CN113917150A

CN113917150A - Tumor marker related to acetylation of K264 site of p62 and application thereof

Info

Publication number: CN113917150A
Application number: CN202111478774.1A
Authority: CN
Inventors: 王海英; 罗建沅; 熊健楠; 李美婷
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2021-09-10
Filing date: 2021-12-06
Publication date: 2022-01-11
Anticipated expiration: 2041-12-06
Also published as: CN113917150B

Abstract

The invention provides a tumor marker related to acetylation of a K264 site of p62 and application thereof, wherein the marker is acetylation of the K264 site of p62, acetylation of the K264 site of p62 is jointly regulated and controlled by acetyltransferase hMOF of p62 and deacetylase SIRT7 of p62, acetylation of the K264 site of p62 is further involved in regulation and control of Base Excision Repair (BER), and the application comprises prediction of patients with high BER repair efficiency and strong tumor repair effect, so that the drug resistance of antitumor drugs is easy to occur. The invention also provides a marker or a method for screening drugs which can improve BER efficiency of tumors and enable the tumors to be easy to relapse and resist drugs. In addition, the invention also provides a specific antibody for detecting acetylation of the K264 site of p62, and a preparation method and application thereof. The invention provides new clues for the treatment medicines and treatment strategies of p62 related diseases, in particular to cancers.

Description

Tumor marker related to acetylation of K264 site of p62 and application thereof

The present invention claims priority from chinese patent application CN202111064291.7 filed on 9/10/2021, and the contents of the specification, drawings and claims of this priority document are incorporated in their entirety into the specification of the present invention and are included as part of the original description of the present invention. Applicants further claim that applicants have the right to amend the description and claims of this invention based on this priority document.

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a tumor marker related to acetylation of a K264 site of p62 and application thereof. The applications include high BER repair efficiency and strong tumor repair effect, so that the prediction of patients with drug resistance of antitumor drugs is easy to occur. The invention also relates to a marker or a method for screening drugs which cause the BER efficiency of tumors to be improved and enable the tumors to be easy to relapse and resist drugs. Additionally, the invention also relates to a specific antibody for detecting acetylation of the K264 site of p62, and a preparation method and application thereof.

Background

Cells maintain genome stability through different DNA damage Repair pathways to different types of damage, including Mismatch Repair (MMR), Base Excision Repair (BER), Nucleotide Excision Repair (NER), Non-Homologous End Joining (NHEJ), and Homologous Recombination (HR). Among these DNA repair pathways, BER is the most evolutionarily highly conserved, most widely effective DNA repair mechanism that can repair most types of DNA damage, such as base oxidation by reactive oxygen species, alkylation modification by alkylating agents, base deamination, purine-or pyrimidine-free sites, and X-ray induced DNA single strand breaks. As a primary defense against damage by cells, BER imbalance is closely associated with a variety of human diseases, including neurodegenerative diseases, aging, tumors, and chemotherapy resistance.

The defect in the BER system is associated with tumor susceptibility, and cancer cell DNA repair is more dependent on BER than normal cells. Inhibiting BER can weaken the repair capacity of tumor cell to DNA damage after the action of anticancer medicine, avoid the generation of medicine resistance and raise the sensitivity of anticancer medicine.

p62, also known as sequestosome 1(SQSTM1), is a multifunctional scaffold protein that plays an important role in cellular activities as a pivotal receptor, and is involved in regulation of oxidative stress, cellular metabolism, inflammatory response, and DNA damage repair, in addition to being an important receptor protein for autophagy. It was found that p62 can regulate DNA Damage Response (DDR) and repair through different mechanisms. There is a large body of evidence that p62 is closely associated with various types of tumors, including breast, esophageal, liver and lung cancers. p62 is highly expressed in most types of tumors and exerts carcinogenic effects.

p62 is used as a pivot of cell signaling, is subject to complex and strict regulation and relates to various regulation modes such as transcription, post-transcription and post-translation modification. Post-translational modification (PTM) of proteins is a major mechanism for regulating protein functions, and reports on post-translational modification of p62 have been increasing in recent years, and it has an important regulatory role in the biological function of p 62. The research finds that p62 is regulated by extensive phosphorylation and ubiquitination modifications, and the modifications mainly affect the affinity of p62 to ubiquitin proteins and the binding capacity of the ubiquitin proteins and are involved in regulating biological processes such as autophagy, cell proliferation and oxidative stress.

The phosphorylation modification of p62 is currently being studied more. Protein Kinase A (PKA) mediated phosphorylation of serine 24 at p62 inhibits the polymerization and interaction of p62 with other proteins. Threonine 138 of p62 is regulated by leucine-rich repeat kinase 2 (LRRK 2), and p62 serves as a substrate of LRRK2 to enhance neurotoxicity. Cyclin-dependent kinase 1 (CDK 1) -mediated phosphorylation of threonine 269 and serine 272 is important for the correct entry and exit of cells from the cell cycle, as well as being essential for maintaining cyclin B1 levels and CDK1 activity.

Besides phosphorylation modifications, ubiquitination modifications of p62 are also important for their regulation. The PB1 domain of p62 forms p62 self-oligomerization through electrostatic interaction between lysine at position 7 and aspartic acid at position 69 for head-to-tail binding. The PB1 domain-mediated p62 aggregation is crucial for p62 to function as selective autophagy. Studies have reported that E3 ubiquitin ligase TRIM21 inhibits p62 aggregation by ubiquitinating lysine 7 of p62 under oxidative stress conditions, thereby modulating Keap1 chelation in response to antioxidant responses. E3 ligase RNF166 mediated lysine ubiquitination 91 and 189, and is involved in the xenobiotic autophagy degradation of intracellular bacteria.

