CN115389764A

CN115389764A - Application of protein FBXO7 in preparation of endometrial cancer diagnostic marker

Info

Publication number: CN115389764A
Application number: CN202210899497.XA
Authority: CN
Inventors: 张辉; 金晓锋; 叶孟; 李锦芸
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2022-07-28
Filing date: 2022-07-28
Publication date: 2022-11-25

Abstract

The invention discloses an application of protein FBXO7 in preparing endometrial cancer diagnostic markers, which is characterized in that the protein FBXO7 is applied in preparing endometrial cancer diagnostic markers and/or therapeutic drugs, and further the protein FBXO7 mutant is applied in preparing endometrial cancer diagnostic reagents; further the application of the protein FBXO7 in the preparation of precise targeted therapeutic drugs for endometrial cancer and discloses the application of a mitochondrion inhibitor Mdivi-1 in the preparation of endometrial cancer with low expression and/or mutation of the FBXO7 protein, which has the advantages of being beneficial to the early diagnosis of endometrial cancer and being capable of designing a targeted therapy means aiming at the endometrial cancer.

Description

Application of protein FBXO7 in preparation of endometrial cancer diagnostic marker

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of protein FBXO7 in preparation of endometrial cancer diagnostic markers.

Background

Endometrial cancer, also known as uterine body cancer, is one of the most common malignant tumors of the female reproductive system. The overall prognosis of endometrial cancer treatment is good, but the prognosis of endometrial cancer at the middle and late stages and recurrent metastasis is very poor, and the average survival time of patients is less than 1 year, so that early and accurate diagnosis is particularly important.

The conventional diagnosis of endometrial cancer mainly depends on diagnostic uterine curettage for histopathological diagnosis, the operation is blindness and traumatic, tiny focuses are easy to miss, and the requirement for early accurate diagnosis cannot be met. In addition, the heterogeneity of endometrial cancer is significant, the repeatability of traditional histopathological evaluation is low, and histological features of tumors are overlapped, so that the clinical diagnosis and treatment needs of endometrial cancer cannot be met. With the development of precise medical treatment, targeted therapy implemented according to the characteristics of specific biomolecules (genes, proteins and the like) of patients gradually becomes a research hotspot, and the precise medical treatment also becomes a new therapy for treating endometrial cancer with guiding significance and wide application prospect. Meanwhile, the classification of diseases is no longer based on traditional pathology, but focuses more on the difference of specific biomolecules.

FBXO7 is an E3 ubiquitin ligase consisting of 522 amino acids with a protein size of approximately 62 kDa. Its studies have shown that the main functions are to maintain mitochondrial homeostasis within the cell and ubiquitination degradation of proteins. At present, no relevant research report about using FBXO7 protein as a diagnosis and treatment marker in endometrial cancer is published at home and abroad.

Disclosure of Invention

The invention aims to solve the technical problem of providing an application of a protein FBXO7 negatively correlated with the prevalence rate of endometrial cancer in preparation of endometrial cancer diagnostic markers.

The technical scheme adopted by the invention for solving the technical problems is as follows: an application of protein FBXO7 in preparing endometrial cancer diagnostic marker and/or therapeutic drug is provided.

Further, the protein FBXO7 mutant is applied to preparation of endometrial cancer diagnostic reagents.

Further, the protein FBXO7 is applied to preparation of precise targeted therapeutic drugs for endometrial cancer.

Application of a mitochondrion inhibitor Mdivi-1 in preparation of endometrial cancer with low expression and/or mutation of FBXO7 protein.

