CN113913523B

CN113913523B - Application of BUD31 as ovarian cancer prevention, diagnosis or prognosis marker

Info

Publication number: CN113913523B
Application number: CN202111387958.7A
Authority: CN
Inventors: 刘招舰; 王子翔; 秦君超; 赵玲; 张锡宇
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2023-04-11
Anticipated expiration: 2041-11-22
Also published as: CN113913523A

Abstract

The invention relates to an application of BUD31 as an ovarian cancer prevention, diagnosis or prognosis marker. The research of the invention proves that: BUD31 is highly expressed in patients with high-grade serous ovarian cancer, functional tests prove that BUD31 can inhibit apoptosis of ovarian cancer cells and promote proliferation and invasion migration of the ovarian cancer cells, and in vivo experiments prove that BUD31 high expression can promote tumor growth. The research result of the invention shows that BUD31 is a splicing factor participating in the occurrence and development of ovarian cancer, can be used as a biomarker and a treatment target for diagnosing high-grade serous ovarian cancer, and has good practical application value. Furthermore, the invention also designs siRNA aiming at BUD31, which is proved to have good effect of inhibiting tumor development and is expected to be applied to the development of high-grade serous ovarian cancer resistant drugs.

Description

Application of BUD31 as ovarian cancer prevention, diagnosis or prognosis marker

Technical Field

The invention belongs to the technical field of high-grade serous ovarian cancer markers, and particularly relates to application of BUD31 as an ovarian cancer prevention, diagnosis or prognosis marker.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Epithelial ovarian cancer is the most common ovarian malignancy, and is divided into different histological types, wherein High-grade serous ovarian cancer (HGSOC) is the most main subtype, lacks precursor lesions, has High malignancy degree and strong invasiveness, and has a five-year survival rate of only about 30% (Vaughan, heated et al.2011). The molecular mechanisms underlying ovarian cancer origin, recurrence and resistance are currently poorly understood (Bell, berchuck et al.2011, jiang, li et al.2019). Therefore, the deep research on the biological mechanism of HGSOC occurrence and development, the identification of early markers and therapeutic targets provide important basis for guiding clinical diagnosis and exploring new accurate therapeutic means.

The abnormality of alternative splicing is closely related to genetic diseases, tumors and other diseases. Research shows that the number of splicing events in tumor tissues is more than 30% than that in normal tissues (Kahles, lehmann et al 2018), and splicing reprogramming in tumor cells can promote malignant transformation, tumor proliferation, invasion and metastasis, drug resistance and other malignant behaviors (Inoue, chenw et al 2019, zhou, wang et al 2019). These aberrant splicing events are precisely regulated by various elements including splicing factors. High throughput sequencing revealed that there were a large number of splicing abnormalities in ovarian cancer and that the spliceosome associated pathways were highly enriched. The inventors analyzed the expression of 134 core splicing factors in ovarian cancer, and found that 20 of them are highly expressed. BUD31 (BUD 31 homolog, G10) is one of the 20 core splicing factors highly expressed in ovarian cancer, and its encoding gene is very well conserved in yeast, nematodes, drosophila and humans. In yeast, a deletion of Bud31 results in stunted budding, slow growth, morphological abnormalities (Masciadri, areces et al 2004); BUD31 is combined with splicing factors such as SF3B1, U2AF1, PRP8 and the like, participates in the catalytic reaction of the first step and the second step of the spliceosome, and plays a key role in the processes of catalyzing the assembly of the spliceosome, stabilizing the relation of a protein-precursor mRNA compound and the like; in addition, it has been reported that buf 31 tightly binds to U2, U5, U6 micronuclein and NTC (Prp 19 complex) early in RNA splicing, catalyzing activation of pre-B complex (Saha, khandelia et al.2012); recent cryo-electron microscopy results indicated that BUD31 is involved in the formation of human secondary spliceosomes (Bai, wan et al 2021). BUD31 is highly expressed in ovarian cancer, but the role of BUD31 in the malignant behavior of the ovarian cancer needs to be elucidated, and the downstream abnormal splicing network needs to be further studied.

Small interfering RNAs (sirnas) are double-stranded RNA molecules of 20-25 nucleotides in length, specifically degrade messenger ribonucleic acid (mRNA) by activating RNA-induced silencing complex (RISC), thereby reducing gene expression levels and regulating abnormally active signaling pathways, thereby affecting the biological behavior of cells (Reynolds, leake et al 2004). In recent years, siRNA has a wide application prospect in the field of drug development due to its characteristics of good specificity, rapid action, easy development, etc., and its mechanism and function and optimized administration mode are reported continuously (Kanasty, dorkin et al.2013).

