Application of pip4k2c gene in detection of breast cancer

Abstract

The application belongs to the technical field of gene diagnosis and treatment, and particularly relates to application of PIP4K2C gene in breast cancer detection, wherein the kit comprises a solid carrier and a detection reagent coated on the solid carrier, and the detection reagent is used for detecting PIP4K2C gene or PIP4K2C protein. The PIP4K2C gene and its protein expression product in the application can be used as a specific marker for diagnosing breast cancer. The small interfering RNA designed according to the PIP4K2C gene in the application can also be used as a gene therapy tool for treating breast cancer, thereby providing a new breast cancer treatment approach.

Description

Technical Field

[0001] This invention belongs to the field of gene diagnosis and treatment technology, specifically involving the application of the PIP4K2C gene in breast cancer detection. Background Technology

[0002] Breast cancer, a heterogeneous malignant tumor, accounts for approximately 30% of all female cancers, making it one of the most common malignant tumors among women both domestically and internationally, and a leading cause of cancer-related deaths in women. In recent years, with the development of medicine and in-depth research, significant progress has been made in the diagnosis and treatment of breast cancer. However, breast cancer still frequently recurs and metastasizes, and its malignancy and mortality rates remain high.

[0003] Currently, the diagnosis of breast cancer in clinical practice mainly relies on pathological examination, and there is a lack of effective specific molecular markers, especially molecular markers for the diagnosis of early breast cancer.

[0004] Therefore, the search for new tumor markers has important scientific and clinical significance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the application of the PIP4K2C gene in breast cancer detection, which can serve as the first effective pathological molecular marker to distinguish between normal breast epithelial cells and cancerous epithelial cells.

[0006] The technical solution provided by this invention is as follows:

[0007] A kit for breast cancer detection, the kit comprising a solid-phase carrier and a detection reagent coated on the solid-phase carrier, the detection reagent being used to detect the PIP4K2C gene or PIP4K2C protein.

[0008] Furthermore, the kit includes reagents for immunohistochemical quantitative detection of PIP4K2C protein, as well as corresponding labels or instructions.

[0009] Furthermore, the kit includes reagents for the quantitative detection of PIP4K2C gene mRNA and corresponding labels or instructions.

[0010] Furthermore, the kit includes PIP4K2C-specific primers and a self-made PIP4K2C-specific antibody.

[0011] Furthermore, the PIP4K2C protein includes both fusion and non-fusion proteins.

[0012] Furthermore, the label or instructions for the kit include the following: when the PIP4K2C antibody immunohistochemical staining of the normal tissue surrounding the test subject shows (0 to +), while the staining of the breast epithelium in the suspicious microscopic area shows (+++ to ++++), it indicates a significantly increased possibility of cancer in the microscopic area; and when the staining of invasive breast cancer epithelial cells is significantly enhanced (+++ to ++++), it indicates a significantly increased possibility of cancer infiltration in the microscopic area.

[0013] Furthermore, the label or instructions for the kit include the following: if the ratio of the mRNA expression level of PIP4K2C relative to the reference protein in the test subject to the mRNA expression level of PIP4K2C relative to the reference protein in adjacent normal tissue is ≥0.5, it indicates that the test subject has a higher probability of developing breast cancer than the general population.

[0014] Furthermore, the breast tissue sample includes breast cancer tissue and adjacent normal tissue.

[0015] Furthermore, the label or instructions for use of the kit include the reference protein being GAPDH.

[0016] The present invention also provides a use of the PIP4K2C protein for the preparation of gene therapy tools that inhibit the growth or proliferation of breast cancer cells, or for the preparation of drugs for the treatment of breast cancer.

[0017] The present invention also provides the application of detection reagents for the PIP4K2C gene or PIP4K2C protein in the preparation of reagents or kits for detecting breast cancer.

[0018] The present invention also provides a method for in vitro non-therapeutic inhibition of breast cancer cell growth, proliferation or invasion, comprising the steps of: designing specific siRNA targeting PIP4K2C and transfecting it into breast cancer cells, thereby inhibiting breast cancer cell growth, proliferation or invasion.

