WO2019169711A1 - EphA8基因在制备抗乳腺癌药物及其诊断试剂盒中的应用 - Google Patents

EphA8基因在制备抗乳腺癌药物及其诊断试剂盒中的应用 Download PDF

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WO2019169711A1
WO2019169711A1 PCT/CN2018/084507 CN2018084507W WO2019169711A1 WO 2019169711 A1 WO2019169711 A1 WO 2019169711A1 CN 2018084507 W CN2018084507 W CN 2018084507W WO 2019169711 A1 WO2019169711 A1 WO 2019169711A1
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epha8
cells
breast cancer
sirna
cell
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倪启超
汪桂华
王旭东
黄剑飞
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南通大学附属医院
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention belongs to the technical field of cancer precision medical drugs, and particularly relates to the application of the EphA8 gene in preparing anti-breast cancer drugs and diagnostic kits thereof.
  • Breast cancer is a malignant tumor that occurs in the mammary epithelial tissue.
  • the incidence rate is the first in female malignant tumors in China. It has become the main killer threatening women's health and life.
  • tumor treatment has entered a new era of molecular targeted therapy.
  • targeted therapy can effectively and selectively kill tumor cells, reduce damage to normal tissues, and have fewer adverse reactions.
  • Breast cancer targeted therapy makes tumor treatment more precise, and the development of targeted drugs for the development and development of breast cancer-related signaling pathways has become a new hotspot in breast cancer treatment research.
  • trastuzumab treatment can benefit the survival of HER2-positive patients, and the combination of PARP and VEGF inhibitors can be used in the treatment of breast cancer. It also shows a good application prospect. It is of great practical clinical significance to explore new biomarkers to predict the progress of breast cancer and to guide precise treatment to improve patients' quality of life and prolong their lives.
  • Eph The human hepatocellular carcinoma receptor (Eph) is the largest family of receptor tyrosine kinases discovered to date. Eph receptors have been found to have EphA1-8, A10, Eph B1-4, and B6. Eph is a membrane-bound protein with a typical RTK topology, a ligand binding region, and an extracellular region with a receptor clustering motif, a single transmembrane domain, and an intracellular region containing a kinase domain. Like other receptor tyrosine kinases, the Eph gene plays an important role in embryonic development, particularly in the development of the nervous and vasculature.
  • Ephs and its ligand Ephrins exhibit complex expression patterns in both tumor cells and tumor stromal cells, and are involved in many aspects of cancer development and progression, including tumor growth, migration and invasion, tumor stem cells, angiogenesis and metastasis.
  • EphA2 is highly expressed in a variety of human tumors and has the characteristics of oncogenes that promote tumor progression and metastasis.
  • EphA2 is highly expressed in HER-2 positive cells. It was found that Ephrin A1/EphA2 signaling pathway regulates the metabolism of glutamine in human HER-2 positive breast cancer cells, suggesting that this signaling pathway may become a new target for breast cancer treatment.
  • EphA4 expression and decreased EphB2 expression are associated with liver metastasis of colorectal cancer.
  • targeted therapies for Ephs/Ephrins signaling are in the experimental stage, mainly by reducing the expression of Ephs or Ephrins.
  • silencing of EphA2 by small interfering RNA inhibits the growth and migration of malignant mesothelioma cells and induces Caspase-9-mediated apoptosis; anti-EphB4 monoclonal antibody inhibits tumor angiogenesis and prevents tumor growth and metastasis; EphB4 monoclonal antibody The combination with bevacizumab showed synergistic anti-tumor effects, although one of these antibodies induced EphB4 degradation, but at the same time it effectively inhibited the growth of EphB4-negative and positive cells; the introduction of an HIV-TAT in gastric cancer cells and Ephrin-B1c-end 331-346 amino acid synthetic peptide, can inhibit RhoA activation, thereby blocking Ephrin-B1 signaling pathway and peritoneal metastasis; EphA2 antisense oligonucleotide inhibits EphA2 expression and inhibits Ephrin-A1 and VEGF-mediated tumor metastas
  • EphA8 acts as a receptor for GPI-anchored Ephrin-A2, A3 and A5, and is rarely reported in tumors. According to the available literature, the relationship between EphA8 expression in breast cancer and its clinical pathological parameters, and the effect of EphA8 on invasion and migration of breast cancer have not been studied.
  • the object of the present invention is to provide an application of the EphA8 gene in the preparation of a medicament for treating breast cancer, and to meet the demand for use of an anti-breast cancer drug.
  • Another object of the present invention is to provide an application of the EphA8 gene in the preparation of a kit for diagnosis or prognosis of breast cancer.
  • EphA8 gene (gene number NM_020526) is used in the preparation of a kit for the diagnosis of breast cancer.
  • EphA8 gene in the preparation of a diagnostic kit for prognosis of breast cancer.
  • EphA8 gene in the preparation of a medicament for the treatment of breast cancer.
  • the drug is designed with the EphA8 gene as a target.
  • the drug includes the following four siRNA sequences:
  • EphA8#2 5'-TCTATGCTGAGATCAAGTTTA-3';
  • EphA8#3 5'-GGAGAAGATGCACTATCAGAA-3';
  • EphA8#4 5'-ACCAGGTTTGCAACGTCATGA-3'.
  • the present invention uses tissue microarray and immunohistochemistry to detect the expression of EphA8 in breast cancer, and found that the expression of EphA8 protein is significantly increased in breast cancer tissues, and the prognosis of patients with high expression of EphA8 Poor.
  • Western blot, CCK8 method, Transwell chamber and other techniques were used to study the effect of down-regulating EphA8 gene expression on the biological behavior of breast cancer cell proliferation and invasion in vitro. It was found that specific siRNA sequence can effectively inhibit human breast cancer cell line MCF- Expression of EphA8 protein in 7.
  • EphA8 siRNA stably transformed strain was constructed, and the nude mice xenograft test further confirmed that the RNA interference inhibited the expression of EphA8, and the growth of subcutaneous xenografts of MCF-7 cells was slowed down.
  • EphA8 is a target for gene diagnosis and treatment of breast cancer cells and will be widely used in the preparation of diagnostic kits for breast cancer and therapeutic drugs.
  • Figure 1 is an immunohistochemical map of breast cancer EphA8 protein; in the figure, A1IHC stained breast cancer tissue EphA8 positive (EnVision method ⁇ 40), A2IHC stained EphA8 positive (EnVision method ⁇ 400), adjacent tissue B1IHC stained EphA8 negative (EnVision method ⁇ 40), B2IHC staining was negative for EphA8 (EnVision method ⁇ 400);
  • Figure 2 is a survival curve of 146 patients with EphA8 high and low expression breast cancer
  • Figure 3 is a graph showing the expression of breast cancer cell line EphA8;
  • Figure 4 is a graph showing the expression of EphA8 in each group of MCF-7 cells after RNAi;
  • Figure 5 is a graph showing cell growth curves of MCF-7 cells after different treatments
  • Figure 6 is a cell cycle diagram of different treatments of MCF-7 cells
  • Figure 7 is a graph showing the results of invasive ability of each group of cells treated differently (x400).