Disclosure of Invention

Based on the above technical background, the present invention aims to clarify the role of buf 31 in the development of high-grade serous ovarian cancer, and to provide an active ingredient useful for inhibiting high-grade serous ovarian cancer.

Based on the technical purpose, the invention provides the following technical scheme:

in a first aspect of the invention, there is provided the use of BUD31 as a prophylactic, diagnostic or prognostic marker for ovarian cancer.

The invention screens splicing factors with abnormal expression in ovarian cancer samples, wherein BUD31 has higher expression in salpingo-umbrella tissues of high-grade serous ovarian cancer and is related to poor prognosis. Furthermore, the invention establishes a tumor model of BUD31 overexpression to prove the cancer promotion effect of BUD31, including improving the anti-apoptosis effect of ovarian cancer cells, enhancing the invasion and migration capacity of the ovarian cells and the like.

Based on the research conclusion, the invention considers that the expression of BUD31 may play an important role in the process of the occurrence and the development of ovarian cancer, and is expected to be applied as a marker for the prevention, the diagnosis or the prognosis of the ovarian cancer. The application mode includes but is not limited to developing the active ingredients with the BUD31 inhibition effect for treating ovarian cancer, or assisting judgment of the development condition of ovarian cancer of patients by detecting the expression content of BUD 31.

In a preferred embodiment of the first aspect, the ovarian cancer is an epithelial tumor; further, a serous tumor; in one embodiment in which ovarian cancer is classified as high-grade and low-grade according to clinical and pathological characteristics, and the first aspect is more effective, the BUD31 is used as a high-grade serous ovarian cancer prevention, diagnosis or prognosis marker.

In a second aspect of the invention, a high-grade serous ovarian cancer prognosis kit is provided, wherein the kit comprises a reagent for detecting the BUD31 expression content in a sample to be detected.

Preferably, the detection sample of the kit is one or more of serum (plasma), whole blood, secretion, cotton swab, pus, body fluid, tissue, organ and paraffin section.

It should be noted that the types of reagents in the above-mentioned kit can be adjusted according to the detection method selected by those skilled in the art, and the detection method that the kit can satisfy includes detecting the ribonucleotide of BUD31 in the ovarian cancer sample based on a high-throughput sequencing method, a quantitative PCR method, liquid phase hybridization, northern hybridization, in situ hybridization or an RNA chip; or detecting the BUD31 protein in the ovarian cancer sample based on enzyme-linked immunosorbent assay, colloidal gold detection, protein immunoblotting, protein chip detection or proteomics.

In a preferred embodiment of the second aspect, the kit detects the content of the BUD31 in the sample to be detected based on a quantitative PCR method, and the kit at least comprises a pair of primers, wherein the primer sequences are as follows:

BUD31 pre-primer: 5 'CATTCAGAGACGGGACACCA-3' (SEQ ID NO: 1);

BUD31 rear primer: 5 'ATGATGCGGCCCACTTCC-3' (SEQ ID NO: 2).

In a third aspect of the invention, the application of the BUD31 inhibitor in preparing an anti-ovarian cancer product is provided.

Preferably, the anti-ovarian cancer product is applied to medicines, health products or model medicaments.

Further, the use of the BUD31 inhibitor as a model agent which is a substance capable of inhibiting the reduction in the expression and/or activity of BUD31 may be performed by at least one of the following (1) to (3):

(1) Inhibiting the proliferation of ovarian cancer cells, or preparing a model agent for inhibiting the proliferation of ovarian cancer cells;

(2) Inhibiting invasion and migration of ovarian cancer cells, or preparing a model medicament for inhibiting invasion and migration of ovarian cancer cells;

(3) Promoting apoptosis of ovarian cancer cells, or preparing model medicament for promoting apoptosis of ovarian cancer cells.

Further, the drug is one of solid small molecule compound, natural drug extract or nucleic acid component; the nucleic acid component comprises DNA or RNA for down regulating gene expression, or RNA substance for exerting gene inhibition based on RNAi pathway, such as miRNA, siRNA or shRNA.

In the research process, the corresponding siRNA is designed aiming at BUD31, and the siRNA is applied to ovarian cancer or high-grade serous ovarian cancer and shows stable knockout effect on mRNA and protein levels through verification of the siRNA.

In the fourth aspect, the invention provides an application of siRNA with a sequence shown in SEQ ID NO. 3 in preparing a high-grade serous ovarian cancer resistant medicament.

In some alternatives of the above embodiments, the siRNA further comprises a product of modification of the above nucleic acid strand or a formulation form of the siRNA; the modifications include modifications for optimizing the properties of the siRNA such as stability, solubility, etc., such as salt forms of the siRNA, e.g., sodium salt, potassium salt, etc., and also fusion peptide forms of the siRNA.