[0019] Furthermore, the nucleotide sequence of the PIP4K2C-specific siRNA is as follows: sense: 5'-UAGGAUUCAUUCUCCAUGUAA-3', anti-sense: 5'-AUCCUAAGUAAGAGGUACAUU-3'.

[0020] Furthermore, the transfection reagents used to transfect breast cancer cells include liposomes. Beneficial effects

[0021] 1. Through extensive and in-depth research, we have discovered that the PIP4K2C gene may be an effective molecular marker for breast cancer diagnosis. The PIP4K2C gene encodes the protein PIP4Kγ, which, along with PIP4Kα and PIP4Kβ, belongs to the type II phosphatidylinositol-5-phosphate 4-kinase (PIP4K) family. It can generate specific PI(4,5)P2 by phosphorylating phosphatidylinositol 5-phosphate (PI5P), which is crucial for peroxisome function, cholesterol transport, and tumorigenesis. Compared to normal breast epithelial cells, the expression level of PIP4K2C is significantly increased in breast cancer cells. PIP4K2C is also significantly highly expressed in breast cancer tissue compared to adjacent normal tissue; therefore, PIP4K2C can serve as an important marker for the pathological diagnosis of breast cancer. Reducing PIP4K2C expression through siRNA interference can inhibit the growth, migration, and invasion of breast cancer cells; therefore, PIP4K2C could become an important gene therapy tool for breast cancer treatment.

[0022] 2. This invention discloses an effective novel molecular marker for breast cancer, PIP4K2C, which can serve as the first effective pathological molecular marker to distinguish between normal breast epithelial cells and cancerous epithelial cells. Furthermore, it discloses for the first time that interfering with the PIP4K2C gene can effectively inhibit the proliferation and invasion of breast cancer cells. This invention also discloses for the first time the application of the PIP4K2C gene in the diagnosis and treatment of breast cancer, providing a new method for breast cancer diagnosis and treatment.

[0023] 3. This invention discloses an effective novel molecular marker for breast cancer, PIP4K2C, which can serve as the first effective pathological molecular marker to distinguish between normal breast epithelial cells and cancerous epithelial cells. Breast cancer and normal breast tissue can be differentiated through PIP4K2C immunohistochemical experiments and mRNA expression assays.

[0024] 4. This invention discloses for the first time that knocking down the PIP4K2C gene can effectively inhibit the growth, proliferation and invasion of breast cancer cells.

[0025] 5. This invention discloses for the first time the use of the PIP4K2C gene in the diagnosis and treatment of breast cancer, providing a new marker for breast cancer diagnosis and new methods and tools for breast cancer treatment. Attached Figure Description

[0026] Figure 1 In Example 1 of this invention, PIP4K2C is highly expressed in breast cancer.

[0027] Figure 1 A represents the real-time quantitative PCR detection in Example 2 of this invention, which showed that the expression level of the PIP4K2C gene in breast cancer cell lines was higher than that in normal breast cell lines.

[0028] Figure 1 B is a schematic diagram of the real-time quantitative PCR detection of PIP4K2C gene expression in cancer tissue and paired adjacent normal tissue of breast cancer patients in Example 2 of the present invention. "Normal" refers to paired adjacent normal tissue, and "Tumor" refers to breast cancer tissue. The expression level of PIP4K2C gene in cancer tissue is higher than that in paired adjacent normal tissue.

[0029] Figure 1 C represents the Western blot experiment in Example 3 of this invention, which showed that the expression of PIP4K2C in the breast cancer cell line was significantly higher than that in the normal breast cell line.

[0030] Figure 1 D represents the difference in PIP4K2C gene expression in breast cancer tissue and paired adjacent normal tissue as shown by the Western blot experiment in Example 3 of this invention. Here, "Normal" refers to paired adjacent normal tissue and "Tumor" refers to breast cancer tissue. The expression level of PIP4K2C gene in the cancer tissue is significantly higher than that in the paired adjacent normal tissue.

[0031] Figure 1 E represents the difference in PIP4K2C gene expression in breast cancer tissue and paired adjacent normal tissue detected by immunohistochemistry in Example 1 of this invention. Here, "Normal" refers to paired adjacent normal tissue and "Tumor" refers to breast cancer tissue. The expression level of PIP4K2C gene in the cancer tissue is significantly higher than that in the paired adjacent normal tissue.

