PRMT5 derived polypeptide and application thereof

Abstract

The invention provides an anti-tumor PRMT5 derived polypeptide and an application thereof. According to a molecular docking simulation result, a PRMT5-derived peptide P20 capable of blocking the combination of PRMT5 and EphA2 is designed, and it is proved that the PRMT5-derived peptide P20 synthesized in vitro can enter tumor cells, block the combination of PRMT5 and EphA2, enable the R816 site of EphA2 not to be methylated, increase the combination of Cbl and EphA2, promote ubiquitination degradation of EphA2, inhibit nasopharyngeal carcinoma cell dryness, increase chemotherapy sensitivity and inhibit in-vivo tumor initiation ability. The obvious anti-tumor effect is realized. Therefore, the PRMT5 derived peptide P20 can be used for preparing antitumor drugs and has important clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drug development and treatment, and specifically relates to an anti-tumor PRMT5 derivative polypeptide and an application thereof. Background Art

[0002] Protein arginine methylation is one of the most common post-translational modifications, mainly referring to the transfer of the methyl group on S-adenosylmethionine to the guanidinium nitrogen atom of the protein arginine side chain under the regulation of protein arginine methyltransferases (PRMTs). PRMTs can be divided into three types according to the different forms of methylated arginine generated: type I (PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, PRMT8) mainly catalyzes the formation of monomethylarginine and asymmetric dimethylarginine, type II (PRMT5, PRMT9) mainly catalyzes the formation of monomethylarginine and symmetric dimethylarginine, and type III (PRMT7) mainly catalyzes the formation of monomethylarginine.

[0003] PRMT5 is a major type II protein arginine methylase, which is widely expressed in eukaryotes. The structure of PRMT5 includes a triosephosphate isomerase (TIM) domain at the N-terminus, a Rossmann-fold domain in the middle, and a β-barrel domain at the C-terminus. PRMT5 usually binds to MEP50 to form a hetero-octamer complex, thereby maintaining its methylase activity. As an epigenetic enzyme, PRMT5 participates in a variety of biological processes, including transcriptional regulation, RNA metabolism, and cell cycle regulation. In addition, PRMT5 is highly expressed in a variety of tumors, promoting tumor cell proliferation and differentiation, invasion and migration, and maintaining tumor cell stemness. Therefore, PRMT5 has attracted attention as an important target for anticancer drugs, and a variety of PRMT5 inhibitors have entered clinical trials. However, no inhibitors have been approved for marketing because their specificity and safety have not been improved.

[0004] EphA2 belongs to the receptor tyrosine kinase (RTK) superfamily. It is a transmembrane glycoprotein composed of 976 amino acids and a molecular weight of 130kD. The EphA2 protein consists of three domains: the N-terminal extracellular domain, the transmembrane domain, and the C-terminal intracellular domain. The C-terminal intracellular domain of EphA2 is mainly composed of the protein kinase region and the SAM (sterile alpha motif) region. A large number of studies have shown that EphA2 is highly expressed in many tumors, and has the effects of promoting tumor cell growth, invasion and metastasis, enhancing tumor angiogenesis, and maintaining tumor stemness. High expression of EphA2 is also closely related to tumor progression and poor prognosis of patients.

[0005] Both PRMT5 and EphA2 are potential anticancer targets, but the interaction between the two proteins and the arginine methylation modification of EphA2 have not been reported so far. The present invention uses immunoprecipitation-high-throughput mass spectrometry analysis technology (targeted proteomics technology) to screen the interacting proteins of nasopharyngeal carcinoma cell EphA2, and finds that PRMT5 is an interacting protein of EphA2, and EphA2 has arginine methylation modification. Immunoprecipitation experiments confirm that PRMT5 is an interacting protein of nasopharyngeal carcinoma cell EphA2, and exogenous PRMT5 and EphA2 also interact in HEK293 cells. GST Pull-down experiments confirm that PRMT5 and EphA2 proteins can interact directly. Protein interaction is a major component of the cell biochemical reaction network and is involved in regulating various physiological functions of cells. For example, protein interaction regulates cell function and its signal transduction by regulating protein modification, stability and subcellular localization. Studies have shown that protein interaction is not only closely related to tumors, but also an important drug target. Peptides that regulate protein interaction are expected to become new anticancer drugs. Therefore, based on the fact that PRMT5 and EphA2 are important tumor proteins and their interaction, blocking the interaction between PRMT5 and EphA2 is likely to lead to the development of new anti-tumor drugs. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-tumor PRMT5-derived polypeptide and its application. The present invention is based on our discovery of a new mechanism: PRMT5 binds to EphA2 through the N-terminal 251-324 amino acids and methylates EphA2 at the R816 site, inhibiting Cbl-mediated ubiquitination and degradation of EphA2, stabilizing EphA2 and promoting tumor development. Further, based on the molecular simulation docking results, a PRMT5 279-298 amino acid derivative polypeptide P20 was designed and invented. The P20 polypeptide can specifically block the interaction between PRMT5 and EphA2, reduce the methylation of the EphA2 R816 site, increase the binding of Cbl to EphA2, promote the ubiquitination and degradation of EphA2, and target downregulate the level of EphA2, thereby inhibiting the stemness of nasopharyngeal carcinoma cells, increasing chemotherapy sensitivity, and inhibiting the ability of tumor initiation in vivo.

[0007] The main contents of the invention are as follows:

[0008] The PRMT5-derived polypeptide is derived from amino acids 279-298 of the PRMT5 protein, and the polypeptide sequence is SYLQYLEYLSQNRPPPNAYE.

[0009] The polypeptides include a series of polypeptides with anti-tumor effects obtained by modifying the N-terminus or C-terminus, deleting, replacing, cyclizing or chiral transformation of amino acids on the basis of the polypeptide sequence.

[0010] The second object of the present invention is to provide a nasopharyngeal carcinoma therapeutic preparation, including the PRMT5-derived polypeptide or a polypeptide series with anti-tumor effect obtained by modifying the N-terminus or C-terminus, deleting, replacing, cyclizing or chiral transformation of amino acids based on the PRMT5-derived polypeptide sequence.

[0011] The third object of the present invention is to provide the use of the anti-tumor polypeptide in the preparation of anti-tumor drugs.

[0012] The tumor type is nasopharyngeal carcinoma.

[0013] The fourth object of the present invention is to provide the use of the polypeptide in preparing a preparation for blocking PRMT5 from binding to EphA2.

[0014] The fifth object of the present invention is to provide the use of the polypeptide in preparing a preparation for reducing the symmetrical methylation of arginine at the R816 site of EphA2.

[0015] The sixth object of the present invention is to provide the use of the polypeptide in preparing a preparation for promoting the binding between EphA2 and Cbl.

[0016] The seventh objective of the present invention is to provide the use of the polypeptide in the preparation of a preparation for promoting the ubiquitination and degradation of EphA2.

[0017] The present invention uses targeted proteomics technology to screen the interacting proteins of nasopharyngeal carcinoma cell EphA2, and finds that PRMT5 is an interacting protein of EphA2. Immunoprecipitation confirms that PRMT5 is an interacting protein of nasopharyngeal carcinoma cell EphA2 and can methylate EphA2. Proximity ligation experiments also confirm that there is an interaction between nasopharyngeal carcinoma cell PRMT5 and EphA2. Exogenous PRMT5 in HEK293 cells can also interact with EphA2 and methylate EphA2. GST Pull-down experiments confirm that PRMT5 and EphA2 proteins can directly interact. In vitro methylation experiments confirm that PRMT5 can methylate EphA2. See Figure 1 .

[0018] The present invention found that PRMT5 can inhibit the ubiquitination degradation of EphA2 in nasopharyngeal carcinoma cell lines, significantly increase the stability of EphA2, and prolong the half-life of EphA2. Figure 2 .

