CN111575382A - Application of protein/gene IFI30 related to breast cancer occurrence and development - Google Patents

Application of protein/gene IFI30 related to breast cancer occurrence and development Download PDF

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CN111575382A
CN111575382A CN202010484939.5A CN202010484939A CN111575382A CN 111575382 A CN111575382 A CN 111575382A CN 202010484939 A CN202010484939 A CN 202010484939A CN 111575382 A CN111575382 A CN 111575382A
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ifi30
breast cancer
expression
protein
staining
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相玉娟
叶春淼
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Abstract

The invention belongs to the technical field of biological medicines, particularly relates to application of a protein/gene IFI30 related to breast cancer occurrence and development, particularly provides application of a protein/gene IFI30 related to breast cancer occurrence and development, and particularly relates to application value of the protein/gene IFI30 as a breast cancer molecular typing marker and application of the protein/gene IFI30 in preparation of a breast cancer molecular typing kit. The invention also provides a method for screening potential drugs for treating breast cancer, and whether the candidate is a potential drug for treating breast cancer is judged by regulating the expression level of the biomarker.

Description

Application of protein/gene IFI30 related to breast cancer occurrence and development
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of a protein/gene IFI30 related to breast cancer occurrence and development.
Background
Breast cancer is one of the most common malignancies in women, a malignancy that usually occurs in the epithelial tissue of the breast gland. According to data statistics, the incidence rate accounts for 7-10% of all malignant tumors in the whole body, and only about 1-2% of breast cancer patients are male. Second only to uterine cancer, the incidence of which is often genetically related, and between the ages of 40-60, the incidence of peri-menopausal women is high, one of the most common malignancies severely affecting women's physical and mental health and even life threatening.
Although significant progress has been made in the systemic treatment of breast cancer, many patients still suffer from tumor recurrence, metastasis and treatment resistance, which severely affects patient survival and quality of life. Therefore, the research on the mechanism involved in the development process of breast cancer is crucial to find a new therapeutic target for breast cancer.
In 2000, Perou et al first proposed the concept of breast cancer molecular typing based on the different molecular characteristics of ER, PR, HER-2 and Ki67 by cDNA chip data analysis of 65 breast cancer tissues, wherein by taxonomic clustering analysis, the expression abundance of 1753 genes in at least 3 tissue samples was changed by more than 4-fold compared with the median abundance, which included IFI 30.
At present, IFI30 is not reported in documents as a breast cancer molecular typing marker and guides prognosis and personalized treatment. The invention finds that the expression of IFI30 in breast cancer tissues is related to the prognosis of patients, and the high expression of IFI30 in the breast cancer tissues can increase the 5-year survival rate of breast cancer patients.
Disclosure of Invention
The invention relates to application of IFI30, in particular to application of protein/gene IFI30 related to breast cancer occurrence and development, and particularly application value of the protein/gene IFI30 as a breast cancer molecular typing marker and application of the protein/gene IFI30 in preparation of a breast cancer molecular typing kit. The invention also provides a method for screening potential drugs for treating breast cancer, and whether the candidate is a potential drug for treating breast cancer is judged by regulating the expression level of the biomarker.
The technical scheme of the invention is as follows:
an application of a protein/gene IFI30 related to the occurrence and development of breast cancer, in particular to an application of a reagent for changing the expression level of IFI30 in evaluating products for the progression, prognosis and treatment responsiveness of breast cancer. Among other things, low expression of IFI30 may increase cell proliferation, colony formation, migration, invasion and decrease apoptosis.
First aspect of the invention: the application of the protein/gene IFI30 related to the occurrence and development of the breast cancer is specifically the application of IFI30 in the preparation of a breast cancer molecular typing marker.
Second aspect of the invention: the application of the protein/gene IFI30 related to the generation and development of the breast cancer is specifically the application of IFI30 in the preparation of a breast cancer IFI30 expression strength immunohistochemical detection kit. The breast cancer IFI30 expression intensity immunohistochemical detection kit is used for detecting the expression level of IFI 30.
Furthermore, the kit detects the expression of IFI30 in the biological sample by an immunohistochemical method.
Further, the kit comprises: an antibody specifically recognizing human IFI30, and an immunohistochemical test reagent.
Further, the biological sample is paraffin-embedded tissue of breast cancer of a patient.
In a third aspect of the invention: the application of the protein/gene IFI30 related to the occurrence and development of the breast cancer, in particular to a detection method of the breast cancer molecular typing kit, which comprises the following steps:
step 1, dewaxing to water
(1) Slicing 4 μm thick paraffin, continuously slicing 2 pieces of each tumor tissue, and baking in an oven at 60 deg.C for 1 hr;
(2) xylene 10min x 3 times;
(3) sequentially passing through 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol, and double distilled water for 5 min;
step 2 antigen retrieval
(1)3％H2O2Methanol solution for 20 min;
(2) double-steaming and washing for 5minx 3 times;
(3) antigen repair (high pressure repair):
the slice is put into EDTA alkaline repair liquid (2mM EDTA, 2mM Na)2HPO4) Boiling in a pressure cooker for 2min under high pressure, and naturally cooling to room temperature;
step 3 Primary antibody incubation
(1) Washing with PBS for 5 min;
(2)50 μ l of 1% BSA coated tissue, blocked for 30min at 37 deg.C;
(3) absorbing the confining liquid, respectively dropwise adding 50 mu l of IFI30 primary antibody, and incubating overnight at 4 ℃ in a wet box;
step 4. incubation with Secondary antibody
(1) Sucking away the primary antibody, and washing with PBS for 5minx 4 times;
(2) dripping 50 μ l secondary antibody with HRP mark, and incubating at 37 deg.C for 30-45 min;
step 5, color development and blue reflection
(1) Washing with PBS for 5minx 4 times;
(2) DAB (1:50) is developed for 3-10min until brick red precipitate appears, and double-distilled water stops developing;
(3) counterstaining with hematoxylin for 10min, and washing with tap water for 20-30min after differentiation with hydrochloric acid and ethanol;
step 6, dewatering sealing sheet
Sequentially adding double distilled water, 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, phenol, and xylene, each for 5 min. Dripping a resin sealing sheet;
step 7. Observation evaluation and scoring
The stained sections were evaluated by 2 pathologists and the expression level of IFI30 in breast cancer cells was observed under a microscope. The 2 pathologists performed in a "back-to-back" manner without any explicit patient clinical information and evaluated the staining results according to the modified harvest scoring criteria. The scoring result is divided into two parts: IFI30 staining intensity IFI30 proportion of cells staining positive. Staining intensity was scored as 0 (cell unstained), 1+ (cell cytoplasm appeared light brown), 2+ (cell cytoplasm appeared moderately stained) and 3+ (cell cytoplasm appeared dark brown) as 0, 1, 2 and 3 points, respectively. The positive staining proportion of the breast cancer cells IFI30 is counted and divided into 0 point (without positive cells), 1 point (< 1/100 cell specific staining), 2 points (1/100-1/10 cell specific staining), 3 points (1/10-1/3 cell specific staining), 4 points (1/3-2/3 cell specific staining) and 5 points (> 2/3 cell specific staining) according to the positive cell proportion. Finally obtaining a staining range score, a staining intensity score and a total score of the staining range score and the staining intensity score.
