Asymmetrically dimethylated RNF34 protein and its application

Abstract

The present invention relates to the field of biotechnology, and specifically to an asymmetric dimethylated RNF34 protein and its application. The present invention provides an RNF34 protein. Key modification sites are identified through protein modification mass spectrometry, point mutagenesis, and immunoprecipitation techniques. It is found that the antigens modified by asymmetric dimethylation at positions 26 and 40 have excellent performance. The RNF34 protein is used to prepare an artificial antigen and immunize animals to obtain an asymmetric dimethylated antibody against the RNF34 protein. The antibody can specifically recognize the asymmetric dimethylation of the R26 and R40 sites of the RNF34 protein in tumor cells. The asymmetric dimethylated antibody against the RNF34 protein can specifically detect the level of RNF34 methylation in tumor tissues, which is used to assess chemotherapy sensitivity and patient prognosis, providing certain assistance for the detection of clinical tumor chemotherapy resistance and improving the efficacy of neoadjuvant chemotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an asymmetrically dimethylated RNF34 protein and applications thereof. Background Art

[0002] Colorectal cancer (CRC) is a common intestinal malignancy, with the third and second highest incidence and mortality rates among all cancers worldwide, respectively. Its incidence is increasing annually and is trending toward younger patients, severely impacting public health. Chemotherapy is an important treatment modality for CRC. However, CRC patients often develop varying degrees of drug resistance during chemotherapy, significantly impacting treatment efficacy. Of particular concern, some patients exhibit significant chemotherapy resistance from the initial stages of treatment, leading to ineffective tumor control and even tumor progression. This primary drug resistance not only complicates treatment but also accelerates disease progression, severely impacting patient survival and quality of life. Therefore, in-depth exploration of the molecular mechanisms underlying chemotherapy resistance is crucial for improving chemotherapy efficacy and overall prognosis in CRC patients.

[0003] Tumor tissues often differ significantly from normal tissues in terms of methylation levels. Abnormally elevated methylation levels in tumor tissues often activate the expression of anti-apoptotic genes and enhance the activity of DNA damage repair pathways, thereby significantly increasing cancer cells' tolerance to chemotherapeutic drugs. Previous studies have found that methylation levels of many non-histone proteins are significantly increased in tumor tissues. For example, the tumor suppressor protein p53 maintains low levels of methylation in normal tissues; however, in tumors, hypermethylation of p53 reduces its activity, allowing cells to evade apoptosis. Furthermore, increased methylation of the NF-κB protein enhances tumor cells' tolerance to stressful environments.

[0004] RNF34 (Ring Finger Protein 34) is an E3 ubiquitin ligase that regulates multiple biological processes through ubiquitin-mediated proteasomal degradation of various target proteins. Studies on colorectal cancer have found that RNF34 is highly expressed in cancer tissues and possesses anti-apoptotic and oncogenic properties. Its overexpression may confer resistance to chemotherapeutic drugs in tumor cells, thereby compromising the efficacy of chemotherapy. Currently, there is a lack of understanding of the functional role and molecular mechanisms of RNF34 protein and its antibodies in chemotherapeutic drug resistance, and this research urgently needs to be addressed. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide an asymmetrically dimethylated RNF34 protein and its application. The present invention provides a technical solution for obtaining an antigen by asymmetrically dimethylating the RNF34 protein and preparing an antibody that specifically detects the methylation modification of the RNF34 protein.

[0006] The present invention provides the use of the R26 and / or R40 sites of the RNF34 protein as targets in the following I and / or II:

[0007] I. Preparation of drugs for diagnosis and / or treatment of colorectal cancer;

[0008] II. Prepare reagents for detecting sensitivity of chemotherapy drugs.

[0009] The present invention uses protein modification mass spectrometry, point mutation and immunoprecipitation techniques to discover that asymmetric dimethylation modification of the R26 and R40 sites of the RNF34 protein plays an important role in enhancing the sensitivity of tumor cells to chemotherapy drugs.

[0010] The present invention provides an RNF34 protein having an amino acid sequence as shown in SEQ ID NO: 1, wherein the RNF34 protein includes at least one asymmetrically dimethylated arginine.

[0011] In some embodiments, position 26 and / or position 40 of the RNF34 protein is an asymmetrically dimethylated arginine.

