WO2021139561A1

WO2021139561A1 - Anti-tumor polypeptide for inhibiting ezh2 activity, and use thereof

Info

Publication number: WO2021139561A1
Application number: PCT/CN2020/140552
Authority: WO
Inventors: 李博; 廖昆; 邓舒烨; 杨晴; 杨时雨
Original assignee: 中山大学
Priority date: 2020-01-10
Filing date: 2020-12-29
Publication date: 2021-07-15
Also published as: CN111394334B; CN111394334A

Abstract

Provided in the present invention are an anti-tumor polypeptide for inhibiting EZH2 activity, and the use thereof. Constructed in the present invention are polypeptide molecules derived from truncated FBP1, all of which have the effect of targeting EZH2 and inhibiting tumors. The polypeptide has a small molecular weight and easily penetrates cell membranes, which can directly inhibit the activity of EZH2 methyltransferase and block the effect of EZH2 promoting the growth of cancer cells to achieve an anti-tumor effect. Same has a good application value and prospects in the development of anti-tumor drugs for solid tumors with high expression of EZH2.

Description

Anti-tumor polypeptide inhibiting EZH2 activity and application thereof

Technical field

The invention belongs to the technical field of biomedicine. More specifically, it relates to an anti-tumor polypeptide that inhibits EZH2 activity and its application.

Background technique

Many cancers are associated with genetic mutations, including some mutations in chromatin remodeling and histone methylation pathways. EZH2 is the active core subunit of Polycomb Inhibitory Complex 2 (PRC2), which also includes EED, SUZ12 and RbAp46/48. The PRC2 complex mainly uses the EZH2 methyltransferase as the core to catalyze the trimethylation modification of the 27th lysine of histone 3, which leads to chromatin tightening and gene silencing. Scientific research has shown that the occurrence and development of a variety of cancers are related to EZH2, and EZH2 is highly expressed in various malignant tumors such as breast cancer, prostate cancer, lung cancer, liver cancer, and kidney cancer. The results of the study indicate that EZH2 is necessary for the malignant proliferation of cancer cells, and the exogenous overexpression of EZH2 in normal cells can significantly promote cell proliferation.

At present, a variety of small molecule drugs have achieved anti-tumor effects by reducing the activity of EZH2, such as GSK126, UNC1999, and tazemetostat. Among them, tazemetostat's treatment of metastatic/locally advanced epithelioid sarcoma that is not suitable for surgical treatment has been granted priority review by the US FDA, indicating that epigenetic drugs represented by EZH2 inhibitors are getting more and more attention.

However, the existing EZH2 inhibitors still have many shortcomings in actual clinical trials and have poor responsiveness in many solid tumors. Therefore, the development of new EZH2 inhibitors has become one of the key issues in the field of tumor drug research.

Summary of the invention

The present invention aims to develop new EZH2 inhibitors to further develop anti-tumor drugs, especially anti-tumor drugs for solid tumors with high expression of EZH2.

The purpose of the present invention is to provide an anti-tumor polypeptide that can inhibit the activity of EZH2.

Another object of the present invention is to provide the application of the polypeptide in the preparation of anti-tumor drugs.

Another object of the present invention is to provide an antitumor drug containing a polypeptide that inhibits EZH2 activity.

The above objectives of the present invention are achieved through the following technical solutions:

We took renal clear cell carcinoma as the research object. The study found that the prerequisite for EZH2 to promote cancer is the absence of fructose-1,6,-bisphosphatase 1 (FBP1). FBP1 is a key enzyme in the gluconeogenesis metabolic pathway, and its expression is down-regulated in a variety of tumors. Experimental studies have shown that FBP1 can inhibit glycolysis and metabolism, thereby preventing tumor cells from obtaining sufficient energy and metabolic intermediate products to inhibit tumor cell growth. In addition, FBP1 can also enter the nucleus to perform non-classical enzymatic activity functions, such as inhibiting hypoxia-inducible factor, Wnt/b-actenin signaling and GTPase activation interacting protein 1, etc., suggesting that the metabolic enzyme FBP1 can be inhibited through a variety of ways Tumor growth. Studies have found that FBP1 can directly affect the activity of PRC2 methyltransferase by binding to EZH2. Therefore, we propose that the key peptides in the binding region of FBP1 and EZH2 can interfere with the function of EZH2 and achieve the effect of inhibiting the growth of tumor cells. The invention can provide new polypeptide molecules that can inhibit EZH2 and help the development of new anti-tumor drugs.