  • Figure 8 is a screen showing the construction of EphA8 shRNA and NC stable strain of MCF-7 cells
  • Figure 9 is a graph showing the results of a nude mouse xenograft test of each group of cells treated differently.
  • the main reagents used in the following examples are: two-step immunohistochemistry kit: Gene Technology Shanghai Co., Ltd.; rabbit anti-human EphA8 polyclonal antibody (for immunohistochemistry): sigma; horseradish peroxidase Labeled goat anti-rabbit secondary antibody (for immunohistochemistry): Dako, USA; Antibody dilution: Beijing Zhongshan Biotechnology Co., Ltd.; 0.01 mol/L citrate buffer (pH 6.0): Beijing Zhongshan Biotechnology Co., Ltd.; DAB: American Sigma; xylene, neutral gum, etc. are provided by the pathology department.
  • DAB working solution 20 mg of DAB powder was dissolved in 50 mL of 0.01 mol/L PBS, filtered with a qualitative filter paper, stored in a brown bottle (prepared before use); 3% H 2 O 2 prepared from 0.01 mol/L PBS solution was added as needed. A few drops help color development.
  • Human breast cancer cell lines HS-578T, LCC, MDA-MB-453, MCF-7 and normal breast cell line MCF-10A were purchased from CTCC; DMEM high glucose, 1640 medium, fetal bovine serum: American HyClone company; EGF, Insulin, trypsin: Gibco, USA; BCA protein assay kit: Biosharp; rabbit anti-human EphA8 polyclonal antibody (for Western blot): proteintech; horseradish peroxidase-labeled goat anti-rabbit secondary antibody (for Westernblot test) : Dako, USA; ECL luminescence kit: Seven Seas; LipofectamineTM 2000: Invitrogen, USA; MTT: Sigma; PI: Biyuntian C1052.
  • RPMI-1640 complete medium Add RPMI-1640 and fetal bovine serum, respectively, and make the final concentration of 90%, 10%, 1 ⁇ , 4 °C.
  • Cell cryopreservation solution RPMI-1640 complete medium, fetal bovine serum and DMSO were prepared at a ratio of 5:4:1 and stored at 4 °C.
  • 1 ⁇ TBST1L Take Tris 2.42g, NaCl 8.0g, Tween-200.5mL, mix and dissolve, dilute to 1L, and store at room temperature.
  • 1 ⁇ transfer Buffer1L glycine 14.4g, Tris 3.03g, add appropriate amount of double distilled water to stir and dissolve, add 200mL of anhydrous methanol, dilute to 1L, mix well (timely prepared).
  • 100 mL of blocking solution Take 5 g of skim milk powder, add 100 mL of 1 ⁇ TBST, and mix and dissolve (if necessary).
  • tissue chip maker Beecher Instruments, USA
  • automated immunohistochemical stainer (2D) LABVISION, USA.
  • Inverted phase contrast microscope Olympus, Japan
  • gel imaging system BIO-RAD, USA
  • multi-function microplate reader Thermo, USA
  • upright, inverted fluorescence microscope Olympus, Japan
  • BD Accuri TM C6 flow cytometer USA Becton Dickinson.
  • Immunohistochemical specimens 146 cases of breast cancer tissues mentioned above, all specimens were confirmed by pathology. Tumor specimens were taken from the central part of intraoperative tumor resection, 61 cases of adjacent paracancerous tissues and 30 cases of benign breast diseases. All specimens were from Nantong. University Library Hospital Pathology Archives. All tissue specimens were routinely fixed with 10% neutral formalin, embedded in paraffin, and the wax blocks were screened without obvious defects. The tissue was made into a tissue chip with a thickness of 4 mm thick and placed in a refrigerator at 4 °C. Store for backup.
  • the donor wax block is selected from the most representative cancer nest area at the marked point, and a tissue block having a diameter of 2 mm is taken, and each core is taken.
  • the tissue array block was heated and fused in a constant temperature oven at 55 ° C for 10 minutes, and cooled to room temperature before rapid melting to fuse the acceptor wax block with the donor tissue.
  • tissue chip was frozen at 4 ° C for about 4 hours, and then the tissue array block was corrected by a fully automatic tissue microtome at a speed of 20 mm/rev, and all the cores were completely exposed.
  • the slicer cuts the tissue array block, and the continuous slice is floated in the cool water to make it naturally unfold, and then the slice is transferred to the 45 °C warm water for about 2 minutes, and after being unfolded, it is pasted. The stripped slides were allowed to dry.
  • the slices were placed in an environment of 60 ° C for 3 minutes, and baked at 58 ° C for 16 hours.
  • the prepared tissue chip is stored in a slice box and placed in a refrigerator at 4 ° C for use.
  • tissue chip Before dewaxing, the tissue chip was placed in an incubator at 60 ° C and baked for about 20 minutes. The dried tissue chip was immersed in xylene twice for 10 minutes. After taking out, the gradient alcohol was dehydrated, 100% ethanol for 10 minutes, 95% ethanol for 10 minutes, 80% ethanol for 10 minutes, and 70% ethanol for 10 minutes. The tissue chip was rinsed with running water.
  • tissue chip was placed on a high temperature resistant slice rack, placed in a citrate buffer solution having a pH of 6.0, and the high temperature antigen was repaired for 5 minutes, and naturally cooled to room temperature, and then washed 3 times with PBS for 5 minutes each time.
  • tissue chip was taken out, rewarmed for 1 hour, and then immersed in PBS buffer for 5 minutes for a total of 3 times, after which the stem was taken out.
  • the results of immunohistochemistry were judged by double-blind method, and the results of staining on the tissue chip were independently evaluated by two experienced pathologists. According to the percentage of the number of tumor cells positive for staining, it is 0-100%, and the staining intensity is scored according to the depth of staining of tumor cells: no coloring is 0, yellow is 1 minute, light brown is 2, and brown is 3 .
  • the final staining score for EphA8 is the product of the staining intensity and the staining area of the positive cells.
  • the demarcation point of the EphA8 expression score is derived from the X-tile software. The scores are as follows: 0 to 139 is low or no expression, and 140 to 300 is high.
  • EphA8 protein is significantly elevated in breast cancer tissues. Patients with high expression of EphA8 have poor prognosis in patients with lower expression.
  • siRNA sequences and negative control siRNA (negative control, NC) for human EphA8 were designed and completed by Invitrogen, and the sequences are as follows:
  • EphA8#1 5'-TTCTGGATCGAGGCCGTCAAT-3'; position on the gene 1353-1373;
  • EphA8#2 5'-TCTATGCTGAGATCAAGTTTA-3'; position on the gene 415-435;
  • EphA8#3 5'-GGAGAAGATGCACTATCAGAA-3'; position on the gene 1862-1882;
  • EphA8#4 5'-ACCAGGTTTGCAACGTCATGA-3'; position on the gene 334-354;
  • the UV lamp is disinfected for 30 minutes, and the required liquid is taken out in advance. After the temperature rises to room temperature, the alcohol disinfection bottle mouth is placed in the ultra-clean table, and the alcohol lamp is turned on.
