Asymmetric dimethylation modified RNF34 protein and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an asymmetric dimethylation modified RNF34 protein and application thereof. The RNF34 protein is provided, key modification sites are found through protein modification mass spectrometry, point mutation and co-immunoprecipitation technologies, it is found that the 26th antigen and the 40th antigen which are subjected to asymmetric dimethylation modification have excellent performance, the RNF34 protein is adopted for preparing an artificial antigen immune animal, an asymmetric dimethylation antibody of the RNF34 protein is obtained, and the RNF34 protein can be used for preparing the asymmetric dimethylation antibody of the RNF34 protein. The antibody can specifically recognize asymmetric dimethylation of R26 and R40 loci of RNF34 protein in tumor cells, and the asymmetric dimethylation antibody of the RNF34 protein can specifically detect the level of RNF34 methylation in tumor tissue and is used for evaluating chemotherapy sensitivity and patient prognosis. Certain help is provided for clinical tumor chemotherapy drug resistance detection and improvement of neoadjuvant chemotherapy curative effect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an asymmetric dimethylation-modified RNF34 protein and an application thereof. Background Art

[0002] Colorectal cancer (CRC) is a common intestinal malignancy. Its morbidity and mortality rate rank third and second among all cancer morbidity and mortality rates worldwide, respectively. Its morbidity rate is increasing year by year and is showing a trend of younger patients, which seriously affects people's health. Chemotherapy is one of the important means of treating colorectal cancer. However, colorectal cancer patients often develop drug resistance to varying degrees during chemotherapy, which greatly affects the treatment effect. It is particularly concerning that some patients show obvious chemotherapy resistance from the beginning of treatment, resulting in the failure to effectively control tumor cells and even tumor progression. This primary drug resistance not only increases the difficulty of treatment for patients, but also accelerates disease progression, seriously affecting the survival rate and quality of life of patients. Therefore, it is necessary to deeply explore and reveal the molecular mechanism leading to chemotherapy resistance, which has important clinical significance for improving the chemotherapy effect and overall prognosis of colorectal cancer patients.

[0003] There are usually significant differences in the methylation modification levels between tumor tissues and normal tissues. Abnormally increased methylation modification levels in tumor tissues often activate the expression of anti-apoptotic genes and enhance the activity of DNA damage repair pathways, thereby significantly improving the tolerance of cancer cells to chemotherapeutic drugs. Previous studies have found that in tumor tissues, the methylation levels of many non-histone proteins are significantly increased. For example, the tumor suppressor protein p53 can maintain low levels of methylation in normal tissues; while in tumors, high methylation of p53 reduces its activity, making it easier for cells to escape apoptosis. In addition, increased methylation of NF-κB protein enhances the tolerance of tumor cells to stressful environments.

[0004] RNF34 is an E3 ubiquitin ligase. RNF34 (Ring Finger Protein 34) is an E3 ubiquitin-protein ligase that regulates multiple biological processes through ubiquitin-mediated proteasomal degradation of various target proteins. In the study of colorectal cancer, it was found that RNF34 is highly expressed in cancer tissues and has anti-apoptotic and carcinogenic properties. Its overexpression may make tumor cells resistant to chemotherapeutic drugs, thereby affecting the efficacy of chemotherapy. At present, there is no functional analysis and molecular mechanism of RNF34 protein and its antibodies in chemotherapeutic drug resistance, and this part of the research needs to be solved urgently. Summary of the invention

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

[0006] The present invention provides the use of R26 and / or R40 sites of 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 chemotherapeutic 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 comprises 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 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 also connected to the N-terminus and the C-terminus of the artificial antigen.

[0016] The present invention provides an asymmetric dimethylated antibody of 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 the RNF34 protein includes a monoclonal antibody and / or a polyclonal antibody.

[0018] The present invention provides a method for preparing an asymmetric dimethylated antibody of the RNF34 protein, comprising immunizing an animal with the RNF34 protein or the artificial antigen, and then isolating the asymmetric dimethylated antibody of 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, the sequence of which is 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 the sensitivity of a chemotherapeutic drug:

[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 of 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 the sensitivity of chemotherapeutic drugs, comprising at least one of the following ① to ⑤:

[0029] ①, the RNF34 protein;

[0030] ②. The artificial antigen

[0031] ③. The asymmetric dimethylation antibody of the RNF34 protein;

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

[0033] ⑤. The cell-penetrating peptide.