[0012] In some specific embodiments, positions 26 and 40 of the RNF34 protein are asymmetrically dimethylated arginines.

[0013] The present invention provides an artificial antigen, which is obtained by coupling a carrier protein with the RNF34 protein.

[0014] In some embodiments, the carrier protein is keyhole limpet hemocyanin.

[0015] In some specific embodiments, cysteine ​​is further linked to the N-terminus and the C-terminus of the artificial antigen.

[0016] The present invention provides an asymmetric dimethylated antibody against RNF34 protein, wherein the asymmetric dimethylated antibody is obtained by immunizing an animal with the RNF34 protein or the artificial antigen;

[0017] The asymmetric dimethylation antibody of RNF34 protein includes monoclonal antibody and / or polyclonal antibody.

[0018] The present invention provides a method for preparing an asymmetric dimethylated antibody against the RNF34 protein, comprising immunizing an animal with the RNF34 protein or the artificial antigen, and then isolating the asymmetric dimethylated antibody against the RNF34 protein from the serum of the immunized animal.

[0019] In some embodiments, the animal comprises a rabbit, and the number of immunizations is ≥ 3 times.

[0020] The present invention provides a cell-penetrating peptide having a sequence of YGRKKRRQRRRA-x, wherein x has an amino acid sequence as shown at positions 21 to 31 and / or positions 35 to 45 of SEQ ID NO: 1.

[0021] In some specific embodiments, the cell-penetrating peptide has an amino acid sequence as shown in SEQ ID NO:4 and SEQ ID NO:5.

[0022] The present invention provides the use of at least one of the following (1) to (5) in the preparation of a drug for diagnosing and / or treating colorectal cancer or a reagent for detecting sensitivity to chemotherapeutic drugs:

[0023] (1) the RNF34 protein;

[0024] (2) The artificial antigen

[0025] (3) an asymmetric dimethylation antibody against the RNF34 protein;

[0026] (4) The asymmetric dimethylated antibody against RNF34 protein prepared by the preparation method;

[0027] (5) The cell-penetrating peptide.

[0028] The present invention provides a drug for diagnosing and / or treating colorectal cancer or a reagent for detecting sensitivity to chemotherapeutic drugs, comprising at least one of the following:

[0029] 1. The RNF34 protein;

[0030] ②. The artificial antigen

[0031] ③. An asymmetric dimethylated antibody against the RNF34 protein;

[0032] ④ The asymmetric dimethylated antibody against RNF34 protein prepared by the preparation method;

[0033] ⑤. The membrane-penetrating peptide.

[0034] The present invention provides RNF34 protein. Key modification sites are found through protein modification mass spectrometry, point mutation and immunoprecipitation technology. It is found that the antigen modified by asymmetric dimethylation at positions 26 and 40 has excellent performance. The RNF34 protein is used to prepare artificial antigens and immunize animals to obtain asymmetric dimethylation antibodies of the RNF34 protein. The asymmetric dimethylation antibodies can specifically recognize the asymmetric dimethylation of the R26 and R40 sites of the RNF34 protein in tumor cells. The asymmetric dimethylation antibodies of the RNF34 protein are used to detect the level of RNF34 methylation in tumor tissues, which is used to evaluate chemotherapy sensitivity and patient prognosis, providing certain help for the detection of clinical tumor chemotherapy resistance and improving the efficacy of neoadjuvant chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The immunogenicity, hydrophilicity and surface accessibility of arginine at positions R26 and R40 of RNF34 protein are shown;

[0036] Figure 2 Anti-RNF34 (R26 me2a 、R40 me2a ) Immunodot assay results of polyclonal antibodies;

[0037] Figure 3 Anti-RNF34 (R26 me2a 、R40 me2a ) Western-Blot identification results of polyclonal antibodies;

[0038] Figure 4 Shows the detection results of TAT transmembrane peptide blocking the methylation of RNF34 protein at R26 and R40 sites;

[0039] Figure 5 Anti-RNF34 (R26 me2a 、R40 me2a ) Immunohistochemical identification results of polyclonal antibodies, where from left to right are primary antibody dilution (left), R26 me2a (Medium) and R40 me2a (right) Immunohistochemistry results;