  • Cryopreservation placed at 4 ° C for 30 min, placed at -20 ° C for 30 min, placed in a -80 ° C refrigerator, can be stored for about 3 months, if long-term preservation, put into a liquid nitrogen tank.
  • Protein extraction (1) The six-well plate was taken out, the culture solution was aspirated, and the cells were washed twice with pre-cooled PBS. (2) Add 150 ⁇ l/well of pre-cooled cell lysate, let stand for 20 min on ice, and shake for 5 min. (3) Centrifuge at 4 ° C, 12000 rpm, 15 min, and take the supernatant to a new EP tube. (4) Measuring protein concentration. (5) The extracted protein was stored at -70 ° C, or the extracted protein was mixed with 5 ⁇ SDSpage loading buffer in a ratio of 4:1, rapidly mixed, boiled at 100 ° C for 5 min, and stored at -20 ° C.
  • SDS-PAGE electrophoresis (1) Align two clean glass plates, put them into clips, and clamp them vertically on the shelf to prepare the glue. (2) Firstly, 12% of the separation glue is filled with the 1mL gun along the gap between the two glass, first fast and then slow, avoiding the generation of air bubbles, glue the glue to about 1.5cm from the upper edge, and slowly add a layer of water on the glue. Leave it for about 30 minutes until the polymerization is complete. Obvious fold lines can be seen between the water and the glue. Pour off the upper layer of water, fill the remaining space with 5% concentrated glue, and insert the comb. Aggregate for about half an hour, unplug the comb and prepare to load.
  • Electrophoresis The initial voltage is 80V, about 40min, and the bromophenol blue enters the separation gel. The voltage is changed to 100V, about 90min. When the bromophenol blue is close to the bottom of the separation gel, the electrophoresis is stopped.
  • Transfer film (1) The electrotransfer pad and the filter paper were wetted with a transfer buffer, and the PVDF film was immersed in methanol for 10 s. (2) Carefully remove the gel from the glass plate, remove all the concentrated gel, and place the gel in a transfer buffer for soaking. (3) Assembling the electric transfer device, from the negative electrode to the positive electrode, sequentially placing 3 layers of electric transfer pads, 1 layer of filter paper, gel, PVDF film, 1 layer of filter paper, 3 layers of electrotransfer pads, PVDF film for positive electrode, gel connection Negative electrode, use a glass rod to drive out the air bubble, put it into the electric converter, cover the electrode and the insulating cover, 300mA, 2h.
  • Membrane blocking labeled antibody and development, exposure: (1) The PVDF membrane was taken out, placed in a TBST blocking solution containing 5% skim milk powder, and incubated for 2 h at room temperature on a shaker. (2) The PVDF membrane was taken out, and the TBST solution was washed 4 times for 5 minutes each time, and washed while washing. (3) Add the primary antibody, mix the primary antibody (EphA8 antibody) and the blocking solution in a ratio of 1:100, and place the membrane in the liquid at 4 ° C overnight (about 20 hours). (4) The PVDF membrane was taken out, and the TBST solution was washed 4 times for 5 minutes each time, and washed while washing.
  • EphA8 in HS-578T, LCC, MDA-MB-453, MCF-7 and MCF-10A cell lines was detected by Western blot. The results showed that EphA8 protein was the highest in MCF-7 cells and the lowest in MCF-10A cells. 3).
  • the FAM-labeled siRNA group cells were observed under light microscope and inverted fluorescence microscope 24 hours after transfection. Green fluorescence was observed in the successfully transfected cells, and FAM-labeled siRNA transfection was determined according to the number of green fluorescent cells. effectiveness. Different concentrations of FAM-labeled siRNA have a certain effect on the transfection rate. According to the design principle of RNA interference experiments, it is required to make the transfection rate as high as possible and the siRNA concentration is relatively low. The experimental results showed that the 12.5 ⁇ l N.C.FAM group had the highest transfection rate, about 80%, and could be used for subsequent experimental tests.
  • siRNA was transfected into MCF-7 cells: (1) Experimental group: EphA8-siRNA-1 group, EphA8-siRNA-2 group, EphA8-siRNA-3 group, EphA8-siRNA-4 group, negative control group and blank control group. (2) One day before transfection, MCF-7 cells in logarithmic growth phase were seeded on a six-well culture plate at a density of 4 to 5 ⁇ 10 4 /well, and 2 mL of RPMI-1640 containing 10% fetal bovine serum was added. base. (3) Cultured in a 37 ° C, 5% CO 2 cell culture incubator, the fusion rate of MCF-7 cells reached about 70% within 24 hours.
  • the experiment was divided into five groups: Normal, siRNA-NC, siRNA-NC + paclitaxel, EphA8 siRNA, EphA8 siRNA + paclitaxel, and each group was provided with 3 duplicate wells.
  • the cell growth curve was drawn from the A450 values of the five groups of cells at different detection time points (24h, 48h, 72h).
  • EphA8-siRNA-3 After 48 hours of transfection, the cells transfected into EphA8-siRNA-3 group showed significant growth inhibition compared with untransfected group and transfected NC-siRNA group (P ⁇ 0.05).
  • the cell proliferation ability of EphA8 siRNA group was significantly lower than that of siRNA- In the NC control group, EphA8-siRNA-3 reduced the proliferative capacity of MCF-7 cells (Fig. 5).
  • the cell concentration was adjusted to 10 5 cells/mL, and inoculated into a 6-well culture plate, 3 ml of the culture solution per well, and cultured at 37 ° C, 5% CO 2 incubator for 24 hours.
  • PI was added to have a concentration of 50 ⁇ g/mL. Incubate for 30 min at 37 ° C in the dark.
  • the experiment was divided into five groups: Normal, siRNA-NC, siRNA-NC + paclitaxel, EphA8 siRNA, EphA8 siRNA + paclitaxel group, and each group was provided with 3 duplicate wells.
  • the chamber was placed in a 24-well plate and incubated at 37 ° C for 1 h before use.
  • the cells were routinely digested, resuspended, and inoculated into a chamber covered with Matrigel gel at 200 ⁇ L/well.
  • the lower chamber was 600 ⁇ L of RPMI-1640 medium containing 20% fetal bovine serum at 37 °C. Incubate for 24 h in a 5% CO 2 incubator.
  • the chamber was taken out, washed twice with PBS, and the adherent cells on the inner side of the upper filter were gently wiped with a cotton swab, and washed twice with PBS.
  • MCF-7 cells were added to 1640 medium, plated in a cell culture dish, and cultured at 37 ° C in a 5% incubator;
  • the piercing point is about 1cm away from the injection point, forming a raised ridge, preventing the liquid from leaking out and disinfecting the skin, and observing the tumor formation;
  • the shRNA NC group had the largest tumor mass and the fastest tumor growth; the tumor decreased and the tumor growth rate slowed after paclitaxel administration; the tumor size and tumor growth rate of the EphA8 shRNA group were significantly lower than that of shRNA NC (Fig. 9).
  • EphA8 is a target for breast cancer gene therapy.