FIG. 1 is a schematic diagram showing IFI-30 expression intensity of breast cancer tissue after immunohistochemical staining, wherein black oval is cell nucleus, cell nucleus periphery is cytoplasm, IFI-30 is stained cytoplasm, and the stained cytoplasm is brown when observed under a microscope, and is divided into 0, 1+, 2+ and 3+ according to the difference of brown intensity, and the darker the cytoplasm gray shown in the figure indicates that DAB is stained more deeply and the intensity is stronger, and the IFI-30 intensity is 0, 1+, 2+ and 3+ in sequence from left to right.
The fourth aspect of the present invention: the application of the protein/gene IFI30 related to the occurrence and development of the breast cancer is specifically the application of the kit for detecting the expression level of IFI30 in the breast cancer tissue in preparing a breast cancer molecular typing kit.
Further, the breast cancer molecular typing kit is used for detecting the expression level of IFI30 in breast cancer tissues.
Further, the breast cancer molecular typing kit detects the expression condition of IFI30 in a biological sample through an immunohistochemical method.
Further, the breast cancer molecular typing kit comprises: an antibody specifically recognizing human IFI30, and an immunohistochemical test reagent.
Further, the biological sample is paraffin-embedded tissue of breast cancer of a patient.
Fifth aspect of the present invention: the application of the protein/gene IFI30 related to the occurrence and development of the breast cancer is particularly the application of IFI30 in screening potential substances for treating the breast cancer.
Further, the IFI30 is applied to screening of potential substances for treating breast cancer, and the method comprises the following steps: treating a system expressing or containing IFI30 with a candidate substance and detecting expression of IFI30 gene/protein in said system; if the candidate substance can increase the expression level of IFI30, the candidate substance is a potential substance for treating breast cancer; further, the candidate substance may increase the expression level of IFI30 by more than 20%, preferably more than 50%, and most preferably more than 80%.
Further, the system is selected from: a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.
Further, the candidate substances include, but are not limited to: an overexpression vector, a specific antibody or an activating small molecule compound designed aiming at the IFI30 gene or an upstream gene or a downstream gene thereof; further, the candidate substance in the present invention is preferably a lentiviral over-expression vector of IFI 30.
The sixth aspect of the present invention: the application of the protein/gene IFI30 related to the occurrence and development of the breast cancer is specifically to provide the application of IFI30 in preparing a pharmaceutical composition for treating the breast cancer.
Further, the pharmaceutical composition comprises an activator of functional expression of IFI30 selected from the group consisting of: contains small molecular substance and specific antibody for specifically activating IFI30 gene/protein expression or activity. The tumor is low in IFI30 protein expression.
Preferably, the pharmaceutical composition comprises a small molecule substance, specific antibody, capable of up-regulating the IFI30mRNA or protein level or other specific binding and promoting expression or activity of IFI30 gene/protein.
Furthermore, the pharmaceutical composition also comprises medicines compatible with the activator and pharmaceutically acceptable carriers and/or auxiliary materials.
Pharmaceutically acceptable carriers include, but are not limited to: buffer, emulsifier, suspending agent, stabilizer, preservative, physiological salt, excipient, filler, coagulant and blender, surfactant, dispersing agent, and defoaming agent.
Seventh aspect of the present invention: there is provided a pharmaceutical composition for treating breast cancer, the pharmaceutical composition comprising:
an activator of functional expression of IFI 30; and
a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers include, but are not limited to: buffer, emulsifier, suspending agent, stabilizer, preservative, physiological salt, excipient, filler, coagulant and blender, surfactant, dispersing agent, and defoaming agent.
The beneficial effect of the invention is that,
the present invention used a breast cancer cell line to verify the biological behavior of IFI30 in vivo and in vitro. The results of the study indicate that low expression of IFI30 in breast cancer cells can increase cell proliferation, colony formation, migration, invasion and decrease apoptosis. In a nude mouse experiment, the tumor formation speed and the tumor size are obviously reduced after the cell line with high IFI30 expression is transplanted subcutaneously into the nude mouse. The results indicate that the IFI30 can be used as an independent biomarker for guiding the prognosis of the breast cancer and a novel molecular typing marker.
Through the research, the invention provides the application of the protein/gene IFI30 related to the breast cancer occurrence and development, in particular the application value as a breast cancer molecular typing marker and the application in preparing a breast cancer molecular typing kit. The invention also provides a method for screening potential drugs for treating breast cancer, and whether the candidate is a potential drug for treating breast cancer is judged by regulating the expression level of the biomarker.
In conclusion, the application of the protein/gene IFI30 related to the occurrence and development of the breast cancer has positive effects on the detection of breast cancer molecular typing, the treatment of the breast cancer, the prognosis and the like.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a photograph of the immunohistochemical staining of IFI-30 protein in breast cancer tissues.
FIG. 2 is a fluorescent microscopic image of breast cancer cells transfected with IFI-30 overexpressing/silencing virus.
FIG. 3 is a WesternBlot development of breast cancer cells after transfection with IFI-30 overexpressing/silencing virus.
FIG. 4 is a bar graph of relative IFI-30 gene expression levels detected by Real-Time PCR after transfection of IFI-30 over-expressed/silenced virus into breast cancer cells.
FIG. 5 is a graph of cell growth after transfection of breast cancer cells with IFI-30 overexpressing/silencing virus.
FIG. 6 is a fluorescent microscope photograph of EDU staining after transfection of breast cancer cells with IFI-30 overexpressing/silencing virus.
FIG. 7 is a diagram of nude mouse tumorigenesis after transfection of IFI-30 overexpression virus into breast cancer cells.
FIG. 8 is a microscopic image of a Transwell cell invasion assay after transfection of IFI-30 overexpression/silencing virus into breast cancer cells.