[0032] Figure 2 This is a test of the knockdown effect of siRNA on the PIP4K2C gene in Example 4 of the present invention.

[0033] Figure 2 A is a schematic diagram showing the results of RT-qPCR detection of PIP4K2C mRNA expression in MDA-MB-468 cells after siRNA transfection in Example 4 of the present invention.

[0034] Figure 2 B represents Example 4 of this invention, which uses Western blot experiments to detect the expression of PIP4K2C protein in MDA-MB-468 cells after 48h, 72h, and 96h of siRNA transfection.

[0035] Figure 2 C is a schematic diagram showing the expression of PIP4K2C mRNA in MCF-7 cells after siRNA transfection by RT-qPCR in Example 4 of the present invention.

[0036] Figure 2D represents the expression of PIP4K2C protein in MCF-7 cells after 48h, 72h, and 96h of siRNA transfection, as detected by Western blot experiment in Example 4 of this invention.

[0037] Figure 3 In Example 5 of this invention, PIP4K2C affects the growth, proliferation, migration, and invasion of breast cancer cells.

[0038] Figure 3 In Example 5 of this invention, silencing PIP4K2C expression via RNA interference inhibited the proliferation of breast cancer cells MDA-MB-468 and MCF-7.

[0039] Figure 3 In Example 6 of this invention, silencing PIP4K2C expression via RNA interference inhibited the migration and invasion of breast cancer cells MDA-MB-468 and MCF-7.

[0040] Figure 4 In Example 7 of this invention, RNA interference with PIP4K2C protein promoted autophagy in breast cancer cells MDA-MB-468 and MCF-7.

[0041] Figure 5 In Example 8 of this invention, RNA interference with PIP4K2C protein affected the level of PI(4,5)P2 in breast cancer cells. Detailed Implementation

[0042] Through extensive and in-depth research, the inventors have discovered for the first time that PIP4K2C is highly expressed in breast cancer tissue, while its expression is low-to-high in adjacent normal breast tissue. Therefore, PIP4K2C can serve as a biomarker for breast cancer detection or as an adjunct to breast cancer detection. This invention was completed based on this finding.

[0043] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or according to the manufacturer's recommendations. The reagents used in the experiments are shown in Table 1.

[0044] Table 1. Reagents used in the experiment

[0045]

[0046]

[0047] Example 1: Immunohistochemical detection of PIP4K2C expression in breast cancer tissue and normal breast tissue

[0048] 1. Sample collection

[0049] Breast cancer tissue and paired normal breast tissue samples were collected. All specimens were obtained with the approval of the organization's ethics committee.

[0050] 2. Immunohistochemical staining

[0051] (1) Preparation of sections: Fix the tissue with 10% buffered formalin solution for 24-48 hours, then place the fixed tissue in alcohol with gradually increasing concentration for dehydration. After removing the water, embed the tissue in paraffin. Use a microtome to cut the paraffin-embedded tissue specimen into thin sections of 4-5 micrometers. After the section preparation is completed, the sections need to be dried in an oven to remove the water from the sections.

[0052] (2) Dewaxing: Take out the sections and place them in xylene I and II for 15 min each, then in 100% ethanol I and II for 10 min each, then in 90% ethanol for 5 min, then in 80% ethanol for 5 min, then in 75% ethanol for 5 min.

[0053] (3) Antigen retrieval: Place the slide into a small container containing 1×Tris-EDTA antigen retrieval solution, and then microwave it for 5 minutes;

[0054] (4) After cooling to room temperature, wash with PBST for 5 min;

[0055] (5) Inactivation of endogenous peroxidase: Add 30% hydrogen peroxide solution and block for 25 min at room temperature;

[0056] (6) Wash with PBST for 5 min;

[0057] (7) Antigen blocking: Remove PBST, add protein-free rapid blocking solution and block at room temperature for 7 min;

[0058] (8) Add primary antibody: Discard the blocking solution, dilute PIP4K2C primary antibody at a ratio of 1:200, add the diluted primary antibody to the slide, and incubate overnight at 4°C in a humidified chamber;

[0059] (9) Remove the slides and wash them three times with PBST for 5 minutes each time at room temperature;