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Abstract

公开了EphA8基因在制备抗乳腺癌药物及其诊断试剂盒中的应用。所述药物以EphA8基因为靶点设计而成。

Description

EphA8基因在制备抗乳腺癌药物及其诊断试剂盒中的应用 技术领域
本发明属于癌症精准医疗药物技术领域，具体涉及EphA8基因在制备抗乳腺癌药物及其诊断试剂盒中的应用。
背景技术
乳腺癌是发生在乳腺上皮组织的恶性肿瘤，发病率居我国女性恶性肿瘤的首位，已成为威胁女性健康和生命的主要杀手。随着细胞、分子水平对肿瘤发病机制进一步的阐明和分子生物学技术的发展，肿瘤治疗进入了一个全新分子靶向治疗时代。与全身化放疗相比，靶向治疗可以高效、选择性地杀伤肿瘤细胞，减少对正常组织损伤，不良反应小。乳腺癌靶向治疗使肿瘤治疗更趋精准化，针对乳腺癌发生、发展有关的信号通路开发靶向药物应用于临床成为乳腺癌治疗研究的新热点。现已研发的一些针对不同靶点的药物在应用中显示了较好的疗效趋势，如曲妥珠单抗治疗使HER2阳性病人生存获益，PARP、VEGF抑制剂的联合应用于乳腺癌的治疗也显示了较好的应用前景。探寻新的生物分子标志物来预测乳腺癌的进展，指导精准治疗，以改善患者生活质量，延长患者生命，具有重要的实际临床意义。
促红细胞生成素人肝细胞癌受体(Eph)是至今发现的最大的受体酪氨酸激酶家族，目前已发现Eph受体有EphA1-8、A10、Eph B1-4、B6。Eph为膜结合蛋白，在结构上具有典型的RTK拓扑结构，具有配体结合区域，其胞外区域具有受体聚簇的基序，单个跨膜结构域和含有激酶结构域的胞内区域。Eph基因与其他受体酪氨酸激酶一样，在胚胎发育阶段发挥重要作用，特别是参与了神经和脉管系统的发育。Ephs及其配体Ephrins在肿瘤细胞和肿瘤基质细胞中均呈现出复杂的表达模式，参与癌症发生和进展的多个方面，包括肿瘤生长，迁移侵袭，肿瘤干细胞，血管生成和转移。EphA2在多种人类肿瘤中高表达，并有促进肿瘤进展和转移等癌基因的特点。HER-2阳性的细胞中EphA2高表达，研究发现Ephrin A1/EphA2信号通路调节人HER-2阳性的乳腺癌细胞谷氨酰胺的代谢，提示该信号通路有可能成为乳腺癌治疗的新靶标。此外，EphA4表达增高和EphB2表达降低与结直肠癌的肝脏转移相关。近年来针对Ephs/Ephrins信号的靶向治疗 正处于实验阶段，主要是通过降低Ephs或Ephrins表达的手段来实现。例如，小干扰RNA沉默EphA2可抑制恶性间皮瘤细胞的生长和迁移并诱导Caspase-9介导的细胞凋亡；应用抗EphB4单克隆抗体抑制肿瘤血管生成，阻止肿瘤生长和转移；EphB4单抗与贝伐单抗的联合应用显示协同抗肿瘤作用，虽然这些抗体中的一种诱导EphB4降解，但同时使用时可有效抑制EphB4阴性和阳性细胞的生长；胃癌细胞内引入一种HIV-TAT和Ephrin-B1c-端331-346氨基酸组成的合成肽，可抑制RhoA的激活，从而阻断Ephrin-B1的信号通路和腹膜转移；EphA2反义寡核苷酸抑制EphA2的表达从而抑制Ephrin-A1和VEGF介导的肿瘤转移。
EphA8作为GPI锚定的Ephrin-A2，A3和A5的受体起作用，在肿瘤中的报道较少。据现有文献，乳腺癌中EphA8表达和它与临床病理参数之间的关系，以及EphA8对乳腺癌侵袭迁移的影响尚未有研究。
发明内容
发明目的：针对现有技术中存在的不足，本发明的目的是提供一种EphA8基因在制备用于治疗乳腺癌药物中的应用，满足抗乳腺癌药物的使用需求。本发明的另一目的是提供一种EphA8基因在制备用于乳腺癌诊断或预后判断的试剂盒中的应用。
技术方案：为了实现上述发明目的，本发明采用的技术方案如下：
EphA8基因(基因号NM_020526)在制备用于乳腺癌诊断的试剂盒中的应用。
EphA8基因在制备用于乳腺癌预后判断的诊断试剂盒中的应用。
EphA8基因在制备用于治疗乳腺癌的药物中的应用。
所述药物以EphA8基因为靶点设计而成。
所述药物包括以下四条siRNA序列：
EphA8#1：5’-TTCTGGATCGAGGCCGTCAAT-3’；
EphA8#2：5’-TCTATGCTGAGATCAAGTTTA-3’；
EphA8#3：5’-GGAGAAGATGCACTATCAGAA-3’；
EphA8#4：5’-ACCAGGTTTGCAACGTCATGA-3’。
有益效果：与现有的技术相比，本发明采用组织芯片和免疫组化技术检测乳癌中EphA8的表达情况，发现EphA8蛋白的表达在乳腺癌组织中明显升高，且 EphA8高表达组患者预后较差。此外通过Westernblot、CCK8法、Transwell小室等技术，在体外试验中研究下调EphA8基因表达对乳腺癌细胞增殖侵袭等生物学行为的影响，发现特异性的siRNA序列可以有效抑制人乳腺癌细胞株MCF-7中EphA8蛋白的表达。构建EphA8siRNA稳转株，裸鼠移植瘤试验进一步验证RNA干扰抑制EphA8表达后，MCF-7细胞皮下移植瘤生长减慢。EphA8是乳腺癌细胞基因诊疗的一个靶点，在用于制备乳腺癌诊断试剂盒和治疗药物中将具有广泛的应用。
附图说明
图1是乳腺癌EphA8蛋白免疫组化图；图中，A1IHC染色乳腺癌组织EphA8阳性(EnVision法×40)，A2IHC染色EphA8阳性(EnVision法×400)，癌旁组织B1IHC染色EphA8阴性(EnVision法×40)，B2IHC染色EphA8阴性(EnVision法×400)；
图2是146例EphA8高、低表达组乳腺癌患者的生存曲线图；
图3是乳腺癌细胞株EphA8的表达结果图；
图4是RNAi后MCF-7细胞各组EphA8的表达结果图；
图5是MCF-7细胞不同处理后细胞生长曲线图；
图6是MCF-7细胞不同处理后细胞周期图；
图7是不同处理的各组细胞侵袭能力的结果图(×400)；
图8是MCF-7细胞EphA8shRNA及NC稳转株构建筛选图；
图9是不同处理的各组细胞的裸鼠移植瘤试验结果图。
具体实施方式
下面结合具体实施例对本发明做进一步的说明。