[0040] Figure 6 Shown is the human-derived tumor xenograft model RNF34 R26 me2a 、R40 me2a Methylation level results;

[0041] Figure 7 The human tumor xenograft model RNF34 (R26 me2a 、R40 me2a) Results of hypomethylation-promoting chemotherapy sensitivity test. The left image shows the photographic results of subcutaneous PDX tumor tissue in mice, and the right image shows the weighing and statistical results of subcutaneous PDX tumor tissue in mice;

[0042] Figure 8 RNF34 TAT transmembrane peptide blocks R26 me2a 、R40 me2a Methylation promotes chemotherapy sensitivity in human tumor xenograft models. The left image shows a photograph of subcutaneous PDX tumor tissue in mice, and the right image shows the weighing results of subcutaneous PDX tumor tissue in mice.

[0043] Figure 9 Shown are the test results of ALT, AST, ALB, TBIL, DBIL and CREA in mice after treatment with TAT transmembrane peptide;

[0044] Figure 10 The figure shows the staining results of the heart, liver, spleen, lung and kidney of mice after treatment with TAT transmembrane peptide;

[0045] Figure 11 WB detection of RNF34 R26 in organoids me2a 、R40 me2a Methylation level results;

[0046] Figure 12 Figure 3 shows the effect of blocking R26 and R40 methylation with a penetrating peptide on the sensitivity to chemotherapy drugs in an organoid model. Figure A shows the statistical results of cell viability after organoid 01# treatment, Figure B shows the microscopic morphological differences before and after treatment of organoid 01#, Figure C shows the statistical results of cell viability after treatment of organoid 03#, and Figure D shows the microscopic morphological differences before and after treatment of organoid 03#.

[0047] Figure 13 RNF34 R26 me2a 、R40 me2a The relationship between methylation level and the efficacy of neoadjuvant chemotherapy for colorectal cancer, TNM stage of colorectal cancer, and prognosis of colorectal cancer. Figure A shows RNF34 R26 me2a Correlation between methylation levels and chemotherapy efficacy. Figure B shows RNF34 R40 me2a Correlation between methylation levels and chemotherapy efficacy. Figure C shows RNF34 R26 me2a 、RNF34R40 me2a ROC curves of methylation levels alone and in combination for predicting chemotherapy sensitivity. Figure D shows RNF34 R26 me2a Survival analysis of methylation levels, Figure E shows RNF34 R40 me2a Survival analysis of methylation levels. DETAILED DESCRIPTION

[0048] The present invention provides asymmetric dimethylation-modified RNF34 proteins and their applications. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It should be noted in particular that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0049] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention will be further described below with reference to the examples.

[0050] Example 1 Peptide Synthesis

[0051] 1. Design and synthesis of peptides with asymmetrical dimethylation of R26 and R40 arginines in RNF34 protein

[0052] The amino acid sequence of the RNF34 protein obtained from Uniprot is as follows. When synthesizing the following RNF34 protein, we performed asymmetric dimethylation modification on the arginine at positions 26 and 40.

[0053] MKAGATSMWASCCGLLNEVMGTGAVRGQQSAFAGATGPFRFTPNPEFSTYPPAATEGPNIVCKACGLSFSVFRKKHVCCDCKKDFCSVCSVLQENLRRCSTCHLLQETAFQRPQLMRLKVKDLRQYLILRNIPIDTCREKEDLVDLVLCHHGLGSEDDMDTSSLNSSRSQTSSFFTRSFFSNYTAPSA TMSSFQGELMDGDQTSRSGVPAQVQSEITSANTEDDDDDDDEDDDDEEENAEDRNPGLSKERVRASLSDLSSLDDVEGMSVRQLKEILARNFVNYSGCCEKWELVEKVNRLYKENEENQKSYGERLQLQDEEDDSLCRICMDAVIDCVLLECGHMVTCTKCGKRMSECPICRQYVVRAVHVFKS (SEQ ID NO:1)

[0054] Results: RNF34 protein contains 372 amino acids.

[0055] At the same time, DNAstar software was used to analyze the immunogenicity, hydrophilicity and surface accessibility of arginine at positions R26 and R40 of RNF34 protein (e.g. Figure 1The results showed that the arginine residues at positions R26 and R40 of the RNF34 protein had good immunogenicity, hydrophilicity, and surface accessibility.