Acetylation has been a gap as to how the main means of post-translational modification of proteins affects the function of p 62. Recently, studies by scholars have found that the acetyltransferase Tip60 and the deacetylase HDAC6 regulate the acetylation of the 420 th and 435 th lysines of p 62. Under the condition of nutrient deficiency, the acetylation levels of the two sites of p62 are remarkably increased, and the combination of p62 and ubiquitin protein is influenced, and the acetylation level plays an important role in autophagy.

Different from the research, the inventor firstly discovers that the acetyltransferase hMOF and the deacetylase SIRT7 jointly regulate the acetylation of the 264 th lysine of p62, and firstly proves that the acetylation of the 264 th lysine of p62 has an important regulation function in BER. These studies will provide new clues for the therapeutic drugs and therapeutic strategies for p 62-related diseases, particularly cancer.

Disclosure of Invention

On the basis of the research, the inventor firstly proposes a tumor marker related to acetylation of a K264 site of p62 and application thereof, wherein acetylation of the K264 site of p62 is jointly regulated and controlled by acetyltransferase hMOF and deacetylase SIRT7, acetylation of the K264 site of p62 further participates in regulation and control of Base Excision Repair (BER), and the application comprises high BER repair efficiency and strong tumor repair effect, so that prediction of patients with antitumor drug resistance is easy to occur. Additionally, the present inventors have also presented for the first time markers or methods for screening drugs that result in an increase in the BER efficiency of tumors, rendering tumors susceptible to relapse and drug resistance.

Specifically, the invention is realized by the following technical schemes:

in a first aspect, the invention provides a tumor marker related to acetylation of K264 site of p62, wherein the tumor marker is acetylation of K264 site of p62, acetylation of K264 site of p62 is jointly regulated by acetyltransferase hMOF of p62 and deacetylase SIRT7 of p62, acetylation of K264 site of p62 is further involved in BER regulation, the tumor marker is used for BER repair with high efficiency and strong tumor repair effect, so that the patient prediction of drug resistance of an anti-tumor drug is easy to appear, and the tumor marker is used for detecting a sample of the patient by a conventional molecular biology method, wherein the sample is from tumor tissue, blood, plasma, serum, urine, feces, sputum or ascites.

Alternatively, in the above marker, the acetyltransferase hmuf of p62 mediates acetylation of the K264 site of p62, the deacetylase SIRT7 of p62 mediates deacetylation of the K264 site of p62, and the conventional molecular biological methods include enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunocytochemistry, co-immunoprecipitation, or immunoblotting.

In a second aspect, the present invention provides a kit for predicting patients who have high BER repair efficiency and strong tumor repair effect and are thus prone to develop anti-tumor drug resistance, the kit comprising the tumor marker related to acetylation of the K264 site of p62 in the first aspect, and an instruction describing a reference data set of levels of the tumor marker in samples of tumor patients and healthy people.

Alternatively, in the marker or the kit, the method for predicting patients who have high BER repair efficiency and strong tumor repair effect and are thus prone to develop anti-tumor drug resistance comprises the following steps:

(1) providing a sample from a patient to be detected, and detecting the level of a tumor marker in the sample, wherein the tumor marker is the acetylation level of the K264 locus of p 62;

(2) comparing the marker levels measured in step (1) with corresponding reference data sets of tumor marker levels for samples of tumor patients and healthy populations; and

(3) and if the level of the tumor marker in the sample of the patient to be detected is within the range of the corresponding tumor marker level reference data set of the tumor patient and the level of the tumor marker in the sample of the patient to be detected is obviously far away from the range of the corresponding tumor marker level reference data set of the healthy population sample, judging the patient to belong to the patient with high BER repair efficiency and strong tumor repair effect, so that the patient with anti-tumor drug resistance is easy to appear.

Alternatively, in the marker or the kit, in the step (3), in the patient with high BER repair efficiency and strong tumor repair effect and thus easy occurrence of anti-tumor drug resistance, the acetylation level of the K264 site of p62 is significantly higher than that of the reference data set of the acetylation levels of the K264 site of the healthy human sample p 62.

In a third aspect, the invention provides a marker for screening drugs which cause the BER efficiency of tumors to be improved and enable the tumors to be easy to relapse and drug resistance, wherein the marker is acetylation of K264 site of p62, acetylation of K264 site of p62 is commonly regulated by acetyltransferase hMOF of p62 and deacetylase SIRT7 of p62, acetylation of K264 site of p62 is further involved in BER regulation, and the marker is used for detecting a sample of a subject to be tested by a conventional molecular biology method, wherein the sample is a tumor cell line or tumor tissue, blood, plasma, serum, urine, feces, sputum or ascites of the subject to be tested.

In a fourth aspect, the present invention provides a method for screening for a drug that results in an increase in BER efficiency of a tumor, rendering the tumor susceptible to relapse and drug resistance, comprising the steps of:

(1) in a test group, a test drug is applied to a test object, and the acetylation level L of the K264 locus of the marker p62 in a sample derived from the test object in the test group is respectively detected_1aIn a control group, a blank control or a vehicle control is applied to the test subject, and the level L of acetylation of the K264 site of the marker p62 in the sample derived from the test subject in the control group is detected_0aWherein the marker is acetylation of K264 site of p62, acetylation of K264 site of p62 is co-regulated by acetyltransferase hMOF of p62 and deacetylase SIRT7 of p62, acetylation of K264 site of p62 is further involved in regulating BER;

(2) respectively comparing L detected in the step (1)_1aAnd level L_0aMaking a comparison if said level L_1aIs statistically significantly higher than said level L_0aDetermining that the BER efficiency of the tumor is improved due to the existence of the drug to be detected, so that the tumor is easy to have the risks of relapse and drug resistance,

wherein, in the step (1) and the step (2), the marker is used for detecting a sample of a subject to be detected by a conventional molecular biology method, wherein the sample is a tumor cell line or is derived from tumor tissue, blood, plasma, serum, urine, feces, sputum or ascites of the subject to be detected.

