CN116004830B - Biomarker for ovarian cancer and application thereof - Google Patents
Biomarker for ovarian cancer and application thereof Download PDFInfo
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- CN116004830B CN116004830B CN202310013928.2A CN202310013928A CN116004830B CN 116004830 B CN116004830 B CN 116004830B CN 202310013928 A CN202310013928 A CN 202310013928A CN 116004830 B CN116004830 B CN 116004830B
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Abstract
The invention discloses a biomarker for ovarian cancer and application thereof, and relates to the technical field of biological medicines. The biomarker is WBP11. The invention carries out related researches on the occurrence and development mechanisms of the ovarian cancer, and determines that the WBP11 is highly expressed in the ovarian cancer by researching the expression levels of WBP11mRNA and protein in oviduct umbrella and ovarian cancer tissues and the relation between the expression condition of the WBP11 and the clinical prognosis of the ovarian cancer, wherein the high expression has correlation with the poor prognosis of the ovarian cancer. The invention further researches the relation between the expression of WBP11 and the expression level of DNA replication related protein MCM7, and the result shows that WBP11 can regulate and control the alternative splicing of MCM7, thereby promoting the occurrence and development of ovarian cancer.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a biomarker for ovarian cancer and application thereof.
Background
Ovarian malignancy is one of common malignant tumors of female reproductive organs, ovarian cancer is hidden from disease, and the early stage lack of specific symptoms and an early screening method are found to be late stage, and the survival rate of five years at the late stage is only about 30%. Prognosis in ovarian cancer patients depends on the staging and early diagnosis of the disease. Thus, early diagnosis is critical to improve prognosis in ovarian cancer patients. The research shows that the tumor markers can be changed a few months before clinical symptoms and imaging changes occur, and accurate judgment can be made for the disease condition of the patient by comprehensively judging and dynamically observing the tumor markers. Currently, tumor markers commonly used in clinic comprise HE4, CA125, and the like, but each marker has certain limitations in terms of sensitivity and specificity.
WW domain binding protein 11 (WBP 11, also known as Npw BP, SIPP1, SNP70 and PPP1R 165) is considered a pre-mRNA splicing factor, co-localized with SC35 in the site nuclear spot where the splicing factor is stored and modified. Studies have shown that it is associated with the PRP19 spliceosome complex. This is a highly dynamic Ribonucleoprotein (RNP) machine that removes non-coding introns from precursor messenger RNAs. WBP11 promotes centromere replication by modulating alternative splicing of TUBGCP 6. WBP11 is reported as an endogenous partner of mammalian PQBP1, both expressed in the developing mesoderm and nervous system, and in xenopus models, loss of gene function leads to defects in mesoderm and neural patterns, accompanied by abnormal gastric and neurogenesis. The deficiency of WBP11 haploid is believed to be a possible cause of a multiple deformity syndrome. Loss of heterozygosity in WBP11 function results in various congenital defects in the heart, bones, gastrointestinal and renal systems of humans and mice. Inhibiting WBP11 inhibits proliferation and migration of gastric cancer cells. However, whether WBP11 can serve as a marker for ovarian cancer remains unknown.
Disclosure of Invention
The invention aims to provide a biomarker for ovarian cancer and application thereof, so as to solve the problems in the prior art, and the biomarker discovers that WBP11 is highly expressed in ovarian cancer patients and is related to poor prognosis of ovarian cancer, and proves that WBP11 can be used as a marker for diagnosis and prognosis prediction of ovarian cancer.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a biomarker for ovarian cancer, which is WBP11.
The invention also provides application of the reagent for detecting the WBP11 expression level in preparing ovarian cancer diagnosis products.
Further, the product is a kit or a reagent.
The invention also provides application of the reagent for detecting the WBP11 expression level in preparing ovarian cancer prognosis diagnosis products.
Further, the product is a kit or a reagent.
The invention also provides application of a substance for inhibiting WBP11 expression in preparing a medicament for treating ovarian cancer.
The invention also provides a medicament for treating ovarian cancer, which comprises a substance for inhibiting WBP11 expression.
Further, the medicament also comprises pharmaceutically acceptable auxiliary materials.