[0056] 2. Peptide synthesis and carrier protein coupling

[0057] After analysis, we asymmetrically dimethylated arginine at positions R26 and R40. The designed antigen peptide sequence specifically recognizing asymmetric dimethylation at R26 is: CGTGAVR(me, ADMA)GQQS (SEQ ID NO: 2), located at aa 19-30. The antigen peptide specifically recognizing asymmetric dimethylation at R40 is: CATGPFR(me, ADMA)FTPNP (SEQ ID NO: 3), located at aa 34-45. The peptides were synthesized by Hangzhou Huaan Biotechnology Co., Ltd.

[0058] The TAT transmembrane peptide sequence used to block methylation at RNF34 R26 is YGRKKRRQRRRAGTGAVRGQQSA (SEQ ID NO: 4), and the TAT transmembrane peptide sequence used to block methylation at RNF34 R40 is YGRKKRRQRRRAATGPFRFTPNP (SEQ ID NO: 5). The TAT transmembrane peptides were synthesized by Anhui Guoping Pharmaceutical.

[0059] Preparation of antigen-coupled products: Synthetic peptides are coupled to KLH to prepare antigens. The additional cysteine ​​C at both ends of the peptide is mainly used for coupling KLH and the preparation of antigen affinity purification columns.

[0060] Example 2 Preparation and purification of rabbit polyclonal antibodies against asymmetric dimethylated arginines at R26 and R40 of the RNF34 protein

[0061] Three New Zealand white rabbits were immunized with each conjugate product prepared in Example 1 using multiple subcutaneous injections of 0.2 ml per injection. A second immunization was performed 14 days after the first immunization, with a 7-day interval between the second and third immunizations. Seven days after the third immunization, a small serum sample was collected from the middle auricular artery of the animal for testing. If the sample passed the test, a booster immunization was administered 7 days later. Seven days after the booster immunization, whole blood was collected for purification using an affinity chromatography column.

[0062] Example 3 Identification of rabbit polyclonal antibodies against asymmetric dimethylated arginines at R26 and R40 of the RNF34 protein

[0063] Anti-RNF34 (R26 me2a 、R40 me2a ) Polyclonal antibody ELISA titer detection:

[0064] R26 of synthesized RNF34 me2a Peptide, R40me2a The peptide and R26 and R40 unmodified peptides were used as detection antigens and coated on enzyme-labeled plates respectively. Non-immune rabbit serum diluted 1:2000 was used as negative control. Rabbit serum and purified antibodies were diluted in multiple ratios and detected by ELISA method to calculate the antibody titer.

[0065] Results: From the perspective of titer, the antibody was purified twice to remove the non-methylated antibody components, and the anti-RNF34 (R26 me2a 、R40 me2a ) The titer of polyclonal antibody is greater than 1:16000, R26 me2a The corresponding absorbance value was 1.155, while in the control group, under the same dilution conditions, the absorbance value of the R26 arginine non-modified peptide was only 0.021 (Table 1); R40 me2a The corresponding absorbance value was 1.261, while in the control group, under the same dilution conditions, the absorbance value of the R40 arginine non-modified peptide was only 0.014 (Table 2).

[0066] Table 1:

[0067]

[0068] Table 2:

[0069] Example 4 Anti-RNF34 (R26 me2a 、R40 me2a ) Immunodot detection of polyclonal antibodies

[0070] 1 mg of R26 and R40 asymmetrically dimethylated modified peptides and non-modified peptides were weighed and dissolved in 1 ml of 0.01 M PBS buffer. 5 μl of the dissolved peptides were spotted on PVDF membranes. The PVDF membranes were blocked with TBST blocking solution containing 5% skim milk powder for 1 h. The corresponding purified anti-RNF34 (R26 me2a 、R40 me2a ) polyclonal antibody (dilution factor 1:1000), incubated at room temperature for 1 hour, and then washed five times (8 minutes / time). HRP-labeled goat anti-rabbit secondary antibody (dilution factor 1:50000) was added, and after incubation at room temperature for 1 hour, the membrane was washed five times (8 minutes / time). The final data were collected by Jena UVP Chemstudio touch multifunctional imager.