Compared with the prior art, the invention has the advantages that: the invention discloses application of protein FBXO7 in preparation of endometrial cancer diagnostic markers and/or therapeutic drugs for the first time. And analyzing the immunohistochemical staining picture, and evaluating the expression of the FBXO7 protein in the endometrial cancer patient sample and the normal sample by scoring of 2 pathologist professionals, wherein the expression of the protein FBXO7 in the endometrial cancer is obviously reduced, so that the FBXO7 protein can be used for assisting in diagnosing the endometrial cancer. Cell function experiments were used to demonstrate the effect of differential expression and mutation of the FBXO7 protein on the occurrence of endometrial cancer and to treat endometrial cancer by targeting the FBXO7 protein-associated downstream signaling molecule pathway. Compared with the histopathological diagnosis of diagnostic curettage, the technology is beneficial to the early diagnosis of endometrial cancer and can design a targeted treatment means aiming at the endometrial cancer.

Drawings

Fig. 1 is a graph showing immunohistochemical confirmation of FBXO7 protein expression in human endometrial cancer tissue and normal endometrial tissue. (A) 102 tissue samples comprising 97 endometrial cancer tissues and 5 normal endometrium samples; selecting representative images of FBXO7 protein immunohistochemical staining (1 case of normal endometrial tissue, 2 cases of endometrial cancer tissue, scale bar: 20 μm); (B) Scoring (Positive; low Positive; negative) and statistical stacked histograms (P < 0.05) for endometrial cancer tissue and normal endometrial tissue by immunohistochemical staining;

FIG. 2 is a clone formation experiment to verify the effect of FBXO7 protein knock-out on the proliferative capacity of AN3 CA cells. (A) A Western Blotting experiment verifies that the FBXO7 protein is knocked out and the AN3 CA cell stable strain of the FBXO7 mutant protein derived from wild FBXO7 protein and endometrial cancer is complemented; (B) clone formation experiment: the proliferation capacity of the AN3 CA cell stable strain with FBXO7 protein knocked out is obviously improved; the complementation wild FBXO7 protein can reverse and knock out the cell malignant phenotype caused by the FBXO7 protein, and the complementation endometrial cancer derived FBXO7 mutant protein cannot reverse; (C) Clonogenic experimental quantitative statistical plots (n.s: no statistical difference;. P < 0.05);

FIG. 3 shows ubiquitination degradation of INF2 protein by FBXO7 protein. The wild FBXO7 protein can degrade INF2 protein, (B) the wild FBXO7 protein can perform ubiquitination modification on the INF2 protein, (C) the wild FBXO7 protein can degrade the INF2 protein, and an FBXO7 mutant from endometrial cancer loses the function of degrading the INF2 protein;

FIG. 4 shows Mdivi-1 treatment of AN3 CA wild-type cell line and FBXO7 protein knock-out AN3 CA cell line. (A) Western Blotting detection of protein expression change of AN3 CA cell strain after treatment of Mdivi-1 finds that Mdivi-1 does not affect protein levels of INF2, FBXO7 and Drp 1; (B) clone formation experiment: mdivi-1 obviously inhibits the multiplication capacity of AN AN3 CA cell stable strain for knocking out FBXO7 protein; (C) clonogenic experimental quantitative statistics (. P < 0.05).

Detailed Description

The invention is described in further detail below with reference to the accompanying examples.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

1. Experimental methods

1. Immunohistochemical staining of endometrial cancer tissue samples

Endometrial cancer tissue chips were purchased from Shaanxi Aira Biotechnology, inc. and comprised 97 samples of endometrial cancer tissue and 5 samples of endometrial normal tissue.

Inclusion criteria were: 1. the pathological diagnosis of the sample is definite, and the sample is confirmed to be endometrial cancer; 2. the patient does not receive the treatment of radiotherapy and chemotherapy before operation; 3. the patient had no other systemic malignancies.

All human tissue specimens related to the invention are subject to medical ethical examination and limited laboratory research.

(1) Antigen retrieval: diluting 50 x sodium citrate antigen repairing liquid to 1 x with deionized water, shaking uniformly, pouring 1 x sodium citrate antigen repairing liquid into a pressure cooker, closing the cooker cover, boiling the liquid in the cooker, placing a slide filled with slices in the pressure cooker, completely immersing the slices in the sodium citrate antigen repairing liquid, screwing the cooker cover, starting timing after the air valve of the pressure cooker starts to uniformly emit air, turning off the power supply of an electromagnetic oven after 8 min, and finishing heating. The pressure cooker is placed under running tap water for cooling, and the cover of the pressure cooker is opened to cool the paraffin section to room temperature.