以下实施例中使用的主要试剂为：二步法免疫组化检测试剂盒：基因科技上海有限公司；兔抗人EphA8多克隆抗体(免疫组化试验用)：sigma公司；辣根过氧化物酶标记山羊抗兔二抗(免疫组化试验用)：美国Dako公司；抗体稀释液：北京中山生物技术有限公司；0.01mol/L柠檬酸缓冲液(pH6.0)：北京中山生物技术有限公司；DAB：美国Sigma公司；二甲苯、中性树胶等由病理科提供。DAB工作液：DAB粉剂20mg溶解于50mL0.01mol/LPBS中，再用定性滤纸过滤，保存在棕色瓶中(使用前配制)；根据需要加入由0.01mol/LPBS液配制 的3％H 2O 2数滴帮助显色。人乳腺癌细胞株HS-578T、LCC、MDA-MB-453、MCF-7和乳腺正常细胞株MCF-10A购自CTCC；DMEM高糖、1640培养基、胎牛血清：美国HyClone公司；EGF、Insulin、胰蛋白酶：美国Gibco公司；BCA蛋白测定试剂盒：Biosharp；兔抗人EphA8多克隆抗体(Westernblot试验用)：proteintech公司；辣根过氧化物酶标记山羊抗兔二抗(Westernblot试验用)：美国Dako公司；ECL发光试剂盒：七海生物；LipofectamineTM2000：美国Invitrogen公司；MTT:Sigma公司；PI：碧云天C1052。RPMI-1640完全培养液：分别加入RPMI-1640与胎牛血清混匀，使其终浓度分别为90％，10％，1×，4℃保存。细胞冻存液：将RPMI-1640完全培养液、胎牛血清和DMSO按5∶4∶1比例配制，4℃保存。1×TBST1L：取Tris2.42g、NaCl8.0g、Tween-200.5mL，混合溶解，定容至1L，常温保存。1×转膜Buffer1L：甘氨酸14.4g、Tris3.03g，加适量双蒸水搅拌溶解，再加200mL无水甲醇，定容至1L，混合均匀(用时配制)。封闭液100mL：取脱脂奶粉5g，加入100mL1×TBST，混合溶解即可(需用时配制)。
以下实施例中使用的主要仪器如下：组织芯片制作仪：美国Beecher Instruments公司；自动免疫组化染色仪(2D)：美国LABVISION公司。倒置相差显微镜：日本Olympus公司；凝胶成像系统：美国BIO-RAD公司；多功能酶标仪：美国Thermo公司；正置、倒置荧光显微镜：日本Olympus公司；BD Accuri TMC6流式细胞仪：美国Becton Dickinson公司。
实施例1
146例乳腺癌组织切片标本、相应61例癌旁组织及30例乳腺良性疾病切片标本，所有切片组织均取自南通大学附属医院甲乳外科2007年1月～2013年12月住院手术治疗患者。所有患者术前均未经过放、化疗，临床数据(包括年龄、分期、肿瘤大小、分化、淋巴结转移、远处转移和TNM分期)均有书面记录。上述146例病例随访截止日期为2016年6月，随访率100％。
免疫组织化学法标本：上述乳腺癌组织146例，所有标本均经病理证实，肿瘤标本取自术中肿瘤切除的中心部位，相应癌旁组织61例及30例乳腺良性疾病，所有标本均来自南通大学附属医院病理科档案库。所有组织标本均常规10％中性福尔马林固定，石蜡包埋，蜡块经筛选无明显缺陷，委托病理科制作成厚度为4mm厚的组织芯片共五张，置于冰箱4℃冷藏室储存备用。
1)组织芯片的制作
所有标本均用10％甲醛固定，石蜡包埋，蜡块经过筛选无明显缺陷。制作成组织芯片。主要流程为：
(1)根据HE染色切片的镜检结果，在蜡块上有代表性的癌巢区域作标记。
(2)1:1混合石蜡与蜂蜡，制作空白受体蜡块。在蜡块上设计10×7孔的组织阵列，然后用组织芯片仪制成TMA空白蜡块。
(3)将供体蜡块在标记的点上选取最有代表性的癌巢区域，取直径2mm的组织块，每例各取1个芯。
(4)将取好的组织芯转移到受体蜡块的孔中，并取相应癌旁组织做对照。
(5)组织阵列块在55℃的恒温烤箱中加热融合10分钟，在快融化之前放至室温冷却，使受体蜡块与供体组织融为一体。
(6)将组织芯片置于4℃条件下冷冻4小时左右，随后用全自动组织切片机对组织阵列块进行修正，速度为20mm/转，等修到所有组织芯完全曝露。
(7)切片机对组织阵列块进行切片，将连续切片分别漂在凉水中，使其自然展开，再将切片转移至45℃温水中展片2分钟左右，待展开后将其贴在经过防脱片处理的载玻片上晾干。
(8)将切片置于60℃的环境下烤片3分钟，58℃继续烤片16小时。
(9)将做好的组织芯片保存于切片盒，置于冰箱4℃冷藏室备用。
2)免疫组化染色(EnVision两步法)
(1)常规脱蜡水化：脱蜡前，将组织芯片放在60℃的恒温箱中，烘烤约20分钟。将已干燥的组织芯片浸于二甲苯中10分钟2次。取出后进行梯度酒精脱水，100％乙醇10分钟，95％乙醇10分钟，80％乙醇10分钟，70％乙醇10分钟，流水冲洗组织芯片。
(2)将组织芯片置于耐高温切片架上，置于pH为6.0的柠檬酸盐缓冲液，高温抗原修复5分钟，自然冷却至室温后用PBS冲洗3次，每次5分钟。
(3)取出蒸馏水中的芯片，滴加30％H 2O 2避光孵育20分钟，以消除内源性过氧化物酶的活性。蒸馏水冲洗，再将芯片置入PBS缓冲液中浸泡5分钟，总共3次，然后取出甩干。
(4)滴加200μL的兔抗人EphA8多克隆抗体工作液(稀释比例为1:50)于 组织芯片上，在4℃条件下过夜。
(5)第二天，取出组织芯片，复温1小时，后置入PBS缓冲液中浸泡5分钟，一共3次，之后取出甩干。
(6)在组织芯片上滴加200μl的二抗工作液，于室温孵育30分钟，将组织芯片置于PBS缓冲液中浸泡5分钟，一共3次，之后取出甩干。
(7)滴加准备好的显色剂DAB工作液，光镜下控制显色程度，显色完全后，立即用蒸馏水冲洗，终止显色。
(8)室温下用苏木素复染2分钟，用蒸馏水流水震洗后甩干。
(9)芯片脱水，透明，封片。
(10)光镜下观察免疫组化染色结果，细胞相应部位出现棕黄色作为阳性表现。
结果判断：免疫组化结果判断采用双盲法，由两位经验丰富的病理科医师对组织芯片上的染色结果进行独立评价。根据染色阳性的肿瘤细胞数目所占百分比计为0～100％，染色强度按肿瘤细胞着色的深浅计分：无着色为0分，黄色计1分，浅棕色计2分，棕褐色计3分。EphA8的最终染色得分为染色强度与阳性细胞染色面积的乘积。EphA8表达分数的分界点由X-tile软件得出。评分如下：0～139为低表达或无表达，140～300为高表达。
所有数据均用统计软件SPSSV.20.0和STATAV.9.0处理，计量资料以均数±标准差表示，组间比较采用单因素方差分析，EphA8表达与乳腺癌患者的预后关系分析用Kaplan-Meier生存分析，所有检验结果P<0.05为差异有统计学意义。