[0060] (10) Add secondary antibody: Add horseradish peroxidase-labeled secondary antibody (HRP Goat Anti-Rabbit IgG (H+L)) to the slide and incubate in a humidified chamber at room temperature for 1 h;

[0061] (11) Remove the slides and wash them three times with PBST for 5 minutes each time at room temperature;

[0062] (12) DAB color development: Prepare DAB substrate solution, drop it onto the slice, observe under a microscope for 3-10 min, and rinse thoroughly with tap water;

[0063] (13) Hematoxylin counterstaining: Place the sections in hematoxylin solution for 2-3 minutes and rinse thoroughly with tap water;

[0064] (14) 0.5%-1% ammonia solution for blueing: Place the slice in a 0.5%-1% ammonia solution emulsion for 30-60 seconds and observe under a microscope;

[0065] (15) Dehydration and transparency: Place in 75% ethanol for 5 min → 80% ethanol for 5 min → 90% ethanol for 5 min → 100% anhydrous ethanol for 5 min → xylene I and II for 15 min each;

[0066] (16) Mounting: Add a drop of neutral resin, cover with a coverslip (do not leave small air bubbles in the tissue area), and let it air dry naturally.

[0067] 3. Results

[0068] The results show that ( Figure 1 E): PIP4K2C protein shows a brownish-yellow granular staining reaction, is highly expressed in breast cancer tissue, and is lowly expressed in adjacent tissue.

[0069] Example 2: RT-qPCR Quantitative Detection of PIP4K2C Gene

[0070] 1. RNA extraction and real-time quantitative PCR

[0071] RNA extraction from breast cancer tissues or cells followed the method outlined in the RNA-easy™ Isolation Reagent extraction kit. Real-time quantitative PCR reactions were performed using the Premix Ex Taq™ (Perfect Real Time) kit reaction system, utilizing the StepOnePlus™ Real-Time PCR System (DRR041A Real-Time PCR Instrument, TaKaRa). The reaction time was 2... -ΔΔC The relative expression level of the PIP4K2C gene in the sample was calculated. Primer sequences for real-time quantitative PCR were designed based on the coding sequence of the PIP4K2C gene (NM_024779.4), as shown below.

[0072] PIP4K2C gene RT-qPCR quantitative detection primer sequences:

[0073] Forward primer: 5'-CCGGGAAGCCAGCGATAAG-3' (SEQ ID No. 3);

[0074] Reverse primer: 5'-AGCTGCACTAGAAACTCCACA-3' (SEQ ID No. 4);

[0075] ACTB gene RT-qPCR quantitative detection primer sequences:

[0076] Forward primer: 5'-CATGTACGTTGCTATCCAGGC-3' (SEQ ID No. 5)

[0077] Reverse primer: 5'-CTCCTTAATGTCACGCACGAT-3' (SEQ ID No. 6).

[0078] The relative expression level of PIP4K2C = PIP4K2C expression level / ACTB expression level.

[0079] 2. Results

[0080] The results show that ( Figure 1 AB): In breast cancer cells, the PIP4K2C gene is significantly highly expressed in all breast cancer cells compared with normal breast epithelial cells; in breast cancer tissue, the PIP4K2C gene is highly expressed in breast cancer tissue, while it is lowly expressed in adjacent normal tissue.

[0081] Example 3: Western Blot Detection of PIP4K2C Protein

[0082] 1. Protein extraction

[0083] (1) Collect cells or tissues, add protein lysis buffer and protease inhibitor. For cells, a cell scraper can be used. For tissues, liquid nitrogen can be used to grind them into powder or a grinder can be used to fully lyse the cells or tissues and collect them into centrifuge tubes.

[0084] (2) Centrifuge at 4℃ for 15 min at 12000 rpm;

[0085] (3) Collect the supernatant and use the BCA method to detect the protein concentration;

[0086] (4) Denature the protein by boiling and store at -80°C.

[0087] 2. SDS-polyacrylamide gel electrophoresis

[0088] 3. Antibody incubation.