(2) Washing: the sections were first washed extensively with deionized water for 5 min, followed by 3 min with PBS phosphate buffer and repeated 3 times.

(3) Blocking endogenous peroxidase: diluting 10% hydrogen peroxide water solution with deionized water to 3% concentration, preparing for use each time, placing paraffin section in a wet box, dripping appropriate amount of 3% hydrogen peroxide water solution on the position of tissue on the glass slide, closing the cover of the wet box, and sealing at 37 deg.C for 10 min.

(4) Washing: washing the slices with PBS phosphate buffer solution for 3 min × 3 times, and throwing off the liquid on the glass slide;

(5) Serum blocking: placing the washed slices in a wet box, dropwise adding a proper amount of 10% donkey serum confining liquid on each slice, sealing for 15 min at room temperature, and then throwing off the confining liquid on the slices;

(6) Primary anti-incubation: wiping off sealing liquid around tissues, drawing circles around the tissues by using a special hydrophobic pen for immunohistochemical staining, placing the slices in a wet box, dropwise adding FBXO7 antibody diluent (antibody brand: proteitech; cargo number: 10696-1-AP; dilution ratio: 1: 100) with proper concentration, soaking the tissues in the antibody diluent, and placing in a refrigerator at 4 ℃ for keeping out of the light overnight;

(7) Washing: washing the slices with PBS phosphate buffer solution for 3 min × 3 times;

(8) And (3) secondary antibody incubation: wiping liquid around the tissue, dripping HRP (horse radish peroxidase) -labeled donkey anti-rabbit secondary antibody with proper concentration on the tissue to enable the antibody to completely cover the tissue, and incubating for 1 h in a 37 ℃ wet box in a dark place;

(9) Washing: washing the slices with PBS phosphate buffer solution for 3 min × 3 times;

(10) DAB color development: and (3) dripping the DAB developing solution to the position of the tissue on the glass slide, placing the glass slide on an inverted microscope to observe the tissue dyeing condition, throwing away the developing solution when the dyeing intensity reaches the optimum, and washing for 3 min multiplied by 3 times by using deionized water.

(11) Hematoxylin counterstaining: dropping a proper amount of improved Lillie-Mayer hematoxylin staining solution at the position of the tissue on the glass slide, staining for 2 min, throwing away the staining solution, and placing the glass slide with the glass slide under running tap water for washing for 10 min to turn blue.

(12) And (3) dehydrating: soaking the slide rack with the slices in 75% ethanol solution for 5 min × 1 time; soaking in 95% ethanol solution for 5 min × 1 times; soaking in absolute ethanol solution for 5 min × 3 times;

(13) And (3) transparency: soaking the slices in xylene solution for 5 min × 2 times;

(14) And (3) sealing: dropping a proper amount of neutral gum at the position of the tissue on the glass slide, clamping a cover glass by using forceps, slightly covering the cover glass on the glass slide, and airing the cover glass in a fume hood to form a sheet;

(15) And (3) scoring: the section is placed on an inverted microscope, the tissue morphology, the cell nucleus staining, the target protein staining and the like are observed under a low-power microscope and a high-power microscope in sequence, and 2 professional pathologist professionals score the picture under the condition of blinding experimental grouping.