[0019] The present invention uses targeted proteomics technology to identify the methylation site of EphA2, and immunoprecipitation experiments confirm that PRMT5 methylates the R816 site of EphA2. Figure 3 .

[0020] The present invention discovered the mechanism by which PRMT5 stabilizes EphA2: PRMT5 reduces the binding of EphA2 to E3 ubiquitin ligase Cbl by methylating the R816 site of EphA2, inhibiting Cbl-mediated ubiquitination and degradation of EphA2, and increasing the stability of EphA2. Figure 4 .

[0021] The present invention determines the interaction region between PRMT5 and EphA2: construct various truncated mutants of PRMT5 and EphA2, and use immunoprecipitation method to analyze the interaction between truncated PRMT5 and EphA2. It is found that the interaction region between PRMT5 and EphA2 is located at the 251-324 amino acids of the N-terminus of PRMT5 and the 707-807 amino acids of EphA2. Further, using the molecular simulation docking method, the 251-324aa domain of PRMT5 and the 707-807aa domain of EphA2 were used for molecular docking, and it was found that EphA2 binds to the 279-298 amino acids of PRMT5. See Figure 5 .

[0022] In summary, the present invention found that PRMT5 binds to EphA2 and methylates the R816 site of EphA2 in nasopharyngeal carcinoma cells, inhibits the ubiquitination degradation of EphA2, and stabilizes EphA2. Among them, the N-terminal amino acids 251-324, especially the 279-298 amino acids, of PRMT5 are the key regions for PRMT5 to bind to EphA2. The research results provide theoretical and experimental basis for the 279-298 amino acid polypeptide of the N-terminal of PRMT5 as a therapeutic polypeptide for nasopharyngeal carcinoma.

[0023] The present invention found that the in vitro synthesized PRMT5 N-terminal 279-298 amino acid polypeptide P20 can enter tumor cells, significantly inhibit the binding of PRMT5 and EphA2 in cells, reduce the methylation of the R816 site of EphA2, promote the binding of Cbl and EphA2, reduce the stability of EphA2, lead to the ubiquitination degradation of EphA2, and reduce the level of EphA2 in a dose-dependent manner. Figure 6 .

[0024] The present invention found that P20 polypeptide can significantly inhibit the stemness characteristics of nasopharyngeal carcinoma cells and increase the chemotherapy sensitivity of cisplatin by promoting the ubiquitination degradation of EphA2 in nasopharyngeal carcinoma cells. Figure 7 .

[0025] The present invention found that the P20 polypeptide can significantly inhibit the stemness characteristics of nasopharyngeal carcinoma cells in vivo, thereby inhibiting the tumor initiation ability of nasopharyngeal carcinoma cells. Figure 8 .

[0026] In summary, the present invention found that the PRMT5-derived peptide P20 can target and reduce EphA2, inhibit the stemness characteristics of NPC, enhance chemotherapy sensitivity, and inhibit the tumor initiation ability of NPC cells in vivo.

[0027] The present invention has found through a series of studies that there is an interaction between PRMT5 and EphA2 in nasopharyngeal carcinoma cells, and PRMT5 stabilizes EphA2 by inhibiting the ubiquitination degradation of EphA2. Among them, the 20 amino acids at the N-terminus 279-298 of PRMT5 are the key EphA2 binding region. In vivo and in vitro experiments have confirmed that the PRMT5 N-terminal amino acid polypeptide at the 279-298 position can enter nasopharyngeal carcinoma cells, inhibit the binding of PRMT5 and EphA2 in cells, promote the degradation of EphA2, inhibit the stemness of nasopharyngeal carcinoma cells, increase chemotherapy sensitivity and inhibit the tumor initiation ability in vivo, and achieve the effect of treating tumors. This polypeptide has obvious anti-tumor effect and no obvious toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 .Discovered and confirmed that PRMT5 interacts with EphA2 protein and methylates EphA2

[0029] A: Mass spectrometry identified PRMT5 as an interacting protein of EphA2;

[0030] B is an immunoprecipitation experiment confirming the interaction between PRMT5 and EphA2 in nasopharyngeal carcinoma cell lines;

[0031] C is the proximity ligation experiment confirming the interaction between PRMT5 and EphA2 in nasopharyngeal carcinoma cell lines;

[0032] D is an immunoprecipitation experiment confirming the interaction between exogenous PRMT5 and EphA2 in HEK293 cells;

[0033] E: GST Pull-down experiment confirms that EphA2 directly interacts with ANXA1.

[0034] F is an immunoprecipitation experiment confirming that knocking down PRMT5 can reduce the symmetrical dimethylation level of EphA2 in nasopharyngeal carcinoma cell lines;

[0035] G is an immunoprecipitation experiment confirming that overexpression of PRMT5 in HEK293 cells can increase the symmetrical dimethylation level of EphA2, while overexpression of enzyme-inactive PRMT5-R368A has no effect on the symmetrical dimethylation level of EphA2;

[0036] H is an in vitro methylation experiment confirming that PRMT5 can directly and symmetrically dimethylate EphA2;

[0037] Among them, Input represents the total protein level, IP represents the immunoprecipitated protein level; GAPDH represents the internal control; IP:EphA2 represents the added antibody EphA2, and the co-precipitated PRMT5 is detected by Western blot (WB); IP:PRMT5 represents the added antibody PRMT5, and the co-precipitated EphA2 is detected by WB; Myc represents Myc tag; Flag represents Flag tag; GST represents GST tag, GST-EphA2 represents EphA2 connected to GST tag, and His-PRMT5 represents PRMT5 connected to His tag.

[0038] Figure 1 The results found and confirmed that PRMT5 interacts with EphA2 in nasopharyngeal carcinoma cells and can methylate EphA2.

[0039] Figure 2 PRMT5 regulates the stability of EphA2 through the ubiquitin-proteasome system

[0040] A is the effect of PRMT5 knockdown on the EphA2 protein level in nasopharyngeal carcinoma cells detected by Western blot. As shown in the figure, knockdown of PRMT5 can reduce the EphA2 protein level;

[0041] B: Knockdown of PRMT5 in nasopharyngeal carcinoma cell lines SUNE1 and HK1 reduced the stability / half-life of EphA2 protein;

[0042] C is Western blot detection of PRMT5 knockdown in SUNE1 and HK1 nasopharyngeal carcinoma cell lines, proteasome inhibitor MG132 can restore the protein level of EphA2;

[0043] D is an immunoprecipitation experiment to detect the effect of PRMT5 knockdown on the ubiquitination level of EphA2 in nasopharyngeal carcinoma cells. The results show that knockdown of PRMT5 promotes the ubiquitination of EphA2 in nasopharyngeal carcinoma cells.

[0044] E is an immunoprecipitation experiment to detect the effects of overexpression of exogenous PRMT5 wild-type and R368A enzyme-inactivated plasmids on exogenous EphA2 ubiquitination in HEK293 cells. The results showed that overexpression of wild-type PRMT5 could inhibit the ubiquitination of EphA2, while overexpression of R368A enzyme-inactivated PRMT5 had no effect on the ubiquitination of EphA2.

[0045] F: Immunoprecipitation experiment confirmed that PRMT5 inhibited the K48 chain polyubiquitination of EphA2.

[0046] Among them, CHX is the protein synthesis inhibitor cycloheximide, MG132 is a proteasome inhibitor, shCtrl represents shRNA interference control, shPRMT5 represents shRNA interference PRMT5, IB: anti-Ub represents ubiquitination detection antibody; EphA2-(Ub)n represents EphA2 polyubiquitination. HA-Ub represents the total ubiquitination plasmid with HA tag, HA-Ubk48 represents the Lys48 ubiquitination plasmid with HA tag, and HA-Ubk63 represents the Lys63 ubiquitination plasmid with HA tag.