146例乳腺癌组织切片标本行免疫组化染色，EphA8蛋白阳性主表达于腺癌组织的细胞质和中，呈棕黄色，而大部分癌旁组织低表达或无表达，见免疫组化照片(图1)。免疫组化染色结果显示146例乳腺癌组织EphA8蛋白表达阳性率为30.8％(45/146)，癌旁组织表达阳性率13.1％(21/94)，乳腺良性疾病表达阳性率16.7％(5/30)，三者比较差异显著，有统计学意义(P＝0.0148)(表1)。Kaplan-Meier生存曲线显示EphA8高表达组比EphA8低表达组总体生存率低(图2)*P<0.05。
表1乳腺癌组织和癌旁组织/乳腺良性疾病组织中EphA8蛋白的表达
临床参数 例数 低表达(％) 高表达(％) Pearsonχ2 P值
乳腺良性疾病 30 25(83.3％) 5(16.7％)    
癌旁组织 61 53(86.9) 8(13.1％)    
乳腺癌 146 101(69.2％) 45(30.8％)    
总数 237     8.431 0.0148*
可见，EphA8蛋白的表达在乳腺癌组织中明显升高。EphA8高表达患者较低表达患者预后差。
实施例2
1)siRNA设计
针对人EphA8的四段不同的siRNA序列及阴性对照siRNA(negative control，NC)，由Invitrogen公司设计并完成，序列如下：
EphA8#1：5’-TTCTGGATCGAGGCCGTCAAT-3’；在基因上的位置1353-1373；
EphA8#2：5’-TCTATGCTGAGATCAAGTTTA-3’；在基因上的位置415-435；
EphA8#3：5’-GGAGAAGATGCACTATCAGAA-3’；在基因上的位置1862-1882；
EphA8#4：5’-ACCAGGTTTGCAACGTCATGA-3’；在基因上的位置334-354；
NC：5’-TTCTCCGAACGTGTCACGT-3’。
2)细胞复苏：
(1)从-80℃超低温冰箱中取出HS-578T、LCC、MDA-MB-453、MCF-7和MCF-10A细胞的冻存管，放到37℃水浴箱中快速摇动，使细胞在1min内完全解冻。
(2)取出冻存管，用酒精消毒后，放入超净台。
(3)吸取细胞悬液至15mL的离心管中，添加10mLRPMI-1640或DMEM培养液，混合均匀，1000rpm离心5min。
(4)吸去上清液，取2mL含10％胎牛血清的培养液混匀细胞，加到细胞培养瓶中，再添加适量培养液，于37℃，5％CO 2的细胞培养箱中培养，次日更换一次培养液，继续培养。
3)细胞传代培养：
(1)紫外灯消毒超净台30分钟，将所需液体提前拿出，待温度升至室温后酒精消毒瓶口放入超净台，打开酒精灯。
(2)将长满细胞的培养瓶中的旧培养液弃去，PBS冲洗一遍，加入1mL含0.02％EDTA+0.25％胰酶消化液，消化3～5分钟，显微镜下观察细胞状态，当细胞变圆，间隙变大时，加入5mL含10％胎牛血清的RPMI-1640或DMEM培养液终止消化，用吸管反复吹打瓶底。
(3)将吹散的细胞悬液分装至新的培养瓶，再加入适量培养液，置于37℃，5％CO 2的细胞培养箱中继续培养。
4)细胞计数法：
(1)将细胞消化后，制备成细胞悬液，用酒精棉球将盖玻片擦干净，放到细胞计数板上。
(2)吹打细胞悬液使其混匀，取10μl轻轻注入盖玻片和计数板的交界处。
(3)在显微镜下读取计数板四角大方格的细胞数，细胞压到线时，只计左侧和上方者，不计右侧和下方。
(4)计算：细胞密度(个/mL)＝(4大格细胞数之和/4)×10 4。
5)细胞冻存：
(1)选择对数生长期的细胞，冻存前24h换液。
(2)常规消化细胞，制备成细胞悬液，离心1000r/min，5min。
(3)吸去上清，加入冻存液，冻存液中细胞的终密度为5×10 6/mL，轻轻吹打混匀。
(4)将细胞悬液分装到无菌的冻存管中，每管1mL。
(5)将冻存管封好，标明细胞名称、冻存时间等信息。
(6)冻存：4℃放置30min，-20℃放置30min，置于-80℃冰箱中，可保存3个月左右，如长期保存则放入液氮罐。
6)HS-578T、LCC、MDA-MB-453、MCF-7和MCF-10A细胞的培养：
(1)按上述复苏方法从-80℃冰箱取出五种乳腺癌及正常乳腺上皮细胞，37℃快速解冻，吸出细胞悬液，用RPMI-1640或DMEM培养液漂洗后，低速离心，加入含10％胎牛血清的RPMI-1640或DMEM培养液混匀细胞，移入25cm 2培养瓶中，37℃，5％CO 2的细胞培养箱中培养。
(2)次日更换细胞培养液，继续培养待细胞生长达90％融合后，细胞传代培养。
(3)经过几次传代后部分细胞可冻存，部分细胞继续培养，准备试验用。
7)Westernblot筛选EphA8高表达乳腺癌细胞株
蛋白质提取：(1)取出六孔板，吸去培养液，用预冷的PBS洗细胞两遍。(2)加预冷的细胞裂解液150μl/孔，冰上静置20min，震荡5min。(3)4℃离心，12000rpm，15min，取上清移至新的EP管。(4)测蛋白质浓度。(5)将提取的蛋白质保存于-70℃，或按4∶1的比例将提取的蛋白质与5×SDSpageloading buffer混合，迅速混匀，100℃煮沸5min，保存于-20℃。
SDS-PAGE电泳：(1)将两片干净的玻璃板对齐后放入夹中卡紧，垂直卡在架子上，准备灌胶。(2)先将12％的分离胶用1mL枪沿两玻璃间隙灌胶，先快后慢，避免产生气泡，注胶至距上缘约1.5cm处，在胶上缓慢加一层水。放置30分钟左右至聚合完全，水和胶之间可见明显折线，倒掉上层的水，剩余空间灌满5％浓缩胶，并插上梳子。约半小时聚合，拔掉梳子，准备上样。(3)每孔上样约为15～20μl，其中一孔加入预染蛋白质Marker。(4)电泳：起始电压80V，约40min，待溴酚蓝进入分离胶，电压改为100V，约90min，当溴酚蓝接近分离胶底部时停止电泳。
转膜：(1)电转移垫和滤纸用转移缓冲液浸湿，PVDF膜在甲醇中浸10s。(2)从玻璃板上小心取下凝胶，去除所有浓缩胶，将凝胶放在转移缓冲液中浸泡。(3)组装电转移装置，由负极到正极，依次放入3层电转移垫、1层滤纸、凝胶、PVDF膜、1层滤纸、3层电转移垫，PVDF膜接正极、凝胶接负极，用玻璃棒赶出气泡，放入电转仪，加盖电极和绝缘盖板，300mA，2h。