FIG. 9 is a bar graph of cells transfected with IFI-30 over-expressing virus against docetaxel ic 50.
FIG. 10 is a bar graph of cell-to-epirubicin, docetaxel, ic50, after transfection of breast cancer cells with IFI-30 overexpressing/silencing virus, where a is epirubicin and b is docetaxel.
FIG. 11 is a graph showing the change in sensitivity to radiation therapy following transfection of breast cancer cells with IFI-30 overexpressing virus.
FIG. 12 is a graph of survival analysis of breast cancer patients grouped by IFI-30 expression range, intensity, total score.
FIG. 13 is a Western Blot image of recombinant IFI-30 protein.
FIG. 14 shows the peptide sequence coverage of Trypsin-digested test samples of recombinant IFI-30 protein.
FIG. 15 shows the peptide sequence coverage of the Chymotrypsin digested test sample of recombinant IFI-30 protein.
FIG. 16 shows the peptide sequence coverage of the test sample digested with Glu-C of recombinant IFI-30 protein.
FIG. 17 shows the integrated amino acid sequence coverage of recombinant IFI-30 protein.
FIG. 18 is a fluorescent microscope image of immunofluorescence staining of breast cancer cells incubated with recombinant IFI-30 protein.
FIG. 19 is a bar graph of the effect on cell proliferation following the addition of recombinant IFI-30 protein to breast cancer cells.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention relates to an application of a protein/gene IFI30 related to breast cancer occurrence and development, in particular to an application of a reagent for changing the expression level of IFI30 in evaluating breast cancer progression, prognosis and treatment reactivity products. Among other things, low expression of IFI30 may increase cell proliferation, colony formation, migration, invasion and decrease apoptosis.
The application specifically relates to the following:
the application of IFI30 in preparing breast cancer molecular typing markers.
The application of the IFI30 in preparing a breast cancer molecular typing kit for detecting the expression level of IFI 30. The kit is used for detecting the expression condition of IFI30 in a biological sample (paraffin embedded tissue of breast cancer of a patient) by an immunohistochemical method. The kit comprises: an antibody specifically recognizing human IFI30, and an immunohistochemical test reagent.
And thirdly, a detection method of the breast cancer molecular typing kit. The method comprises the following specific steps: step 1, dewaxing to water
(1) Slicing 4 μm thick paraffin, continuously slicing 2 pieces of each tumor tissue, and baking in an oven at 60 deg.C for 1 hr;
(2) xylene 10min x 3 times;
(3) sequentially passing through 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol, and double distilled water for 5 min;
step 2 antigen retrieval
(1)3％H2O2Methanol solution for 20 min;
(2) double-steaming and washing for 5minx 3 times;
(3) antigen repair (high pressure repair):
the slice is put into EDTA alkaline repair liquid (2mM EDTA, 2mM Na)2HPO4) Boiling in a pressure cooker for 2min under high pressure, and naturally cooling to room temperature;
step 3 Primary antibody incubation
(1) Washing with PBS for 5 min;
(2)50 μ l of 1% BSA coated tissue, blocked for 30min at 37 deg.C;
(3) absorbing the confining liquid, respectively dropwise adding 50 mu l of IFI30 primary antibody, and incubating overnight at 4 ℃ in a wet box;
step 4. incubation with Secondary antibody
(1) Sucking away the primary antibody, and washing with PBS for 5minx 4 times;
(2) dripping 50 μ l secondary antibody with HRP mark, and incubating at 37 deg.C for 30-45 min;
step 5, color development and blue reflection
(1) Washing with PBS for 5minx 4 times;
(2) DAB (1:50) is developed for 3-10min until brick red precipitate appears, and double-distilled water stops developing;
(3) counterstaining with hematoxylin for 10min, and washing with tap water for 20-30min after differentiation with hydrochloric acid and ethanol;
step 6, dewatering sealing sheet
Sequentially adding double distilled water, 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, phenol, and xylene, each for 5 min. Dripping a resin sealing sheet;
step 7. Observation evaluation and scoring
The stained sections were evaluated by 2 pathologists and the expression level of IFI30 in breast cancer cells was observed under a microscope. The 2 pathologists performed in a "back-to-back" manner without any explicit patient clinical information and evaluated the staining results according to the modified harvest scoring criteria. The scoring result is divided into two parts: IFI30 staining intensity IFI30 proportion of cells staining positive. Staining intensity was scored as 0 (cell unstained), 1+ (cell cytoplasm appeared light brown), 2+ (cell cytoplasm appeared moderately stained) and 3+ (cell cytoplasm appeared dark brown) as 0, 1, 2 and 3 points, respectively. The positive staining proportion of the breast cancer cells IFI30 is counted and divided into 0 point (without positive cells), 1 point (< 1/100 cell specific staining), 2 points (1/100-1/10 cell specific staining), 3 points (1/10-1/3 cell specific staining), 4 points (1/3-2/3 cell specific staining) and 5 points (> 2/3 cell specific staining) according to the positive cell proportion. Finally obtaining a staining range score, a staining intensity score and a total score of the staining range score and the staining intensity score.
And fourthly, the application of the kit for detecting the IFI30 expression level in the breast cancer tissue in preparing a breast cancer molecular typing kit. The breast cancer molecular typing kit is used for detecting the expression level of IFI30 in breast cancer tissues, and further detecting the expression condition of IFI30 in biological samples (paraffin embedded tissues of breast cancer of patients) through an immunohistochemical method by the breast cancer molecular typing kit. The breast cancer molecular typing kit comprises: an antibody specifically recognizing human IFI30, and an immunohistochemical test reagent.
And fifthly, the IFI30 is applied to screening potential substances for treating breast cancer. The method comprises the following steps: treating a system expressing or containing IFI30 with a candidate substance and detecting expression of IFI30 gene/protein in said system, said system selected from the group consisting of: a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system. Such candidate substances include, but are not limited to: an overexpression vector, a specific antibody or an activating small molecule compound designed aiming at the IFI30 gene or an upstream gene or a downstream gene thereof; further, the candidate substance in the present invention is preferably a lentiviral over-expression vector of IFI 30.
If the candidate substance can increase the expression level of IFI30, the candidate substance is a potential substance for treating breast cancer; further, the candidate substance may increase the expression level of IFI30 by more than 20%, preferably more than 50%, and most preferably more than 80%.
And sixthly, the application of the IFI30 in preparing the pharmaceutical composition for treating the breast cancer is provided.