In a specific embodiment provided by the invention, the 3' of the siRNA with the sequence shown in SEQ ID NO. 3 has dTdT modification to improve the stability; the sequence is 5 '-UGUUGGCUCAAUCCdTdT-3'.

Further, the medicament is one of solid oral preparation, liquid oral preparation or injection; further, the injection is injectable implant, emulsion, liposome, microcapsule, microsphere or nanoparticle formulation, and the administration dose of the active ingredient in the drug may vary depending on the body weight, age and sex, health condition, diet, administration time, administration route, excretion rate and severity of disease of a patient.

The beneficial effects of one or more technical schemes are as follows:

(1) Provides effective molecular markers for diagnosis, disease evaluation and prognosis judgment of ovarian cancer: the invention combines database analysis, and by the verification of clinical samples and tissue chips, the method of qPCR, immunohistochemical staining and the like is adopted to prove that the BUD31 is highly expressed in ovarian cancer patients, and the high expression of the BUD31 is related to poor prognosis of ovarian cancer.

(2) Provides a new target for treating ovarian cancer: the invention proves that the over-expression of BUD31 inhibits the apoptosis of cells and promotes the proliferation of ovarian cancer cells through in vivo and in vitro functional tests. In addition, the reduced BUD31 expression can induce the apoptosis of ovarian cancer cells and inhibit malignant biological behaviors such as tumor growth, clone formation and the like. Based on the method, a drug which specifically targets BUD31 can be designed, and the expression of BUD31 is reduced, so that the aim of treating ovarian cancer is fulfilled.

(3) The invention discloses that BUD31 influences apoptosis by regulating BCL2/Bax pathway for the first time, and influences cracked levels of Caspase3 and PARP to participate in the process of apoptosis. The invention finds a new effective method for the targeted regulation of an apoptosis signal path, namely inducing apoptosis by the siRNA of the targeted BUD31 and applying the method in the basic medical research.

(4) The invention designs and provides the siRNA acting on BUD31 based on the function of BUD31 in the development of ovarian cancer, and the siRNA has good effect of inhibiting the development of ovarian cancer and is expected to be applied to the development of anti-ovarian cancer medicaments as nucleic acid medicaments by verification.

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 is a graph showing the expression level of splicing factors such as BUD31 in high-grade serous ovarian cancer in example 1;

wherein, FIG. 1A is the gene expression data in TCGA and GTEx databases, which is used to analyze the expression of 134 core splicing factors in ovarian cancer;

FIG. 1B is an analysis of the mRNA expression of BUD31 based on TCGA and GTEx databases;

FIG. 1C is a qPCR validation of BUD31 mRNA expression in ovarian cancer tissues and normal oviduct controls;

FIG. 1D is an analysis of protein expression of BUD31 based on CPTAC database;

FIG. 1E is a graph showing the protein profile of BUD31 verified by using the tissue chip prepared by the collection of subjects.

FIG. 2 is a graph of the clinical significance of BUD31 in example 1 in high-grade serous ovarian cancer;

FIG. 2A is a graph showing the relationship between BUD31 high expression and overall survival time of a patient by using clinical information analysis corresponding to a tissue chip;

FIG. 2B is a graph of the relationship between BUD31 high expression and the progression-free survival of a patient using clinical information analysis corresponding to tissue chips;

FIG. 2C is a graph of BUD31 high expression versus overall survival of patients analyzed using the Kaplan-Meier Plotter database;

FIG. 2D is a graph of high BUD31 expression versus progression free survival of patients analyzed using the Kaplan-Meier Plotter database.

FIG. 3 is a study on the inhibitory effect of the BUD 31-targeting siRNA sequence described in example 2;

wherein, fig. 3A is the inhibition rate at the RNA level of the three sirnas constructed in example 2;

FIG. 3B shows the inhibition rate of three siRNAs constructed in example 2 at the protein level.

FIG. 4 is a graph of BUD31 promoting ovarian cancer cell proliferation in example 4;

wherein, FIG. 4A is a graph showing the result of proliferation of ovarian cancer cells overexpressing BUD31 measured by the EdU assay;

FIG. 4B is a graph showing the results of proliferation of ovarian cancer cells overexpressing BUD31 as determined by the MTT assay;

FIG. 4C shows that BUD31 promotes the clonogenic capacity of ovarian cancer cells, and a plate cloning experiment proves that BUD31 can promote the clonogenic capacity of ovarian cancer cells and enhance the malignancy of the cancer cells;

fig. 4D is a cell morphology following knockdown or overexpression of bun 31 in HEY and OVCAR3 ovarian cancer cell lines viewed under light microscopy.