Alternatively, in the above method, the acetyltransferase hmuf of p62 mediates acetylation of the K264 site of p62, the deacetylase SIRT7 of p62 mediates deacetylation of the K264 site of p62, and the conventional molecular biological method includes enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunocytochemistry, co-immunoprecipitation, or immunoblotting.

Alternatively, in the above method, the test subject is a tumor cell line or an animal having a tumor.

Preferably, the animal is a mammal.

More preferably, the mammal is a human, a non-human primate or a rodent.

In a fifth aspect, the present invention provides a method for preparing a specific antibody for detecting acetylation of K264 site of p62, the method comprising the steps of:

(1) artificially synthesizing IDVEHGGKRSRLTP sequence p62-K264 polypeptide, and performing acetylation treatment on a K locus;

(2) and (2) immunizing a rabbit with the polypeptide synthesized in the step (1) to obtain polyclonal antiserum, measuring the antibody titer in the serum by an enzyme-linked immunosorbent assay (ELISA), and purifying the antibody to obtain the specific antibody.

Alternatively, in the above production method, preferably, in step (1), the acetylase used for the acetylation treatment of the K site is hmf;

in the step (2), the specific process of immunizing rabbits is as follows: primary immunization, emulsifying 600 mu g p62-K264 polypeptide and an equal volume of Freund's complete adjuvant in each rabbit, and then injecting the mixture into multiple points (preferably, 6 points) subcutaneously, respectively, after 28 days, 42 days and 56 days of the primary immunization, and emulsifying 400 mu g p62-K264 polypeptide and an equal volume of Freund's incomplete adjuvant in each rabbit, and then injecting the mixture into multiple points (preferably, 4 points) subcutaneously; taking the ear blood after the third boosting immunization, separating the serum to be used as the serum after the immunization, measuring the antibody titer in the serum by adopting ELISA, collecting the whole blood by a cardiac puncture method on the 2 nd day after the titer is detected after the antibody titer reaches the index, and collecting the serum to obtain the polyclonal antiserum;

in step (2), ELISA was used to determine antibody titer in serum by indirect ELISA, and the antigen affinity column used for antibody purification was CnBr-activated Sepharose 4B.

In a sixth aspect, the invention provides a specific antibody for detecting acetylation of K264 site of p62, wherein the antibody is prepared by the preparation method of the fifth aspect, and the detection sensitivity of the antibody against p62-K264 polypeptide is 3.3 ng/ml.

In a seventh aspect, the present invention provides the use of a specific antibody according to the sixth aspect above in the preparation of a reagent for detecting acetylation of the K264 site of p 62.

Preferably, the reagent is used for in vivo or in vitro detection of acetylation of the K264 site of p62 described in the first to fourth aspects above.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention provides a tumor marker related to acetylation of a K264 site of p62 and application thereof for the first time, wherein the application comprises high BER repair efficiency and strong tumor repair effect, so that the prediction of a patient with anti-tumor drug resistance is easy to occur.

(2) The inventor also provides a marker or a method for screening the medicine which leads to the increase of BER efficiency of the tumor and enables the tumor to be easy to relapse and resist for the first time.

(3) The inventor also prepares a specific antibody for detecting acetylation of the K264 site of p 62.

(4) The invention provides new clues for the treatment medicine and the treatment strategy of p62 related diseases, particularly cancer.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1: hMOF acetylates p 62. (A) HCT116 cells were collected, lysed and subjected to immunoprecipitation with a pantysine acetylated antibody, and immunoblotting to detect p62 protein. (B) Co-transfecting unloaded HA-P300, HA-CBP, Flag-PCAF, Myc-hMOF and Flag-P62 into HCT116 cells, collecting the cells after 60 hours, extracting cell proteins, adding Flag-M2 beads into protein lysate for immunoprecipitation experiments, and detecting the acetylation level of P62 by immunoblotting experiments. (C) Respectively transfecting unloaded Myc-hMOF and Flag-p62 into HCT116 cells, collecting the cells after 48 hours, extracting cell proteins, adding Flag-M2 beads into protein lysate for immunoprecipitation experiments, and detecting the acetylation level of p62 by immunoblotting experiments. (D) Myc-hMOF is transfected into HCT116 cells, the cells are collected after 48 hours to extract protein, the protein is enriched by agarose beads, hMOF protein is used as enzyme, GST-p62 purified from the bacteria body is used as substrate, in vitro acetylation experiment is carried out, and the acetylation level of p62 is detected by immunoblotting experiment.

FIG. 2: p62 is capable of interacting with hMOF. (A) Respectively transfecting the unloaded Myc-hMOF and the Flag-p62 plasmid into HCT116 cells, collecting the cells after 48 hours to extract protein, carrying out an immunoprecipitation experiment by using an Myc antibody, and detecting the corresponding protein by using an immunoblotting experiment. (B) Collecting HCT116 cells, cracking the cells, then carrying out co-immunoprecipitation experiment by using hMOF antibody, and detecting corresponding protein by immunoblotting experiment. (C) And (3) co-incubating TF-His-p62 protein purified from the bacterial body with GST protein and GST-hMOF protein purified from the bacterial body, and detecting corresponding protein by an immunoblotting experiment. Coomassie brilliant blue staining showed specific bands of purified protein.

FIG. 3: hMOF-mediated acetylation of p62K 264. (A) Mass spectrometry was performed to analyze the site of acetylation of p 62. (B) The Flag-p62-WT, Flag-p62-K189R and Flag-p62-K264R plasmids and the unloaded and Myc-hMOF plasmids are respectively transfected into HCT116 cells, the cells are collected after 48 hours to extract protein, Flag-M2 beads are added into protein lysate for an immunoprecipitation experiment, and the p62 acetylation level is detected by an immunoblotting experiment. (C) The amino acid sequence around the K264 position in the p62 sequence of human and other species and its conservation. (D) His-p62-WT and His-p62-K264R proteins purified from the bacterial body and GST-hMOF protein purified from the bacterial body are respectively subjected to in vitro acetylation experiments under the condition that acetyl coenzyme A exists, and the acetylation level of p62 is detected by immunoblotting. Coomassie brilliant blue staining showed specific bands of purified protein.