Therefore, designing small-molecule peptides based on the key peptides in the binding region of FBP1 and EZH2 can inhibit the activity of EZH2, thereby preventing tumors.

The design principles of the small molecule polypeptide sequence are:

Wherein X means that the 5'end of the LVAAGYALYGSATMLVLAMDCGVNCFMLDP fragment in the binding region of FBP1 and EZH2 continues to be extended by 0-20 amino acids.

That is, the anti-tumor polypeptide developed by the present invention that can inhibit the activity of EZH2 has the shortest sequence shown in SEQ ID NO. 1: MLVAAGYALYGSATMLVLAMDCGVNCFMLDP

Further, this polypeptide fragment can be lengthened at the 5'end according to the above design principles, such as SEQ ID NO. 2: MKSTDEPSEKDALQPGRNLVAAGYALYGSATMLVLAMDCGVNCFMLDP

The nucleic acid sequences encoding the polypeptides shown in SEQ ID NO. 1 and SEQ ID NO. 2 are shown in SEQ ID NOs. 3 and 4, respectively.

(SEQ ID NO.3) Nucleic acid sequence encoding small molecule polypeptide AA2: ATGCTGGTGGCAGCCGGCTACGCACTGTATGGCAGTGCCACCATGCTGGTCCTTGCCATGGACTGTGGGGTCAACTGCTTCATGCTGGACCCG

(SEQ ID NO.4) Nucleic acid sequence encoding the small molecule polypeptide AA1: ATGAAATCAACTGATGAGCCTTCTGAGAAGGATGCTCTGCAACCAGGCCGGAACCTGGTGGCAGCCGGCTACGCACTGTATGGCAGTGCCACCATGCTGGTCCTTGCCATGGACTGTGGGGTCAACTGCTTCATGCTGGACCCG

The aforementioned small molecule polypeptides can specifically inhibit the activity of the epigenetic factor EZH2, block the cancer-promoting effect of EZH2 in solid tumors, and achieve the effect of inhibiting the growth of tumor cells.

The specific polypeptide activity verification scheme is as follows:

First, after knocking down EZH2 in renal clear cell cancer cell lines, the content of H3K27me3 decreased significantly, and the proliferation rate of renal clear cell cancer cells was also significantly reduced, which proved the necessity of EZH2 for the growth of renal cancer cells (Figure 1). Then we explored the potential interaction between FBP1 and EZH2 based on our previous studies on the gluconeogenic metabolism enzyme FBP1. Specifically, HEK-293T was transiently transfected with tagged FBP1 or EZH2 plasmids, and then the protein immunoprecipitation experiment was performed. The results show that FBP1 and EZH2 do combine with each other (Figure 2).

In order to analyze the interaction region between FBP1 and EZH2, the 7 exons of FBP1 were deleted from cDNA by mutation technology, and 7 FBP1 mutants lacking different exons were constructed. Then these FBP1 mutants were transiently transfected into cells expressing EZH2, and then co-immunoprecipitation experiments were performed. The results showed that exon 4 of FBP1 and EZH2 combined with each other. Through further structural analysis and mutation experiment verification, it was finally found that the serine at position 169 on FBP1 was the key site of the interaction. The FBP1S169A mutation can severely disrupt the interaction with EZH2 (Figure 3).

Based on the above research, we speculate that the polypeptide fragment near S169 on the FBP1 protein can inhibit its methyltransferase activity and exert its anti-tumor function by binding to EZH2. Therefore, we intercepted two polypeptide fragments (AA1 and AA2) with relatively complete secondary structure near residue S169 based on the structure of FBP1, and constructed corresponding expression vectors (Figure 4). After that, the two polypeptides were expressed separately, and the effects of AA1 and AA2 on EZH2 activity were detected by quantitative PCR. The results showed that in the two kinds of kidney cancer cells, AA1 and AA2 had different degrees of significant inhibitory effects on EZH2 activity (Figure 5).

The two polypeptide fragments (AA1 and AA2) were packaged by virus and stably transfected into renal cancer cells to test their inhibitory effects on tumor cell proliferation. The CCK-8 experiment showed that the inhibitory effects of peptides AA1 and AA2 on renal cancer cells were consistent with the inhibitory effects on EZH2 activity: AA1 and AA2 both significantly inhibited the growth of RCC4 renal cancer cells, while in RCC10 renal cancer cells, AA2 played a more important role. Significant inhibitory effect (Figure 5). The above results suggest that the two polypeptides have a significant inhibitory effect on the proliferation of renal clear cell carcinoma.