[0047] Figure 2 The results showed that PRMT5 increased the stability of EphA2 protein by inhibiting the ubiquitination degradation of EphA2 protein.

[0048] Figure 3 PRMT5 methylates EphA2 at R816

[0049] A is the methylation site of EphA2 identified by mass spectrometry;

[0050] B is an immunoprecipitation experiment to detect the effect of PRMT5 on the symmetric dimethylation of EphA2 after overexpression of EphA2 methylation site mutant plasmid in HEK293 cells. The results show that among the methylation sites identified by mass spectrometry, overexpression of R816 site mutant plasmid has no effect of PRMT5 on the symmetric dimethylation of EphA2.

[0051] Figure 3 The results showed that PRMT5 caused symmetrical dimethylation of the R816 site of EphA2.

[0052] Figure 4 PRMT5 methylates the R816 site of EphA2, reducing the binding of Cbl to EphA2 and thus stabilizing EphA2.

[0053] A is an immunoprecipitation experiment to detect the effects of overexpression of EphA2 wild-type and R816K inactivated mutant plasmids on EphA2 methylation and the binding of EphA2 and Cbl in HEK293 cells. The results showed that compared with wild-type EphA2, inactivation of the EphA2 R816 site reduced the symmetrical dimethylation of EphA2 and promoted the binding of EphA2 and Cbl;

[0054] B is an immunoprecipitation experiment to detect the effect of overexpression of EphA2 wild-type and R816K inactivated mutant plasmids on EphA2 ubiquitination in HEK293 cells. The results showed that compared with EphA2 wild-type, EphA2 R816 site inactivation increased EphA2 ubiquitination levels;

[0055] C is an immunoprecipitation experiment to detect the effects of overexpression of wild-type PRMT5 and R368A enzyme-inactivated mutant plasmids on EphA2 methylation and the binding of EphA2 and Cbl in HEK293 cells. The results showed that overexpression of wild-type PRMT5 increased EphA2 methylation and decreased binding with Cbl, while overexpression of PRMT5 R368A enzyme-inactivated plasmid did not affect EphA2 methylation and the binding of EphA2 and Cbl;

[0056] D is an immunoprecipitation experiment to detect the effects of overexpression of wild-type PRMT5 and R368A enzyme-inactivated mutant plasmids on the ubiquitination level of EphA2 in HEK293 cells. The results showed that overexpression of wild-type PRMT5 reduced the ubiquitination level of EphA2, while overexpression of PRMT5 R368A enzyme-inactivated plasmid did not affect the ubiquitination level of EphA2;

[0057] E is the effect of overexpression of EphA2 wild-type and R816K inactivation mutant plasmids on EphA2 protein stability / half-life in HEK293 cells detected by Western blot. The results showed that EphA2 R816 mutation reduced EphA2 protein stability / half-life;

[0058] F is the effect of EphA2 R816 mutation on EphA2 methylation and the binding of EphA2 to Cbl in nasopharyngeal carcinoma SUNE1 and HK1 cell lines detected by immunoprecipitation experiments. The results showed that EphA2 R816 mutation reduced the symmetrical dimethylation of EphA2 and increased the binding of EphA2 to Cbl.

[0059] G is an immunoprecipitation experiment to detect the effect of EphA2 R816 mutation on the ubiquitination level of EphA2 in nasopharyngeal carcinoma SUNE1 and HK1 cell lines. The results showed that EphA2 R816 mutation increased the ubiquitination level of EphA2;

[0060] H is an immunoprecipitation experiment to detect the effects of overexpression of PRMT5 wild-type and R368A enzyme-inactive mutant plasmids on EphA2 methylation and the binding of EphA2 to Cbl in nasopharyngeal carcinoma SUNE1 and HK1 cell lines with PRMT5 knockdown. The results showed that knockdown of PRMT5 reduced EphA2 methylation and increased binding to Cbl. Knockdown of PRMT5 and overexpression of PRMT5 wild-type restored the methylation level of EphA2 and the binding of EphA2 to Cbl, while overexpression of PRMT5 R368A enzyme-inactive mutant could not restore the methylation level of EphA2 and the binding of EphA2 to Cbl;

[0061] I is an immunoprecipitation experiment to detect the effect of overexpression of PRMT5 wild-type and R368A enzyme-inactive mutant plasmids on the ubiquitination level of EphA2 in nasopharyngeal carcinoma SUNE1 and HK1 cell lines with PRMT5 knockdown. The results showed that knockdown of PRMT5 increased the ubiquitination level of EphA2. Knockdown of PRMT5 and overexpression of PRMT5 wild-type restored the ubiquitination level of EphA2, while overexpression of PRMT5 R368A enzyme-inactive mutant could not restore the ubiquitination level of EphA2;

[0062] J is Western blot detection of EphA2 R816K mutation in nasopharyngeal carcinoma SUNE1 and HK1 cell lines, which reduces EphA2 protein stability / half-life;

[0063] K: Western blot detection of EphA2 R816K mutation in SUNE1 and HK1 nasopharyngeal carcinoma cell lines, proteasome inhibitor MG132 can restore the protein level of EphA2.

[0064] Figure 4 The results showed that PRMT5 methylated the R816 site of EphA2. Methylation of the R816 site of EphA2 reduced the binding of Cbl to EphA2, inhibited the ubiquitination degradation of EphA2 protein, and increased the stability of EphA2 protein.

[0065] Figure 5 . Determine the binding region of EphA2 and PRMT5

[0066] A is a schematic diagram of the constructed EphA2 truncation or deletion mutants;

[0067] B and C are immunoprecipitation experiments to detect the binding of EphA2 truncated or deleted mutants to PRMT5. The results show that EphA2 with amino acid deletions at positions 707-807 cannot bind to PRMT5;

[0068] D is a schematic diagram of the constructed PRMT5 truncation or deletion mutants

[0069] E and F are immunoprecipitation experiments to detect the binding of PRMT5 truncated or deleted mutants to EphA2. The results show that PRMT5 with amino acid deletion at positions 251-324 cannot bind to EphA2;

[0070] G is a molecular docking simulation diagram, and the results show that amino acids 279-298 of PRMT5 bind to EphA2.

[0071] Figure 5 The results showed that amino acids 707-807 of EphA2 bind to PRMT5, and amino acids 279-298 of PRMT5 bind to EphA2.

[0072] Figure 6 PRMT5-derived peptide P20 promotes the ubiquitination and degradation of EphA2 by blocking the binding of PRMT5 to EphA2

[0073] A is a cell penetration experiment of PRMT5-derived peptide P20, i.e., PRMT5 amino acids 279-298. The results showed that FITC-P20 with green fluorescence can enter nasopharyngeal carcinoma SUNE1 and HK1 cell lines;

[0074] B is an immunoprecipitation experiment to detect the effect of P20 on the binding of PRMT5 and EphA2. The results showed that P20 could significantly reduce the binding of PRMT5 and EphA2;

[0075] C is Western blot detection of P20 reducing the protein expression level of EphA2 in nasopharyngeal carcinoma SUNE1 and HK1 cell lines;

[0076] D is Western blot detection of P20 reducing the protein stability / half-life of EphA2 in nasopharyngeal carcinoma SUNE1 and HK1 cell lines;

[0077] E is Western blot detection of the protein level of EphA2 in nasopharyngeal carcinoma SUNE1 and HK1 cell lines treated with P20, in which the proteasome inhibitor MG132 can restore the protein level of EphA2;

[0078] F is an immunoprecipitation experiment to detect the effect of P20 on EphA2 methylation and the binding of EphA2 and Cbl. The results showed that after P20 treatment, EphA2 methylation decreased and the binding of EphA2 and Cbl increased;

[0079] G is an immunoprecipitation experiment to detect the effect of P20 on the ubiquitination level of EphA2. The results showed that after P20 treatment, the ubiquitination level of EphA2 increased;

[0080] H is the effect of P20 on the binding of EphA2 and Cbl detected by cell immunofluorescence. The results showed that after P20 treatment, the co-localization of EphA2 and Cbl in cells increased, and the binding increased.