The pharmaceutical composition comprises an activator of functional expression of IFI30 selected from the group consisting of: contains small molecular substance and specific antibody for specifically activating IFI30 gene/protein expression or activity. The tumor is low in IFI30 protein expression.
The pharmaceutical composition comprises small molecule substances, specific antibodies, which can up-regulate the IFI30mRNA or protein level or other substances which can specifically bind to IFI30 gene/protein and promote the expression or activity thereof. The pharmaceutical composition also comprises medicines compatible with the activator and a pharmaceutically acceptable carrier and/or auxiliary material.
Pharmaceutically acceptable carriers include, but are not limited to: buffer, emulsifier, suspending agent, stabilizer, preservative, physiological salt, excipient, filler, coagulant and blender, surfactant, dispersing agent, and defoaming agent.
A pharmaceutical composition for treating breast cancer, the pharmaceutical composition comprising:
an activator of functional expression of IFI 30; and
a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers include, but are not limited to: buffer, emulsifier, suspending agent, stabilizer, preservative, physiological salt, excipient, filler, coagulant and blender, surfactant, dispersing agent, and defoaming agent.
The following is a further description by specific experimental examples.
Experiment 1 use of IFI30 to overexpress lentivirus and interfere with lentivirus infection of breast cancer cells
(1) The full-name sequence of IFI30 and the corresponding shRNA are respectively cloned under lentiviral vectors GV287 and GV115 to form an IFI30 over-expression lentiviral vector (GV287-IFI-30Ubiquitin-IFI30-3FLAG-SV40-EGFP) and an RNA interference lentiviral vector (GV115-shIFI-30hU6-shIFI 30-CMV-EGFP).
(2) 4 breast cancer cell lines (MCF-7, MDA-MB-231, SK-BR-3 and BT-474 cells) were seeded in a 24-well plate, 4-5 ten thousand cells per well, and after 24 hours of seeding, the cells were cultured by replacing with a conventional medium containing viruses, in this experiment, the re-infection index (MOI) of the lentivirus used was 20, and the Polybrene used was at a concentration of 5. mu.g/mL.
(3) After 12h of transfection, the virus medium was changed to normal medium and the culture was continued.
(4) And when the cells are continuously cultured for 72h, observing the fluorescence expression abundance by using a microscope, wherein the infection efficiency is about 80 percent, and continuously subculturing the successfully transfected cells for detecting the cell phenotype.
(5) The transfection effect was verified by observing GFP fluorescence using a fluorescence microscope and detecting the expression of IFI30 protein using Western Blot. See in particular fig. 1, fig. 2, fig. 3, fig. 4.
FIG. 1 is a photograph of the immunohistochemical staining of IFI-30 protein in breast cancer tissues.
After the cells shown in FIG. 2 were transfected with IFI-30 overexpressing/silencing virus, the cells fluoresced at 507nm under 488nm excitation light (the gray light in the EGFP column is indicated as fluorescence), and the cells that fluoresced indicated that they had been successfully transfected with the overexpressing/silencing virus.
FIG. 3 shows the IFI-30 gene expression of breast cancer cells after IFI-30 overexpression/silencing virus transfection of breast cancer cells by the WesternBlot method, GILT is the name of the protein expressed by IFI-30, beta-actin is a reference protein, and the darker the black band in FIG. 3 represents the higher content. The upper part of the picture is used for detecting the expression quantity of MDA-MB-231, SK-BR-3, BT-474 and MCF-7 cell basic IFI-30 protein (GILT), the result shows that except MDA-MB-231 cell expression is negative, the expression of other cells is positive, after 4 cells are transfected with IFI-30 overexpression and silencing virus, the expression of cell IFI-30 protein is performed below the picture, the left column is overexpression, IFI-30 LV is transfected overexpression virus, CON is transfected control virus, the right column is silencing, IFI-30 RNAi LV is transfected silencing virus, and CON is transfected control virus.
FIG. 4 shows the results of Real-Time PCR, and the IFI-30mRNA expression levels of 4 breast cancer cell lines and a common mammary epithelial cell are detected on the top of the image. In the lower part of the graph, after 4 breast cancer cells are transfected with IFI-30 overexpression and lentivirus silencing, the mRNA expression level of IFI-30 is changed.
The experimental result shows that after the breast cancer cells over-express and interfere with the IFI30 gene, the mRNA level and the protein (GILT) expression level of the IFI30 in the cells are obviously up-regulated and down-regulated.
Experiment 2 CCK-8 was used to detect proliferation of breast cancer cells after transfection of IFI30 Gene
(1) Stably transfected breast cancer cells were seeded in 96-well plates, with 2000 or 3000 cells per well.
(2) Add 10. mu.L of CCK-8 to each well and incubate at 37 ℃ for 3 hours.
(3) MK-3 microplate reader (Thermo) measures absorbance at 450 nm.
(4) After eliminating the absorbance of the blank (normal medium), the data were compared to the absorbance of 0D to summarize the diffusion rate.
The experimental results show that the proliferation level of the breast cancer cells is reduced after the IFI30 gene is over-expressed, and the proliferation capacity of the breast cancer cells is increased after IFI30 is silenced, as shown in figure 6.
Experiment 3 BeyoClick was usedTMDetection of proliferation condition of breast cancer cells after IFI30 gene transfection by EdU-555 cell proliferation detection kit
(1) The cancer cells infected with lentivirus were inoculated into 24-well plates, each well being inoculated with 20000-30000 cells in 500ul medium.
(2) After cells adhered to the wall overnight, 20uM of 2X EDU working solution was prepared, which was preheated at 37 ℃ and 500ul of the solution was added to a 24-well plate and cells were cultured for 2 hours.
(3) The culture medium was removed, 1ml of 4% paraformaldehyde was added, and the mixture was fixed at room temperature for 15 minutes.
(4) The fixative was removed and the cells were washed 3 times with 1ml of wash solution (3% BSA in PBS) per well for 3 minutes each.
(5) The wash solution was removed and each well was incubated with 1ml of the permeate (0.3% Triton X-100 PBS) for 15 minutes at room temperature.
(6) The permeate was removed and the cells were washed 2 times with 1ml of wash solution per well for 3 minutes each time.
(7) 0.5ml of Click reaction solution (Beyoclick EDU-555 kit) was added to each well, incubated at room temperature for 30 minutes in the dark, the reaction solution was removed, and the wash solution was washed 3 times for 3 minutes each.
(8) Each well used 1: 1000 dilution of Hoechst staining solution dyeing 10 minutes, suction to remove the staining solution after washing liquid washing 3 times, each time 3 minutes.