FIG. 4: specificity and efficiency of p62K264 acetylated antibody. (A) The specificity of the acetylated p62K264 antibody was determined by dot blot hybridization. (B) The Flag-p62-WT and Flag-p62-K264R plasmids and Myc-hMOF plasmids are respectively transfected into HCT116 cells, the cells are collected after 48 hours to extract protein, Flag-M2 beads are added into protein lysate to carry out an immunoprecipitation experiment, and the p62K264 acetylation level is detected by an immunoblotting experiment.

FIG. 5: p62 was able to interact with SIRT 7. (A) The unloaded, GFP-SIRT7 and Flag-p62 plasmids were co-transfected into HCT116 cells, and after 48 hours, the cells were harvested for protein extraction, and the corresponding proteins were detected by immunoprecipitation with GFP antibody and immunoblotting. (B) HCT116 cells are collected, and after the cells are lysed, a co-immunoprecipitation experiment is carried out by using a SIRT7 antibody, and the corresponding protein is detected by an immunoblotting experiment. (C) His-SIRT7 protein purified from the bacterial body is incubated with GST protein purified from the bacterial body and GST-p62 protein, and the corresponding protein is detected by an immunoblotting experiment. Coomassie brilliant blue staining showed specific bands of purified protein.

FIG. 6: SIRT7 mediates p62 deacetylation. (A) The SIRT7 siRNA is transfected into HCT116 cells, the protein is extracted from the cells after 72 hours, and the p62 antibody is used for an immunoprecipitation experiment, and the acetylation level of p62 is detected by an immunoblotting experiment. (B) Collecting SIRT7 wild type (SIRT7 WT) and SIRT7 gene knock-out (SIRT7 KO) HCT116 cells, respectively, cracking the cells, extracting proteins, performing immunoprecipitation experiment with p62 antibody, and detecting acetylation level of p62 by immunoblotting. (C) The unloaded, Flag-SIRT7 cells were transfected into HCT116 cells, and the cells were harvested 48 hours later for protein extraction, and immunoprecipitation experiments were performed with p62 antibody and immunoblotting experiments were performed to detect the level of p62 acetylation.

FIG. 7: SIRT7 mediates deacetylation of p62K 264. (A) Flag-p62 was transfected into HCT116 cells, treated with 1. mu.M TSA for 12 hours and 5mM NAM for 16 hours, the cell extract protein was collected, and Flag-M2 was added to the protein lysate for immunoprecipitation assay, and immunoblotting assay was used to detect the acetylation level of p62K 264. (B) The method comprises the steps of transfecting Flag-p62-WT and Flag-p62-K264R into HCT116 cells, treating the cells with 5mM NAM for 16 hours, collecting cell extract protein, adding Flag-M2 beads into protein lysate for immunoprecipitation experiments, and detecting the acetylation level of p62K264 by immunoblotting experiments. (C) The unloaded, GFP-SIRT7 and Flag-p62 plasmids were co-transfected into HCT116 cells, treated with 5mM NAM for 16 hours, the cells were collected for protein extraction, and Flag-M2 beads were added to the protein lysate for immunoprecipitation experiments, which detected the acetylation level of p62K 264. (D) His-p62 protein purified from bacteria and GST-hMOF protein purified from bacteria in vitro are subjected to acetylation in the presence of acetyl coenzyme A, and then purified from cells to obtain SIRT7 protein in NAD⁺In the presence ofIn vitro deacetylation experiments were performed and immunoblotting experiments were performed to detect the level of acetylation of p62K 264. Coomassie brilliant blue staining showed specific bands of purified protein.

FIG. 8: MMS was able to specifically increase the acetylation level of p62K 264. (A) At a rate of 40J/m²HCT116 cells were treated with UV, 5mmol/L HU, 1. mu. mol/L cisplatin and 0.01% MMS, respectively, and after 4 hours the cell extract proteins were collected and subjected to immunoprecipitation with p62 and immunoblotting to detect the level of acetylation of p62K 264. (B) The method comprises the steps of transfecting Flag-p62-WT and Flag-p62-K264R into HCT116 cells, treating the cells with 0.01% MMS for 4 hours, collecting cell extract protein, adding Flag-M2 beads into protein lysate for immunoprecipitation experiments, and detecting the acetylation level of p62K264 by immunoblotting experiments. (C) HCT116 cells were treated with different doses (0.005%, 0.01%, 0.015%) of MMS for 4 hours, and the cell extracts were collected and subjected to immunoprecipitation with p62 and immunoblotting to detect the acetylation level of p62K 264. (D) HCT116 cells were treated with 0.01% MMS for various periods of time (1, 2, 4 hours), respectively, and the cells were harvested for protein extraction, immunoprecipitation with p62, and immunoblotting to detect the level of acetylation of p62K 264. (E) LoVo, HeLa and HepG2 cells were treated with different doses (0.005%, 0.01%, 0.015%) of MMS for 4 hours, respectively, and total protein was extracted from the cells collected and tested for acetylation level of p62K264 by immunoblotting assay.