[0071] The results are as follows Figure 2 Shown: RNF34 (R26 in the present invention me2a 、R40 me2a) The polyclonal antibody basically recognized the R26 asymmetric arginine dimethylated peptide and the R40 asymmetric arginine dimethylated peptide on the PVDF membrane, and could hardly recognize the R26 and R40 arginine non-methylated peptides.

[0072] Example 5 Anti-RNF34 (R26 me2a 、R40 me2a ) Western-blot identification of polyclonal antibodies

[0073] 1. Construct Flag-tagged expression vectors for wild-type RNF34 and RNF34 R26, R40, and R26 / R40 site mutations (the mutants mutate arginine at positions 26 and 40 to lysine). RNF34-WT, RNF34-R26K, RNF34-R40K, and RNF34-R26 / R40K (2RK) were transiently transfected into human embryonic kidney HEK293T cells, respectively. Cell proteins were collected 48 hours after transfection and purified by incubation with Flag magnetic beads. The purified proteins were eluted and denatured, then subjected to 10% SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking with skim milk powder, purified anti-RNF34 (R40 arginine dimethyl) polyclonal antibody (dilution 1:1000) was added and incubated at room temperature for 1 hour. The membrane was then washed five times (8 minutes each time). HRP-labeled goat anti-rabbit secondary antibody (dilution 1:50,000) was added and incubated at room temperature for 1 hour, then the membrane was washed five times (8 minutes each time). The final data were acquired using a Jena UVPChemstudio touch multifunctional imager.

[0074] The results are as follows Figure 3 Shown: the anti-RNF34 (R26 me2a 、R40 me2a ) polyclonal antibody can detect a protein band of approximately 42 kD in HEK293T cell lysates, consistent with the molecular weight of the RNF34 protein. RNF34 protein expressed in cells transfected with the wild-type plasmid is significantly methylated at arginine 40. RNF34 protein expressed in cells transfected with the R26 and R40 mutant plasmids also exhibits a small amount of methylation. This is due to asymmetric dimethylation at both sites. Mutating only one of these sites in RNF34 still results in asymmetric dimethylation, while RNF34 protein expressed in cells transfected with the 2RK mutant plasmid, which mutates both R26 and R40, exhibits almost no asymmetric dimethylation.

[0075] 2. The RNF34-WT plasmid was transiently transfected into human embryonic kidney HEK293T cells. After transfection, the methylation sites of R26 and R40 were blocked with the TAT transmembrane peptides constructed above. HEK293T cell proteins were collected 48 hours after transfection and RNF34 protein was purified by incubation with Flag magnetic beads. The purified protein was eluted and denatured, and then subjected to 10% SDS-PAGE electrophoresis. After electrophoresis, it was transferred to a PVDF membrane and blocked with skim milk powder. Purified anti-RNF34 (R40 arginine dimethylation) polyclonal antibody (dilution 1:1000) was added and incubated at room temperature for 1 hour. The membrane was then washed five times (8 minutes each time). HRP-labeled goat anti-rabbit secondary antibody (dilution 1:50,000) was added and incubated at room temperature for 1 hour. The membrane was then washed five times (8 minutes each time). The final data were collected using a Jena UVP Chemstudio touch multifunctional imager.

[0076] The results are as follows Figure 4 As shown in the results, the TAT transmembrane peptides at R26 and R40 sites of the present invention can successfully block the methylation of RNF34 protein, and the methylation of R26 and R40 sites is inversely proportional to the TAT concentration.

[0077] Example 6 Anti-RNF34 (R26 me2a 、R40 me2a ) Immunohistochemical identification of polyclonal antibodies

[0078] The pathological sections were placed in a 60°C oven for 2 h, then dewaxed with xylene, hydrated with graded alcohol, and antigen repaired by high-pressure boiling with citric acid. The sections were blocked and the primary antibody (primary antibody diluent, R26 me2a 、R40 me2a ), secondary antibody, DAB color development, hematoxylin, differentiation, blueing, neutral gum sealing, and observation under a microscope.

[0079] The results are as follows Figure 5 As shown: It can be seen that R26 in the present invention me2a 、R40 me2a It mainly appeared in the cell nucleus, which was consistent with the localization of RNF34, and was a positive result. me2a 、R40 me2a ) After detection with polyclonal antibodies, no specific staining was found in the cell nucleus, indicating a negative result.