The invention discloses the following technical effects:
the invention carries out related researches on the occurrence and development mechanisms of the ovarian cancer, and determines that the WBP11 is highly expressed in the ovarian cancer by researching the expression levels of WBP11mRNA and protein in oviduct umbrella and ovarian cancer tissues and the relation between the expression condition of the WBP11 and the clinical prognosis of the ovarian cancer, wherein the high expression has correlation with the poor prognosis of the ovarian cancer. The invention further researches the relation between the expression of WBP11 and the expression level of MCM7 (DNA replication related protein), and the result shows that WBP11 can regulate and control the alternative splicing of MCM7, thereby promoting the occurrence and development of ovarian cancer. The invention proves that WBP11 can be applied to the diagnosis of ovarian cancer and the judgment of prognosis, and provides a new choice for molecular diagnosis markers of ovarian cancer.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing that WBP11 is highly expressed in a sample of ovarian cancer patients and that the high expression is positively correlated with a poor prognosis for ovarian cancer patients; wherein, A is that compared with oviduct umbrella of a control group, WBP11mRNA expression is increased in ovarian cancer tissue; b is that compared with the oviduct umbrella of a control group, the WBP11 protein expression is increased in ovarian cancer tissues; c is a representative diagram of immunohistochemical detection of clinical tissue samples, and WBP11 expression is increased in ovarian cancer tissues; d is compared with a oviduct umbrella of a control group, and the immunohistochemical result of the clinical tissue sample shows that the proportion of WBP11 in ovarian cancer is increased; e is the positive correlation of WBP11 high expression in ovarian cancer and bad prognosis according to the result of immunohistochemical scoring of clinical samples;
FIG. 2 is a graph showing detection results of WBP11mRNA in ovarian cancer cell lines HEY, SKOV3 and OVCAR8 in which the siRNA silences WBP 11;
FIG. 3 is immunoblotting results of ovarian cancer cell lines HEY, SKOV3 and OVCAR8 with siRNA silencing WBP 11;
FIG. 4 is the relative cell viability of ovarian cancer cell lines HEY (A), SKOV3 (B) and OVCAR8 (C) with siRNA silencing WBP 11;
FIG. 5 is a clonogenic potential assay result of ovarian cancer cell lines HEY (A), SKOV3 (B) and OVCAR8 (C) with siRNA silencing WBP 11;
FIG. 6 is a graph showing the results of the migration invasive potential assays of ovarian cancer cell lines HEY, SKOV3 and OVCAR8 in which the siRNA silences WBP 11;
FIG. 7 is a graph showing the results of siRNA silencing MCM7 total mRNA (B) and immunoblots (A) of ovarian cancer cell lines HEY, SKOV3 and OVCAR8 of WBP11, MCM7 intron retention/MCM 7 total mRNA ratio (C);
FIG. 8 is a graph showing the detection of MCM7 mRNA in siRNA-silenced ovarian cancer cell lines HEY and OVCAR8 of MCM 7;
FIG. 9 is immunoblotting results of siRNA silencing of ovarian cancer cell lines HEY and OVCAR8 of MCM 7;
FIG. 10 is the relative cell viability of ovarian cancer cell lines HEY and OVCAR8 of siRNA silencing MCM 7;
FIG. 11 shows the results of the clonogenic potential assays of ovarian cancer cell lines HEY (A) and OVCAR8 (B) of siRNA silencing MCM 7;
FIG. 12 shows the results of migration ability assays of ovarian cancer cell lines HEY and OVCAR8 of siRNA silencing MCM 7;
fig. 13 is the results of invasive capacity assays of ovarian cancer cell lines HEY and OVCAR8 of siRNA-silenced MCM 7.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, 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. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Term interpretation:
reagents for detecting WBP11 expression levels: the kit comprises reagents required for detecting the WBP11 content by a common detection method such as an immunoblotting method, an immunohistochemical method, an enzyme-linked immunosorbent assay method, an RT-qPCR detection method, immunofluorescence detection and the like.
Antibody information: WBP11 (Abcam, ab 75987), β -actin (Sigma, a 5441), LC3B (Abcam, ab 51520), p62 (Abcam, ab 109012), PARP (CST, 9542S), caspase-3 (CST, 29629S), ki67 (Abcam, ab 15580).
The ovarian cancer cell line HEY used in the following examples was from the university of western united states Wei Jianjun laboratory benefit; OVCAR8 was from the maryland laboratory benefit of the university of science and technology; SKOV3 was purchased from the cell bank of the national academy of sciences.