Therefore, the application of the polypeptide in the preparation of EZH2 inhibitors and the application in the preparation of anti-tumor drugs should all fall within the protection scope of the present invention.

In addition, anti-tumor drugs containing the above-mentioned polypeptides should also fall within the protection scope of the present invention.

Specifically, the tumor is a solid tumor with high expression of EZH2. Such as kidney cancer, liver cancer, breast cancer, lung cancer, colorectal cancer, etc.

The present invention has the following beneficial effects:

The present invention is based on the protein interaction between FBP1 and EZH2 obtained through research. FBP1 can interfere with the binding of EZH2 and EED, affect the integrity of the PRC2 complex, thereby reducing the activity of histone methyltransferase in the PCR2 complex, and The identified corresponding peptide fragments and specific sites of the interaction between FBP1 and EZH2 provide a design scheme for small molecule peptides that inhibit EZH2 activity and thus anti-tumor, and construct peptide molecules derived from FBP1 truncated bodies, all of which have targets To EZH2 and inhibit the role of tumors. Moreover, the peptide has a small molecular weight and is easy to penetrate cell membranes. It can directly inhibit the activity of EZH2 methyltransferase, block the effect of EZH2 in promoting the growth of cancer cells, and achieve the effect of resisting renal clear cell carcinoma and other solid tumors with high EZH2 expression. The anti-tumor drugs for solid tumors with high expression of EZH2 have very good application value and prospects.

Description of the drawings

Figure 1 shows that knocking down EZH2 inhibits the growth of renal clear cell carcinoma cells.

Figure 2 shows the interaction between EZH2 and FBP1 protein.

Figure 3 shows the interaction site between EZH2 and FBP1.

Figure 4 is a diagram of the polypeptide structure and encoding gene vector.

Figure 5 shows the inhibition of EZH2 activity after polypeptide expression.

Figure 6 shows the inhibition of the growth of renal tumor cells after polypeptide expression.

Detailed ways

The present invention will be further described below with reference to the drawings and specific embodiments of the specification, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

Various materials and reagents used in the present invention: HEK-293T, RCC4, RCC10 and UMRC2, the above cells use DuLbecco's modified eagle medium (DMEM) medium, plus 10% fetal bovine serum (Hyclone) and 1% Penicillin-Streptomycin Solution (Hyclone) cultured under 5% carbon dioxide, 37°C. Other reagents include PEI (Polyethylenimine, Linear, MW 25000, Polysciences Inc.), streptavidin-bound magnetic beads (Promega, Z5482), Cell Counting Kit-8 (Biyuntian), GSK126 (MCE), FBP1 (Abcam ), EZH2 (CST), HA (CST), Puromycin (Sigma), Q5 High-Fidelity DNA Polymerase (NEB), RNA extraction kit (AXYGEN), reverse transcription kit (TAKARA), qPCR detection kit (full format gold).

The various experimental operation steps of the following examples are as follows:

(1) Plasmid construction:

The mRNA of HEK-293T cells was extracted, reverse transcribed into cDNA, and the cDNA was used as a template to amplify EZH2 and FBP1 genes by designing primers and applying PCR technology. After ligating the vector and the amplified fragment, it was transformed into E. coli competent In the cell Stabl3, positive clones were selected on an agar plate containing ampicillin and sent to Shenggong Bioengineering (Shanghai) Co., Ltd. for first-generation sequencing verification.

(2) Virus packaging and infection:

HEK-293T cells were inoculated on a 10cm culture dish at 50% confluence. After the cells were attached for 24 hours, the cells were combined with the lentivirus expression plasmid (10μg), virus packaging plasmid (psPAX2, 5μg) and envelope plasmid ( pMD2.G, 2μg) co-transfected. Following the manufacturer’s recommendations, 48 hours after transfection, the virus supernatant was collected and filtered through a 0.45 μm pore size. For virus infection, inoculate the target cells into a 6-well plate, add an appropriate amount of the collected virus solution and polybrene at a final concentration of 8μg/ml for 24 hours, then trypsinize the cells into a 10cm culture dish, add antibiotics Screen the stably transfected cells for subsequent experiments.