[0081] Figure 6 The results confirmed that the PRMT5-derived peptide P20 reduced the methylation of EphA2 by reducing the binding of PRMT5 to Cbl, promoting the binding of EphA2 to Cbl, and reducing the protein stability of EphA2.

[0082] Figure 7 .PRMT5-derived peptide P20 inhibits the stemness of nasopharyngeal carcinoma cells by targeting EphA2 degradation and enhances the sensitivity of nasopharyngeal carcinoma cells to chemotherapy

[0083] A: ALDH was detected by ALDEFLUOR kit combined with flow cytometry (FACS) + The results showed that 0, 5 and 10 μM P20 peptides were used to treat nasopharyngeal carcinoma cell lines for 24 h, and P20 peptides dose-dependently reduced ALDH in nasopharyngeal carcinoma SUNE1 and HK1 cells. + Percentage of cells that were transfected with exogenous EphA2 to restore ALDH + The left figure is a representative flow cytometry result, and the right figure is a statistical analysis figure. +DEAB is the quality control group: DEAB is a specific inhibitor of ALDH, so the ALDH of the DEAB treatment group (+DEAB) is + The cell percentage is zero.

[0084] B is an in vitro spheroidization experiment to detect the in vitro spheroidization ability of nasopharyngeal carcinoma cells. The results showed that 0, 5 and 10 μM P20 peptides were used to treat nasopharyngeal carcinoma cell lines for 24 hours, and P20 dose-dependently downregulated the number of spheroids of nasopharyngeal carcinoma SUNE1 and HK1 cells, and transfection of exogenous EphA2 could restore the spheroidization ability of cells. The left figure is a representative cell spheroidization result, and the right figure is the statistical analysis result of counting the number of cell spheres with a diameter greater than 100 μm under an inverted microscope. The scale bar is 100 μm.

[0085] C is the detection of CD133 in nasopharyngeal carcinoma cells by flow cytometry + The results showed that 0, 5 and 10 μM P20 peptides were used to treat nasopharyngeal carcinoma cell lines for 24 h, and P20 peptides dose-dependently reduced the expression of CD133 in nasopharyngeal carcinoma SUNE1 and HK1 cells. + The percentage of cells that were transfected with exogenous EphA2 could restore CD133 + The percentage of cells.

[0086] D is WB detection of the expression levels of NPC stem cell-related markers (Sox2, Nanog, ALDH1A1 and c-Myc). The results showed that 0, 5 and 10 μM P20 peptides were used to treat NPC cell lines for 24 hours, and P20 peptide dose-dependently downregulated the expression levels of tumor stemness markers Sox2, Nanog, ALDH1A1 and c-Myc in NPC SUNE1 and HK1 cells, and transfection of exogenous EphA2 could restore the expression of these four proteins.

[0087] E is the MTT experiment to detect the effect of P20 on the viability of nasopharyngeal carcinoma cells. The results showed that P20 combined with cisplatin treatment could further reduce the viability of nasopharyngeal carcinoma SUNE1 and HK1 cells compared with cisplatin alone, that is, P20 could enhance the sensitivity of nasopharyngeal carcinoma cells to cisplatin;

[0088] F is a cell clone formation experiment to detect the effect of P20 on the proliferation of nasopharyngeal carcinoma cells. The results showed that the number of clones formed by nasopharyngeal carcinoma SUNE1 and HK1 cells after P20 treatment was significantly reduced compared with the control group, and P20 combined with cisplatin treatment can further reduce the number of clones formed, that is, P20 can inhibit the proliferation ability of nasopharyngeal carcinoma cells, and can cooperate with cisplatin to inhibit the proliferation of nasopharyngeal carcinoma cells.

[0089] Figure 7 The results confirmed that the PRMT5-derived peptide P20 could significantly inhibit the stemness characteristics of NPC cells by targeting and degrading EphA2, and enhance the chemosensitivity of NPC cells to cisplatin.

[0090] Figure 8 PRMT5-derived peptide P20 inhibits tumor initiation and stemness characteristics of nasopharyngeal carcinoma in vivo

[0091] A is a general picture of the transplanted tumor in mice treated with P20, and B is a statistical table of tumor occurrence. The results showed that compared with the PBS control group, P20 treatment can reduce the tumor incidence of NPC cells and inhibit the tumor initiation ability of NPC cells in vivo;

[0092] C is the growth curve of mouse transplanted tumor. The results showed that compared with the PBS control group, the volume of transplanted tumors was significantly reduced after P20 treatment, that is, P20 can inhibit the growth of mouse transplanted tumors;

[0093] D is the weight of mouse transplanted tumor. The results showed that P20 treatment could reduce the weight of tumor compared with the PBS control group;

[0094] E: Detection of ALDH in transplanted tumors using the ALDEFLUOR kit combined with flow cytometry (FACS) + The results showed that P20 treatment could downregulate ALDH in transplanted tumors. + The percentage of cells;

[0095] F is the detection of CD133 in transplanted tumor by flow cytometry + The results of the percentage of cells showed that P20 treatment could downregulate CD133 in transplanted tumors. + The proportion of cells;

[0096] G is Western blot detection of the expression levels of stem cell-related markers in transplanted tumors. The results showed that the protein expression levels of Sox2, Nanog, ALDH1A1, and c-Myc in transplanted tumors were significantly reduced after P20 treatment.

[0097] H is the immunohistochemical staining of EphA2, PRMT5, Sox2, Nanog, ALDH1A1 and c-Myc in mouse subcutaneous transplanted tumors. I is the statistical analysis of the immunohistochemical scores of each index. The results showed that P20 significantly reduced the expression of the above indexes in transplanted tumor tissues.

[0098] Figure 8 The results confirmed that PRMT5-derived peptide P20 could inhibit the tumorigenicity of NPC cells in vivo. DETAILED DESCRIPTION

[0099] The following embodiments are intended to further illustrate the present invention, but are not intended to limit the present invention.

[0100] Figure 1A is the mass spectrometry identification of PRMT5 as an interacting protein of EphA2. Nasopharyngeal carcinoma SUNE1 cells were cultured in 1640 medium containing 10% fetal bovine serum. After 48 hours, the cells were collected and the protein was extracted by adding an appropriate amount of NP-40 lysis buffer. 1.2 mg of protein lysate was added with 30 μl of Protein A / G beads and 2 μg of EphA2 antibody at 4°C overnight. The beads were collected and 30 μl of 2×SDS loading buffer was added. The mixture was boiled for 5 min and SDS-PAGE electrophoresis was performed. The gel was stained with Coomassie brilliant blue. Protein bands were cut from the Coomassie stained gel for enzymatic digestion and mass spectrometry analysis to identify the interacting proteins of EphA2. Figure 1 A is the mass spectrometry identification result of PRMT5.

[0101] Figure 1 B is an immunoprecipitation experiment to detect the interaction between PRMT5 and EphA2 in nasopharyngeal carcinoma cell lines SUNE1 and HK1. IP:EphA2: The immunoprecipitation antibody is EphA2, and the co-precipitated PRMT5 is detected by WB; IP:PRMT5: The immunoprecipitation antibody is PRMT5, and the co-precipitated EphA2 is detected by WB. Immunoprecipitation steps: Cell protein was extracted using NP-40 lysis buffer, 1.2 mg of cell protein was added to 30 μl protein A / G beads and 2 μg of corresponding antibodies and incubated at 4°C for 12 hours, centrifuged at 1000 rpm for 3 minutes, and the supernatant was removed. The beads were washed 3 times with NP-40 lysis buffer, the supernatant was removed, 30 μl 2×SDS loading buffer was added, boiled for 5 minutes, and the interacting proteins were detected by Western blot.