[0089] (1) After completing the transfer and blocking operations, incubate overnight with PIP4K2C primary antibody;

[0090] (2) TBST cleaning 3 times, 7 minutes each time;

[0091] (3) Incubate with horseradish peroxidase-labeled secondary antibody (HRP Goat Anti-Rabbit IgG (H+L)) for 1 h;

[0092] (4) TBST cleaning 3 times, 7 minutes each time.

[0093] 4. Development

[0094] 5. Use chemiluminescence (ECL) to catalyze the substrate to emit light and detect proteins.

[0095] 6. Results

[0096] The results show that ( Figure 1 CD): In breast cancer cells, PIP4K2C is significantly highly expressed in all breast cancer cells compared with normal breast epithelial cells; in breast cancer tissue, PIP4K2C is highly expressed in breast cancer tissue, but lowly expressed in adjacent normal tissue.

[0097] Example 4: PIP4K2C was knocked down using siRNA, and the interference effect was detected by Western Blot and RT-qPCR.

[0098] 1. Cell Culture

[0099] Human breast cancer cell lines MDA-MB-468 and MCF-7 were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin in an incubator at 37°C, 5% CO2, and 90% relative humidity. The medium was changed every 2-3 days, and cells were routinely passaged using 0.25% trypsin containing EDTA. Cells were counted and prepared into a cell suspension, seeded in 6-well plates to ensure a cell count of 5 × 10⁶ cells / well. 5 Cells per well, incubate overnight, and observe cell status and density the next day. Transfection can be performed when the cell density is 70-90%.

[0100] 2. Synthesis of PIP4K2C small interfering RNA

[0101] PIP4K2C small interfering RNA was synthesized by Ribo Biotech, and its nucleotide sequence is as follows:

[0102] sense: 5'-UAGGAUUCAUUCUCCAUGUAA-3' (SEQ ID No.7),

[0103] anti-sense: 5'-AUCCUAAGUAAGAGGUACAU-3' (SEQ ID No. 8).

[0104] When transfecting cells, the PIP4K2C small interfering RNA was named siRNA-PIP4K2C, and its negative control RNA was siRNA-NC (purchased from Ribo Biotech).

[0105] 3. Transfection

[0106] For MDA-MB-468 and MCF-7: The blank control group (transfected with siRNA-NC) and the experimental group (transfected with siRNA-PIP4K2C) were both transfected at a concentration of 50 nmol / ml. Transfection of the vectors was performed using Lipofectamine™ 3000 Reagent, following the manufacturer's instructions.

[0107] 4. Cells were removed 24h, 48h, 72h, and 96h after transfection. Cell growth was observed under a microscope, and RNA and protein were extracted from the cells.

[0108] 5. The RT-qPCR and Western Blot methods are the same as above.

[0109] 6. Results

[0110] The results show that ( Figure 2 For MDA-MB-468 and MCF-7, compared with the control group, the mRNA and protein levels of PIP4K2C in the experimental group were significantly reduced after transfection with siRNA, indicating that the siRNA-mediated PIP4K2C knockdown effect was significant.

[0111] Example 5: Detection of the effect of PIP4K2C gene on breast cancer cell proliferation using the CCK8 assay.

[0112] 1. Cell Culture

[0113] The culture methods and conditions for human breast cancer cell lines MDA-MB-468 and MCF-7 are the same as above.

[0114] 2. Observe cell growth under a microscope. After the cells have reached confluence, add trypsin to digest them. After digestion, centrifuge to remove the trypsin, add cell culture medium and mix well to suspend the cells, and then count the cells.

[0115] 3. Dilute the cell suspension and then seed it into 96-well plates. Add approximately 200 μL of cell suspension to each well, maintaining a cell count of approximately 4000. The experimental group and control group each have four replicates. Six 96-well plates were used for six detection time points: day 0, day 1, day 2, day 3, day 4, and day 5.

[0116] 4. After 24 hours, remove the 96-well plate and observe the cells under a microscope. After cell adhesion, the control group and experimental group were transfected with 50 nmol / ml siRNA-NC and siRNA-PIP4K2C, respectively. The transfection method was the same as before.

[0117] 5. Before transfection, remove the first 96-well plate, add 10 μL of CCK8 detection solution to each well, and continue to incubate the 96-well plate in the cell culture incubator for 2 hours. Use a microplate reader to detect the absorbance value of each well at a wavelength of 450 nm and record the data.