FIG. 5 is a graph showing that BUD31 inhibits apoptosis of ovarian cancer cells in example 4;

wherein, FIG. 5A is a flow cytometry detection apoptosis graph, which proves that the apoptosis proportion of the ovarian cancer cell line over expressing BUD31 is lower than that of the control group, and the apoptosis proportion of the ovarian cancer cell line with the BUD31 knocked down is higher than that of the control group;

FIG. 5B is a western blot detection cell apoptosis-related analysis protein expression diagram, wherein the knockdown of BUD31 can promote the expression of Bax protein, reduce the expression of Bcl-2 protein to induce ovarian cancer cells to undergo apoptosis, and the overexpression of BUD31 can reduce the expression level of cleaved Caspase3 and PARP proteins to play an anti-apoptosis role;

fig. 5C is a confocal microscope image of the morphology of ovarian cancer cells, which shows that the morphology of ovarian cancer cells becomes round and shriveled and the structural disorder of tubulin alpha is caused after the BUD31 is knocked down by using siRNA.

FIG. 6 is a graph of the enhancement of ovarian cell invasion migration by BUD31 in example 4;

in the crystal violet staining image shot under an optical microscope in fig. 6A, a Transwell experiment proves that the invasion and metastasis capacity of ovarian cancer cells can be enhanced by over-expressing the BUD31, and the invasion and metastasis capacity of the ovarian cancer cells can be reduced by knocking down the BUD 31;

FIG. 6B is an expression diagram of BUD31 capable of affecting EMT pathway-related proteins, and over-expression of BUD31 can promote expression of proteins such as Snail, N-Cadherin, vimentin, slug, ZEB1 and the like, so as to promote invasion and migration capacity of ovarian cancer cells.

FIG. 7 is a graph of BUD31 promoting the in vivo tumorigenesis of ovarian cancer cells in example 5;

wherein, FIG. 7A is an imaging diagram of a nude mouse abdominal cavity tumor formation living body;

FIG. 7B is a fluorescent signal histogram of an imaging image of a tumor-forming living body in the abdominal cavity of a nude mouse;

fig. 7A and 7B results show that knockdown of BUD31 can inhibit the celiac tumorigenic capacity of ovarian cancer cells;

FIG. 7C is an open abdominal anatomical view of abdominal cavity neoplasia in nude mice, both the number and volume of abdominal cavity neoplasia are significantly reduced after BUD31 knockdown;

FIG. 7D is a photograph of nude mice bearing tumor;

FIG. 7E is a subcutaneous tumorigenicity map of nude mice, showing significantly greater volume of BUD 31-overexpressing ovarian cancer cells after subcutaneous tumorigenicity in nude mice;

FIG. 7F is a statistical chart of subcutaneous tumor formation volumes of nude mice with ovarian cancer cells, wherein BUD31 can promote the in vivo growth of tumors;

FIG. 7G is hematoxylin-eosin staining pattern of subcutaneous tumorigenic section of nude mouse with ovarian cancer cells;

FIG. 7H is an immunohistochemical staining chart of a subcutaneous tumorigenic section of a nude mouse with ovarian cancer cells, wherein the expression of a protein marker Ki67 representing the proliferation rate is obviously reduced after the BUD31 is knocked down;

FIG. 7I is TUNEL staining diagram of naked mouse subcutaneous tumor section with ovarian cancer cells, and the apoptosis cell ratio is obviously increased after BUD31 knockdown.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

In the following examples, ovarian cancer cells are isolated from ascites of patients with high-grade serous ovarian cancer, primary cells OVBWZX are obtained by separation and purification, and auxiliary verification is performed by experiments such as in vitro proliferation and clone formation.

Example 1 expression and clinical significance of BUD31 in ovarian cancer

In the present example, the expression levels of 134 core splicing factors in 545 ovarian cancer samples in the TCGA database and 180 normal ovarian tissues in the GTEx database were analyzed by bioinformatics techniques, and 37 splicing factors among them were found to be abnormally expressed (fig. 1A), wherein buf 31 was highly expressed in the ovarian cancer samples in the TCGA database (fig. 1B), 9 samples of collected ovarian normal pileus tissues and 23 samples of high-grade serous ovarian cancer tissues were used, tissue RNA was extracted, and the relative expression level of buf 31 mRNA was detected by qPCR, and it was found that the expression level of buf 31 mRNA in the high-grade serous ovarian tissues was significantly higher than that of normal pileus tissues (P < 0.05) (fig. 1C). In addition, the bun 31 protein level was highly expressed in ovarian cancer samples in the CPTAC database (fig. 1D), tissue chips were made by collecting an additional 73 normal oviduct tissues and 149 HGSOC tissues, and the bun 31 protein level was significantly elevated in HGSOC using immunohistochemical staining (P < 0.01) (fig. 1E). Based on the corresponding clinical information of the tissue chips, high expression of BUD31 and shorter overall survival correlation were found (FIGS. 2A and 2B), while further validation of BUD31 and poor prognosis correlation was made based on the Kaplan-Meier Plotter online database (http:// kplot.com) (FIGS. 2C and 2D).