(9) And (3) observing red and blue fluorescence of the cells by using a fluorescence microscope, wherein the maximum excitation wavelength of the red is 555nm, the maximum emission wavelength of the red is 565nm, the maximum excitation wavelength of the blue is 346nm, and the maximum emission wavelength of the blue is 460nm, photographing, and counting the proportion of the proliferating cells, namely the proportion of the red cells.
The experimental result is shown in fig. 6, the right column is cell nucleus stained by hoechst, gray color shows cell nucleus staining, the middle column is cell nucleus staining, gray bright spot shows fluorescence of EDU, the left column is fusion graph of hoechst and EDU staining, the higher the proportion of the bright spot in the cell nucleus is, the higher the cell proliferation rate is, the right histogram is statistical graph of the EDU positive rate, the result shows that after the breast cancer cell over-expresses IFI30 gene, the EDU staining rate of the breast cancer cell is reduced, proliferation is slowed, and after IFI30 gene is interfered, the EDU staining rate is increased, and proliferation is quickened.
Application of IFI30 gene in inhibiting breast cancer cell nude mouse tumorigenesis in experiment 4
Nude mouse tumorigenesis experiment
(1) After MDA-MB-231 cells were transfected with IFI30 gene, the over-expressed cells were expanded.
(2) After the cells are proliferated to 70-90% confluency, digesting the resuspended cells, centrifuging, washing the cells with ice PBS, counting, resuspending the cells with PBS, adjusting the cell concentration to 8 × 107 cells/ml, and mixing the cell suspension with matrigel 1: 1 and mixing.
(3) Injecting the cell suspension to the axilla of the forelimb of a 4-week-old Balb/c nu/nu female nude mouse, measuring the long diameter and the short diameter of the tumor every week, anesthetizing the mouse after 6-8 weeks, removing the neck, killing the neck, dissecting and taking out the subcutaneous tumor.
(4) Tumor weights were measured using a balance and photographed, and 2 independent sample t-test statistical analyses were performed on experimental and control tumor volumes and weights using SPSS21.0 software.
The experimental results are shown in fig. 7, wherein the left side of the graph is the nude mice used in the experimental group and the control group, the upper right side is the tumor of the nude mice taken down, and the lower right side is the two groups of tumor volume change curves. The result shows that the subcutaneous tumor formation of the nude mice is increased more slowly and the tumor formation rate is lower after the IFI30 gene is over-expressed by the breast cancer cells.
Experiment 5 application of IFI30 gene in inhibiting migration and invasion of breast cancer cells
Cell migration and invasion Capacity assays
(1) The migratory and invasive ability of the cells was tested using the transwell assay. For cell invasion experiments, the upper chamber of the transwell plate was coated with 100. mu.L of diluted matrigel (medium and matrigel diluted 1:6-1: 8).
(2) The transfected cells were resuspended in serum-free medium containing 0.2% BSA and plated on the upper layer of a tranwell plate with a pore size of 8 μm, the lower layer of the plate containing normal medium containing 10% serum, and then the cells were cultured.
(3) After 24h of cell culture, the matrigel and cells on the upper surface of the polycarbonate membrane were gently wiped off with a wet cotton swab. The upper chamber was carefully removed, fixed with 4% paraformaldehyde for 30min, and then stained with 0.5% crystal violet. Cells attached to the lower surface of the polycarbonate membrane were counted in 6 fields at random under a high power microscope (200 times) and averaged.
The experimental results are shown in fig. 8, the left side is a statistical graph of the cells which are stained after the cells pass through the transwell chamber, and the right side is a statistical graph of the passed cells, and the results show that after the IFI30 gene is over-expressed in the breast cancer cells, the migration and invasion capacity of the cells is reduced, and after the gene is silenced, the migration and invasion capacity of the cells is enhanced.
Experiment 6 application of IFI30 gene in increasing triple negative breast cancer chemotherapy sensitivity
(1) The triple negative breast cancer cell MDA-MB-231 after the over-expression of the IFI30 is inoculated in a 96-well plate, 7000 cells are evenly inoculated in each well, and the cells are cultured for 24 h.
(2) Docetaxel was diluted with MDA-MB-231 complete medium at final concentrations of 0. mu.g/mL, 2.5. mu.g/mL, 5. mu.g/mL, 7.5. mu.g/mL, 10. mu.g/mL and 20. mu.g/mL of drug used in the cells.
(3) MDA-MB-231 cells in a 96-well plate are washed for 1 time by PBS, and cell culture solution containing epirubicin with different concentrations is added to be respectively cultured for 24 hours.
(4) Before the culture is finished, a CCK8 detection reagent is prepared, and 10 mu L of CCK8 detection reagent is added into each 100ul of culture solution.
(5) The cells were washed 2 times with PBS, the configured CCK8 detection reagent was added, 3 blank sub-wells were set, and incubation was continued for 2 h.
(6) The OD value at 450nm was measured by a microplate reader.
(8) Spss21.0 software calculates IC50 and the cell inhibition rate of each group of cells, 3 times of obtained experimental data are analyzed by using the sps 21.0 software through a paired t test method, and the difference with p <0.05 has statistical significance.
The results are shown in FIG. 9, MDA-MB-231 cells showed a decrease in docetaxel ic50 after IFI30 overexpression, indicating that IFI-30 can increase breast cancer cell chemotherapy sensitivity.
Application of IFI30 gene in increasing sensitivity of Luminal type breast cancer chemotherapy in experiment 7
(1) Luminal type breast cancer cells MCF-7 with over-expressed IFI30 and silenced LUMINal type breast cancer cells are respectively inoculated in a 96-well plate, 7000 cells are evenly inoculated in each well, and the cells are cultured for 24 h.
(2) Anthracycline and taxane chemotherapeutic drug stocks (epirubicin and docetaxel) were diluted separately with MCF-7 cell complete medium and the final concentrations of the drugs used in the cells were epirubicin (0 μ g/mL, 2.5 μ g/mL, 5 μ g/mL, 7.5 μ g/mL, 10 μ g/mL and 20 μ g/mL) and docetaxel (0 μ g/mL, 5 μ g/mL, 7.5 μ g/mL, 10 μ g/mL, 15 μ g/mL and 20 μ g/mL), respectively.
(3) And washing the cells in the 96-well plate for 1 time by using PBS, respectively adding cell culture solution containing epirubicin and docetaxel, and continuously culturing for 24 hours.
(4) Before the culture is finished, a CCK8 detection reagent is prepared, and 10ul of the CCK8 detection reagent is added into every 100ul of culture solution.