[0080] RNF34 R26 of the present invention me2a 、R40 me2a The polyclonal antibody can specifically recognize the asymmetric dimethylation modifications of R26 and R40 of the RNF34 protein and can be used to detect the methylation level of these sites in tumor cells, providing a tool for exploring the chemotherapy resistance of tumor cells and providing certain help for the improvement of later chemotherapy regimens.

[0081] Example 7 Anti-RNF34 (R26 me2a 、R40 me2a ) polyclonal antibodies to detect methylation modification levels in patient-derived tumor xenograft models (PDX)

[0082] Tumor tissue was obtained surgically and implanted into the armpit of immunodeficient nude mice in the form of fragments. When the tumor grew to a diameter of about 1 cm, it was peeled off from the first-generation host (P0). A portion of the tissue was minced into about 3 mm × 3 mm × 3 mm and implanted into new mice (P1). A portion was fixed with paraformaldehyde and preserved for further testing, or preserved in freezing solution for revival and seed preservation. This process was repeated until PDX (P3) was bred. PDX (P3) tumor tissue was frozen and ground to extract tissue protein, and subjected to 10% SDS-PAGE electrophoresis. After electrophoresis, it was transferred to a PVDF membrane, blocked with skim milk powder, and purified anti-RNF34 (R26 me2a 、R40 me2a ) polyclonal antibody (dilution factor 1:1000), incubated at room temperature for 1 hour, and then washed five times (8 minutes / time). HRP-labeled goat anti-rabbit secondary antibody (dilution factor 1:50000) was added and incubated at room temperature for 1 hour, and then washed five times (8 minutes / time). The final data were collected by Jena UVP Chemstudio touch multifunctional imager.

[0083] The results are as follows Figure 6 Shown: the anti-RNF34 (R26 me2a 、R40 me2a ) The polyclonal antibody can detect a protein band of approximately 42KD in PDX tumor tissue lysate, which is consistent with the molecular weight of RNF34 protein. The degree of RNF34 methylation in tumor tissues from different patients varies.

[0084] Example 8 Human tumor xenograft model RNF34 (R26 me2a 、R40 me2a ) Hypomethylation promotes chemotherapy sensitivity

[0085] 1. Select an example of RNF34 (R26 me2a 、R40 me2a ) hypermethylated PDX tissue and one case of RNF34 (R26 me2a 、R40 me2a) hypomethylated PDX tissues were divided into four groups. One group did not receive any intervention, two groups were treated with 5-fluorouracil and oxaliplatin respectively, and the last group was treated with 5-fluorouracil and oxaliplatin combined. The weight of the mice, the size of the tumor tissue and the response to chemotherapy were observed. After the treatment, the subcutaneous PDX tumor tissue of the mice was removed, photographed, recorded and weighed.

[0086] The results are as follows Figure 7 As shown: the present invention anti-RNF34 (R26 me2a 、R40 me2a ) polyclonal antibodies to detect the methylation level of PDX tumor tissue, RNF34 R26 me2a 、R40 me2a The tumor tissue of the low-expression PDX model grows more slowly than that of the high-expression PDX model, is more sensitive to chemotherapy drugs, and has lower tumor tissue size and weight.

[0087] 2. RNF34 TAT transmembrane peptide blocks R26 me2a 、R40 me2a Methylation promotes chemotherapy sensitivity in human tumor xenograft models.

[0088] One case of RNF34 R26 was selected based on the aforementioned PDX me2a 、R40 me2a Highly expressed PDX tissues were divided into two major groups, one as a solvent control group and the other as a 5-fluorouracil and oxaliplatin combined treatment group. The two major groups were further divided into four subgroups, which were treated with NC-Pe penetrant peptide, R26-Pe penetrant peptide, R40-Pe penetrant peptide, and R26-Pe+R40-Pe penetrant peptide respectively. The weight of the mice, tumor tissue size and response to chemotherapy were observed. After the treatment, the subcutaneous PDX tumor tissue of the mice was removed, photographed, recorded and weighed.

[0089] Results: In the control group, R26-Pe and R40-Pe alone could inhibit tumor growth compared with the NC-Pe group. The combined use of R26-Pe and R40-Pe further inhibited tumor growth. Moreover, the treatment with R26-Pe and R40-Pe made the tumor more sensitive to chemotherapy drugs (e.g. Figure 8 shown).

