A competitive CTCF protein and its construction method and application

Abstract

The present invention relates to the field of biomedicine and molecular biology technology, specifically a competitive CTCF protein and its construction method and application. By selecting a highly conserved zinc finger protein CTCF, retaining the DNA binding domain of the insulating protein CTCF, using GFP to replace the functional domain of CTCF, obtaining a c-CTCF gene sequence, and then connecting it to a vector, expressing and constructing a competitive CTCF protein in cells, using a competitive c-CTCF gene sequence to change the CTCF affinity of the entire genome, competitively blocking functional genes in esophageal cancer cells that are susceptible to CTCF binding affinity regulation, and accurately capturing the action targets that specifically affect the occurrence of esophageal cancer through changes in the malignancy of tumor cells.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine and molecular biology, and specifically to a competitive CTCF protein and a construction method and application thereof. Background Art

[0002] CCCTC binding factor (CTCF) is a classic transcriptional regulator that contains 11 C2H2 zinc fingers. About 20,000-40,000 CTCF binding sites have been found in the human genome. Disruption of CTCF binding sites in the genome can lead to abnormal gene expression. At present, most studies on the treatment of CTCF directly knock out or inhibit the expression of CTCF. However, this method has a certain degree of carcinogenicity, the knockout rate cannot be controlled, and it is even more difficult to determine the functional targets regulated by CTCF selective binding. For example, after knocking out the CTCF group at a specific site by the CRISPR method, CTCF binding in the whole genome is reduced, the expression of the oncogene FGF4 is promoted, and the tumorigenesis of gastrointestinal stromal tumors (GIST) is promoted. It is worth noting that the study found that by establishing CTCF semi-heterozygous deletion mice, long-term reduction of CTCF binding will change the transcription of cancer pathways and gene expression patterns.

[0003] Verve-101 is the first in vivo base editing therapy to enter clinical trials. Clinical trials began in New Zealand and the UK last year, but its application for clinical trials in the United States was suspended by the FDA in November last year. Two more serious adverse events in the experiment caused some concern. Two of the 10 participants experienced cardiovascular events, one of whom died of a heart attack 15 weeks after treatment, and the other had a heart attack 1 day after treatment. However, our CiPR technology does not knock out or eliminate CTCF, but uses competitor c-CTCF to change the affinity of CTCF throughout the genome, competitively blocking functional genes in esophageal cancer cells that are susceptible to CTCF binding affinity regulation, and accurately capturing the action targets that specifically affect the occurrence of esophageal cancer through changes in the malignancy of tumor cells, bringing hope to targeted tumor treatment. Since many targets are regulated by TFs, the use of similar CiPR technology is likely to provide another option for accurately identifying functional targets mediated by the selective binding of these TFs. Summary of the invention

[0004] The purpose of the present invention is to provide a competitive CTCF protein and a construction method and application thereof to solve the problems raised in the above background technology.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A competitive CTCF protein is obtained by embedding a c-CTCF gene sequence in a vector and expressing the competitive CTCF protein through cells.

[0007] More optimally, the c-CTCF gene sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO.2.

[0008] More optimally, the vector is a pCMV plasmid vector.

[0009] More optimally, the method for constructing the competitive CTCF protein is as follows: the gene sequence of the zinc finger protein CTCF is selected as the basis, the DNA binding domain of the insulating protein CTCF is retained, the functional domain of CTCF is replaced with GFP, the c-CTCF gene sequence is obtained, and then it is connected to a vector and expressed in cells.

[0010] More optimally, the functional domain is used to recognize the DNA binding site of endogenous CTCF.

[0011] More optimally, the DNA binding site is the connection site between the c-CTCF gene sequence and the vector.

[0012] More optimally, the competitive CTCF protein is used as a targeted therapeutic drug for esophageal cancer.

[0013] Compared with the prior art, the beneficial effects achieved by the present invention are: in order to more accurately find the functional targets regulated by the selective binding of CTCF, the present invention selects the highly conserved zinc finger protein CTCF, retains the DNA binding domain of the insulating protein CTCF, replaces the functional domain of CTCF with GFP, and does not form dimers only through protein-protein interactions, thereby constructing a c-CTCF gene sequence; then the constructed c-CTCF gene sequence is overexpressed in an esophageal cancer cell line, competitively binds to the connection site of endogenous CTCF and regulates gene expression, and uses competitor c-CTCF to change the CTCF affinity of the entire genome, competitively blocks the functional genes in esophageal cancer cells that are susceptible to CTCF binding affinity regulation, and accurately captures the action targets that specifically affect the occurrence of esophageal cancer through changes in the malignancy of tumor cells, bringing hope for targeted treatment of esophageal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0015] Figure 1 It is a schematic diagram of the c-CTCF gene sequence after the construction of the competitive CTCF protein;

[0016] Figure 2 It is a schematic diagram of the overall technology of c-CTCF gene expression;

[0017] Figure 3 It is the Western blot analysis of c-CTCF gene expression in KYSE450 cells and KYSE510 cells and the comparison with the negative control group;

[0018] Figure 4 It is the observed colony image after the c-CTCF gene was expressed in KYSE450 cells and KYSE510 cells and the comparison with the negative control group;

[0019] Figure 5 is the colony formation rate after expression of c-CTCF gene in KYSE450 cells and the comparison with the negative control group;

[0020] Figure 6 is the colony formation rate after expression of c-CTCF gene in KYSE510 cells and the comparison with the negative control group;

[0021] Figure 7 It is the proliferation ability of cells after the expression of c-CTCF gene in KYSE450 cells and the comparison with the negative control group;

