medium was changed every 3 days until the monoclonal grows to a proper density.
(2) Cells were harvested, medium was removed, cells were washed 1 time with chilled PBS to remove PBS, and 4% paraformaldehyde solution was added to fix at room temperature for 15 minutes.
(3) Removing paraformaldehyde, and adding 0.1-1% crystal violet dye solution for dyeing for 30 minutes at room temperature.
(4) And (3) rinsing for a plurality of times by PBS, washing off crystal violet dye solution of cell background on the pore plate, airing and photographing.
(5) 1mL of 30% acetic acid was added to the well plate to dissolve crystal violet dye attached to cells, the acetic acid-crystal violet mixture was sucked into a 1.5mL tube, diluted 10 times with water, and the absorbance was measured at 590nm by an enzyme-labeled instrument.
sh-circFOXK2-1：5’-GACAGCCCGAAGGTGCACATT-3’(SEQ ID NO:4)；
sh-circFOXK2-2：5’-AGACAGCCCGAAGGTGCACAT-3’(SEQ ID NO:5)；
sh-circFOXK2-3：5’-GAAGGTGCACATTCAGGTTTT-3’(SEQ ID NO:6)；
Control shRNA:5'-TTTGACTGCAGGTACGGCCTGAG-3' (SEQ ID NO: 8).
FIG. 3 shows the results of detection of the effect of circFOXK2 siRNA knockdown on MCF7 cell growth.
FIG. 4 shows the results of detection of the effect of PCD2.1 plasmid over-expression of circFOXK2 on MCF7 cell growth.
FIG. 5 shows the effect of siRNA knockdown of circFOXK2 on MCF7 cell growth by overexpression of circFOXK2 on PCD2.1 plasmid.
FIG. 6 shows cell cycle analysis of the circFOXK2 postflow in siRNA knockdown MCF7 cells.
FIG. 7 shows cell cycle analysis of PCD2.1 plasmid over-expression of circFOXK2 in MCF7 cells.
FIG. 8 is a graph showing the effect of siRNA knockdown circFOXK2 on scoring of MCF7 cells.
FIG. 9 is a graph showing the effect of siRNA knockdown circFOXK2 on MCF7 cell transwell.
FIG. 10 is the effect of circFOXK2 shRNA knockdown on MCF7 cell growth.
FIG. 11 is the effect of circFOXK2 shRNA knockdown on MCF7 cell colony formation.
FIG. 12 is a graph showing the effect of siRNA knockdown circFOXK2 on MDA-MB-231 cell proliferation.
FIG. 13 is a graph showing the cell cycle effect of siRNA knockdown circFOXK2 on MDA-MB-231 cells.
FIG. 14 is a graph showing the effect of siRNA knockdown circFOXK2 on the ability of MDA-MB-231 cells to migrate.
FIG. 15 is a graph showing the effect of siRNA knockdown circFOXK2 on colony formation in MDA-MB-231 cells.
From the above results, it was found that after knocking down the circFOXK2, the estrogen receptor positive breast cancer cell line MCF-7 and the triple negative breast cancer cell line MDA-MB-231 cells were blocked in the G0/G1 phase, the division of the cells was inhibited, and the migration thereof, the clone formation was significantly affected.
Example 4
In vivo cell experiments to knock down the effect of circRNA-FOXK2 on breast cancer cell lines
Animal experiments verify the effect of circFOXK2 on breast cancer, injecting sh-CTL, sh-circFOXK2-1 and sh-circFOXK2-2 infected MCF7 cells subcutaneously into female BALB/C nude mice for xenograft experiments, tumor bearing mice were divided into three groups: shRNA control group, 2. Two independent shRNA knockdown groups of 6 each injected 10 6 Individual cells/mL, after 4 weeksTumor size and weight were assessed.
Fig. 16 shows tumor size and weight results, tumor weight (±s.e.m), < P <0.05, < P <0.01. In vivo cell results show that knocking down the circFOXK2 can significantly promote the anti-tumor effect of death of breast cancer cells.