2. Screening and identification of AN3 CA cell stable strain for knocking out FBXO7 protein and complementing wild FBXO7 protein and FBXO7 mutant protein derived from endometrial cancer

(1) The CD513B-CAs9-sgFBXO7 plasmid was transfected into AN3 CA cells, and 48 hours later, the cells were digested into a uniform cell suspension, and the cells were counted. Taking 1 six-hole plate, and planting the cell suspension into the six-hole plate according to 1000 cells per hole;

(2) Placing the six-hole plate into a constant-temperature incubator for culturing for 10 days, when cell colonies are formed by naked eyes, digesting single cell colonies by pancreatin, transferring the cell colonies into a 24-hole plate, placing the cell colonies into the constant-temperature incubator for continuous culture, and taking the monoclonal cells in each hole as a group;

(3) After the 24-pore plate is uniformly paved with the cells, transferring each group of cells to a 12-pore plate according to the method, after the pore plate is uniformly paved with the cells, transferring the cells to a 6-pore plate according to the same method, when the cell density reaches 80%, carrying out passage on each group of cells in the six-pore plate to a new 6-pore plate according to the passage 1 to 3, and placing the cells in a constant-temperature incubator for continuous culture;

(4) When the cell density reaches 80%, taking a hole of cells from each group of cells to carry out Western Blotting identification;

(5) Wild FBXO7 protein and FBXO7 mutant protein derived from endometrial cancer are transiently transfected and supplemented in an endometrial cancer cell strain with the knockout of the FBXO7 protein, and the identification is carried out by Western Blotting.

3. Clone formation experiments

(1) Taking AN AN3 CA wild type cell strain with good growth, knocking out AN AN3 CA cell stable strain by FBXO7 protein, digesting adherent cells into uniform cell suspension, and counting the cells;

(2) Taking several 6-well cell plates, adding 2X 10 cells per well ³ Each treatment group is provided with 3 compound holes and placed in a constant-temperature incubator, the liquid is changed once every 3 days, and the cells are cultured for 10 to 15 days;

(3) When cell colonies can be seen by naked eyes in the six-hole plate, removing culture medium in the holes, rinsing with PBS buffer solution for 2 times, adding a proper amount of 4% paraformaldehyde into each hole to fix cells, and standing for 30 min;

(4) Discarding the fixing solution, rinsing with PBS buffer solution for 2 times, adding 800 muL of 0.1% crystal violet dye into each well, placing on a shaking table for dyeing for 10 min, recovering the dye, rinsing with PBS buffer solution for 3 times, placing in an oven at 37 ℃ overnight, taking a picture after drying, and counting data.

4. Ubiquitination degradation of INF2 protein by FBXO7 protein

(1) Instantly transfecting FLAG-INF2 recombinant protein plasmids in HEK293T cells, instantly transfecting Myc-FBXO7 recombinant protein plasmids with gradually increased gradient on the basis, and finally detecting that the protein level of the exogenous FBXO7 and the protein level of the exogenous INF2 are obviously negatively correlated by Western blotting;

(2) Transient transfection of FLAG-INF2, myc-FBXO7 and HA-Ub plasmids in HEK293T cells, 8 hours before collection of cell lysates, MG132 inhibition of INF2 ubiquitin-proteasome pathway degradation, followed by co-immunoprecipitation of INF2 using FLAG antibody-rich commercial M2 beads, and finally Western blotting using HA antibody to detect that INF2 is ubiquitinated modified by FBXO 7;

(3) The FLAG-INF2 recombinant protein plasmid is transiently transfected in HEK293T cells, myc-FBXO7 wild type and endometrial cancer source mutant recombinant protein plasmids with ascending gradient are transiently transfected on the basis, and finally Western blotting is used for detecting that the endometrial cancer source FBXO7 mutant cannot degrade INF2.

5. Mdivi-1 drug therapy

And taking the AN3 CA wild type cell strain with good growth, knocking out the AN3 CA cell stable strain by the FBXO7 protein, and performing a clone formation experiment. Mdivi-1 was dissolved in DMEM high glucose complete medium at a final concentration of 1. Mu.M. When the cells cloned to form a plated plate adhere to the wall, the culture medium is replaced by a DMEM high-glucose complete culture medium containing pure DMSO (Mdivi-1 initial solvent, solvent toxicity interference is eliminated) and a DMEM high-glucose complete culture medium containing Mdivi-1, the culture medium is changed once every 3 days, and the culture is carried out for 10 to 15 days. The effect of Mdivi-1 on the proliferative capacity of stable strains of endometrial cancer cells knocked out of the FBXO7 protein was observed.