Example 1
1. Detection of WBP11mRNA and protein expression levels in Brevibacterium and ovarian cancer tissues
mRNA and protein levels of P11mRNA and protein were detected by RT-qPCR and Western Blot methods using TRIzol (15596018, invitrogen) and Western and IP cell lysates (P0013, biyun) respectively, to extract mRNA and protein from oviduct umbrella and ovarian cancer tissues. 73 oviduct umbrella tissues and 143 ovarian cancer tissues collected by Qilu Hospital of Shandong university are selected, tissue chips are manufactured, and the WBP11 protein level is detected by an immunohistochemical method. The immunohistochemical staining is carried out by adopting a kit (Zhonghua gold bridge, goods number: PV-9000), and the specific steps are as follows:
a. tissue chip xylene dewaxing and gradient ethanol hydration;
b. adopting EDTA antigen retrieval liquid to carry out antigen retrieval;
c. the peroxidase blocking agent (reagent a) was incubated for 10min to block the activity of endogenous peroxidase;
d. serum (reagent B) was blocked for 30min;
wbp11 antibody (1:500 dilution) incubated overnight at 4 ℃;
f. incubating the biotin-labeled secondary antibody (reagent C) for 30min at room temperature;
g. reacting streptomycin avidin-peroxidase (reagent D) for 20min at room temperature;
color development of DAB solution, counterstaining of mature hematoxylin for 5min, color separation of 1% (v/v) ethanol hydrochloride for 3s, and blue returning of running water for 15min;
i. sequentially dehydrating gradient ethanol, transparency to xylene for 5min, sealing with neutral resin, storing, and photographing under microscope.
Results: as shown in fig. 1 a and B, WBP11mRNA and protein were significantly increased in ovarian cancer tissue compared to control oviduct umbrella tissue. FIGS. 1C and D show that WBP11 is expressed in ovarian cancer tissue significantly higher than paradox in clinical tissue samples.
2. Detection of WBP11 in relation to clinical prognosis of ovarian cancer
Statistical analysis was performed on the immunohistochemical results of fig. 1C, and ovarian cancer patients were classified into WBP11 high-expression groups and WBP11 low-expression groups, and the relationship between WBP11 expression levels and the total survival rate of ovarian cancer patients was counted in correspondence with survival information of these ovarian cancer patients.
Results: as shown in fig. 1E, high WBP11 expression levels are indicative of low survival.
3. Biological function of WBP11 in ovarian cancer cells
The method comprises the steps of performing WBP11 expression inhibition on ovarian cancer cell lines HEY, SKOV3 and OVCAR8 cultured in vitro by adopting an siRNA silencing technology, extracting cellular proteins, and analyzing and verifying the WBP11 expression quantity by utilizing an immunoblotting method, wherein the results are shown in figures 2-6. As shown in fig. 2 and 3, WBP11 expression levels of cancer cells with suppressed WBP11 expression were significantly reduced using siRNA silencing techniques, indicating successful siRNA silencing.
(1) SiRNA silencing technique
Cells with good logarithmic growth phase were inoculated in a 6cm dish in an appropriate amount and cultured overnight. The cells are uniformly distributed in the next day, the density is 50% -60%, and transfection can be performed. EP tube containing siRNA powder was centrifuged at 5000rpm at 4℃for 1 min, and a proper amount of DEPC water was added according to the instructions to be sufficiently dissolved and then placed on ice for use. Preparing a transfection complex system: tube A, opti-MEM 250. Mu.L+Lipo 200010. Mu.L, tube B, opti-MEM 250. Mu.L+target or control siRNA 10. Mu.L (interference sequence: si-NC: UUCUCCGAACGUGUCACGUtt; siWBP11#1: CCGUGAUAAUGGUGAGAGAGT; siWBP11#2: GGAUCAAGAUAAGCAUGAUtt), tube A, tube B were mixed upside down, the action was gentle, and incubated for 5 minutes at room temperature; then, the tube A and the tube B are evenly mixed and kept stand for 20 minutes, the original culture medium in the culture dish is sucked, the mixture of the tube A and the tube B is added, 1.5mL of Opti-MEM is supplemented, and the culture dish is placed in a cell culture box for 6 hours for culture. After 6 hours, the mixed solution in the cell culture dish is discarded, 3mL of complete culture medium is added for continuous culture, RNA can be extracted after 24 hours for detecting interference efficiency by qRT-PCR experiment, and protein can be extracted after 48 hours for carrying out WesternBlot experiment or cell function experiment.