[0102] Figure 1 C is the proximity ligation experiment to detect the interaction between PRMT5 and EphA2 in nasopharyngeal carcinoma cell lines SUNE1 and HK1. Prepare cell slides, fix with 4% paraformaldehyde for 10 minutes, block with blocking solution for 40 minutes, and incubate PRMT5 and EphA2 antibodies at 1:100 at 4℃ overnight. Incubate the corresponding MINUS and PLUS probes at 37℃ for 1 hour, then incubate with ligase at 37℃ for 30 minutes, and incubate with polymerase at 37℃ for 100 minutes. Finally, stain the nuclei with DAPI for 10 minutes, seal the slides, and observe the results with a confocal microscope.

[0103] Figure 1D is an immunoprecipitation experiment to detect the interaction between exogenous PRMT5 and EphA2 proteins in HEK293 cells. HEK293 cells were co-transfected with Flag-tagged PRMT5 and Myc-tagged EphA2 expression plasmids. After 48 hours, the whole cell protein was extracted with NP-40 lysis buffer, and the interaction between PRMT5 and EphA2 was examined by immunoprecipitation. IP:Flag: The immunoprecipitation antibody was Flag (PRMT5), and the co-precipitated Myc (EphA2) was detected by WB. IP:Myc: The immunoprecipitation antibody was Myc (EphA2), and the co-precipitated Flag (PRMT5) was detected by Western blot;

[0104] Figure 1 E is a GST Pull-down experiment to detect the direct interaction between EphA2 and PRMT5 protein: First, the GST-EphA2 fusion protein and His-PRMT5 fusion protein were expressed and purified in prokaryotes. Then the Pull-down experiment was performed. The specific experimental steps are as follows: In a 1.5ml EP tube, 5μg of GST-EphA2 fusion protein and control GST protein were added, as well as 5μg of His-PRMT5 fusion protein, 20μl Glutathione Sepharose-4B beads, and GST-Pull down buffer was added to a total volume of 500μl. The beads were combined overnight at 4℃, centrifuged at 1000 rpm for 3min, and the supernatant was removed. 500μl GST-Pull down buffer was added to wash the beads 3 times. After the liquid above the Glutathione Sepharose 4B beads was completely aspirated, 60μl 1×SDS loading buffer was added, boiled for 10min, and the interaction between the two proteins was detected by Western blot using a tag antibody.

[0105] Figure 1 F is the immunoprecipitation detection of the methylation level of EphA2 in nasopharyngeal carcinoma cell lines SUNE1 and HK1 with knockdown of PRMT5. IP: EphA2: The immunoprecipitation antibody is EphA2. Cell protein was extracted with NP-40 lysis buffer, 1.2 mg of cell protein was added with 30 μl protein A / G beads and 2 μg EphA2 antibody, incubated at 4°C for 12 hours, centrifuged at 1000 rpm for 3 minutes, and the supernatant was removed. The beads were washed 3 times with NP-40 lysis buffer, the supernatant was removed, 30 μl 2×SDS loading buffer was added, boiled for 5 minutes, and the methylation level of EphA2 was detected by Western blot.

[0106] Figure 1G is the effect of overexpression of wild-type and enzyme-inactive mutant PRMT5 on EphA2 methylation in HEK293 cells detected by immunoprecipitation. HEK293 cells were transfected with wild-type and enzyme-inactive mutant PRMT5, and co-transfected with EphA2. After 48 hours, the whole cell protein was extracted with NP-40 lysis buffer, and EphA2 was immunoprecipitated and the methylation level of EphA2 was detected by Western blot.

[0107] Figure 1 H is an in vitro methylation experiment to detect the direct methylation effect of PRMT5 on EphA2. HEK293 cells were transfected with wild-type and enzyme-inactive mutant PRMT5, respectively. PRMT5 protein was co-immunoprecipitated, and 5 μg of purified GST-EphA2 protein was added and co-incubated in methyl buffer containing 1 μM methyl donor SAM at 37°C for 75 min. The methylation level of EphA2 was detected by Western blot.

[0108] The present invention uses targeted proteomics technology to screen the interacting proteins of nasopharyngeal carcinoma cell EphA2, and finds that PRMT5 is the interacting protein of EphA2. Immunoprecipitation confirms that PRMT5 is the interacting protein of nasopharyngeal carcinoma cell EphA2, and PRMT5 can methylate EphA2.

[0109] Figure 2 A: PRMT5 knockdown SUNE1 and HK1 nasopharyngeal carcinoma cell lines were established using the shRNA interference method, and the total cell protein was extracted. Western blot was used to detect the effect of PRMT5 on EphA2 protein levels.

[0110] Figure 2 B shows the PRMT5 knockdown in SUNE1 and HK1 nasopharyngeal carcinoma cell lines. The cells were treated with 20 μg / ml of the protein synthesis inhibitor cycloheximide (CHX) for 4 h, 8 h, 12 h and 16 h, and then the cells were collected and the total protein was extracted. The level of EphA2 was detected by Western blot, and the half-life of EphA2 was analyzed.

[0111] Figure 2 C: Changes in EphA2 protein levels detected by Western blot after the addition of proteasome inhibitor MG132 in PRMT5-knockdown SUNE1 and HK1 nasopharyngeal carcinoma cell lines.

[0112] Figure 2D is an immunoprecipitation experiment to detect the effect of knocking down PRMT5 on the ubiquitination level of EphA2 in SUNE1 and HK1 nasopharyngeal carcinoma cells. The whole protein of the cells was extracted using RIPA lysis buffer, and the EphA2 antibody was immunoprecipitated, and the EphA2 polyubiquitination (Ub) level was detected by Western blot.

[0113] Figure 2 E is the effect of overexpression of wild-type and enzyme-inactive mutant PRMT5 on the ubiquitination level of EphA2 in HEK293 cells detected by immunoprecipitation. HEK293 cells were transfected with wild-type and enzyme-inactive mutant PRMT5, and co-transfected with EphA2. After 48 hours, the whole cell protein was extracted with NP-40 lysis buffer, and EphA2 was immunoprecipitated and the polyubiquitination (Ub) level of EphA2 was detected by Western blot.

[0114] Figure 2 F is an immunoprecipitation experiment to detect the type of EphA2 ubiquitination affected by PRMT5. HEK293 cells were co-transfected with the plasmids shown in the figure. After 48 hours, total cell protein was extracted using NP-40 lysis buffer, immunoprecipitated with EphA2 antibody, and the level of EphA2 polyubiquitination (Ub) was detected by Western blot.

[0115] 1. Establishment of PRMT5 knockdown SUNE1 and HK1 nasopharyngeal carcinoma cell lines by shRNA interference method: HEK293 cells were co-transfected with lentiviral vector plasmids carrying PRMT5 interference sequences and lentiviral packaging plasmids. After 48 hours, the supernatant virus solution was collected and added to nasopharyngeal carcinoma SUNE1 and HK1 cells for virus infection. After 72 hours, the cells were screened and cultured with culture medium containing appropriate concentrations of puromycin. Drug-resistant clones were identified by Western blot to establish PRMT5 stable knockdown cell lines.

[0116] 2. Transient cell transfection: The day before transfection, cells were seeded into six-well plates. When the cell confluence reached about 90%, 2 μg of each plasmid to be transfected was added to 100 μl serum-free DMEM medium, mixed evenly and allowed to stand for 5 minutes; Lipofectamine 2000 was added to 100 μl serum-free DMEM medium (plasmid: Lipofectamine 2000 was 1:1.5), mixed gently and allowed to stand for 5 minutes, then the medium containing plasmid and Lipofectamine 2000 was gently mixed, allowed to stand for 15 minutes and added to the six-well plate. After 6-8 hours, fresh DMEM medium containing 10% FBS was replaced and culture was continued. Subsequent experiments were carried out 48 hours after transfection.