FIG. 9: acetylation of p62K264 promoted BER. (A) HCT116 cells were treated with 1mmol/L hydrogen peroxide for various periods of time (10, 20, 30 minutes), total protein was collected from the cells and the level of acetylation of p62K264 was determined by immunoblotting. (B) A p62 stable knockout HCT116 cell line is constructed by using a CRISPR-Cas9 gene editing technology, and then HCT116 cell lines of p62-WT, p62-K264R and p62-K264Q which stably express Flag tags are established on the basis of the cell line. Cells were collected and total protein was extracted and immunoblotting was performed to detect expression of p62 protein. (C) And quantitatively detecting the AP locus numbers of three cells, namely p62-WT, p62-K264R and p 62-K264Q. Statistical results in the figure represent p <0.05, expressed as mean ± SD. (D) Single cell gel electrophoresis experiments were performed by treating three cells, p62-WT, p62-K264R and p62-K264Q, with 0.01% MMS for 20 minutes, and treating three cells of another group with 0.01% MMS for 20 minutes and releasing for 1 hour. (E) Statistics of% of tailing DNA under the above (D) treatment conditions. At least 100 cells of each group of experiments were analyzed for nuclear comet tail. Statistical results in the figure represent p < 0.001.

FIG. 10: acetylation of p62K264 promoted cell survival after MMS treatment. (A) Three cells, p62-WT, p62-K264R and p62-K264Q, were treated with different doses of MMS for 4 hours for colony formation experiments. (B) The number of clones under the treatment conditions (A) above was quantified, survival rate was calculated, and a histogram was prepared. Data are expressed as mean ± SD, statistical results in the figure represent p < 0.05.

FIG. 11: the purity of the purified antibody was checked by SDS-PAGE. M, protein Marker (from top to bottom: 170KD/130KD/95KD/72KD/55KD/43KD/34KD/26KD/17KD in turn); an antigen affinity purified antibody of JXR 4541.

Detailed Description

In order to better understand the present invention, some terms involved in the present invention are further explained below.

It is particularly noted that in the present invention, the term "marker" also referred to as "biological marker" refers to a measurable indicator of the biological state of an individual. Such a biological marker can be any substance in an individual so long as it is associated with a particular biological state (e.g., a disease, condition, or disorder) of the individual being tested, e.g., a nucleic acid (e.g., DNA or RNA) marker, a protein marker, a cytokine marker, a chemokine marker, a carbohydrate marker, an antigen marker, an antibody marker, a functional marker, and the like. Biological markers are measured and evaluated, often to examine normal biological processes, pathogenic processes, or therapeutic intervention pharmacological responses, and are useful in many scientific fields.

In the present invention, the term "plasma" refers to the liquid component of whole blood. Depending on the separation method used, the plasma may be completely free of cellular components, or may contain varying amounts of platelets and/or small amounts of other cellular components.

In the present invention, the term "marker level reference data set" refers to a reference value or range of normal values for a sample of a tumor patient or a healthy population. The skilled person will appreciate that, in the case of a sufficiently large number of samples, the absolute or normal range of values for each biomarker in the patient or healthy population as described above can be determined and calculated. In addition, it can be calculated by a statistical method.

The invention is further illustrated with reference to specific examples. It should be understood that the specific embodiments described herein are illustrative only and are not limiting upon the scope of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products which are not known to manufacturers and are available from normal sources.

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

Examples

hMOF mediated acetylation of p62K264

1.1hMOF is capable of acetylating p62

First, it was confirmed whether or not p62 could be modified by acetylation. HCT116 cells were harvested, lysed, immunoprecipitated with a pantysine-acetylated antibody, and detected with p62 antibody, and p62 was found to be acetylated (FIG. 1A). To determine the acetyltransferase of P62, several common acetyltransferases were selected, including P300, CBP, PCAF and hmuf, co-transfected with P62 into HCT116 cells, and the cells were harvested after 60 hours and tested for changes in the acetylation level of P62 protein. Experimental results show that hmfo can significantly increase the acetylation level of p62 (fig. 1B).

The hMOF was then overexpressed in HCT116 cells, and the results showed that the acetylation level of p62 gradually increased with increasing hMOF expression levels (FIG. 1C). To further demonstrate that hMOF can directly acetylate p62, Myc-hMOF plasmid was transfected into HCT116 cells, the cells were harvested 48 hours later, hMOF proteins were immunoprecipitated, and in vitro acetylation experiments were performed with GST-p62 purified from the bacteria as a substrate. As shown in fig. 1D, hmf was able to acetylate p62 in the presence of acetyl-CoA (Ac-CoA), indicating that hmf was able to acetylate p62 directly in vitro. All the above results indicate that hMOF is an acetyltransferase of p 62.

1.2p62 being able to interact with hMOF

To further validate that hMOF could catalyze acetylation of p62, it was examined whether p62 and hMOF could interact. The ability of p62 to interact with hMOF was confirmed by an exogenous co-immunoprecipitation experiment (FIG. 2A), while the ability of the two to interact was confirmed by an endogenous co-immunoprecipitation experiment using antibodies to hMOF (FIG. 2B). It follows from this that: there is a clear interaction between p62 and hMOF in cells, both from endogenous and exogenous sources. In addition, GST pull down results in vitro showed that the interaction of p62 and hmf was direct (fig. 2C). Taken together, the above results indicate that p62 is capable of interacting with hMOF.

1.3hMOF mediated acetylation of p62K264

To further determine the acetylation site of hMOF-specific catalytic p62, Myc-hMOF and Flag-p62 were co-transfected into HCT116 cells, and then Flag-p62 protein was affinity-enriched with agarose beads, and the pooled peptide fragments were subjected to combined mass spectrometry to generate acetylation sites. The analysis results showed that the position where hMOF acetylated p62 was probably lysine 189 and 264 (FIG. 3A). To further confirm the acetylation site of p62 by hMOF, lysine (K) at positions 189 and 264 of p62 was mutated to arginine (R) to give p62-K189R and p62-K264R mutant plasmids, and then the acetylation level of p62 protein after site mutation was examined. After co-transfection of p62-WT, p62-K189R, p62-K264R plasmids with Myc-hMOF, respectively, into HCT116 cells, detection with a pantysine acetylation antibody revealed only a significant decrease in the acetylation level of p62-K264R (FIG. 3B), suggesting that K264 is the major acetylation site of hMOF regulation of p62, and that this is also evolutionarily conserved (FIG. 3C). In vitro acetylation experiments were next performed. The results show that hmf can significantly increase the acetylation level of p62-WT, but not the acetylation level of the K264R mutant. Indicating that hmf directly catalyzes the acetylation of p62K264 (fig. 3D).