(3) Real-time fluorescent quantitative PCR:

According to the manufacturer's instructions, the RNA Midiprep kit (Axygen, AP-MN-MS-RNA-250) was used to extract total RNA from the cells. Using RNA as a template, cDNA was generated by reverse transcriptase PCR in a reverse transcription kit (Takara, RR036A). Use TB Green Premix Ex Taq qPCR kit (Transgen Biotech, AQ101) for real-time quantitative PCR. The ribosomal component 18S is used as a housekeeping gene for standardization. The primer sequence is self-designed or obtained from PrimerBank (https://pga.mgh.harvard.edu/primerbank/).

(4) Western blotting:

After adding protease inhibitor lysate to RIPA buffer (1% Triton X-100 or NP-40) to lyse the cells, use BCA protein to quantify and then add SDS loading buffer, heat at 95°C for 10 minutes, and then sample the protein with the same quality (10-50μg) SDS-PAGE electrophoresis. After electrophoresis, the protein on the PAGE gel was transferred to the PVDF membrane through the membrane transfer system, and the primary antibody and secondary antibody were incubated respectively. The antibodies used include: EZH2 (Cell Signaling Technology, 5246), FBP1 (Abcam, ab109732), HA-Tag (Cell Signaling Technology, 3724).

(5) Co-immunoprecipitation:

After adding the protease inhibitor lysis buffer to the 1% NP-40 IP lysis buffer to lyse the cells, the BCA protein quantification method was used to determine the protein quality in the lysis buffer. Save 50μL of lysate for input control, and take the remaining protein lysate of the same quality and add it to streptavidin-bound magnetic beads (Promega, Z5482), incubate in a 4℃ refrigerator with slow rotation for 4h, and then use IP The lysate was washed with the magnetic beads 4 times, and finally the protein bound to the magnetic beads was eluted with 2×SDS loading buffer for Western blotting.

(6) Cell proliferation experiment:

The cells were seeded into a 96-well plate at a density of 1000 cells/well by counting, and cultured in an incubator overnight. Change the fresh medium every 3 days, and use Cell Counting Kit-8 (Biyuntian) to detect cell proliferation.

(7) Statistical analysis method:

The software analysis uses SPSS, and the experimental results are expressed as mean±standard error (Mean±SD). In the experiment, the difference between the two groups divided into two groups is compared by Student’s-T test, and P<0.05 is a statistically significant difference. Among them, P<0.05 is marked as *; P<0.01 is marked as **; P<0.001 is marked as ***; n.s. means that the difference is not statistically significant.

Example 1 Knockdown of EZH2 inhibits the growth of renal clear cell carcinoma

1. Infect the renal clear cell carcinoma cells UMRC2 and 786-O with lentiviral particles carrying two kinds of EZH2 shRNA. Part of the cells were detected by Western blotting with EZH2 antibody, and the other part of the cells were screened by antibiotics and inoculated into a 96-well plate, cultured in a cell incubator and subjected to a CCK-8 experiment to detect cell growth. After culturing for 24 hours, take a 96-well plate for CCK-8 detection, which is used as a calibration for the initial amount of inoculated cells. After that, the cells in the 96-well plate were replaced with new medium every 3 days, and a 96-well plate was taken every one to two days to detect the proliferation of the cells with CCK-8. The method of using CCK-8 is to dilute the stock solution of CCK-8 with the culture medium 1:10, then remove the culture medium from the plate, add 100μL of the diluted solution, place it in a 37℃ incubator for 2 hours, and finally use an enzyme label The meter detects the absorbance value of OD 450nm.

2. The results showed that the EZH2 protein in two renal clear cell carcinoma cell lines was knocked down by EZH2shRNA (Figure 1A). The level of H3K27me3 decreased with the knockdown of EZH2. Both EZH2 shRNAs with different sequences significantly inhibited the growth of renal clear cell carcinoma cells (Figure 1B), suggesting that EZH2 expression is necessary for the growth of renal cancer cells.