(5) The cells were washed 2 times with PBS, the configured CCK8 detection reagent was added, 3 blank sub-wells were set, and incubation was continued for 2 h.
(6) The OD value at 450nm was measured by a microplate reader.
(8) Spss21.0 software calculates IC50 and the cell inhibition rate of each group of cells, 3 times of obtained experimental data are analyzed by using the sps 21.0 software through a paired t test method, and the difference with p <0.05 has statistical significance.
The results are shown in FIG. 10, after IFI30 overexpression, the sensitivity of MCF-7 cells to epirubicin and docetaxel is reduced by ic50, and chemotherapy is enhanced, while after IFI30 is silenced, the sensitivity of MCF-7 cells to epirubicin and docetaxel is increased by ic50, and chemotherapy is reduced.
Experiment 8 application of IFI30 gene in increasing breast cancer radiotherapy sensitivity
(1) The breast cancer cells MDA-MB-231 after IFI30 overexpression are inoculated in a 96-well plate, and cultured for 24h by BT-474.
(2) Cells were given 5Gy, 10Gy radiation therapy post-adherent.
(3) After 5 days, the prepared CCK8 detection reagent was added and 3 blank auxiliary wells were set and incubation was continued for 3 h.
(6) The OD value at 450nm was measured by a microplate reader.
(8) Spss21.0 software calculates the inhibition rate of each group of cells, and 3 times of obtained experimental data are analyzed by using the spss21.0 software through a paired t test method, and the difference is statistically significant when p is less than 0.05.
As shown in FIG. 11, after over-expressing IFI30 in breast cancer cells under 5Gy and 10Gy radiotherapy irradiation, MDA-MB-231 and BT-474 cells were less active and had increased sensitivity to radiotherapy.
Experiment 9 increased expression of IFI30 on breast cancer tissue improves prognosis in breast cancer patients
(1) Selecting 714 patients who do not undergo preoperative chemotherapy and directly undergo breast cancer surgery in the second hospital of Shandong university and the civil hospital of Linyi city in 2006 from 1 month to 2014 month, slicing tumor tissues of the 714 patients, performing immunohistochemical staining of IFI30, evaluating staining results by two pathologist experts, and determining the intensity and range scores of IFI30 staining.
(2) The IFI30 staining was judged as the proportion of cells positive for specific staining and the staining intensity. The specific staining range is graded as 0-5 to 6, 0 is no positive cell, 1 is less than 1% positive staining, 2 is 1% -10% positive staining, 3 is 10% -33% positive staining, 4 is 33% -66% positive staining, 5 is more than 66% positive staining, the intensity is 0 is uncolored, 1 is light brown in cytoplasm, 2 is medium staining in cytoplasm, and 3 is dark brown in cytoplasm. By adding the range and intensity scores, a total score is obtained, with 2 or less scores being expressed as IFI30, and 3-8 scores being negative for artificial GILT expression, and a score of high expression, defined as positive.
(3) Follow-up is carried out on all patients in the group, follow-up data of 5 years after breast cancer of the patients are collected, the survival state of the patients is determined, and the prognosis of the patients is analyzed by using a K-M survival analysis method in SPSS21.0 software according to the expression condition of IFI30 protein in breast cancer tissues of the patients, so that the influence of IFI30 expression on the disease-free survival period and the total survival period of the patients in 5 years is determined.
The results are shown in fig. 12, and after grouping according to the staining range, intensity and total score of the IFI30 protein, patients with negative IFI30 expression in breast cancer tissues have significantly reduced disease-free survival and overall survival compared with patients with positive expression, and have statistical significance.
Experiment 10 basic study of IFI30 recombinant protein as a drug for treating breast cancer
A first part: synthesis and purification of IFI30 recombinant protein
(1) Construction of cells that synthesize IFI30 recombinant protein
1. The sequence of IFI30 was subjected to codon optimised gene synthesis and cloned into a vector.
2. Human cells Expi293F were selected as cells expressing IFI 30. The cells were in serum-free Expi293TMGrowth in expression medium. The cell culture conditions were 37 ℃ and 8% CO 2. And the flask containing the cells was placed on an orbital shaker.
3. One day prior to transfection, Expi293F cells were seeded at 2.0 × 106 cells/ml into culture flasks.
4. On the day of transfection, Expi293TMHeating of expression MediumTo 25-37 ℃. The DNA and reagents were thawed.
5. In maintenance flasks, CELL density and viability were determined using VI-CELL. The cell density at transfection should be 2.0X 106 to 3.0X 106 cells/ml.
6. The plasmid, reagents and Opti-MEM I were pre-warmed to room temperature. Then, an appropriate amount of the plasmid was added to Opti-MEM I, and mixed well.
7. An appropriate amount of Expifectamine 293 was added to Opti-MEM I and incubated at room temperature for 5 minutes
8. The two solutions were mixed by inversion 3-5 times and incubated at room temperature for 20-30 minutes.
9. The DNA/reagent mixture is added to the cells.
10. Appropriate volumes of enhancer were added to the cell culture 16-18 hours after transfection.
11. Cell culture supernatants collected on day 6 were used for purification.
(2) His tag protein purification step
1. Cell culture fluid was harvested on day 6 post transfection.
2. The cell culture solution was centrifuged at about 4000g for 30 minutes to remove cell pellets and cell debris. The supernatant was retained.
3. 1M imidazole was added to a final concentration of 10 mM. 5M NaCl was added to a final concentration of 10 mM. Adding 0.2M NiSO 4; the final concentration was 0.2 mM. The pH of the supernatant was measured and adjusted up to 8.0 with 1M Tris. The filtered supernatant was placed on ice or in a 4 ℃ freezer.
4. The cell culture supernatant at 4 ℃ was loaded onto a fully equilibrated column using the AKTA system. The flux was collected to prevent poor protein binding.
5. Equilibrating the column with 3-5 column volumes of buffer;
6. the protein was fractionated by the AKTA system at 5%, 10%, 50%. Eluted protein was collected at a rate of 3-5ml/min according to the absorbance of UV 280.
7. The eluted fractions were analyzed by SDS-PAGE and Western blot analysis.
8. Buffer exchange was performed according to SDS-PAGE and Western bot results.
Binding buffer: 25mM Tris-HCl, 300mM NaCl, pH 8.0.
Elution buffer: 25mM Tris-HCl, 300mM NaCl, 1M imidazole, pH 8.0.