[0117] The present invention finds that PRMT5 can inhibit the ubiquitination degradation of EphA2 in nasopharyngeal carcinoma cell lines, significantly maintain the stability of EphA2, and prolong the half-life of EphA2.

[0118] Figure 3 A is the preliminary identification of the methylation site of EphA2 by mass spectrometry. Nasopharyngeal carcinoma SUNE1 cells were cultured in 1640 medium containing 10% fetal bovine serum. After 48 hours, the cells were collected and the protein was extracted by adding an appropriate amount of NP-40 lysis buffer. 1.2 mg of protein lysate was added with 30 μl Protein A / G beads and 2 μg EphA2 antibody at 4°C overnight. The beads were collected and 30 μl 2×SDS loading buffer was added. The mixture was boiled for 5 min and SDS-PAGE electrophoresis was performed. The gel was stained with Coomassie brilliant blue. Protein bands were cut from the Coomassie stained gel for enzymatic digestion and mass spectrometry analysis to identify the methylation sites of EphA2.

[0119] Figure 3 B is an immunoprecipitation experiment to detect the methylation sites where PRMT5 causes symmetrical dimethylation of EphA2. Inactivation mutant plasmids of EphA2 methylation sites identified by mass spectrometry were constructed and transfected into HEK293 cells, and PRMT5 was co-transfected at the same time. Immunoprecipitation was performed with Myc antibody, and the symmetrical dimethylation level of EphA2 was detected by Western blot.

[0120] The present invention determines the site where EphA2 undergoes symmetrical dimethylation, and the symmetrical dimethylation is catalyzed by PRMT5.

[0121] Figure 4 A is an immunoprecipitation experiment to detect the effects of exogenous EphA2 wild-type and R816K inactivated mutant plasmids on EphA2 methylation and the binding of EphA2 and Cbl. HEK293 cells were transfected with EphA2 wild-type and R816K mutant plasmids, respectively, and co-transfected with HA-tagged Cbl plasmids. Myc antibody immunoprecipitation was performed, and Western blot was used to detect Epha2 methylation and the binding of Cbl and EphA2.

[0122] Figure 4 B is an immunoprecipitation experiment to detect the effect of overexpression of exogenous EphA2 wild-type and R816K inactivated mutant plasmids on the ubiquitination level of EphA2. HEK293 cells were transfected with EphA2 wild-type and R816K mutant plasmids, respectively, and co-transfected with HA-tagged Cbl plasmids. Myc antibody immunoprecipitation was performed, and the level of Epha2 polyubiquitination (Ub) was detected by Western blot.

[0123] Figure 4C is an immunoprecipitation experiment to detect the effects of overexpression of exogenous PRMT5 wild-type and R368A enzyme-inactivated mutant plasmids on EphA2 methylation and the binding of EphA2 and Cbl. HEK293 cells were transfected with PRMT5 wild-type and R368A enzyme-inactivated mutant plasmids, respectively, and co-transfected with HA-tagged Cbl and Myc-tagged EphA2 plasmids. Myc antibody immunoprecipitation was performed, and Western blot was used to detect Epha2 methylation and the binding of Cbl and EphA2.

[0124] Figure 4 D is an immunoprecipitation experiment to detect the effect of overexpression of exogenous PRMT5 wild-type and R368A enzyme-inactivated mutant plasmids on the ubiquitination level of EphA2. HEK293 cells were transfected with PRMT5 wild-type and R368A enzyme-inactivated mutant plasmids, respectively, and co-transfected with HA-tagged Cbl and Myc-tagged EphA2 plasmids. Myc antibody was used for immunoprecipitation, and Western blot was used to detect the level of Epha2 polyubiquitination (Ub).

[0125] Figure 4 E is HEK293 cells overexpressing EphA2 wild-type and R816K mutant plasmids, respectively. The cells were treated with 20 μg / ml of the protein synthesis inhibitor cycloheximide (CHX) for 4 h, 8 h, 12 h and 16 h, and then the cells were collected and the total protein was extracted. The level of EphA2 was detected by Western blot, and the half-life of EphA2 was analyzed.

[0126] Figure 4 F is the immunoprecipitation detection of EphA2 methylation level and the binding of EphA2 to Cbl in nasopharyngeal carcinoma SUNE1 and HK1 cell lines with wild-type and R816K mutation of EphA2. SUNE1 and HK1 nasopharyngeal carcinoma cell lines with EphA2 knockdown were constructed, and the two cell lines were infected with EphA2 wild-type and R816K mutant lentiviruses, respectively, to construct nasopharyngeal carcinoma SUNE1 and HK1 cell lines with EphA2 wild-type and R816K mutation, and the whole cell protein was extracted, and the EphA antibody was immunoprecipitated, and the methylation of Epha2 and the binding of Cbl to EphA2 were detected by Western blot.

[0127] Figure 4 G is the ubiquitination level of EphA2 in nasopharyngeal carcinoma SUNE1 and HK1 cell lines with wild-type and R816K mutation of EphA2 detected by immunoprecipitation. Total cell protein was extracted, immunoprecipitated with EphA antibody, and the level of Epha2 polyubiquitination (Ub) was detected by Western blot.

[0128] Figure 4H is the effect of transfection of wild-type PRMT5 and R368A enzyme-inactive mutant plasmids on EphA2 methylation and the binding of EphA2 to Cbl in PRMT5-knockdown nasopharyngeal carcinoma SUNE1 and HK1 cell lines detected by immunoprecipitation. Wild-type and R368A enzyme-inactive mutant PRMT5 were transfected into PRMT5-knockdown SUNE1 and HK1 cell lines, respectively, and EphA2 antibody was used for immunoprecipitation. Epha2 methylation and the binding of Cbl to EphA2 were detected by Western blot.

[0129] Figure 4 I shows the effect of transfection of wild-type PRMT5 and R368A enzyme-inactive mutant plasmids on the ubiquitination level of EphA2 in PRMT5-knockdown nasopharyngeal carcinoma SUNE1 and HK1 cell lines by immunoprecipitation. Wild-type and R368A enzyme-inactive mutant PRMT5 were transfected in PRMT5-knockdown SUNE1 and HK1 cell lines, respectively, and EphA2 antibody was immunoprecipitated, and the level of EphA2 polyubiquitination (Ub) was detected by Western blot.

[0130] Figure 4 J: In EphA2 wild-type and R816K mutant nasopharyngeal carcinoma SUNE1 and HK1 cell lines, cells were treated with 20 μg / ml of the protein synthesis inhibitor cycloheximide (CHX) for 4 h, 8 h, 12 h and 16 h, and the cells were collected and the total cell protein was extracted. The level of EphA2 was detected by Western blot, and the half-life of EphA2 was analyzed.

[0131] Figure 4 K: Changes in EphA2 protein levels detected by Western blot in EphA2 wild-type and R816K mutant nasopharyngeal carcinoma SUNE1 and HK1 cell lines after the addition of proteasome inhibitor MG132.

[0132] The present invention determines that PRMT5 promotes the combination of EphA2 and Cbl, stabilizes the protein level of EphA2, and prolongs the half-life of EphA2 protein by methylating the R816 site of EphA2.

[0133] Figure 5 A is a schematic diagram of the constructed EphA2 truncation and deletion mutants. All mutant plasmids were ordered from GeneCare Genetics.

[0134] Figure 5B and C are immunoprecipitation detection of the binding site of PRMT5 and EphA2. EphA2 1-555aa, 556-605aa, 606-906aa, 907-976aa, 606-706aa, 707-807aa, 808-812aa, 813-817aa, 818-906aa deletion mutants were constructed, wild-type and deletion mutant EphA2 with Myc tag were transfected into HEK293 cells, and PRMT5 with Flag tag was co-transfected at the same time. Immunoprecipitation was performed with Flag tag antibody, and Myc (EphA2) level was detected by Western blot.