Example 2 construction of BUD31 overexpression/knockdown plasmids and design of synthetic siRNA

1. Plasmid construction:

construction of an overexpression plasmid uses human placenta cDNA as a template, PCR amplification is carried out on a nucleotide sequence of a full-length coding region of BUD31, a restriction enzyme cutting site and a protective base are connected, the PCR fragment and a vector after SgfI and MLuI restriction enzyme amplification are utilized, agarose gel electrophoresis is carried out to verify the restriction enzyme cutting effect, a gel recovery kit is used for purifying a product, finally, a target gene sequence is connected to a shuttle vector pENTER through T4 DNA ligase, and Sanger sequencing verification is successful. The knockdown grains induced by BUD31 doxycycline were purchased from gecky corporation.

2. Design and synthesis of siRNA:

the full length of the sequence of the mature mRNA of the human BUD31 (serial number: NM-003910) is obtained through an NCBI database of the national center for biotechnology information, the functional structure domain and the conserved sequence region of the BUD31 are combined, on-line siRNA design software is used for designing, a BLAST sequence similarity search program on the NCBI is used for comparing a target sequence, and the final target BUD 31-targeting siRNA is obtained through screening. In the research process of the embodiment, a plurality of siRNAs are designed aiming at BUD31, and the efficiency of the siRNAs is verified on the level of RNA and protein by utilizing qPCR and western blot respectively. In transient transfection studies, three siBUD31#1, siBUD31#2 and siBUD31#3 with high feasibility were selected in this example, wherein the siBUD31#1 is the sequence shown in SEQ ID NO: 3. In addition, dTdT suspension was added at the end of the siRNA to increase the stability of the siRNA. And simultaneously selecting a negative control siRNA (siNC), wherein the siRNA is synthesized by Sharp Biotechnology Co., ltd, guangzhou.

Overexpression and knocking-out of low plasmid and siRNA are carried out to transiently transfect functional cells 293T, and RNA is harvested after 48h, and protein is harvested after 72h, and overexpression and knocking-out efficiency of BUD31 at the RNA and protein level are respectively detected.

Example 3 construction of BUD 31-knockdown cell lines and transient transfection

In this example, siBUD31#1, siBUD31#2 and siBUD31#3 constructed in example 2 were examined for transfection efficiency.

1. Production of viral particles

(1) HEK293T cells in good condition were digested with pancreatin, collected and counted in a 3X 10 format ⁶ Inoculating the density of each cell/dish into a 100mm cell culture dish, placing a cell culture solution before transfection into a cell culture box to culture overnight, and observing that plasmid transfection is carried out when the cell density reaches 70-80%;

(2) Preparing a transfection complex: diluting 4 μ g of doxycycline-induced knockdown BUD31 plasmid, 4 μ g of backbone plasmid psPAX2 and 8 μ g of backbone plasmid pMD2.G with 1.6mL of serum-free OPTI-MEM medium, mixing, standing, adding 36 μ L of liposome lipo2000, blowing uniformly, and incubating at room temperature for 5 min;

(3) Mixing the two solutions, blowing uniformly to avoid generating bubbles, incubating at room temperature for about 20 minutes to form a DNA-liposome mixture, adding the DNA-liposome mixture into a culture dish with well-paved cells, placing the mixture into a carbon dioxide incubator, and culturing at a constant temperature of 37 ℃;

(4) After 3 hours of culture, 5mL of Opti-MEM culture medium is added into the culture dish, after about 6 hours of continuous culture, 6mL of fresh antibiotic-free DMEM complete culture medium is replaced, and the culture dish is put back into the incubator for continuous culture;

(5) After 24h of culture, collecting the supernatant containing the virus particles, temporarily storing the supernatant at 4 ℃, and adding 6mL of fresh complete DMEM medium into the dish;

(6) After 48h incubation the supernatants were collected again and the two supernatants were pooled and the virus fluid was filtered using a 0.22 μm filter and added to Polybrene at a ratio of 1.

2. Infecting host cells

(1) The cells to be infected are arranged according to a 3X 10 ⁴ The cells were seeded in 6-well plates at a density of one cell/mL and cultured overnight;

(2) The next day of plating, adding 1mL of virus solution into each hole for infection, horizontally shaking the culture plate at a constant speed in a cross shape to uniformly mix the virus solution, and putting the culture plate into an incubator for continuous culture;

(3) After 48h of infection, the supernatant was aspirated, the medium containing 2. Mu.g/mL puromycin was replaced for selection, the growth state of the cells was observed every 2 days, the medium containing puromycin was replaced every 3 to 4 days, and cells stably transfected with the desired plasmid were obtained after 2 weeks of continuous selection. After screening, the content of BUD31 was determined using western blot and qPCR.