[0090] 3. Biosafety Assessment of RNF34 TAT Cell-penetrating Peptide

[0091] The nude mice were divided into two groups, with 6 mice in each group. One group was intraperitoneally injected with normal saline, and the other group was intraperitoneally injected with RNF34 R26-TAT penetrating peptide and RNF34 R40-TAT penetrating peptide at a dose of 5 mg / kg, once a day. After four weeks of treatment, the blood, heart, liver, spleen, lung and kidney of the mice were collected for testing to confirm the biosafety of TAT penetrating peptide.

[0092] Results: The main liver and kidney function indicators of mice serum: ALT, AST, ALB, TBIL, DBIL, CREA were all within the normal range (such as Figure 9 There was no obvious damage to the heart, liver, spleen, lung and kidney in the mice (as shown in Figure 10 shown).

[0093] Example 9 RNF34 TAT transmembrane peptide blocks R26 me2a 、R40 me2a Methylation promotes chemotherapy sensitivity in colorectal cancer organoids

[0094] Patients diagnosed with colorectal cancer for the first time were recruited from the Department of Colorectal Surgery, Affiliated Tumor Hospital of Guangxi Medical University. They had not received interventional, radiotherapy, chemotherapy, or targeted drug treatments before surgery. Colorectal cancer surgical specimens were collected from the patients to construct organoid models. The tissues were transferred to PBS supplemented with penicillin and washed twice, then placed in a sterile culture dish containing PBS. Sterile tweezers were used to remove all necrotic tissue and non-epithelial tissue (such as muscle or fat) as much as possible. The tissues were minced into approximately 0.5-2 mm³ in the culture dish with sterile tissue scissors. The minced tissues were transferred to a 15 mL centrifuge tube and resuspended in 50 times the volume of the minced tissue suspension in a constant temperature shaking incubator at 37°C and 50-100 rpm for 60 minutes. Fetal bovine serum was added to terminate the digestion after the digestion was completed. The tissue suspension was filtered using a 100 μm cell strainer, and the filtrate was transferred to a centrifuge tube and centrifuged at 200-300 g. Centrifuge at 400 nm for 3–5 minutes. Aspirate the supernatant, retain the pellet, resuspend, and count. Add the appropriate amount of organoid-specific Matrigel, calculating the ratio of 8,000–30,000 cells to 20–30 μL of Matrigel per well. Mix thoroughly on ice and place on ice. Use a pipette to transfer the Matrigel-cell mixture to the center of the bottom well of a cell culture plate (apply 20–30 μL of the mixture to each well of a 24-well cell culture plate). Place the plate in a 37°C, 5% CO2 incubator to allow the suspension to solidify for 30 minutes. Once the Matrigel has solidified and no longer flows, slowly add organoid-specific culture medium along the sides of the wells.

[0095] After the organoids were successfully constructed, WB was used to detect RNF34 R26 in the organoids. me2a 、R40 me2a Methylation level, select RNF34R26me2a 、R40 me2a TAT blocking experiment was performed on two organoids with high methylation expression, RNF34 R26 me2a 、R40 me2a Two organoids with high methylation expression were passaged into 96-well plates and treated with TAT transmembrane peptide and chemotherapy drugs alone or in combination.

[0096] Results: Western blot detection of RNF34 R26 in organoids me2a 、R40 me2a Arginine methylation level, two patients with high methylation were selected for chemotherapy drugs and peptides alone or in combination (WB test results as shown in Figure 11 As shown in the figure, 01#~04# are organoid models successfully constructed from different patient sources). CCK8 results showed that R26-TAT and R40-TAT transmembrane peptides have inhibited organoid growth to a certain extent. The combined use of 5-fluorouracil and oxaliplatin significantly inhibited the activity of organoids. R26-TAT and R40-TAT transmembrane peptides increased the sensitivity of organoids to chemotherapy drugs (such as Figure 12 shown).