[0034] The present invention provides RNF34 protein, and key modification sites are found through protein modification mass spectrometry, point mutation and immunoprecipitation technology, and it is found that the antigen modified by asymmetric dimethylation at the 26th and 40th positions has excellent performance, and the RNF34 protein is used to prepare artificial antigens to immunize animals to obtain asymmetric dimethylation antibodies of the RNF34 protein, and the asymmetric dimethylation antibodies can specifically recognize the asymmetric dimethylation of the R26 and R40 sites of the RNF34 protein in tumor cells, and 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, and provide certain help for the detection of clinical tumor chemotherapy resistance and the improvement of 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 test results of polyclonal antibodies;

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

[0038] Figure 4 The results show that TAT transmembrane peptide at R26 and R40 sites blocked the methylation of RNF34 protein;

[0039] Figure 5 Anti-RNF34(R26 me2a 、R40 me2a ) The immunohistochemical identification results of polyclonal antibodies, where from left to right in the figure are primary antibody dilution (left), R26 me2a (Medium) and R40 me2a (Right) Immunohistochemistry effect diagram;

[0040] Figure 6 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 the detection of hypomethylation promoting chemotherapy sensitivity, where the left picture shows the photographic results of subcutaneous PDX tumor tissue in mice, and the right picture shows the weighing 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 picture shows the photographic results of subcutaneous PDX tumor tissue in mice, and the right picture shows the weighing statistics of subcutaneous PDX tumor tissue in mice.

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

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

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

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

[0047] Fig.13 RNF34 R26 me2a 、R40 me2a The relationship between methylation level and the efficacy of neoadjuvant chemotherapy for colorectal cancer, TNM staging of colorectal cancer, and prognosis of colorectal cancer. Figure A shows RNF34 R26 me2a Correlation between methylation level and chemotherapy efficacy, Figure B is RNF34 R40 me2a Correlation between methylation level and chemotherapy efficacy, Figure C is 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 to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all 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 of this article 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 is further described below in conjunction with the embodiments.

[0050] Example 1 Peptide Synthesis

[0051] 1. Design and synthesis of asymmetric dimethylated peptides of R26 and R40 arginines of RNF34 protein

[0052] The amino acid sequence of RNF34 protein obtained according to 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: The RNF34 protein contained 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 immunogenicity, hydrophilicity and surface accessibility of the arginine residues at positions R26 and R40 of the RNF34 protein were good.

[0056] 2. Peptide synthesis and carrier protein coupling

[0057] After analysis, we performed asymmetric dimethylation modification on arginine at R26 and R40, and designed the antigenic peptide sequence for specific recognition of asymmetric dimethylation of R26: CGTGAVR(me, ADMA)GQQS (SEQ ID NO: 2), located at 19-30aa, and the antigenic peptide sequence for specific recognition of asymmetric dimethylation of R40: CATGPFR(me, ADMA)FTPNP (SEQ ID NO: 3), located at 34-45aa. The peptides were synthesized by Hangzhou Huaan Biotechnology Co., Ltd.

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

[0059] Preparation of antigen coupling products: Synthetic peptides are coupled to KLH to prepare antigens. The additional cysteine ​​C at both ends of the polypeptide 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 RNF34 protein

[0061] Each coupling product prepared in Example 1 was used to immunize 3 New Zealand white rabbits, and multiple subcutaneous injections were used, with 0.2 ml per point. A second immunization was performed on the 14th day after the first immunization, and the interval between the second immunization and the third immunization was 7 days. A small sample of serum was collected from the middle ear artery of the animal on the 7th day after the third immunization. If the test was qualified, a booster immunization was performed 7 days later. Whole blood was collected 7 days after the booster immunization for purification by affinity chromatography column.

[0062] Example 3 Identification of rabbit polyclonal antibodies against asymmetric dimethylation of arginine at R26 and R40 of 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 non-modified peptides were used as detection antigens and coated on ELISA 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] Weigh 1 mg of each of the R26 and R40 asymmetric dimethyl modified peptides and non-modified peptides and dissolve them in 1 ml of 0.01 M PBS buffer. Take 5 μl of the dissolved peptides and spot them on the PVDF membrane. Block the PVDF membrane with TBST blocking solution containing 5% skim milk powder for 1 h. Add 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 min / time), HRP-labeled goat anti-rabbit secondary antibody (dilution factor 1:50000) was added, incubated at room temperature for 1 hour, and then washed five times (8 min / time), and the final results were collected by Jena UVP Chemstudio touch multifunctional imager.

[0071] The results are as follows Figure 2 As shown: RNF34 (R26 me2a 、R40 me2a) The polyclonal antibody basically recognized the R26 asymmetric arginine dimethylated polypeptide and the R40 asymmetric arginine dimethylated polypeptide on the PVDF membrane, and could hardly recognize the R26 and R40 arginine non-methylated polypeptides.