[0118] 6. After 24 hours, remove the second 96-well plate, add 10 μL of CCK8 detection solution to each well, and continue to incubate the 96-well plate in the cell culture incubator for 2 hours. Use a microplate reader to detect the absorbance value of each well at a wavelength of 450 nm and record the data.

[0119] 7. Repeat step 7 after 2, 3, 4 and 5 days, and finally calculate the absorbance value at each time point to create a growth curve.

[0120] 8. Statistical Analysis

[0121] All experiments were performed in four replicates. Statistical analysis was conducted using SPSS 21.0 software. The differences between the two experiments were analyzed using a t-test, and a p-value < 0.05 was considered statistically significant.

[0122] 9. Results

[0123] The results show that ( Figure 3 A) For MDA-MB-468 and MCF-7, compared with the control, the cell proliferation of the experimental group was significantly inhibited after transfection with siRNA, and the difference was statistically significant (P<0.05), indicating that the reduction of PIP4K2C has the effect of inhibiting cell proliferation.

[0124] Example 6: Effect of PIP4K2C gene on breast cancer cell migration and invasion detected by cell scratch assay.

[0125] Detecting the effects of the IPIP4K2C gene on breast cancer cell migration and invasion using Transwell chambers

[0126] 1. Cell scratch

[0127] (1) The cell culture procedure is the same as above, using 1×10 5 MDA-MB-468 and MCF-7 cells were seeded into 6-well plates at a density of 1 cell / well, respectively.

[0128] (2) When the cell concentration is close to 100%, make a straight line in each well with the tip of a 200 μL pipette;

[0129] (3) Transfect cultured cells with siRNA, following the same transfection steps as above;

[0130] (4) Observe under a microscope and record the scratches at 0 h, 24 h and 48 h by taking pictures.

[0131] 2. Transwell experiment

[0132] (1) The cell culture and transfection steps are the same as above;

[0133] (2) Preparation of Transwell chambers;

[0134] (3) Under sterile conditions, the Matrigel was thawed in an ice bath and diluted 20-fold with PBS. 50 μL of the solution was then spread onto the polycarbonate membrane of the Transwell chamber. The membrane was incubated at 37°C for 4 hours. After the Matrigel gel polymerized, it was removed and the supernatant was gently aspirated. 50 μL of serum-free culture medium containing BSA was added to each well to hydrate the basement membrane, and the membrane was incubated at 37°C for 30 minutes.

[0135] (4) Prepare cell suspension

[0136] Cells were digested, and after digestion was terminated, they were centrifuged to remove the supernatant. They were then resuspended in serum-free medium and counted.

[0137] (5) Cell inoculation

[0138] Add 200 μL of cell suspension to a Transwell chamber and adjust the cell density to 5 × 10⁻⁶. 4 Cells per well. Add 500 μL of DMEM medium containing FBS to the lower well of a 24-well plate. Incubate the cells in a cell culture incubator for 24 hours.

[0139] (6) Staining

[0140] Fix the chambers with 4% paraformaldehyde for 15-20 min, then rinse with PBS. Place the chambers in crystal violet staining solution and stain for 15 min, then rinse with PBS. Gently wipe away the non-transferred cells inside the chambers with a cotton swab, air dry, and observe and count them under a microscope.

[0141] 3. Results

[0142] The results show that ( Figure 3 For MDA-MB-468 and MCF-7, compared with the control, the experimental group showed significantly reduced cell migration and invasion ability after transfection with siRNA-PIP4K2C, indicating that PIP4K2C affects the migration and invasion of breast cancer.

[0143] Example 7: Western Blot method for detecting the effect of PIP4K2C gene on autophagy in breast cancer cells.

[0144] 1. The cell culture and transfection steps are the same as above;

[0145] 2. After 48 hours, MDA-MB-468 and MCF-7 cells were removed and their growth was observed under a microscope. Once the cells reached confluence, protein extraction could be performed.

[0146] 3. The steps for Western blotting are the same as above;

[0147] 4. Results

[0148] The results show that ( Figure 4 For MDA-MB-468 and MCF-7, compared with the control, the autophagy level of cells in the experimental group was significantly enhanced after transfection with siRNA-PIP4K2C.