To further verify that acetylated antibodies specific to p62K264 were customized (see section 5 of the examples section for specific methods of preparation of said antibodies), experimental results showed that the customized acetylated p62K264 antibody had better specificity and efficiency (fig. 4A). Further experimental validation was then performed using a custom-made p62K264 acetylated antibody. After co-transfection of p62-WT, p62-K264R and hMOF into HCT116 cells, respectively, the acetylation level of p62K264 was detected with specific antibodies. The experimental results show that the tailored specific antibody was able to detect acetylation of p62-WT but not of the p62-K264R mutant (FIG. 4B). Combining the above results, it is concluded that: hmf mediates primarily acetylation of p62K 264.

SIRT7 mediated deacetylation of p62K264

2.1p62 being able to interact with SIRT7

The ability of p62 to interact with SIRT7 was confirmed by foreign co-immunoprecipitation experiments (FIG. 5A). Further performing an endogenous co-immunoprecipitation experiment using the SIRT7 antibody confirmed that there is also an endogenous interaction experiment between the two, confirmed that there is also an endogenous interaction between the two (fig. 5B), from which it was concluded: neither endogenous nor exogenous interactions were present between intracellular p62 and SIRT 7. Similarly, GST pull down experiments in vitro confirmed that the interaction between p62 and SIRT7 was direct (fig. 5C). Taken together, the above results indicate that p62 is capable of interacting with SIRT 7.

2.2SIRT7 mediated deacetylation of p62

To further explore the possibility of SIRT7 mediating p62 deacetylation, the level of acetylation of endogenous p62 was examined in HCT116 cells knocking down and knocking out SIRT7, respectively. The results show that both knock-down and knock-out SIRT7 in HCT116 cells had elevated levels of p62 acetylation compared to control cells (fig. 6A and 6B). HCT116 cells were further tested for the level of acetylation of endogenous p62 when SIRT7 was overexpressed. The results show that after overexpression of SIRT7, the level of acetylation of p62 was reduced (fig. 6C). Taken together, the above results demonstrate that SIRT7 is capable of deacetylating p 62.

2.3SIRT7 mediated deacetylation of p62K264

To verify whether SIRT7 regulates the deacetylation of p62K264, a Flag-p62 plasmid was transfected into HCT116 cells, which were treated with Trichostatin a (TSA) (an inhibitor of group i, ii iv histone deacetylases) and Nicotinamide (nim) (an inhibitor of group iii histone deacetylases Sirtuins). The cells were then lysed and the level of acetylation of p62K264 was measured by co-immunoprecipitation experiments using Flag-M2 beads. The results show that Flag-p62 significantly increased the level of K264 acetylation after NAM treatment, whereas TSA treatment did not change significantly (fig. 7A). Further, p62-WT and p62-K264R plasmids were transfected into HCT116 cells, and the acetylation level of p62K264 was examined after NAM treatment, and it was found that the acetylation level of p62-WT was significantly increased after NAM treatment, while p62-K264R was not increased (FIG. 7B). The results of the experiments fully indicate that acetylation of p62K264 is regulated by class III deacetylase Sirtuins.

To further verify that acetylation of K264 is regulated by SIRT7, Flag-p62 and GFP-SIRT7 were co-transfected into HCT116 cells and the level of acetylation of p62K264 was measured. The results show that after over-expressing SIRT7, the acetylation level of p62K264 was significantly reduced, while the inhibitor NAM of SIRT7 was able to block the reduction of acetylation (fig. 7C), suggesting that SIRT7 could remove acetylation of p62K 264. In order to verify that SIRT7 directly catalyzes the deacetylation of p62K264, an in vitro deacetylation experiment was performed. Firstly, the His-p62 protein purified from bacteria and GST-hMOF protein are incubated together in the presence of acetyl coenzyme A to obtain His-p62 with high acetylation level, and then the His-p62 protein and Flag-SIRT7 protein purified from cells are incubated in NAD⁺In vitro deacetylation experiments were performed in the presence. The results show that p62 can be deacetylated in vitro by S IRT7 (fig. 7D), confirming that SIRT7 is directly involved in the deacetylation of p62K 264. Taken together, the above results indicate that SIRT7 mediates deacetylation of p62K 264.

MMS significantly increased acetylation level of p62K264

It is speculated that acetylation of p62K264 may be involved in regulating DNA damage repair in the nucleus. Therefore, we first investigated whether p62 acetylation is regulated by DNA damage stress. HCT116 cells were treated with different types of DNA damaging stress including ultraviolet radiation (UV), Hydroxyurea (HU), Cisplatin (Cisplatin) and mesylate (MMS), respectively, and then tested for the acetylation level of p62K 264. Experimental results showed that MMS significantly increased the acetylation level of p62K264, whereas other treatments had no significant effect (fig. 8A).

Further, the Flag-p62-WT and Flag-p62-K264R plasmids were transfected into HCT116 cells, respectively, and the acetylation level of p62K264 was examined after MMS treatment. The results show that MMS treatment increased the acetylation level of wild-type p62 without having a significant effect on the acetylation level of the K264R mutant (fig. 8B). Next, MMS dose-and time-dependent experiments were performed on HCT116 cells and it was found that the acetylation level of p62K264 exhibited dose-and time-dependent increases under treatment with MMS (fig. 8C and 8D). In order to verify the universality of the phenomenon, other cell lines including colorectal cancer cell LoVo, cervical cancer cell HeLa and liver cancer cell HepG2 were selected for experiments, and experimental results consistent with the HCT116 cell line were obtained (FIG. 8E). These results indicate that MMS-induced increase in acetylation levels of p62K264 is a common phenomenon.