Example 2 Interaction between FBP1 and EZH2

1. Experiment 1:

First, HEK-293T cells were stably infected with BirA expressing biotin ligase, and after obtaining cells stably expressing BirA, they were then infected with EZH2 (EZH2-Avi) virus packaged with a biotin label. Finally, the cells were transfected with PEI. Transfection reagent for transient transfection of FBP1 plasmid. The 293T cells stably expressing BirA and transiently transfected with FBP1 were used as the control group, and the cells stably expressing BirA, EZH2-Avi and transiently transfected with FBP1 were used as the experimental group of cells. The two groups of cells were lysed with IP cell lysate, and after BCA After protein quantification, take 50uL cell lysate as input control, and add the remaining cell lysate to streptavidin magnetic beads with the same protein quality for protein immunoprecipitation. The final sample is used for Western blot detection , The result is shown in Figure 2A. Another experiment carried out at the same time was to infect BirA-expressing cells with FBP1 (FBP1-Avi) packaged with biotin tags, and then use PEI transfection reagent for transient transfection of EZH2 plasmid. Then 293T stably expressing BirA and transiently transfected with EZH2 were used as the control group, and cells stably expressing BirA, FBP1-Avi and transiently transfected with EZH2 were used as the experimental group of cells. The two groups of cells were lysed with IP cell lysate. After BCA protein quantification method, take 50uL cell lysate as input control, and add the remaining cell lysate to streptavidin magnetic beads according to the same protein quality for protein immunoprecipitation. The final sample is used for western blotting The test results are shown in Figure 2B.

2. Experiment 2:

In order to further explore the specific area of the interaction between FBP1 and EZH2, 7 exons of FBP1 were deleted by PCR mutation technology, and 7 FBP1 mutants lacking different exons were constructed. Then these FBP1 mutants were transiently transfected into EZH2-Avi-expressing cells, and streptavidin magnetic beads were used for protein immunoprecipitation. The results showed the interaction between FBP1 mutants that lacked exon 4 and EZH2 Significantly weakened, as shown in Figure 3A. Then, FBP1 exon 4 and EZH2 protein were analyzed on the SPPIDER (http://sppider.cchmc.org/) interaction site, and the most likely site Serine 169 (S169) was selected for follow-up research. The serine at position 169 of FBP1 was mutated to alanine (S169A) by PCR. After that, EZH2-avi was stably transfected into HEK-293T cells containing BirA, and FBP1WT and S169A mutant plasmids were transiently transfected with PEI transfection reagent. After quantification by BCA protein quantification method, 50uL cell lysate was taken as input control, and the remaining cell lysate was added to streptavidin magnetic beads with the same protein quality for protein immunoprecipitation, and the final sample was used for protein Blot detection, the experimental results show that the 169th serine of FBP1 is an important site for interaction with EZH2, as shown in Figure 3B.

Example 3 Construction of an anti-tumor polypeptide that inhibits EZH2

Based on the experimental results of Example 2, it is known that the 169th serine of exon 4 of FBP1 is an important binding site of EZH2. According to the crystal structure of FBP1 (PDB: 5et6) (Figure 4A), two peptides based on the EZH2-FBP1 interaction were designed, namely MKS TDE PSE KDA LQP GRN LVA AGY ALY GSA TML VLA MDC GVN CFM LDP (named AA1) and MLV AAG YAL YGS ATM LVL AMD CGV NCF MLD P (named AA2). AA1 is all the amino acids in exon 4 of FBP1, while AA2 only contains three amino acids in the beta-fold structural sequence. Afterwards, the nucleic acid coding sequence of the polypeptide fragment was cloned into a plasmid by PCR technology. The amplification primer sequences are:

AA1: Upstream: 5’-ctagTctagaatgctggtggcagccggctacg-3’

Downstream: 5’-cgcGGATCccgggtccagcatgaagca-3’

AA2: Upstream: 5’-CTAGTctagaatgaaatcaactgatgagCCTTCTGAG-3’

Downstream: 5’-ATATAGGATCccgggtccagcatgaagcAG-3’

The vector map is shown in Figure 4B.

The polypeptide expression plasmid is stably expressed in renal cancer cells through virus packaging and infection methods, and then the control group and the experimental group's RNA are extracted separately through the AXYGEN RNA extraction kit, and then the TAKARA reverse transcription kit is used The mRNA was reverse transcribed, and finally the expression of small molecule polypeptides was detected by quantitative PCR. The ΔCT values of AA1 and AA2 of RCC4 were 5.25 and 5.00 (corrected with ACTB as the internal reference gene). The ΔCT values of AA1 and AA2 in RCC4 were 5.96 and 6.04, respectively (with ACTB as the internal reference gene correction). The primer sequence for detecting expression is as follows:

Upstream primer: 5-ctggtggcagccggctacgCAC-3

Downstream primer: 5-CGTAGAATCGAGACCGAGGAGA-3

The results indicate that the polypeptides AA1 and AA2 are expressed in both kidney cancer cells.