3. Transient transfection

(1) Cell plating: cells with good status were generally passaged twice and plated for transfection. Trypsinization-medium resuspension of cells-cell count, different cell densities depending on cell size, and transfection purpose. When plasmids are transfected, the cell density is preferably controlled to be 60% -80%.

(2) Preparing a transfection complex: take a six-hole plate as an example

siRNA and plasmid transfection methods: mu.g DNA, 4. Mu.L jetPRIME and 200. Mu.1 jetPRIME Buffer were added to a 1.5m1EP tube, vortexed for 10 seconds and incubated at room temperature for 10 minutes. The presence of air bubbles is avoided.

(3) Adding the transfection mixed solution into a 6-well plate inoculated with cells with proper density, adding the mixed solution of 200 mu 1 per well of culture medium at 2m1, gently mixing in a cross shape, and placing in a cell culture box.

(4) Whether to remove the culture medium containing the transfection reagent is determined according to the cell state and the requirements of subsequent experiments (whether to add medicine). In general, RNA is extracted from cells collected 48h after transfection, and protein is extracted from cells collected 72h after transfection. The BUD31 content was determined by western blotting and qPCR.

The inhibition efficiency of siBUD31#1 on the RNA level can reach 84 percent through verification of the embodiment. The siRNA is applied to ovarian cancer or high-grade serous ovarian cancer, in vitro experiments prove that siBUD31#1 can inhibit malignant behaviors of ovarian cancer cells from multiple aspects, and EdU experiments and MTT experiments prove that siBUD31#1 can inhibit tumor cell proliferation in ovarian cancer cell lines OV90 and primary cells OVBWZX from ascites of patients with high-grade serous ovarian cancer (figures 4A and 4B). Furthermore, siBUD31#1 was demonstrated to inhibit clonality of the tumor cell line OV90 (FIG. 4C).

Example 4 Effect of BUD31 on malignant behavior of ovarian cancer

1. EdU detection proliferation rate: selecting cells in logarithmic growth phase, digesting with trypsin, counting cells, and 2 × 10 ⁴ Each cell was seeded into a 24-well plate, and the next day, after 20 minutes of incorporation with an EdU pseudolite containing a 1.

2. Plate cloning experiment: selecting cells in logarithmic growth phase, digesting with trypsin, counting the cells, inoculating 1000 cells per well into a 6-well plate, culturing for about 10-14 days, fixing with methanol for 15 minutes, and staining with crystal violet for 30 minutes. Observed under the mirror and counted.

3. MTT growth curve: cells in the logarithmic growth phase were selected, trypsinized, counted, 800 cells per well were seeded in a 96-well plate, continuously assayed for 5 days, 10. Mu.L MTT was added per well and incubated for 4 hours, the supernatant was discarded and dissolved in 100. Mu.L DMSO for 10 minutes, the OD at 570nm was measured and a cell growth rate curve was plotted.

4. Transwell cell invasion assay: diluted according to Matrigel gel DMEM = 110, the mixture was added to a Transwell cell at 50 μ L per well, after the gel was solidified, 200 μ L of cell and serum-free DMEM suspension (a 2780 ten thousand cells/well, HEY 15 ten thousand cells/well) was added to the upper chamber, 700 μ L of DMEM containing 30% fbs was added to the lower chamber, and after 48 hours of incubation, methanol was fixed and counted under the microscope after crystal violet staining.

5. Detecting apoptosis by a flow cytometer: well-growing cells were seeded one day in a 6cm dish, digested with pancreatin without EDTA and collected, washed once with PBS, resuspended in 100. Mu.L of 1 XBinding Buffer, 5. Mu.L of Annexin V-PE and 7-AAD dye were added, incubated at room temperature in the dark for 10 minutes, and then 400. Mu.L of 1 XBinding Buffer was added to detect the number of apoptotic cells.

To investigate the effect of BUD31 on the malignant behavior of ovarian cancer, this example was verified using BUD31 knockdown cell lines, BUD31 overexpression plasmids and siRNA. In EdU experiments shown in FIGS. 4A-C, BUD31 promotes the proliferation of ovarian cancer cells OVBWZX, MTT experiments show that BUD31 accelerates the growth of the ovarian cancer cells, and plate cloning experiments prove that BUD31 can promote the cloning capacity of the ovarian cancer cells and enhance the malignancy degree of the cancer cells. FIG. 4D shows that overexpression of BUD31 increases the survival rate of ovarian cancer cells, and that the morphology and growth state of the cells are changed after BUD31 is knocked down.