(2) Cell proliferation assay
The control group and the WBP11 silencing cell line were inoculated into 96-well plates, and the treated test cells were plated in 96-well plates (1X 10 per well 3 And) were incubated for 2h with a medium containing 10wt% CCK-8 (C0041, biyun) at a wavelength of 450nm, respectively at 0h, 24h,48h,72h, 96h and 120 h. The effect of knockdown WBP11 on the clonogenic capacity of cells was examined using a plate cloning experiment. As shown in fig. 4 and 5, knockdown of WBP11 significantly inhibited proliferation and clonogenic capacity of ovarian cancer cell lines.
(3) Transwell experiment
The Transwell chamber was used for cell migration ability analysis, the coated matrigel Transwell chamber was used for invasion ability analysis, and the pore size was 0.8. Mu.m. The control and WBP11 silencing cell lines were resuspended in serum-free medium and inoculated into a chamber (about 3 x 10 4 cells/well), and culturing for a corresponding time according to the characteristics of the cells. Cells not passing through the upper layer of the cell were removed with a cotton swab, and cells passing through the lower layer were fixed with methanol for 15min, stained with 0.1wt% crystal violet for 20min, and observed under a microscope. 5 fields were randomly observed and the number of cells penetrating the membrane was counted. As shown in fig. 6, knockdown of WBP11 significantly inhibited the invasive migratory capacity of ovarian cancer cell lines.
4. Detection of malignant biological behavior of WBP11 at cellular level to promote ovarian cancer by modulation of MCM7 alternative splicing
In HEY, SKOV3 and OVCAR8 cell lines, the expression level of MCM7 is detected by adopting a siRNA silencing technology (interference sequences are si-NC: UUCUCCGAACGUGUCACGUtt; siMC7#1: AGAGGGAAGUGGUAAAUAAtt; siMCM7#2: UCUCUCUGAAGACCCAAtt) to knock down WBP11 and utilizing q-RT PCR and Western Blot. As shown in fig. 7, knockdown of WBP11, MCM7 RNA and protein levels decreased, and MCM7 intron retention increased significantly.
5. Biological function of MCM7 in ovarian cancer cells
Cell proliferation experiments and Transwell experiments are carried out on the cell of the knockdown MCM7, and the method is the same as the biological functions of three WBP11 in ovarian cancer cells, as shown in figures 8-13, the knockdown MCM7 obviously inhibits the proliferation capacity, the clone formation and the invasion migration capacity of an ovarian cancer cell line.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (5)
1. The application of a reagent for detecting WBP11 expression level in preparing an ovarian cancer diagnosis product.
2. The use according to claim 1, wherein the product is a kit or a reagent.
3. Application of reagent for detecting WBP11 expression level in preparing ovarian cancer prognosis diagnosis product.
4. Use according to claim 3, wherein the product is a kit or reagent.
5. Use of a substance that inhibits WBP11 expression in the manufacture of a medicament for treating ovarian cancer, wherein the substance is an siRNA, and the siRNA has an interfering sequence of CCGUGAUAAUGGUGAGAGATT or GGAUCAAGAUAAGCAUGAUTT.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2004035803A2 (en) * | 2002-10-01 | 2004-04-29 | Epigenomics Ag | Method and nucleic acids for the treatment of breast cell proliferative disorders |
| CN113230405A (en) * | 2021-05-08 | 2021-08-10 | 中国医学科学院肿瘤医院 | Application of agent for inhibiting activity of protein kinase CLK in preparation of medicine for treating or improving esophageal squamous cell carcinoma |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004035803A2 (en) * | 2002-10-01 | 2004-04-29 | Epigenomics Ag | Method and nucleic acids for the treatment of breast cell proliferative disorders |
| CN113230405A (en) * | 2021-05-08 | 2021-08-10 | 中国医学科学院肿瘤医院 | Application of agent for inhibiting activity of protein kinase CLK in preparation of medicine for treating or improving esophageal squamous cell carcinoma |
Non-Patent Citations (1)
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| The miR-29c-KIAA1199 axis regulates gastric cancer migration by binding with WBP11 and PTP4A3;Lina Wang等;Oncogene;第3134-3150页 * |
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