[0135] Figure 5 D is a schematic diagram of the constructed PRMT5 truncation and deletion mutants. All mutant plasmids were ordered from GeneCare Gene Corporation.

[0136] Figure 5 E and F are immunoprecipitation detection of the binding site of EphA2 and PRMT5. PRMT5 1-324aa, 325-637aa, 465-637aa, 550-637aa, 1-100aa, 101-200aa, 201-250aa, 251-324aa deletion mutants were constructed, and wild-type and deletion mutant PRMT5 with Flag tag were transfected into HEK293 cells, and Myc-tagged EphA2 was co-transfected at the same time. Myc tag antibody was used for immunoprecipitation, and the Flag (PRMT5) level was detected by Western blot.

[0137] Figure 5 G is the molecular docking simulation diagram of PRMT5 binding to EphA2

[0138] The present invention found that EphA2 lacking amino acids 707-807 cannot bind to PRMT5, while PRMT5 lacking amino acids 251-324 cannot bind to EphA2, and preliminarily determined the key regions for the interaction between PRMT5 and EphA2: amino acids 707-807 of EphA2 and amino acids 251-324 of PRMT5. Molecular simulation further determined that amino acids 279-298 of PRMT5 bind to EphA2.

[0139] Figure 6 A is a peptide sequence diagram. The amino acid sequence of 279-298 at the N-terminus of PRMT5 is the target peptide, the sequence is SYLQYLEYLSQNRPPPNAYE, and the purity is more than 96%. The designed peptide was synthesized by Suzhou Qiangyao Biological Company.

[0140] Figure 6A is a peptide cell penetration experiment: 96-well plates were used, 4×104 SUNE1 or HK1 nasopharyngeal cancer cells were inoculated in each well, and 5 μM FITC-labeled PRMT5-derived peptide P20 was added. After the peptides were added to the cell culture medium for 6 hours, they were fixed with 4% paraformaldehyde, stained with DAPI for nuclei, and observed and photographed under a fluorescence microscope. The results showed that P20 could enter the cells.

[0141] Figure 6 B is the effect of P20 on the binding of PRMT5 and EphA2 in nasopharyngeal carcinoma SUNE1 and HK1 cell lines detected by immunoprecipitation.

[0142] SUNE1 and HK1 were treated with 10 μM P20 for 24 h, and the whole cell protein was extracted. EphA2 antibody was used for co-immunoprecipitation, and the PRMT5 level was detected by Western blot.

[0143] Figure 6 C is Western blot detection of P20 reducing EphA2 protein level in nasopharyngeal carcinoma SUNE1 and HK1 cell lines. SUNE1 and HK1 were treated with 2.5μM, 5μM, and 10μM P20 for 24h, and the total protein of cells was extracted. Western blot was used to detect the expression level of EphA2.

[0144] Figure 6 D shows that in nasopharyngeal carcinoma SUNE1 and HK1 cell lines, cells were treated with 10 μM P20 for 24 h, and then treated with 20 μg / ml protein synthesis inhibitor cycloheximide (CHX) for 4 h, 8 h, 12 h and 16 h. The cells were collected and the total cell protein was extracted. The level of EphA2 was detected by Western blot, and the half-life of EphA2 was analyzed.

[0145] Figure 6 E: In nasopharyngeal carcinoma SUNE1 and HK1 cell lines, the cells were treated with 10 μM P20 for 24 h, and then the proteasome inhibitor MG132 was added for 24 h. The changes in EphA2 protein levels were detected by Western blot.

[0146] Figure 6 F shows that nasopharyngeal carcinoma SUNE1 and HK1 cell lines were treated with 10 μM P20 for 24 h, and the whole cell protein was extracted. EphA2 antibody was used for co-immunoprecipitation, and Western blot was used to detect the methylation level of Epha2 and the binding of Epha2 to Cbl.

[0147] Figure 6G: In nasopharyngeal carcinoma SUNE1 and HK1 cell lines, cells were treated with 10 μM P20 for 24 h, total cell protein was extracted, and EphA2 antibody was used for co-immunoprecipitation. The ubiquitination level of EphA2 was detected by Western blot.

[0148] Figure 6 H is the effect of P20 on the colocalization of EphA2 and Cbl detected by cell immunofluorescence. Nasopharyngeal carcinoma SUNE1 cells were treated with 10 μM P20 for 24 h, fixed with 4% paraformaldehyde for 10 min, blocked with BSA blocking solution for 1 h, incubated with EphA2 (mouse) and Cbl (rabbit) antibodies at a dilution of 1:100, incubated at 4°C overnight, washed 3 times with PBS, incubated with the corresponding mouse (rabbit) secondary antibody (1:100), incubated at 37°C for 1 h, stained with DAPI nuclei, sealed, and observed and photographed with a confocal fluorescence microscope.

[0149] Example 1: Anti-nasopharyngeal carcinoma effect of PRMT5 N-terminal 279-298 amino acid peptide

[0150] The target peptide sequence used in the embodiment of the present invention is the N-terminal amino acid sequence of PRMT5 at positions 279-298: Ser-Tyr-Leu-Gln-Tyr-Leu-Glu-Tyr-Leu-Ser-Gln-Asn-Arg-Pro-Pro-Pro-Asn-Ala-Tyr-Glu, the single-letter sequence is SYLQYLEYLSQNRPPPNAYE, the purity is more than 95%, and it is synthesized by Suzhou Qiangyao Biological Company.

[0151] Figure 7 A: In nasopharyngeal carcinoma SUNE1 and HK1 cell lines, P20 was treated with 0μM, 5μM, and 10μM for 24h, respectively. The P20 / ResEphA2 group was transfected with EphA2 plasmid after 10μM P20 treatment, and the activity of ALDH was detected by ALDEFLUOR detection kit (StemCell Technologies) combined with flow cytometry. Single cells were mixed with ALDEFLUOR assay buffer containing ALDH substrate BODIPY aminoacetaldehyde (BAAA) and incubated at 37°C in the dark for 30min. Diethylaminobenzaldehyde (DEAB) was used as a negative control. The FITC channel was selected for flow cytometry, and data analysis was performed using Flow Jo 10.4 software. The left side is a representative flow cytometry graph, and the right side is the results of statistical analysis of each group.

[0152] Figure 7B shows that nasopharyngeal carcinoma SUNE1 and HK1 cell lines were treated with 0 μM, 5 μM, and 10 μM P20 for 24 h, respectively. The P20 / ResEphA2 group was transfected with EphA2 plasmid after treatment with 10 μM P20. The cells were digested, resuspended in stem cell culture medium, counted on a counting plate, and 1×10 3 Cells were placed in an incubator at 37°C and 5% CO2, and fresh stem cell culture medium was replaced in time. After 10-12 days of culture, the number of spheres with a diameter greater than 100 μm was counted under an optical inverted microscope and photographed. The left side is a representative picture of cell spheres, and the right side is the result of statistical analysis of the number of spheres in each group.

[0153] Figure 7 C: NPC SUNE1 and HK1 cell lines were treated with 0μM, 5μM, and 10μM P20 for 24h, respectively. The P20 / ResEphA2 group was transfected with EphA2 plasmid after 10μM P20 treatment, and CD133 was detected by flow cytometry. + The single cells were mixed with PBS buffer containing APC-CD133 antibody, incubated at 4°C in the dark for 40 min, and the APC channel was selected for detection on a flow cytometer. Flow Jo 10.4 software was used for data analysis.

[0154] Figure 7 D shows that nasopharyngeal carcinoma SUNE1 and HK1 cell lines were treated with 0 μM, 5 μM, and 10 μM P20 for 24 h, respectively. The P20 / ResEphA2 group was transfected with EphA2 plasmid after treatment with 10 μM P20, and the total cell protein was extracted. Western blot was used to detect the expression levels of Sox2, NANOD1, ALDH1A1, and c-Myc.