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

[0073] 1. Construct the wild-type RNF34 and RNF34 R26, R40 and R26 / R40 site mutant expression vectors with Flag tag (the mutant mutated the arginine at position 26 and 40 to lysine). Then 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 the 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 PVDF membrane and blocked with skim milk powder. Purified anti-RNF34 (R40 arginine dimethylation) polyclonal antibody (dilution factor 1:1000) was added, and the membrane was incubated at room temperature for 1 hour, then washed five times (8 min / time), and HRP-labeled goat anti-rabbit secondary antibody (dilution factor 1:50000) was added and incubated at room temperature for 1 hour, then washed five times (8 min / time). The final results were collected by Jena UVP Chemstudio touch multifunctional imager.

[0074] The results are as follows Figure 3 Shown: Anti-RNF34 (R26 me2a 、R40 me2a ) polyclonal antibody can detect a protein band of about 42KD in HEK293T cell lysate, which is consistent with the molecular weight of RNF34 protein. The arginine at position 40 of RNF34 protein expressed by cells transfected with wild-type plasmid is obviously methylated, and RNF34 protein expressed by cells transfected with R26 and R40 mutant plasmids also shows a small amount of methylation. The reason is that both sites are asymmetric dimethylated. If one of the sites is mutated alone, RNF34 will still have asymmetric dimethylation modification. RNF34 protein expressed by cells transfected with 2RK mutant plasmids with mutations of R26 and R40 at the same time has almost no asymmetric dimethylation.

[0075] 2. The RNF34-WT plasmid was transiently transfected into human embryonic kidney HEK293T cells. After transfection, the TAT transmembrane peptides of R26 and R40 constructed above were used to block the methylation of the sites respectively. HEK293T cell proteins were collected 48 hours after transfection, and the 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 PVDF membrane, blocked with skim milk powder, and purified anti-RNF34 (R40 arginine dimethylation) polyclonal antibody (dilution factor 1:1000) was added. After incubation at room temperature for 1 hour, the membrane was washed five times (8 min / time), and HRP-labeled goat anti-rabbit secondary antibody (dilution factor 1:50000) was added. After incubation at room temperature for 1 hour, the membrane was washed five times (8 min / time). The final results were collected by Jena UVP Chemstudio touch multifunctional imager.

[0076] The results are as follows Figure 4 As shown: 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, antigen repaired by high-pressure boiling with citric acid, blocked, and subjected to 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, which was 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 this site 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 antibody detection of methylation modification levels in patient-derived tumor xenograft models (PDX)

[0082] Tumor tissue was obtained through surgery 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, the tumor was peeled off from the first-generation host (P0), and part of the tissue was minced into about 3 mm × 3 mm × 3 mm and implanted into new mice (P1). Part of the tissue was fixed with paraformaldehyde for further testing, or preserved with cryopreservation solution for recovery 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 10% SDS-PAGE electrophoresis was performed. After electrophoresis, it was transferred to PVDF membrane, blocked with skim milk powder, and purified anti-RNF34 (R26 me2a 、R40 me2a ) polyclonal antibody (dilution factor 1:1000), incubate at room temperature for 1 hour, then wash the membrane five times (8 min / time), add HRP-labeled goat anti-rabbit secondary antibody (dilution factor 1:50000), incubate at room temperature for 1 hour, then wash the membrane five times (8 min / time), and the final results are collected by Jena UVP Chemstudio touch multifunctional imager.

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

[0086] The results are as follows Figure 7 As shown: Anti-RNF34 (R26 me2a 、R40 me2a ) Polyclonal antibodies were used 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 chemosensitivity in human tumor xenograft models.

[0088] One case of RNF34 R26 was selected based on the aforementioned PDX me2a 、R40 me2a The highly expressed PDX tissues were divided into two major groups, one was the solvent control group, and the other was the 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, 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, 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 of R26-Pe and R40-Pe made the tumor more sensitive to chemotherapy drugs (e.g. Figure 8 as 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, with 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 Fig. 9 There was no obvious damage to the heart, liver, spleen, lung and kidney in the mice (as shown in Fig.10 as shown).