膜的封闭、标记抗体及显影、曝光：(1)取出PVDF膜，放入含5％脱脂奶粉的TBST封闭液中，在摇床上室温孵育2h。(2)取出PVDF膜，TBST液洗4遍，每次5min，边洗边摇。(3)加一抗，按1∶100的比例将一抗(EphA8抗体)和封闭液混匀，将膜置入此液体中，4℃过夜(20小时左右)。(4)取出PVDF膜，TBST液洗4遍，每次5min，边洗边摇。(5)加二抗，按1∶2000的比例将二抗(辣根过氧化物酶标记二抗和封闭液混匀，将膜置入此液体中，室温孵育2h。(6)取出PVDF膜，TBST液洗4遍，每次5min，边洗边摇。(7)按照化学 发光试剂盒说明书，将A、B液等体积混合，滤纸吸干PVDF膜，将膜正面朝上放在塑料薄膜上，在膜上滴加配好的A、B混合液，凝胶成像系统拍照、保存。
四种乳腺癌细胞的筛选：(1)按上述方法分别提取四种乳腺癌细胞的蛋白。(2)用Westernblot检测四种乳腺癌细胞中EphA8的表达情况。(3)筛选出高表达细胞。
Westernblot检测HS-578T、LCC、MDA-MB-453、MCF-7和MCF-10A细胞株EphA8表达情况，结果表明EphA8蛋白在MCF-7细胞表达量最高，在MCF-10A细胞表达量最低(图3)。
8)LipofectamineTM2000转染siRNA
转染效率的判定：(1)转染前一天，取对数生长期的MCF-7细胞，按4～5×10 4/孔的密度接种在六孔培养板上，加入2mL含10％胎牛血清的RPMI-1640培养基。(2)37℃，5％CO 2的细胞培养箱中培养，24h内MCF-7细胞融合率达到约70％。(3)以250μlRPMI-1640稀释5μlLipofectamineTM2000，轻轻混匀。(4)以250μL RPMI-1640分别稀释0μL、2.5μL、6.25μL、10μL、12.5μL的N.C.FAM，轻轻混匀。(5)室温孵育5min后，混合稀释的siRNA和稀释的LipofectamineTM2000，轻轻混合均匀，在室温下孵育20min，以便允许复合物siRNA-LipofectamineTM2000的形成。(6)6孔板内培养液弃去，PBS洗涤细胞两遍，将混匀的复合物加入到每个包含细胞的孔中，轻轻前后摇动培养板混合，使其充分混匀，每孔加2mL RPMI-1640，放入培养箱。(7)6小时后换液，加入2mL含10％胎牛血清的RPMI-1640培养基。(8)24小时后加DAPI，每孔加0.1mL。染色2分钟，然后用PBS洗涤细胞三次，去除残余荧光，弃上清，然后置于倒置荧光显微镜下观察、拍照，得出siRNA转染最佳效率所需要的浓度。整个过程用锡箔纸包裹，注意避光。
转染后24小时将FAM标记的siRNA组细胞置于光学显微镜和倒置荧光显微镜下观察，转染成功的细胞内可看见绿色的荧光，根据绿色荧光的细胞数量来判定FAM标记的siRNA转染的效率。不同浓度的FAM标记的siRNA对转染率有一定影响，根据RNA干扰实验的设计原则，要求尽可能使转染率相对较高，siRNA浓度相对较低。实验结果显示，12.5μlN.C.FAM组的转染率最高，大约为80％，可以用于进行后续的实验检测。
siRNA转染MCF-7细胞：(1)实验分组：EphA8-siRNA-1组，EphA8-siRNA-2组，EphA8-siRNA-3组，EphA8-siRNA-4组，阴性对照组和空白对照组。(2)转染前一天，取对数生长期的MCF-7细胞按4～5×10 4/孔的密度接种在六孔培养板上，加入2mL含10％胎牛血清的RPMI-1640培养基。(3)37℃，5％CO 2的细胞培养箱中培养，24h内MCF-7细胞融合率达到约70％。(4)DEPC水稀释siRNA，1OD加DEPC水125μL。(5)250μLRPMI-1640稀释5μL LipofectamineTM2000，轻轻混匀。(6)以250μL RPMI-1640稀释12.5μLsiRNA，轻轻混匀。(7)室温孵育5min后，混合稀释的siRNA和稀释的LipofectamineTM2000，轻轻混合，在室温下孵育20min，以便允许复合物siRNA-LipofectamineTM2000的形成。(8)6孔板内培养液弃去，PBS洗涤细胞两遍，将混匀的复合物加入到每个包含细胞的孔中，轻轻前后摇动培养板混合，使其充分混匀，每孔加2mLRPMI-1640，放入培养箱。(9)6小时后换液，加入2mL含10％胎牛血清的RPMI-1640培养基，37℃、5％CO 2培养48小时，至细胞生长到孔板面积约80～90％时收取细胞，进行下一步Western blot检测。
Westernblot筛选转染效率最高的一组siRNA：(1)按上述方法分别提取EphA8-siRNA-1组，EphA8-siRNA-2组，EphA8-siRNA-3组EphA8-siRNA-4组，阴性对照组和空白对照组细胞的蛋白。(2)用Westernblot检测EphA8的表达情况。(3)筛选出转染效率最高的一组siRNA序列用于后续的细胞生物学检测。
Westernblot法检测四段不同的siRNA序列对MCF-7细胞EphA8表达情况的干扰效率，shRNA-1、shRNA-2、shRNA-3、shRNA-4、shRNA-NC和CK组的rt-PCR结果表明，shRNA3对EphA8沉默效率最高(**:P<0.01)，EphA8-siRNA-3在转染后48小时对MCF-7细胞的干扰效率最佳，以此作为后续研究的干扰序列(图4)。
9)CCK8法观察细胞增殖
(1)实验分五组：Normal，siRNA-NC，siRNA-NC+紫杉醇，EphA8siRNA，EphA8siRNA+紫杉醇，每组设3个复孔。
(2)取培养至对数生长期细胞，常规消化，重悬细胞，调整细胞浓度为3×10 4/mL，按100μL/孔接种于96孔板中，铺2块板，每块板每组细胞设3个复孔，37℃，5％CO 2的细胞培养箱中培养。
(3)待细胞贴壁后，使用脂质体LipofectamineTM2000介导转染(转染步骤同上)，取转染后3h时换液时间为CCK8实验0h，分别检测24h、48h、72h、96h四个时间段细胞增殖情况。
(4)先吸尽孔内液体，每孔加入90μL RPMI-1640和10μL CCK8液，37℃、5％CO 2培养3小时，多功能酶标仪测定各孔A450值。
(5)以五组细胞在不同检测时间点(24h、48h、72h)的A450值绘制细胞生长曲线。