[0155] Figure 7 E is the MTT assay to detect the cell viability of SUNE1 and HK1. 1×10 3 Cells were inoculated in 96-well plates, and different concentrations of cisplatin (0, 1, 2.5, 5, 10, 15, 20) μg / mL were added to the cells for 48 h. Then, 20 μL MTT solution was added to each well and incubated for 4 h. The culture was terminated, the supernatant was discarded, and 150 μL DMSO was added to each well. The cells were shaken at room temperature for 10 min. After the cells were fully dissolved, the absorbance value at 490 nm was read on a microplate reader [cell viability = (A 实验组 / A 对照组 )×100%].

[0156] Figure 7 F is a clone formation experiment to detect the effect of P20 on the proliferation of nasopharyngeal carcinoma cells SUNE1 and HK1. 3The cells were inoculated in a six-well plate. After the initial formation of clones, the cells were treated with a 10 μM polypeptide. When the number of single clone cells observed under a microscope exceeded 50, the culture was stopped. The cell clones were stained with crystal violet, photographed and counted. The left side is a picture of the plate clones formed by cells, and the right side is the result of statistical analysis of the number of clones in each group.

[0157] Figures A, C, and F were analyzed using Student-T test, and Figure E was analyzed using Two-way ANOVA. **P<0.01, ***P<0.001, ****P<0.0001, ns, no statistical difference.

[0158] The present invention discovered that the PRMT5-derived peptide P20 can inhibit the stem cell-like characteristics of nasopharyngeal carcinoma cells and increase sensitivity to cisplatin.

[0159] Figure 8 A is a general picture of the transplanted tumor in mice treated with P20, B is a statistical table of tumor occurrence, C is the growth curve of the transplanted tumor in mice, and D is the tumor weight of the transplanted tumor in mice. 5 / ml, 1×10 4 / ml, 1×10 3 The nasopharyngeal carcinoma cells were implanted in the back of NOD / SCID mice at each concentration of 16 mice. 3 At the same time, the mice at each concentration were randomly divided into two groups. One group was intraperitoneally injected with P20 peptide (10 mg / kg) every day, and the other group was intraperitoneally injected with an equal amount of PBS. The tumor volume was measured every day and the formation of new tumors was observed. On the 12th day after treatment, the mice were euthanized, the tumors were removed, and the weight of the tumors was weighed. The tumor volume was calculated using the formula: volume = 1 / 2 × shortest diameter 2 × Longest diameter.

[0160] Figure 8 E: ALDH was detected by ALDEFLUOR kit combined with flow cytometry (FACS) + The percentage of cells, Figure 8 F is the detection of CD133 in transplanted tumor by flow cytometry + The above-mentioned mouse subcutaneous transplanted tumor was used as a sample. The transplanted tumor tissue was cut into pieces using ophthalmic scissors and placed in a six-well plate containing tumor digestion enzymes (2% collagenase IV + 1% hyaluronidase + 0.5% DNase I). Digestion was performed at 37°C, and the mixture was mixed by blowing every 30 minutes. The cell digestion was observed under a microscope. After about 1 hour, the cells were filtered using a 70μm filter to obtain a single cell suspension. The single cell suspension was centrifuged at 4°C, 1200rpm for 5 minutes, the supernatant was removed, the red blood cells were lysed, and the cells were resuspended and counted. 3×10 6The number of cells / tube is used for subsequent staining. TM Dead cells were labeled with FixableViability Kit and blocked with CD16 / CD32 antibodies, and mouse cells were labeled with FITC-Anti-Mouse H2kd antibody for 30 minutes in the dark.

[0161] Figure 8 E The assay was performed according to the instructions of the ALDEFLUOR Detection Kit (Stem Cell Technologies). Figure 8 F. Mix the single cells with PBS buffer containing APC-CD133 antibody and incubate at 4°C in the dark for 40 min. After detection on the machine, use Flow Jo 10.4 software for data analysis.

[0162] Figure 8 G is to detect the expression level of stem cell markers in transplanted tumors. The above mouse subcutaneous transplanted tumors were used as samples, and tissue proteins were extracted by grinding. Western blot was used to detect the expression levels of Sox2, NANOD1, ALDH1A1, and c-Myc in the transplanted tumors. (All four proteins are tumor stemness markers)

[0163] Figure 8 H is the immunohistochemical staining experiment. Figure 8 I is the statistical analysis of the immunohistochemical scores of each index. The tumor tissue removed from the subcutaneous part of the mouse was fixed with 4% formalin, embedded in paraffin, and sliced. After the paraffin sections were dewaxed and hydrated, the sections were immersed in 3% hydrogen peroxide for 10 minutes to block the endogenous peroxidase activity. In order to block the nonspecific binding of the antibody, the sections were incubated with 10% non-immune goat serum at room temperature for 15 minutes. Subsequently, the sections were incubated with EphA2 antibodies, PRMT5 antibodies, Sox2 antibodies, Nanog antibodies, ALDH1A1 antibodies, and c-Myc antibodies at 4°C overnight, and then incubated with biotin secondary antibodies at room temperature for 30 minutes and stained with DAB (3,3-diaminobenzidine). Finally, the nucleus was stained with hematoxylin. In the negative control, normal mouse or rabbit IgG was used instead of the primary antibody. The photos were observed under a microscope. A positive reaction was defined as a positive reaction showing brown signals on the cytoplasm and / or cell membrane. The staining intensity was divided into none (0), weak (1), moderate (2), and strong (3); the percentage of stained cells was none = 0, stained cells <30% = 1, stained cells 30-60% = 2, and stained cells >60% = 3. The staining score was the staining intensity + the percentage of stained cells.

[0164] Figure C was analyzed by Two-way ANOVA, and Figures D, E, F, and H were analyzed by Student-T test, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no statistical difference;

[0165] The present invention finds that the PRMT5-derived peptide P20 can inhibit the stemness characteristics of nasopharyngeal carcinoma cells in vivo, thereby inhibiting their tumor initiation ability.

Claims

1. PRMT5-derived peptide is derived from amino acids 279-298 of PRMT5 protein, and the peptide sequence is SYLQYLEYLSQNRPPPNAYE.

2. The polypeptide according to claim 1, Features: It includes a series of polypeptides with anti-tumor effects obtained by modifying the N-terminus or C-terminus, deleting, replacing, cyclizing or chiral transformation of amino acids based on the polypeptide sequence.

3. A nasopharyngeal carcinoma therapeutic preparation, comprising the PRMT5-derived polypeptide according to claim 1 or 2, or a polypeptide series with anti-tumor effect obtained by modifying the N-terminus or C-terminus, deleting, replacing, cyclizing or chiral transformation of amino acids based on the PRMT5-derived polypeptide sequence.

4. Use of the anti-tumor polypeptide according to claim 1 or 2 in the preparation of anti-tumor drugs.

5. The use according to claim 4 or 5, wherein the anti-tumor include: Inhibit the stemness of nasopharyngeal carcinoma cells, increase chemotherapy sensitivity and inhibit tumor initiation ability in vivo.

6. The use according to claim 4 or 5, wherein the tumor type is nasopharyngeal carcinoma.

7. Use of the polypeptide according to claim 1 or 2 in the preparation of a preparation for blocking PRMT5 from binding to EphA2.

8. Use of the polypeptide according to claim 1 or 2 in the preparation of a preparation for reducing the symmetrical methylation of arginine at the R816 site of EphA2.

9. Use of the polypeptide according to claim 1 or 2 in preparing a preparation for promoting the binding between EphA2 and Cbl.

10. Use of the polypeptide according to claim 1 or 2 in the preparation of a preparation for promoting ubiquitination and degradation of EphA2.

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