Buffer exchange
9. The Desalting column (HiPrep 26/10 desaling 53ml, GE,17-5087-02) was washed with 5 column volumes of distilled water at a rate of 7-10ml/min to remove the storage reagent (20% ethanol);
10. washing the desalting column with 3-5 times of 0.2M NaOH solution at 7-10 ml/min; then washing the mixture for 2 hours by using NaOH solution at the speed of 0.5 ml/min; step 21 is repeated again to remove the NaOH;
11. by using>5 column volumes of desalting buffer (PBS, pH)
Figure BDA0002518797860000171
) And (4) removing the salt from the complete equilibrium desalting column.
12. Loading the sample into a desalting column at a speed of 7-10 ml/min;
13. immediately after loading, the sample was eluted from the desalting column at a rate of 7-10 ml/min. And collecting an elution peak.
14. After quality control by SDS-PAGE, Western blotting, concentration determination, the samples were stored in a refrigerator at-80 ℃.
The results are shown in FIG. 13 as Western Blot results, in which M2 column is a molecular weight marker, P column is a His-tag positive control, 1 column is purified protein in a reduced state, 2 column is purified protein in a non-reduced state, and His-tag mouse anti-marker was used for visualization.
The endotoxin content was determined to be 2.5 EU/mg.
The protein concentration was 0.1 mg/ml.
(3) UPLC-MS liquid chromatography-mass spectrometry amino acid coverage rate detection analysis
And (3) analyzing the amino acid coverage rate of the test sample by using a UPLC-MS liquid chromatography-mass spectrometry method. Firstly, carrying out enzymolysis on a test sample to form a peptide fragment mixture; then injecting UPLC through an automatic sample injector, separating the sample on a chromatographic column by pushing a mobile phase through an infusion pump, and carrying out signal acquisition through an ultraviolet detector to form chromatographic peaks with different retention times; and finally, carrying out mass-to-charge ratio detection through a mass spectrometer, and processing a spectrogram acquired by the mass spectrometer through data analysis software to obtain the amino acid sequence coverage rate of the test sample.
As a result: the coverage of the test sample IFI30GILT was determined by the LC-MS method. The amino acid coverage rate of Trypsin enzyme digestion is 72.0 percent, and a sequence coverage map is shown in figure 14; the amino acid coverage rate of enzyme digestion of Chymotrypsin is 91.0%, and the sequence coverage map is shown in FIG. 15; the amino acid coverage of Glu-C cleavage was 96.5%, and the sequence coverage is shown in FIG. 16. After sequence alignment and combination, the amino acid coverage of the test sample after enzyme digestion by the three enzymes is 100.0%, and the sequence coverage is shown in figure 17.
A second part: the IFI30 protein synthesized and purified has the effect of inhibiting breast cancer cell proliferation, firstly, the IFI30 protein synthesized and purified can enter cells to select MDA-MB-231 cells not expressing IFI30 protein for experiment
1. Preparing a slide: the glass slide is placed in 0.1% gelatin, soaked in 37 ℃ gelatin, taken out and placed on a sterilized paper towel to be dried, and collected on a culture dish for later use. When it is to be used, one of the cells is taken out and put in a well of a 24-well plate, and the seed cells (MDA-MB-2314 ten thousand cells/well)
2. After the cells were attached, the experimental group was added with a culture medium containing 10. mu.g/ml concentrated IFI30 protein, and the control group was added with a culture medium containing no IFI30 protein.
After 3.24 hours the medium was aspirated and washed once with PBS
4.4% PFA fixed cells, 2ml per well, 15min
PBS wash 2 times, after wash 1, place slides in new six well plates
6. Add 120ul of blocking solution to each well, and block for 30 min.
PBS washing 1 time
8. 110ul primary antibody (IFI30 antibody) was added to each well and left at room temperature for 1h
PBS washing 2 times
10. Adding 110ul secondary antibody (which can emit red fluorescence after being excited) into each well, and incubating for 1h at room temperature
PBS Wash 2 times
12. Addition of DAPI (nuclei stained blue)
13. Confocal microscope observation and photographing
As a result, it was found that many IFI30 proteins were introduced into the cells of the experimental group and stained red (white bright spots in gray scale), as shown in FIG. 18, which confirmed that the synthesized and purified IFI30 protein could be introduced into the breast cancer cells.
In FIG. 18, the light gray is dapi staining, the fluorescence microscopic is blue, the bright spot in the figure is IFI-30 immunofluorescent staining, and the fluorescence microscopic is red.
Second, IFI30 protein inhibition cell proliferation assay
1. Breast cancer cells (MDA-MB-231, T47D) which hardly expressed IFI30 protein were seeded in a 96-well plate, and 1500 or 5000 cells were uniformly seeded per well.
2. IFI30 protein was added to the experimental group, an equal amount of PBS was added to the control group,
3. add 10. mu.L of CCK-8 to each well and incubate at 37 ℃ for 3 hours.
MK-3 microplate reader (Thermo) measures absorbance at 450 nm.
5. After eliminating the absorbance of the blank (normal medium), the data were compared with the absorbance of 0D to summarize the proliferation rate.
The experimental result shows that the cell proliferation level of the breast cancer cells is reduced after the IFI30 protein is added.
The results are shown in FIG. 19.
The invention demonstrates that altering the expression level of IFI30 in breast cancer cells can affect a variety of biological behaviors including breast cancer cell proliferation, clonality, migration, invasion and apoptosis, suggesting that IFI30 can guide molecular typing and prognostic assessment of breast cancer.
Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (10)

1. The application of a protein/gene IFI30 related to the occurrence and development of breast cancer is characterized in that,
in particular to the application of the reagent for changing the expression level of IFI30 in evaluating the progress, prognosis and treatment response products of breast cancer.
2. The use according to claim 1, wherein the use of IFI30 in the preparation of a molecular typing marker for breast cancer.
3. The use of claim 1, wherein the IFI30 is used in the preparation of a breast cancer IFI30 expression intensity immunohistochemical detection kit.
4. The use of claim 3, wherein the kit is used for detecting the expression of IFI30 in a biological sample by immunohistochemical method; preferably, the kit comprises: an antibody specifically recognizing human IFI30, an immunohistochemical test reagent; preferably, the biological sample is paraffin-embedded tissue of breast cancer of a patient.