[0149] Example 8: Detection of the effect of PIP4K2C gene on breast cancer cells PI(4,5)P2 using immunofluorescence method

[0150] 1. Place cell spreaders in a 24-well plate, count the cells, and then seed them into the 24-well plate;

[0151] 2. The cell culture and transfection steps are the same as above;

[0152] 3. After 48 hours of transfection, remove the 24-well plate and observe the cells under a microscope. Fix the cells with 4% paraformaldehyde for 30 minutes.

[0153] 4. Wash 3 times with PBS;

[0154] 5. Break the membrane using 0.25% Triton X-100 for 15 minutes;

[0155] 6. Wash 3 times with PBS;

[0156] 7. Block with 1% BSA for 30 minutes;

[0157] 8. Wash 3 times with PBS;

[0158] 9. Antibody incubation: Incubate overnight using PI(4,5)P2 antibody;

[0159] 10. Wash 3 times with PBS;

[0160] 11. Incubate with fluorescent secondary antibody (Goat anti-rabbit IgG H&L (Alexa Fluor® 488)) for 4 hours;

[0161] 12. Wash 3 times with PBS;

[0162] 13. Stain the nucleus with DAPI reagent for 5-15 minutes;

[0163] 14. Wash 3 times with PBS;

[0164] 15. The sealing procedure is the same as above;

[0165] 16. Results.

[0166] The results show that ( Figure 5 For MDA-MB-468 and MCF-7, compared with the control, the PI(4,5)P2 level of cells was significantly reduced in the experimental group after transfection with siRNA-PIP4K2C.

[0167] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

[0168] PIP4K2C gene sequence (SEQ ID No. 1) (Gene accession number: NM_024779.4):

[0169]

[0170] PIP4K2C protein sequence (SEQ ID No. 2):

[0171] MASSSVPPATVSAATAGPGPGFGFASKTKKKHFVQQKVKVFRAADPLVGVFLWGVAHSINELSQVPPPVMLLPDDFKASSKIKVNNHLFHRENLPSHFKFKEYCP QVFRNLRDRFGIDDQDYLVSLTRNPPSESEGSDGRFLISYDRTLVIKEVSSEDIADMHSNLSNYHQYIVKCHGNTLLPQFLGMYRVSVDNEDSYMLVMRNMFSHRL PVHRKYDLKGSLVSREASDKEKVKELPTLKDMDFLNKNQKVYIGEEEKKIFLEKLKRDVEFLVQLKIMDYSLLLGIHDIIRGSEPEEEAPVREDESEVDGDCSLT GPPALVGSYGTSPEGIGGYIHSHRPLGPGEFESFIDVYAIRSAEGAPQKEVYFMGLIDILTQYDAKKKAAHAAKTVKHGAGAEISTVHPEQYAKRFLDFITNIFA.

[0172] PIP4K2C gene RT-qPCR quantitative detection primer sequences:

[0173] Forward primer: 5'-CCGGGAAGCCAGCGATAAG-3' (SEQ ID No. 3);

[0174] Reverse primer: 5'-AGCTGCACTAGAAACTCCACA-3' (SEQ ID No. 4);

[0175] ACTB gene RT-qPCR quantitative detection primer sequences:

[0176] Forward primer: 5'-CATGTACGTTGCTATCCAGGC-3' (SEQ ID No. 5)

[0177] Reverse primer: 5'-CTCCTTAATGTCACGCACGAT-3' (SEQ ID No. 6).

[0178] The nucleotide sequence of PIP4K2C small interfering RNA is as follows:

[0179] sense: 5'-UAGGAUUCAUCUCCAUGUAA-3' (SEQ ID No. 7),

[0180] anti-sense: 5'-AUCCUAAGUAAGGGUACAUU-3' (SEQ ID No. 8).

Claims

1. The application of siRNA in the preparation of products for inhibiting the growth, proliferation, or invasion of breast cancer cells, characterized in that, The siRNA is a specific siRNA designed for the PIP4K2C gene, and the nucleotide sequence of the siRNA is as follows: sense: 5'-UAGGAUUCAUUCUCCAUGUAA-3', anti-sense: 5'-AUCCUAAGUAAGAGGUACAUU-3'.

Images