Example 5
In vitro cell experiments prove the curative effect of the therapeutic strategy taking the circRNA-FOXK2 gene as a target spot on breast cancer
The high expression of circFOXK2 in breast cancer cells and clinical samples, and its functional importance in regulating breast cancer cell growth and tumorigenesis, prompted us to investigate whether targeting circFOXK2 could inhibit breast cancer cell growth.
This example was performed in vitro cell experiments to assess the ability of antisense oligonucleotides (ASOs) to specifically target and degrade cirFOXK2. ASO (ASO cirFOXK 2) and negative control (ASO NC) targeting the cirFOXK2 BKS sequence were designed and transfected into MCF-7 and MDA-MB-231 cells, RT-qPCR was used to measure ASO down-regulation efficiency, and to evaluate cell proliferation, clonogenic, scratch healing and transwell assays to evaluate growth against breast cancer.
ASO targeting the cirFOXK2 BJS sequence: 5'-ACAGCCCGAAGGTGCACATT-3' (SEQ ID NO: 9);
negative control ASO NC:5'-CATTCCCTGAAGGTTCCTCA-3' (SEQ ID NO: 10).
The experimental results are shown below:
FIG. 17 shows the expression of the specifically targeted circFOXK2 ASO (ASO circFOXK 2) and the negative control ASO (ASO NC) transfected in MCF-7 and MDA-MB-231 cells, respectively, and the RT-qPCR analysis. RT-qPCR analysis showed that ASO-circFOXK2 significantly reduced circFOXK2 itself at the mRNA level.
FIG. 18 is the effect of transfected negative control ASO (ASO NC) or ASO specifically targeting circFOXK2 (ASO circFOXK 2) on MCF-7 and MDA-MB-231 cell proliferation. Cell proliferation experiments showed that ASO-circFOXK2 inhibited the growth of MCF7 cells and MDA-MB-231 cells.
FIG. 19 is the effect of transfected negative control ASO (ASO NC) or ASO specifically targeting circFOXK2 (ASO circFOXK 2) on the MCF-7 cell scratch healing assay; FIG. 20 shows the scratch healing assay of MDA-MB-231 cells transfected with a negative control ASO (ASO NC) or ASO specifically targeting circFOXK2 (ASO circFOXK 2). Cell scoring experiments showed that ASO-circFOXK2 attenuated the wound healing rate of MCF7 cells and MDA-MB-231 cells.
FIG. 21 shows an ASO-CTL and ASO-circFOXK2 pair MCF-7 cell colony formation assay; FIG. 22 shows the test of ASO-CTL and ASO-circFOXK2 against MDA-MB-231 cell colony formation. The cloning experiments showed that ASO-circFOXK2 inhibited colony formation in MCF7 cells and MDA-MB-231 cells.
FIG. 23 shows ASO-CTL and ASO-circFOXK2 vs. MCF-7 cell transwell assay; FIG. 24 shows ASO-CTL and ASO-circFOXK2 vs MDA-MB-231 cell transwell assay. Transwell experiments show that ASO-circFOXK2 inhibits the infiltration capacity of MCF7 cells and MDA-MB-231 cells.
Example 6
In vivo cell experiments prove that the therapeutic strategy taking circRNA-FOXK2 gene as target spot has therapeutic effect on breast cancer
In vivo animal experiments evaluate the anti-tumor growth effect of ASO cirFOXK2. The method comprises the following steps: BALB/c nude mice (4-5 weeks old, weight 18-20 g) were used for xenogenic breast cancer transplant model construction only 10. Each mouse was vaccinated with 5X 10 5 100. Mu.L PBS MCF-7 cells, when the tumor size is about 100mm 2 Tumor-bearing mice were randomly divided into two groups of ASO cirFOXK2 and negative control (ASO NC), 5 each, each group was intratumorally injected with 5 nm/100. Mu.L ASO NC or ASO cirFOXK2 every 5 days, tumors were obtained after 8 treatments, tumor ASO treatment growth was evaluated and RT-qPCR detection of cirFOXK2.