FIG. 5A shows that the proportion of apoptotic cells is increased after flow cytometry detection and the BUD31 is knocked down by siBUD31#1, which indicates that siBUD31#1 can effectively induce apoptosis of ovarian cancer cells OVBWZX, and over-expression of BUD31 improves anti-apoptosis capability of the ovarian cancer cells. FIG. 5B is a western blot demonstrating that apoptosis-related protein expression levels are affected by BUD31, that the ratio of BCL2 to Bax decreases after knocking down BUD31, and that Caspase3 and PARP in cleaved state increase, indicating that siBUD31#1 affects apoptosis of ovarian cancer cells through the BCL2/Bax signaling pathway. FIG. 5C shows that tumor cells were morphologically shrunken and the results for alpha tubulin were perturbed, thus inducing apoptosis, after interfering BUD31 expression with siRNA, as photographed by confocal microscopy.

In the aspect of invasion and migration capacity, a Transwell experiment shown in fig. 6A proves that the malignancy of cancer cells can be remarkably enhanced by over-expressing BUD31, and the malignancy of ovarian cancer cells can be reduced by knocking down BUD 31. FIG. 6B shows that BUD31 can influence the expression of EMT pathway-related proteins, and over-expression of BUD31 can promote the expression of key proteins such as Snail, N-Cadherin, vimentin, slug, vimentin and the like, so as to promote the invasion and migration capability of ovarian cancer cells.

Example 5 confirmation of the role of BUD31 in ovarian cancer by nude mouse tumorigenesis

1. Abdominal neoplasia and in vivo imaging

6 BALB/c Nude female mice of 4 weeks old and 6 experimental groups respectively, wherein the control group and the experimental group are injected with HEY cells expressing luciferase, each injection is 300 ten thousand cells, the cells are diluted in 200 mu L PBS and injected in the abdominal cavity, nude mice of the experimental group drink 5% sucrose aqueous solution containing doxycycline (1.2 g/L), and the control group is only 5% sucrose aqueous solution. After 2 weeks, the cells were anesthetized and injected with D-fluorescein sodium salt and visualized using a small animal in vivo imaging instrument. Mice were then sacrificed and dissected open abdomen and observed for tumor number and volume.

2. Subcutaneous neoplasia

6 BALB/c Nude female mice control and experimental groups each at 4 weeks of age. The control group was injected with ID8-NC cells, the experimental group was injected with ID8-BUD31 cells, 300 ten thousand cells each, and the cells were diluted in 200. Mu.L of PBS and injected subcutaneously. Nude mice were sacrificed under anesthesia 2 weeks later, dissected and tumor tissues were taken and soaked in 10% formalin solution, then paraffin-embedded and sectioned, HE-stained and immunohistochemically stained to observe the morphology and number of tumor tissue cells.

3. HE dyeing step

Baking slices: baking the slices at 60 ℃ for 30 minutes.

Dewaxing: xylene I for 15 minutes; xylene for 15 minutes; 5 minutes with 100% alcohol; 5 minutes with 100% alcohol; 95% alcohol for 5 minutes; 80% alcohol for 5 minutes; 5 minutes with 75% alcohol, and 2-3 times of rinsing in tap water.

Hematoxylin staining was performed for 10 min, and the slide was gently rinsed with water.

Hydrochloric alcohol for 1-2 seconds, ammonia water for 5-10 seconds, and tap water for 2-3 times.

Eosin staining for 20 minutes, rinsing in tap water for 2-3 times.

And (3) dehydrating: 75% alcohol for 10s;80% alcohol for 10s;95% alcohol for 10s;100% alcohol for 10s;100% alcohol for 10s; xylene for 3 minutes; xylene was again present for 3 minutes.

4. Immunohistochemical staining

The dewaxing step was as above.

Antigen retrieval: heating EDTA antigen repairing solution with high microwave fire for boiling for 10 min, immediately adding the dehydrated slices, heating with low microwave fire for 15 min, and cooling to room temperature; incubating with 3% hydrogen peroxide in a wet box at 37 ℃ for 15-20 minutes, and washing with PBS for 3 times and 3 minutes in a shaking way; sealing goat serum for 1 hour; adding primary antibody at 4 ℃ overnight, adding reaction enhancing solution at 37 ℃, incubating for 15-20 minutes, and washing for 3 times and 3 minutes by using PBS (phosphate buffer solution) in a shaking way; the mixture of secondary antibody is added to incubate for 10-15 minutes at 37 degrees, and washed 3 times by 3 minutes in a PBS shaker.