Acetylation of p62K264 promotes BER

MMS causes alkylation damage to cellular DNA and repair through BER. It is therefore speculated that acetylation of p62K264 may be involved in regulating the BER of DNA. Since DNA oxidative damage was also repaired by this pathway, HCT116 cells treated with hydrogen peroxide were found to have increased levels of acetylation of p62K264, consistent with the results of MMS treated cells (fig. 9A). A p62 stable knockout HCT116 cell line is constructed by using a CRISPR-Cas9 gene editing technology, and then cell lines of p62-WT, p62-K264R (lacking lysine acetylation) and p62-K264Q (simulating lysine acetylation) which stably express a Flag tag are established on the basis of the cell (FIG. 9B). When a cell develops DNA base damage, the saccharifying enzyme recognizes and removes the damaged DNA bases, thereby forming a single abasic site (AP site). Therefore, assessing the level of intracellular AP sites is a useful indicator of BER. The AP locus level was examined for three cell lines, p62-WT, p62-K264R and p 62-K264Q. The experimental results showed that the number of AP sites of p62-WT cells and p62-K264Q cells was significantly less than that of p62-K264R cells (fig. 9C), which indicates that the DNA damage degree of p62-WT cells and p62-K264Q cells was more mild compared to p62-K264R cells, suggesting that acetylation of p62K264 promotes the BER process.

The single cell gel electrophoresis experiment, also called comet experiment, is a classic experiment for detecting the damage repair capability of cell DNA. After DNA damage occurs to cells, because of different sizes of DNA broken fragments, comet-like tailing can be formed during electrophoresis, and the DNA damage repair capacity of the cells can be quantitatively compared by counting the tailing degree. Basic single cell gel electrophoresis experiments were used to assess DNA damage repair capacity. Cells from p62-WT, p62-K264R and p62-K264Q were treated with MMS to induce DNA damage, then cultured for an additional period of time to allow repair, and finally harvested for comet trail. Compared with p62-K264R cells, p62-WT cells and p62-K264Q cells showed less% of tailing DNA in comet experiments, and the tailing degree was more slight. This indicates that p62-WT cells and p62-K264Q cells were more efficient in cell repair compared to p62-K264R (FIGS. 9D and 9E).

If a tumor cell is not properly repaired when it is damaged by DNA, it will cause cell death and affect the ability to clone. HCT116 cells of p62-WT, p62-K264R and p62-K264Q were treated with a gradient dose of MMS, respectively, and the viability of the cells was examined by colony formation assay. The results show that the survival of p62-K264R cells was significantly lower than that of p62-WT and p62-K264Q, and when acetylation of p62K264 was at a low level, the survival of cells decreased and showed MMS dose dependence, reflecting the enhanced sensitivity of tumor cells to MMS treatment (FIGS. 10A and 10B). It is shown that the p62-K264R mutation reduces the repair efficiency of DNA damage, thereby reducing the survival rate of tumor cells. The results are combined to draw the conclusion that: acetylation of p62K264 promoted BER.

5. Preparation of specific antibody for detecting acetylation of K264 site of p62

The part of the experiment entrusted the development of yeast rice, and if necessary provided a complete experimental report, the animals used in this experiment were New Zealand big ear white rabbits, animal numbers JXR4541 and JXR4542, and the experimental animals were from the Kyoho laboratory animal farm in Hakkaido, Beijing.

5.1 methods for the preparation of antibodies

The preparation method of the specific antibody for detecting acetylation of K264 site of p62 comprises the following steps:

In the above production method, in the step (1), the acetylase used for the acetylation treatment of the K site is hmf;

5.2 results of the experiment

5.2.1 Indirect ELISA for serum titer detection

Table 1: results of indirect ELISA for serum titer (OD value)

The result of detection (judgment standard OD Yang/OD Yin >2.1, and OD value >0.1)

Antiserum titer No. JXR4541 experimental animals 1: 243000; antiserum titer 1 for experimental animal numbered JXR 4542: 243000.

5.2.2SDS-PAGE detection of antibody purity after purification

The results of the experiment are shown in FIG. 11.

5.2.3 Indirect ELISA method for detecting titer of purified antibody

Table 2: result of antibody titer after indirect ELISA method detection and purification

The experimental result shows that the detection sensitivity of the antibody of the invention for acetylated polypeptide is about 3.3ng/mL, and the detection sensitivity for non-acetylated polypeptide is about 111ng/mL, so that the antibody of the invention has higher specificity.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. A tumor marker related to the acetylation of the K264 site of p62, characterized in that: the tumor marker is the acetylation of the K264 site of p62, and the acetylation of the K264 site of p62 is subject to the acetyl transfer of p62 The enzyme hMOF and the deacetylase SIRT7 of p62 are jointly regulated. The acetylation of the K264 site of p62 is further involved in the regulation of base excision repair (BER). The tumor marker is used for high BER repair efficiency and strong tumor repair effect. Therefore, it is easy to predict the occurrence of anti-tumor drug resistance in patients, and the tumor markers are used for the detection of patient samples from tumor tissue, blood, plasma, serum, urine, feces, sputum or ascites;

The acetyltransferase hMOF of p62 mediates the acetylation of the K264 site of the p62, and the deacetylase SIRT7 of the p62 mediates the deacetylation of the K264 site of the p62, and the conventional molecular biology methods include Enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunocytochemistry, co-immunoprecipitation or immunoblotting.

2. A test kit for predicting patients with high BER repairing efficiency and strong tumor repairing effect, and thus prone to appear anti-tumor drug resistance, characterized in that: the test kit comprises the combination of claim 1 and p62 A tumor marker associated with acetylation at the K264 site, and an instruction manual describing a reference data set for the level of the tumor marker in samples from tumor patients and healthy people.