2. Analysis of Experimental results

The invention ensures the completeness, accuracy and correctness of experimental data. Immunohistochemistry pictures are scored by 2 professional pathologist professionals without knowing the grouping of experiments, scoring results are mapped and analyzed by GraphPad Prism software, all data statistical tests take bilateral probabilities, statistical tests are carried out according to a test standard of alpha =0.05, if P is less than 0.05, the difference between the two groups is considered to have statistical significance, otherwise, the difference is considered to have no statistical significance. The clone formation experiment result is analyzed through Image J software, the analysis result is mapped and analyzed through GraphPad Prism software, all data statistical tests adopt bilateral probability, statistical tests are carried out according to the test level of alpha =0.05, if P is less than 0.05, the difference between two groups is considered to have statistical significance, otherwise, the difference is considered to have no statistical significance.

We found that in endometrial cancer tissue and normal endometrial tissue, FBXO7 protein is expressed in endometrial cancer tissue significantly lower than normal endometrial tissue, and that the weak positive rate of FBXO7 protein in normal endometrial tissue is 80% (4/5) and the positive rate is 20% (1/5); in endometrial cancer tissues, the negative rate was 62.8% (61/97) and the weak positive rate was 37.2% (36/97) (FIG. 1A, B;. P < 0.05).

The colony formation experiment shows that compared with wild type AN3 CA cells, the number of the cell clone colonies of which the FBXO7 protein is knocked out is obviously increased. These results suggest that detection of relative expression of FBXO7 protein is of higher value for diagnosis of endometrial cancer (fig. 2A, B, C;. P < 0.05).

Meanwhile, the wild-type FBXO7 protein is found to carry out ubiquitination degradation on INF2 protein, while the FBXO7 protein from endometrial cancer cannot degrade the INF2 protein (fig. 3A, B and C), drp1 is recruited by the INF2 protein in the initial stage of mitochondrial division and then mitochondrial division is triggered, so that the FBXO7 is suspected to inhibit the occurrence of the endometrial cancer by inhibiting the mitochondrial division, and the abnormally reduced or mutated FBXO7 protein in endometrial cancer patients can cause the hyperfunction of mitochondrial division in cells.

Therefore, the mitochondrial fission inhibitor Mdivi-1 is used for treating the endometrial cancer with low expression of FBXO7 protein or mutation, and Mdivi-1 is found to remarkably inhibit the proliferative capacity of AN AN3 CA cell stable strain with the FBXO7 protein knocked out (figures 4A, B, C;. P < 0.05), which indicates that the Mdivi-1 is feasible in accurately targeting treatment of the endometrial cancer with low expression of FBXO7 protein or mutation.

The feasibility of the FBXO7 protein as a diagnostic marker of endometrial cancer is proved by detecting the differential expression of the FBXO7 protein in endometrial cancer and a clone formation experiment of AN AN3 CA cell strain with the FBXO7 protein knocked out. And the FBXO7 protein is applied to the precise target treatment of endometrial cancer by finding a downstream signal molecule path of the FBXO 7.

The above description is not intended to limit the invention, nor is the invention limited to the examples set forth above. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.

Claims

1. An application of protein FBXO7 in preparing endometrial cancer diagnostic marker and/or therapeutic drug is provided.

2. Use of a protein according to claim 1, wherein: the protein FBXO7 mutant is applied to the preparation of endometrial cancer diagnostic reagents.

3. The use of a protein according to claim 1, wherein: the protein FBXO7 is applied to the preparation of the precise targeted therapeutic drug for endometrial cancer.

4. Application of a mitochondrion inhibitor Mdivi-1 in preparation of endometrial cancer with low expression and/or mutation of FBXO7 protein.