[0093] Example 9 RNF34 TAT cell-penetrating peptide blocks R26 me2a 、R40 me2a Methylation promotes chemosensitivity in colorectal cancer organoids

[0094] Patients who were first diagnosed with colorectal cancer in the Department of Colorectal Surgery of the Affiliated Tumor Hospital of Guangxi Medical University and had not received interventional, radiotherapy, chemotherapy or targeted drugs before surgery were collected. Colorectal cancer surgical specimens of patients were collected to construct organoid models. The tissues were transferred to PBS supplemented with penicillin and streptomycin 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 about 0.5-2 mm³ in size with sterile tissue scissors in the culture dish, and the minced tissues were transferred to a 15 mL centrifuge tube and resuspended in a digestion solution 50 times the volume of the minced tissue suspension. The tissues were digested for 60 minutes in a constant temperature shaking incubator at 37°C and 50-100 rpm. Fetal bovine serum was added after digestion to terminate digestion. The tissue suspension was filtered using a 100 μm cell filter, and the filtrate was transferred to a centrifuge tube and centrifuged at 200-300 g. Centrifuge for 3-5 minutes at 10000 rpm, remove the supernatant, retain the precipitate, resuspend and count, add the corresponding amount of organoid-specific matrix gel according to the ratio of 8000-30000 cells per well to 20-30 μL matrix gel, mix well on ice, and place on ice after mixing. Use a pipette to transfer the matrix gel and cell mixture to the center of the bottom of the cell culture well plate (20-30 μL of mixed suspension per well of a 24-well cell culture plate). Place the culture plate in a 37°C, 5% CO2 cell culture incubator to solidify for 30 minutes. After the matrix gel solidifies and no longer flows, slowly add the organoid-specific culture medium along the well wall.

[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 chemotherapeutic 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 and in combination (WB test results are as follows Fig.11 As shown in the figure, 01#~04# are successfully constructed organoid models from different patient sources). CCK8 results showed that R26-TAT and R40-TAT transmembrane peptides have inhibited the growth of organoids 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 Fig.12 as 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 clinical pathological baseline data (including age, gender, tumor site, pathological type, pathological stage, tumor size, surgery, chemotherapy, etc.) were collected from the colorectal anus ward. Inclusion criteria: patients had a first colonoscopy with clamped tumor pathological tissue, and the pathological report diagnosed colorectal adenocarcinoma, had not received surgery, intervention, radiotherapy, chemotherapy or targeted drugs before endoscopic biopsy, had no other history of tumors, and had regular 4 cycles of FOLFOX chemotherapy after diagnosis. Patients received CT examinations of the tumor site or distant metastatic organs before and after chemotherapy in this center. According to the changes in the primary lesions and metastatic lesions, the efficacy was divided into four levels: complete remission (CR), partial remission (PR), stable disease (SD) and progressive disease (PD).

[0099] Detection of RNF34 R26 in cancer tissues by immunohistochemistry me2a 、R40 me2a Analysis of arginine methylation levels at R26 and RNF34 in cancer tissues me2a 、R40 me2a The correlation between arginine methylation level at RNF34 and R26 and the efficacy of neoadjuvant chemotherapy in patients 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 Fig.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. Application of RNF34 protein R26 and / or R40 sites as targets in the following Ⅰ and / or Ⅱ: Ⅰ. Preparation of drugs for diagnosis and / or treatment of colorectal cancer; Ⅱ. Prepare reagents for detecting sensitivity of chemotherapy drugs.

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

3. An artificial antigen, characterized in that It is obtained by coupling a carrier protein with the RNF34 protein described in claim 2.

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

5. The method for preparing the asymmetric dimethylated antibody of RNF34 protein according to claim 4, characterized in that: The method comprises immunizing an animal with the RNF34 protein according to claim 2 or the artificial antigen according to claim 3, and then isolating an asymmetric dimethylated antibody of the RNF34 protein from the serum of the immunized animal.

6. The preparation method according to claim 5, characterized in that: The animal includes a rabbit, and the number of immunizations is ≥ 3 times.

7. A cell-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.

8. The cell-penetrating peptide according to claim 7, characterized in that The cell-penetrating peptide has an amino acid sequence as shown in SEQ ID NO:4 and SEQ ID NO:

5.

9. 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: (1) The RNF34 protein according to claim 2; (2) The artificial antigen according to claim 3 (3) The asymmetric dimethylation antibody of the RNF34 protein according to claim 4; (4) An asymmetric dimethylated antibody of RNF34 protein prepared by the preparation method of claim 5 or 6; (5) The cell-penetrating peptide according to claim 7 or 8.

10. A drug for diagnosing and / or treating colorectal cancer or a reagent for detecting sensitivity to chemotherapeutic drugs, characterized in that: Include at least one of the following ①~⑤:

1. The RNF34 protein according to claim 2; ②. The artificial antigen according to claim 3 ③. The asymmetric dimethylated antibody of RNF34 protein according to claim 4; ④. An asymmetric dimethylated antibody of RNF34 protein prepared by the preparation method according to claim 5 or 6; 5. The cell-penetrating peptide according to claim 7 or 8.

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