[0097] Example 10 RNF34 R26 me2a 、R40 me2a Relationship between methylation level and the efficacy of neoadjuvant chemotherapy and prognosis of colorectal cancer

[0098] After approval by the Ethics Committee of the Guangxi Zhuang Autonomous Region Cancer Prevention and Treatment Institute, colorectal cancer pathological tissue specimens and baseline clinical pathological data (including age, sex, tumor location, pathological type, pathological stage, tumor size, surgery, chemotherapy, etc.) were collected from the colorectal and anal wards. Inclusion criteria: patients had a first colonoscopy with tumor pathological tissue clamped, and the pathology report diagnosed colorectal adenocarcinoma. They had not received surgery, interventional therapy, radiotherapy, chemotherapy, or targeted drug treatment before endoscopic biopsy, and had no history of other cancers. After diagnosis, patients had received regular four cycles of FOLFOX chemotherapy. CT scans of the tumor site or distant metastases were performed at our center before and after chemotherapy. Efficacy was categorized into four levels: complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD), based on changes in the primary and metastatic lesions.

[0099] Detection of RNF34 R26 in cancer tissues using immunohistochemistry me2a 、R40 me2a Analysis of arginine methylation levels at position R26 in cancer tissues me2a 、R40 me2a The correlation between the arginine methylation level at position R26 and the efficacy of neoadjuvant chemotherapy was evaluated by ROC curve.me2a 、R40 me2a The value of arginine methylation level alone and in combination in predicting the efficacy of neoadjuvant chemotherapy; Kaplan-Meier method was further used to analyze RNF34 R26 me2a 、R40 me2a The relationship between arginine methylation level and patient prognosis.

[0100] The results are as follows Figure 13 Shown: Immunohistochemical analysis of 51 colonoscopy-clamped tumor pathological tissues from patients with colorectal cancer, RNF34 R26 me2a 、R40 me2a Arginine methylation level is associated with the efficacy of neoadjuvant chemotherapy, ROC curve analysis RNF34 R26 me2a 、R40 me2a The AUC of arginine methylation level combined with the prediction of neoadjuvant chemotherapy efficacy was greater than that of single prediction, and RNF34 R26 me2a 、R40 me2a The arginine methylation level can predict the patient's prognosis, and the higher the expression, the worse the patient's prognosis.

[0101] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. RNF34 protein, characterized in that It has an amino acid sequence as shown in SEQ ID NO: 1, wherein the 26th and / or 40th positions of the RNF34 protein are asymmetrically dimethylated arginines.

2. Artificial antigen, characterized in that The protein is obtained by coupling a carrier protein with the RNF34 protein according to claim 1.

3. An asymmetric dimethylation antibody against RNF34 protein, characterized in that: The asymmetric dimethylated antibody is obtained by immunizing an animal with the RNF34 protein according to claim 1 or the artificial antigen according to claim 2; The asymmetric dimethylation antibody of RNF34 protein includes monoclonal antibody and / or polyclonal antibody.

4. A membrane-penetrating peptide, characterized in that The sequence thereof is YGRKKRRQRRRA-x, wherein x has the amino acid sequence shown at positions 21 to 31 and / or positions 35 to 45 of SEQ ID NO:

1.

5. The cell-penetrating peptide according to claim 4, characterized in that The cell-penetrating peptide has the amino acid sequence shown in SEQ ID NO: 4 and SEQ ID NO:

5.

6. Use of at least one of the following (1) to (4) in the preparation of a drug for determining the prognosis of a colorectal cancer patient or a reagent for detecting the sensitivity of a chemotherapy drug: (1) The RNF34 protein according to claim 1; (2) The artificial antigen according to claim 2 (3) The asymmetrically dimethylated antibody against the RNF34 protein according to claim 3; (4) The cell-penetrating peptide according to claim 4 or 5.

7. A drug for determining the prognosis of colorectal cancer patients or a reagent for detecting the sensitivity of chemotherapy drugs, characterized in that: Include at least one of the following ① to ④:

1. The RNF34 protein according to claim 1; ②. The artificial antigen according to claim 2 ③. The asymmetrically dimethylated antibody against the RNF34 protein according to claim 3; ④. The cell-penetrating peptide according to claim 4 or 5.

8. Use of the cell-penetrating peptide according to claim 4 or 5 in the preparation of a medicament for treating colorectal cancer.

9. A drug for treating colorectal cancer, characterized in that: The invention comprises the cell-penetrating peptide according to claim 4 or 5.

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