FIG. 25 is a subcutaneous oncological map of MCF7 cell transfected control ASO or ASO-circFOXK2 transplanted nude mice; fig. 26 is a tumor weight statistic and the expression level of circFOXK2 in transplanted tumors, P <0.01. As expected, after transfection of MCF7 cells with ASO-circFOXK2, nude mice were transplanted subcutaneously, both breast cancer cell tumor growth size and tumor quality were significantly reduced.
In summary, the present invention provides a novel breast cancer marker, the circRNA-FOXK2 gene, the expression level of which in breast cancer cells is related to the occurrence, development and prognosis of the disease. The therapeutic strategy based on the breast cancer marker is also provided, and the interference of the circRNA-FOXK2 gene can be helpful for blocking the growth, metastasis and invasion capacity of breast cancer cells, so that a new thought and direction are provided for the treatment of breast cancer.
The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.
Claims (10)
- Application of circRNA-FOXK2 gene as target in preparing or screening breast cancer therapeutic drug.
- 2. The use according to claim 1, characterized in that the expression level of the circRNA-FOXK2 gene in tumor cells is reduced by developing a tumor therapeutic drug or preparation capable of targeting the circRNA-FOXK2 gene using the circRNA-FOXK2 gene as a target for RNA interference;preferably, the circRNA-FOXK2 gene is used as a target for RNA interference, and the target sequence is shown as SEQ ID NO. 1.
- 3. A nucleic acid molecule drug for treating breast cancer, comprising an isolated nucleic acid molecule or a pharmaceutically acceptable salt thereof that reduces the expression level of the circRNA-FOXK2 gene in a tumor cell;preferably, the isolated nucleic acid molecule that reduces the expression level of the circRNA-FOXK2 gene in a tumor cell comprises: the circRNA-FOXK2 gene siRNA, circRNA-FOXK2 gene shRNA, circRNA-FOXK2 gene ASO, miRNA or a combination of at least two of them.
- 4. The nucleic acid molecule drug for treating breast cancer according to claim 3, wherein the sequence of the circRNA-FOXK2 gene siRNA comprises any one of SEQ ID NOs 2 to 3;preferably, the sequence of the circRNA-FOXK2 gene shRNA comprises any one of SEQ ID NO 4-6;preferably, the sequence of the circRNA-FOXK2 gene ASO comprises the sequence shown in SEQ ID NO. 7.
- 5. The nucleic acid molecule drug for treating breast cancer according to claim 3 or 4, wherein the circRNA-FOXK2 gene ASO further comprises a modified nucleotide, wherein the modification is selected from the group consisting of: any one or a combination of at least two of thio modification, phosphothio modification, cholesterol modification or morpholino ring modification.
- 6. A lentiviral vector medicament for treating breast cancer, comprising a circRNA-FOXK2 gene interfering lentiviral vector comprising a gene fragment encoding a shRNA of the circRNA-FOXK2 gene in a nucleic acid molecule medicament for treating breast cancer according to any one of claims 3-5, or a pharmaceutically acceptable salt thereof.
- 7. A lentivirus medicament for treating breast cancer is characterized by comprising circRNA-FOXK2 gene interference lentivirus or pharmaceutically acceptable salt thereof, wherein the circRNA-FOXK2 gene interference lentivirus is formed by virus packaging of a circRNA-FOXK2 gene interference lentivirus vector in accordance with claim 6 under the assistance of a lentivirus packaging plasmid and a cell line.
- 8. A pharmaceutical composition for treating breast cancer, comprising any one or a combination of at least two of the nucleic acid molecule drug for treating breast cancer of any one of claims 3 to 5, the lentiviral vector drug for treating breast cancer of claim 6, or the lentiviral drug for treating breast cancer of claim 7.
- 9. The pharmaceutical composition for treating breast cancer of claim 8, further comprising a pharmaceutically acceptable carrier.
- 10. Use of a nucleic acid molecule drug for treating breast cancer according to any one of claims 3 to 5, a lentiviral vector drug for treating breast cancer according to claim 6, a lentiviral drug for treating breast cancer according to claim 7, or a pharmaceutical composition for treating breast cancer according to claim 8 or 9, or a combination of at least two thereof, for the preparation of a breast cancer treatment product.
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