5. The use of claim 3, wherein the detection method of the breast cancer molecular typing kit comprises the following steps:
step 1, dewaxing to water
(1) Slicing 4 μm thick paraffin, continuously slicing 2 pieces of each tumor tissue, and baking in an oven at 60 deg.C for 1 hr;
(2) xylene 10min x 3 times;
(3) sequentially passing through 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol, and double distilled water for 5 min;
step 2 antigen retrieval
(1)3％H2O2Methanol solution for 20 min;
(2) double-steaming and washing for 5minx 3 times;
(3) antigen repair (high pressure repair):
the slice is put into EDTA alkaline repair liquid (2mM EDTA, 2mM Na)2HPO4) Boiling in a pressure cooker for 2min under high pressure, and naturally cooling to room temperature;
step 3 Primary antibody incubation
(1) Washing with PBS for 5 min;
(2)50 μ l of 1% BSA coated tissue, blocked for 30min at 37 deg.C;
(3) absorbing the confining liquid, respectively dropwise adding 50 mu l of IFI30 primary antibody, and incubating overnight at 4 ℃ in a wet box;
step 4. incubation with Secondary antibody
(1) Sucking away the primary antibody, and washing with PBS for 5minx 4 times;
(2) dripping 50 μ l secondary antibody with HRP mark, and incubating at 37 deg.C for 30-45 min;
step 5, color development and blue reflection
(1) Washing with PBS for 5minx 4 times;
(2) DAB (1:50) is developed for 3-10min until brick red precipitate appears, and double-distilled water stops developing;
(3) counterstaining with hematoxylin for 10min, and washing with tap water for 20-30min after differentiation with hydrochloric acid and ethanol;
step 6, dewatering sealing sheet
Sequentially adding double distilled water, 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, phenol, and xylene, each for 5 min. Dripping a resin sealing sheet;
step 7. Observation evaluation and scoring
The stained sections were evaluated by 2 pathologists and the expression level of IFI30 in breast cancer cells was observed under a microscope. The 2 pathologists performed in a "back-to-back" manner without any explicit patient clinical information and evaluated the staining results according to the modified harvest scoring criteria. The scoring result is divided into two parts: IFI30 staining intensity IFI30 proportion of cells staining positive. Staining intensity was scored as 0 (cell unstained), 1+ (cell cytoplasm appeared light brown), 2+ (cell cytoplasm appeared moderately stained) and 3+ (cell cytoplasm appeared dark brown) as 0, 1, 2 and 3 points, respectively. The positive staining proportion of the breast cancer cells IFI30 is counted and divided into 0 point (without positive cells), 1 point (< 1/100 cell specific staining), 2 points (1/100-1/10 cell specific staining), 3 points (1/10-1/3 cell specific staining), 4 points (1/3-2/3 cell specific staining) and 5 points (> 2/3 cell specific staining) according to the positive cell proportion. Finally obtaining a staining range score, a staining intensity score and a total score of the staining range score and the staining intensity score.
6. The use according to claim 1, in particular, the use of the kit for detecting the expression level of IFI30 in breast cancer tissues for the preparation of a breast cancer molecular typing kit; preferably, the breast cancer molecular typing kit is used for detecting the expression level of IFI30 in breast cancer tissues; preferably, the breast cancer molecule typing kit detects the expression of IFI30 in a biological sample by an immunohistochemical method; preferably, the breast cancer molecular typing kit comprises: an antibody specifically recognizing human IFI30, an immunohistochemical test reagent; preferably, the biological sample is paraffin-embedded tissue of breast cancer of a patient.
7. The use according to claim 1, wherein IFI30 is used in screening for potential agents for the treatment of breast cancer; preferably, the method comprises the following steps: treating a system expressing or containing IFI30 with a candidate substance and detecting expression of IFI30 gene/protein in said system; if the candidate substance can increase the expression level of IFI30, the candidate substance is a potential substance for treating breast cancer; preferably, the candidate substance increases the expression level of IFI30 by more than 20%, preferably more than 50%, most preferably more than 80%; preferably, the system is selected from: a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system; preferably, the candidate substances include, but are not limited to: an overexpression vector, a specific antibody or an activating small molecule compound designed aiming at the IFI30 gene or an upstream gene or a downstream gene thereof; preferably, the candidate substance of the present invention is preferably a lentiviral over-expression vector for IFI 30.
8. The use according to claim 1, wherein the use of IFI30 in the preparation of a pharmaceutical composition for the treatment of breast cancer;
preferably, the pharmaceutical composition comprises an activator of functional expression of IFI30 selected from the group consisting of: contains small molecular substance and specific antibody for specifically activating IFI30 gene/protein expression or activity. The tumor is low in IFI30 protein expression;
preferably, the pharmaceutical composition comprises small molecule substances, specific antibodies, which can up-regulate the IFI30mRNA or protein level or other substances capable of specifically binding to and promoting the expression or activity of IFI30 gene/protein;
preferably, the pharmaceutical composition also comprises medicines compatible with the activator and pharmaceutically acceptable carriers and/or auxiliary materials;
preferably, pharmaceutically acceptable carriers include, but are not limited to: buffer, emulsifier, suspending agent, stabilizer, preservative, physiological salt, excipient, filler, coagulant and blender, surfactant, dispersing agent, and defoaming agent.
9. A pharmaceutical composition for treating breast cancer, comprising:
an activator of functional expression of IFI 30; and a pharmaceutically acceptable carrier.
10. The pharmaceutical composition of claim 9, wherein the pharmaceutically acceptable carrier includes, but is not limited to: buffer, emulsifier, suspending agent, stabilizer, preservative, physiological salt, excipient, filler, coagulant and blender, surfactant, dispersing agent, and defoaming agent.
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CN113209300A (en) * 2021-05-17 2021-08-06 北京大学人民医院 Application of GILT as effect target spot for improving chemotherapy drug sensitivity of acute myeloid leukemia patient
CN115449555A (en) * 2022-10-26 2022-12-09 山东大学 Application of ADGRA2 as breast cancer chemotherapy curative effect and prognosis evaluation biomarker

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CN107475430A (en) * 2017-09-28 2017-12-15 郑州大学第附属医院 A kind of the glioblastoma auxiliary diagnosis based on IFI30 genes, prognostic evaluation kit and its application method

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113209300A (en) * 2021-05-17 2021-08-06 北京大学人民医院 Application of GILT as effect target spot for improving chemotherapy drug sensitivity of acute myeloid leukemia patient
CN115449555A (en) * 2022-10-26 2022-12-09 山东大学 Application of ADGRA2 as breast cancer chemotherapy curative effect and prognosis evaluation biomarker
CN115449555B (en) * 2022-10-26 2023-10-13 山东大学 Application of ADGRA2 as biomarker for breast cancer chemotherapy efficacy and prognosis evaluation

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