3. The tumor marker according to claim 1 or the test kit according to claim 2, characterized in that: it is used for the treatment of patients with high BER repairing efficiency and strong tumor repairing effect, so that it is easy to develop antitumor drug resistance. The method of prediction includes the following steps:

(1) Provide a sample derived from the patient to be tested, and detect the level of a tumor marker in the sample, where the tumor marker is the acetylation level of the K264 site of p62;

(2) comparing the marker levels measured in step (1) to a corresponding tumor marker level reference data set for samples from tumor patients and healthy populations; and

(3) If the level of the tumor marker in the sample of the patient to be tested is within the range of the corresponding tumor marker level reference data set of the tumor patient, and the level of the tumor marker in the sample of the patient to be tested is significantly far away from the healthy population If the corresponding tumor marker level of the sample refers to the range of the data set, the patient is determined to be a patient with high BER repair efficiency and strong tumor repair effect, so that resistance to anti-tumor drugs is prone to occur;

Preferably, in step (3), in patients with high BER repair efficiency and strong tumor repair effect, and thus prone to anti-tumor drug resistance, the acetylation level of the K264 site of p62 is significantly higher than that of the healthy population sample p62 The K264 acetylation level reference data collection.

4. A marker for screening a drug that causes tumor BER efficiency to improve and makes tumor prone to recurrence and drug resistance, wherein the marker is acetylation at the K264 position of p62, and the K264 position of the p62 is acetylated. Point acetylation is jointly regulated by the acetyltransferase hMOF of p62 and the deacetylase SIRT7 of p62, and the acetylation of the K264 site of p62 is further involved in the regulation of BER, and the marker is used for the test object by conventional molecular biology methods. The sample is detected, the sample is a tumor cell line, or from the tumor tissue, blood, plasma, serum, urine, feces, sputum or ascites of the object to be tested, preferably, the p62 acetyltransferase hMOF mediates The K264 site of p62 is acetylated, and the deacetylase SIRT7 of p62 mediates the deacetylation of the K264 site of p62. The conventional molecular biology methods include enzyme-linked immunosorbent assay (ELISA), immunoassay. Histochemistry, immunocytochemistry, co-immunoprecipitation or immunoblotting.

5. A method for screening a drug that causes tumor BER efficiency to improve so that tumor is prone to recurrence and drug resistance, comprising the following steps:

(1) In the test group, administer the drug to be tested to the test object, and detect the acetylation level L _1a of the K264 site of the marker p62 in the sample derived from the test object in the test group, respectively. In the group, a blank control or a vehicle control was administered to the subject to be tested, and the acetylation level L _0a of the K264 site of the marker p62 in the sample derived from the subject to be tested in the control group was respectively detected, wherein the marker It is the acetylation of the K264 site of p62, and the acetylation of the K264 site of p62 is jointly regulated by the acetyltransferase hMOF of p62 and the deacetylase SIRT7 of p62, and the acetylation of the K264 site of p62 is further involved in regulating BER;

(2) respectively comparing L _1a and level L _0a detected in step (1), if the level L _1a is statistically significantly higher than the level L _0a , then it is determined that the presence of the drug to be tested causes The increased efficiency of tumor BER makes the tumor prone to the risk of recurrence and drug resistance,

Wherein, in step (1) and step (2), the marker is used to detect the sample of the object to be tested by conventional molecular biology methods, and the sample is a tumor cell line, or from the tumor tissue of the object to be tested, blood, plasma, serum, urine, feces, sputum or ascites;

Preferably, the acetyltransferase hMOF of p62 mediates the acetylation of the K264 site of the p62, the deacetylase SIRT7 of p62 mediates the deacetylation of the K264 site of the p62, and the conventional molecular biology Chemical methods include enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunocytochemistry, co-immunoprecipitation or immunoblotting.

6. The method according to claim 5, wherein the test object is a tumor cell line or an animal suffering from a tumor, preferably, the animal is a mammal, more preferably, the mammal is Human, non-human primate or rodent.

7. A preparation method of a specific antibody for detecting the acetylation of the K264 site of p62, characterized in that: the preparation method comprises the following steps:

(1) artificially synthesize the IDVEHGGKRSRLTP sequence p62-K264 polypeptide, and perform acetylation at the K site;

(2) using the polypeptide synthesized in step (1) to immunize rabbits to obtain polyclonal antiserum, measure the antibody titer in the serum by enzyme-linked immunosorbent assay (ELISA), and purify the antibody to obtain the specific antibody.

8. preparation method according to claim 7, is characterized in that:

In step (1), the acetylase used in the acetylation treatment of the K site is hMOF;

In step (2), the specific process of immunizing the rabbits is as follows: for the first immunization, each rabbit is emulsified with 600 μg p62-K264 polypeptide plus an equal volume of Freund's complete adjuvant and then injected subcutaneously at multiple points (preferably, 6 points), respectively, at the first time. After 28 days, 42 days, and 56 days after immunization, three booster immunizations were performed. Each rabbit was emulsified with 400 μg p62-K264 polypeptide plus an equal volume of incomplete Freund's adjuvant and then subcutaneously injected at multiple points (preferably, 4 points); the third booster immunization After the ear blood was collected, the serum was separated as the post-immunization serum, and the antibody titer in the serum was determined by ELISA. to obtain the polyclonal antiserum;

In step (2), indirect ELISA is used to measure the antibody titer in the serum by ELISA, and the antigen affinity column used for the purification of the antibody is CnBr-activated Sepharose 4B.

9. A specific antibody for detecting the acetylation of the K264 site of p62, wherein the antibody is prepared by the preparation method of claim 7 or claim 8, and the antibody is directed against the p62-K264 polypeptide The detection sensitivity was 3.3ng/ml.

10. Use of the specific antibody of claim 9 in the preparation of a reagent for detecting the acetylation of the K264 site of p62, wherein the reagent is used for the reagent described in any one of claims 1 to 6 In vivo or in vitro detection of acetylation at the K264 site of p62.