[0022] Figure 8 It is the proliferation ability of cells after the expression of c-CTCF gene in KYSE510 cells and the comparison with the negative control group;

[0023] Fig. 9 This is a comparative observation diagram of the migration ability of the colonies after the c-CTCF gene was expressed in KYSE450 cells and KYSE510 cells and the negative control group;

[0024] Fig.10 is the comparison of tumor volume between the c-CTCF expression group and the negative control group;

[0025] Fig.11 is the comparison of tumor weight of the c-CTCF expression group with that of the negative control group;

[0026] Fig.12 is the comparison of the survival rate of the c-CTCF expression group with that of the negative control group;

[0027] Fig.13 This is a comparative observation diagram of the luciferase signal of the c-CTCF overexpression group and the negative control group;

[0028] Fig.14 This is a comparative observation diagram of lung metastatic nodules in the c-CTCF overexpression group and the negative control group. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1: (1) Connecting the c-CTCF gene sequence to the pCMV plasmid vector to obtain a competitive protein vector;

[0031] (2) Dilute the competitive protein carrier (2.5 μg) with serum-free Opti-MEM (125 μL), mix gently, and then incubate at room temperature for 5 min to obtain a Lipo3000-competitive protein mixture;

[0032] (3) Lipo3000-competitive protein mixture was added to KYSE450 and KYSE510 cells, respectively. KYSE450 cells were numbered A and KYSE510 cells were numbered B. The cells were placed in a 37°C CO 2 Cultured in an incubator, the culture medium was concentrated using a lentivirus kit after 72 hours to obtain competitive protein lentivirus A and B;

[0033] (4) Prepare polybrene for blood-free and antibiotic-free culture medium: add 1 μl of 10 mg / ml polybrene stock solution to 1999 μl of blood-free and antibiotic-free culture medium and stir evenly;

[0034] (5) The original culture medium of the competitive protein lentivirus was discarded and added to a 24-well plate containing a blood-free and antibiotic-free polybrene medium. After 6 hours, a new blood-free and antibiotic-free polybrene medium was replaced. As the cells continued to be transfected and proliferated, 2 ng / mL of puromycin was added for screening when the cells were passaged to obtain cells A and B that stably expressed the c-CTCF gene ( Figure 2 ).

[0035] Experimental detection: According to Example 1, the feasibility of competitive CTCF protein for targeted treatment of esophageal cancer was verified, and the following experiments were set up for verification: specifically:

[0036] 1. Western blotting showed that c-CTCF gene was successfully expressed in KYSE450 and KYSE510 ESCC cells ( Figure 3 );

[0037] 2. In order to study whether tumor characteristics change after overexpression of c-CTCF gene, we first used in vitro cell proliferation assay and in vitro clone formation assay to detect cell proliferation. Compared with the negative control (NC) group, the results showed that the clone formation ability of KYSE450 cells and KYSE510 cells expressing c-CTCF gene was significantly reduced, and only a small number of cells A and B ( Figure 4 );

[0038] 3. Colony quantification showed that the colony formation rates of KYSE450 cells and KYSE510 cells decreased by 30% and 50%, respectively ( Figure 5 , Figure 6 ), similarly, the proliferation capacity of KYSE450 cells and KYSE510 cells expressing c-CTCF gene was also significantly decreased ( Figure 7 , Figure 8 ); The ability of c-CTCF gene to change tumor migration was detected using an in vitro migration assay. Compared with the NC group, the migration ability of ESCC cells was significantly inhibited after c-CTCF gene expression ( Fig. 9 );

[0039] 4. In order to study whether the c-CTCF gene inhibits tumor formation in vivo, a nude mouse subcutaneous xenograft model was established. The tumor volume of the c-CTCF expression group ( Fig.10 ) and weight ( Fig.11 ) were significantly reduced; in addition, we also observed that the survival rate of the c-CTCF expression group was higher than that of the NC group ( Fig.12 );

[0040] 5. To detect the lung seeding ability after c-CTCF gene overexpression, KYSE450 cells were labeled with firefly luciferase. Then, athymic nude mice were injected with tail veins, and systemic metastasis was examined by bioluminescence and fluorescence imaging. The results showed that the luciferase signal in the c-CTCF overexpression group was significantly reduced ( Fig.13 ); 5 weeks later, the animals were sacrificed and their lungs were collected for further analysis. Lung histology showed that the lung metastatic nodules in the c-CTCF overexpression group were smaller ( Fig.14 ).

[0041] Conclusion: In summary, it is shown that c-CTCF protein is an effective competitive peptide that can inhibit the growth and migration of ESCC cells. The expression of c-CTCF gene plays a key role in reducing the growth and metastasis of ESCC cells. Its application in the targeted treatment of esophageal cancer can play a good positive role.

[0042] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0043] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

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Claims

1. A competitive CTCF protein, characterized in that: The c-CTCF gene sequence is embedded in the vector and expressed in cells to obtain competitive CTCF protein; wherein the c-CTCF gene sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. The method for constructing a competitive CTCF protein according to claim 1, characterized in that: The competitive CTCF protein is constructed by selecting the CTCF gene sequence based on the zinc finger protein, retaining the DNA binding domain of the insulating protein CTCF, replacing the functional domain of CTCF with GFP, obtaining the c-CTCF gene sequence, and then connecting it to a vector for expression in cells.

3. The use of a competitive CTCF protein according to claim 1, characterized in that: The competitive CTCF protein is used in the preparation of targeted therapeutic drugs for esophageal cancer.

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