Adding color development liquid DAB, dyeing with hematoxylin, soaking in hydrochloric acid alcohol for 2 seconds, and soaking in ammonia water for 7-10 seconds; dehydration was performed (same procedure as above).

5. TUNEL staining

Dewaxing step as above, using the one-step TUNEL apoptosis assay kit (green, sample soaked in citric acid buffer solution ph6.0, microwave medium high fire for about 8 minutes, left at room temperature, preparation of protease K working solution, adding 10 μ L10 × protease K to 90 μ L PBS per sample, i.e. ready to use, dripping 100 μ L protease K working solution on each tissue section, reacting at 37 ℃ for 30 minutes, immersing the section in PBS for 3 × 5 minutes, preparing 3% hydrogen peroxide confining solution, sealing at room temperature for 10 minutes, dripping 40 μ L TdT enzyme reaction solution on each sample, reacting at 37 ℃ for 30-60 minutes in dark, dripping 50 μ L streptavidin-fluorochein labeling solution, reacting at 37 ℃ in dark for 30 minutes, counterstaining solution for 30 minutes, reacting at room temperature for 10 minutes in dark, washing off DAPI staining solution, adding appropriate amount of blocking tablet (glycerol: =6 PBS), and observing under microscope.

Compared with a control group, the experimental group over-expressing BUD31 can obviously enhance the tumor forming capability of the nude mice, the tumor forming rate and the tumor forming size are obviously higher than those of the control group, and the experimental result has statistical significance. After knocking down the BUD31, the living body imaging shows that the fluorescence signal emitted by the tumor cells is obviously lower than that of the control group, and the abdominal cavity tumor formation and the number are also smaller than those of the control group. BUD31 is a cancer promotion gene, can promote the proliferation and invasion migration of ovarian cancer cells, inhibit the apoptosis of the ovarian cancer cells, and can obviously promote the tumor forming capability of nude mice.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Shandong university

Application of <120> BUD31 serving as ovarian cancer prevention, diagnosis or prognosis marker

<130> 2010

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<400> 1

cattcagaca cgggacacca 20

<210> 2

<211> 18

<212> DNA

<213> Artificial sequence

<400> 2

atgatgcggc ccacttcc 18

<210> 3

<211> 19

<212> RNA

<213> Artificial sequence

<400> 3

uguuggcuca aucaacucc 19

Claims

The application of the BUD31 inhibitor in the preparation of anti-ovarian cancer products is characterized in that,

the BUD31 inhibitor is siBUD31#1,

the siBUD31#1 is a sequence shown as SEQ ID NO. 3.
2. Use of a BUD31 inhibitor according to claim 1 for the preparation of an anti-ovarian product, including but not limited to a pharmaceutical, nutraceutical or model agent.
3. Use of a BUD31 inhibitor according to claim 2 for the preparation of an anti-ovarian cancer product, wherein said BUD31 inhibitor is used as a model agent which is a substance capable of inhibiting the reduction of the expression and/or activity of BUD31, and wherein at least one of the following (1) to (3) is used:

(1) Inhibiting proliferation of ovarian cancer cells, or preparing a model agent for inhibiting proliferation of ovarian cancer cells;

(2) Inhibiting invasion and migration of ovarian cancer cells, or preparing a model medicament for inhibiting invasion and migration of ovarian cancer cells;

(3) Promoting apoptosis of ovarian cancer cells, or preparing model medicament for promoting apoptosis of ovarian cancer cells.
4. The use of a BUD31 inhibitor in the preparation of an anti-ovarian cancer product as claimed in claim 2, wherein the drug is one of but not limited to a solid small molecule compound, a natural drug extract or a nucleic acid component.
5. Use of the BUD31 inhibitor according to claim 4 for the preparation of an anti-ovarian cancer product, wherein the nucleic acid component comprises DNA or RNA that down-regulates gene expression, or RNA species that exert gene inhibitory effects based on RNAi pathways, such as miRNA, siRNA or shRNA.
6, the siRNA with the sequence shown in SEQ ID NO.
7. Use of siRNA of the sequence as set forth in claim 6 for the preparation of a medicament against high-grade serous ovarian cancer, wherein said siRNA further comprises a product obtained by modifying said nucleic acid strand or a formulation form of siRNA; the modifications include salt forms of the siRNA, and also include fusion peptide forms of the siRNA;

the high-grade serous ovarian cancer resistant medicament is one of solid oral preparations, liquid oral preparations or injections.
8. Use of siRNA of sequence as shown in claim 7 in preparation of medicine for treating high-grade serous ovarian cancer, wherein said injection is injectable implant, emulsion, liposome, microcapsule, microsphere or nanoparticle preparation.