Example 4 Anti-tumor polypeptide inhibits the activity of EZH2

Take the reverse transcription product in Example 3 and detect the expression of EZH2 downstream target genes by quantitative PCR to evaluate the effect of AA1 and AA2 on EZH2 activity. In RCC4 renal cancer cells, the inhibitory effect of AA1 on EZH2 activity was very significant (Figure 5A). In RCC10 kidney cancer cells, the activity of AA2 on EZH2 was higher than that of AA1 (Figure 5B).

Example 5 Anti-tumor polypeptide inhibits the activity of EZH2

The two peptide fragments (AA1 and AA2) were respectively packaged by virus and stably transfected into renal cancer cells RCC4 and RCC10. After antibiotic screening, the renal cancer cells in the logarithmic growth phase were inoculated with 1000 cells per well. In a 96-well plate, 3 replicate wells are inoculated for each treatment, and the inoculated cells are cultured in a cell incubator. After culturing for 24 hours, take a 96-well plate for CCK-8 detection as the starting amount of inoculated cells for calibration. The method of using CCK-8 is to dilute the stock solution of CCK-8 with the culture medium 1:10, then remove the culture medium from the plate, add 100μL of the diluted solution, place it in a 37℃ incubator for 2 hours, and finally use an enzyme label The meter detects the absorbance value of OD 450nm. After that, the cells in the 96-well plate were replaced with new medium every 3 days, and a 96-well plate was taken every two days to detect the proliferation of the cells with CCK-8. The inhibitory effect of polypeptides AA1 and AA2 on renal cancer cells (Figure 6) is consistent with the inhibitory effect on EZH2 activity (Figure 5). Both AA1 and AA2 significantly inhibited the cell growth of RCC4, and the inhibitory effect of AA1 was stronger than that of AA2 (Figure 6A). In contrast, in RCC10 cells, AA2 exerted a more significant inhibitory effect (Figure 6B). The above results suggest that the effects of the two polypeptides on the proliferation of renal clear cell carcinoma are cell-specific, but at least one of them has a significant inhibitory effect and can be used as a starting point for the development of potent EZH2 inhibitors.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, etc. made without departing from the spirit and principle of the present invention Simplified, all should be equivalent replacement methods, and they are all included in the protection scope of the present invention.

Claims

An anti-tumor polypeptide capable of inhibiting the activity of EZH2, characterized in that it is designed according to the key peptides in the binding region of FBP1 and EZH2, and the design principle is: M+X+LVAAGYALYGSATMLVLAMDCGVNCFMLDP;

Wherein X means that the 5'end of the LVAAGYALYGSATMLVLAMDCGVNCFMLDP fragment in the binding region of FBP1 and EZH2 continues to be extended by 0-20 amino acids.
The anti-tumor polypeptide capable of inhibiting EZH2 activity according to claim 1, characterized in that its sequence is shown in SEQ ID NO. 1: MLVAAGYALYGSATMLVLAMDCGVNCFMLDP.
The anti-tumor polypeptide capable of inhibiting the activity of EZH2 according to claim 1, characterized in that its sequence is shown in SEQ ID NO. 2: MKSTDEPSEKDALQPGRNLVAAGYALYGSATMLVLAMDCGVNCFMLDP.
The nucleic acid sequence encoding the anti-tumor polypeptide shown in SEQ ID NO.1 is characterized in that it is shown in SEQ ID NO.3.
The nucleic acid sequence encoding the anti-tumor polypeptide shown in SEQ ID NO.2 is characterized in that it is shown in SEQ ID NO.4.
Use of the polypeptide of any one of claims 1 to 3 in the preparation of an EZH2 inhibitor.
The use of the polypeptide of any one of claims 1 to 3 in the preparation of anti-tumor drugs.
The application according to claim 7, wherein the tumor is a solid tumor with high expression of EZH2.
An anti-tumor drug, characterized in that it contains the polypeptide of any one of claims 1-3.
The medicine according to claim 9, wherein the tumor is a solid tumor with high expression of EZH2.