转染48小时后，转染EphA8-siRNA-3组的细胞与未转染组、转染NC-siRNA组相比出现明显生长抑制(P<0.05)，EphA8siRNA组细胞增殖能力显著低于siRNA-NC对照组，EphA8-siRNA-3可降低MCF-7细胞的增殖能力(图5)。
10)流式细胞仪检测细胞周期
(1)待细胞长满后调整细胞浓度为10 5cells/mL，接种于6孔培养板，每孔3ml培养液，37℃，5％CO 2培养箱内培养24h。
(2)按实验分组处理细胞，48h后进行周期检测。
(3)将孔板中的细胞经胰酶消化后，收集至离心管中，1000rpm离心3min，弃去培养液。
(4)将细胞沉淀用2ml PBS洗涤1次。
(5)离心去PBS，加入冰预冷的70％的乙醇，4℃固定过夜。
(6)1000rpm离心3min弃去固定液，PBS洗细胞，1000rpm离心3min。
(7)加入500μL的PBS含100μg/mL RNase A，37℃孵育30min。
(8)30min后加入PI使其浓度为50μg/mL。避光37℃孵育30min。
(9)冷PBS溶液洗涤细胞，1000rpm，3min离心。
(10)取200μL的单细胞悬液，流式上机检测，CELL Quest软件分析。
由流式细胞检测结果可知：shRNA敲降后S期增加，G1期减少；紫杉醇给药后S期明显增加，G1期明显减少(图6)。
11)Transwell观察细胞侵袭能力
(1)实验分五组：Normal，siRNA-NC，siRNA-NC+紫杉醇，EphA8siRNA，EphA8siRNA+紫杉醇组，每组设3个复孔。
(2)取培养至对数生长期细胞，常规消化，重悬细胞，以2.5×10 5/孔接种 于六孔板上，待细胞融合达70％～90％时使用脂质体LipofectamineTM2000介导转染(转染步骤同上)。
(3)取Transwell小室，上室面覆以Matrigel胶(Matrigel胶：RPMI-1640培养基＝1：4)50μl/孔，将小室放入24孔板，使用前37℃孵育1h。
(4)转染48h后，常规消化，重悬各组细胞，以200μL/孔接种于覆盖Matrigel胶的小室内，下室为含20％胎牛血清的RPMI-1640培养液600μL，37℃、5％CO 2培养箱中孵育24h。
(5)孵育结束后，取出小室，PBS洗两遍，用棉签轻轻拭去上室滤膜内侧面贴壁细胞，PBS洗两遍。
(6)将滤膜用4％多聚甲醛固定10分钟，吸去固定液，将膜风干，每孔加入500μL考马斯亮蓝染液，室温置放30min，除去染色液，PBS洗两遍，将上室取出，自然干燥。
(7)正置荧光显微镜下计数膜背面迁移的细胞数，计数每张膜的中央部分和周围部分随机3个视野，实验重复三次，计算平均值。
瞬时转染48h后的各组细胞，通过计数穿过Transwell小孔细胞数目比较迁移和侵袭能力，转染EphA8-siRNA组的细胞与Normal，siRNA-NC组相比迁移和侵袭能力受到抑制(P<0.05)(图7)。shRNA敲降EphA8后MCF-7细胞的侵袭能力下降，紫杉醇也明显抑制了细胞的侵袭能力，shRNA与紫杉醇联合应用具有协同作用。
以上所有数据均用统计软件SPSSV.20.0和STATAV.9.0处理，计量资料以均数±标准差表示，组间比较采用单因素方差分析，P<0.05为差异有统计学意义。
实施例3构建EphA8shRNA及NC稳转株，体内试验验证siRNA干扰EphA8表达对人乳腺癌细胞功能的影响
1)MCF-7细胞EphA8shRNA及NC稳转株构建
(1)MCF-7细胞加入1640培养基，铺于细胞培养皿中，于37℃，5％培养箱培养；
(2)待细胞长满后，将细胞消化后分于6孔板中，达到80％融合后按上述转染方法，分别转入NC质粒和shRNA-3质粒；
(3)24h观察细胞状况，通过5μg/mL的嘌呤霉素筛选含有重组载体的细 胞。
(4)单克隆分选(图8)。
2)EphA8shRNA及NC稳转株裸鼠皮下移植瘤试验
(1)在SPF环境下下观察裸鼠3天，状况良好，准备接种细胞；
(2)超净台内，75％酒精消毒裸鼠注射部位皮肤，在裸鼠前肢腋下注射50μl的正常MCF-7细胞和稳转EphA8shRNA的MCF-7细胞悬液；
(3)刺入点距注射点约1cm，形成一隆起皮丘，防止液体漏出后消毒皮肤，观察成瘤情况；
(4)每3天测量一次裸鼠瘤体大小，用游标卡尺测量瘤体最长直径，最短直径，计算肿瘤体积V(mm 3)＝πab 2/6；
(5)当细胞成瘤至100mm 3后，按照下列分组
A:shRNA-NC稳转株组：即腹腔注射100μL的PBS；B:shRNA-NC稳转株+紫杉醇组：即腹腔注射30mg/kg的紫杉醇C:EphA8shRNA稳转株组：即腹腔注射100μl的PBS；D:EphA8shRNA稳转株+紫杉醇组：即腹腔注射30mg/kg的紫杉醇；每日观察裸鼠活动、摄食及饮水情况变化。每天测量一次记录各组裸鼠肿瘤大小和体重。
shRNA NC组的瘤块最大，肿瘤生长最快；紫杉醇给药后肿瘤减小，肿瘤生长速度减缓；EphA8shRNA组的瘤块大小和肿瘤生长速度明显低于shRNA NC(图9)。
可见，特异性的siRNA序列可以有效抑制人乳腺癌细胞株MCF-7中EphA8蛋白的表达。体内外实验证实RNA干扰抑制EphA8表达后，MCF-7细胞的增殖减慢，周期改变，细胞侵袭能力降低，EphA8是乳腺癌基因治疗的一个靶点。

Claims (5)

  1. EphA8基因在制备用于乳腺癌诊断的试剂盒中的应用。
  2. EphA8基因在制备用于乳腺癌预后判断的诊断试剂盒中的应用。
  3. EphA8基因在制备用于治疗乳腺癌的药物中的应用。
  4. 根据权利要求3所述的应用，其特征在于，所述药物以EphA8基因为靶点设计而成。
  5. 根据权利要求3或4所述的应用，其特征在于，所述药物包括以下四条siRNA序列：
    EphA8#1：5’-TTCTGGATCGAGGCCGTCAAT-3’；
    EphA8#2：5’-TCTATGCTGAGATCAAGTTTA-3’；
    EphA8#3：5’-GGAGAAGATGCACTATCAGAA-3’；
    EphA8#4：5’-ACCAGGTTTGCAACGTCATGA-3’。
PCT/CN2018/084507 2018-03-05 2018-04-25 EphA8基因在制备抗乳腺癌药物及其诊断试剂盒中的应用 WO2019169711A1 (zh)

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