A method for inhibiting expression of an itgav gene

Abstract

The application discloses a method for inhibiting ITGAV gene expression and belongs to the technical field of gene expression. The application links WHDH1 with ITGAV, and the expression of ITGAV, which is a downstream target gene of WDHD1 and is unidirectionally regulated by WDHD1, can be indirectly inhibited by inhibiting WDHD1.

Description

Technical Field

[0001] This invention belongs to the field of gene expression technology, specifically relating to a method for inhibiting ITGAV gene expression. Background Technology

[0002] Integrin subunit αV (ITGAV) is a member of the integrin α chain family and can bind to up to five β subunits to form αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8 integrin receptors. ITGAV interacts with transforming growth factor-β1 and inhibits E-calcin expression. Downregulation of ITGAV is likely to inhibit certain biological functions by interfering with the binding of ITGAV and β subunits, thereby suppressing tumor progression. In previous studies, ITGAV has been shown to affect angiogenesis, cell proliferation, migration, and invasion progression in malignant tumors such as rectal cancer, skin cancer, and laryngeal cancer. Furthermore, studies have shown that the loss of ITGAV and p53 can induce squamous cell carcinoma by activating the Akt pathway, but also prevents the remodeling of the tumor microenvironment and inhibits tumor growth.

[0003] As a member of the integrin family, ITGAV is believed to be involved in angiogenesis and epithelial-mesenchymal transition, which is also considered the root cause of ITGAV's ability to promote the proliferation and metastasis of head and neck squamous cell carcinoma, breast cancer, hepatocellular carcinoma, and osteosarcoma. With further research, autophagy and the Hippo pathway have also been used to explain the functional changes caused by ITGAV. In adipose-derived stem cells, a reduction in TAZ, a key mediator of the Hippo pathway, has been found in ITGAV knockout cells. The relationship between the Hippo pathway and cell proliferation, metastasis, and cytoskeleton formation has been confirmed, which seems to be another explanation for the influence of ITGAV on metastasis, proliferation, and angiogenesis. ITGAV regulates Toll-like receptor signaling by promoting the recruitment of the autophagy component microtubule-associated protein 1 to endosomes containing Toll-like receptors.

[0004] WDHD1 was initially discovered through gene screening and identified as a gene involved in chromosome transmission during cell division. WDHD1 contains several distinct domains, including the N-terminal WD40 repeat domain, the Sep B domain, and the C-terminal HMG domain. Studies have shown that proteins containing the WD40 repeat domain promote protein-protein interactions and play a linking or regulatory role in various cellular processes. Furthermore, WDHD1 contains the highly conserved SepB domain, which plays a crucial role in DNA replication. The HMG domain is the DNA-binding group in the HMGB proteome. Numerous studies have demonstrated that WDHD1 participates in regulating various pathophysiological processes. WDHD1 plays a key role in DNA replication, cell cycle progression, sister chromatin cohesion, DNA damage repair, and embryonic development.

[0005] There is limited research on ITGAV and WDHD1, and no methods have been found to suppress ITGAV using WDHD1. Summary of the Invention

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method to inhibit ITGAV gene expression and suppress WDHD1 expression in cells.

[0008] In one implementation of any aspect, the expression of WDHD1 in cells is suppressed by gene knockdown.

[0009] In any embodiment of any aspect, WDHD1 expression in cells is inhibited by a WDHD1 inhibitor (such as badoxifen acetate, CH3, etc.).

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0011] This invention links WHDH1 with ITGAV, and indirectly inhibits ITGAV expression by inhibiting WDHD1. ITGAV is a downstream target gene of WDHD1 and is unidirectionally regulated by WDHD1. Inhibiting WDHD1 can indirectly inhibit ITGAV expression. Attached Figure Description

[0012] Figure 1 Figure A shows the molecular docking of WDHD1 and ITGAV in CNE-1 and C666-1 cell lines.

[0013] Figure 1 B shows the immunofluorescence colocalization of WDHD1 and ITGAV in CNE-1 and C666-1 cell lines.

[0014] Figure 1 C represents the immunoprecipitation of WDHD1 and ITGAV in CNE-1 and C666-1 cell lines;

[0015] Figure 2 Figure A shows the results of RT-qPCR detection in the si-ITGAV cell line.

[0016] Figure 2 Figure B shows the results of Western blot analysis of the si-ITGAV cell line.

[0017] Figure 3 The protein expression of ITGAV in the WDHD1 stable cell line and the protein expression of WDHD1 in the Si-ITGAV cell line were shown.

[0018] Figure 4Figure A shows the proliferation activity and cell survival fraction of si-ITGAV detected by CCK8 assay;

[0019] Figure 4 B represents the radiosensitivity of si-ITGAV as determined by a plate clone assay. Detailed Implementation

[0020] The present invention is further illustrated below by way of embodiments, but these embodiments are not intended to limit the invention to their scope. Experimental methods for adjustments not specifically described in the following embodiments are selected according to the product manual. All other embodiments obtained by those skilled in the art based on the described embodiments of the invention without inventive effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art.

[0021] Example:

[0022] 1. Experimental Materials and Methods

[0023] 1.1 Experimental Materials

[0024] (1) Experimental cells

[0025] The human nasopharyngeal carcinoma cell lines CNE-1 and C666-1 were both obtained from the Department of Radiation Oncology, First Affiliated Hospital of Guangxi Medical University.

[0026] (2) Experimental reagents

[0027]

[0028] (3) Experimental consumables and instruments

[0029]

[0030] (4) siRNA and primer sequences

[0031]

[0032] 1.2 Experimental Methods

[0033] 1.2.1 Identification of ITGAV, a downstream target gene of WDHD1

[0034] Molecular docking: Molecular docking is the process of simulating molecular recognition in a computer. Its purpose is to find the optimal binding conformation of a protein and its ligands. This study used the ZDock rigid docking method to verify at the molecular level whether WDHD1 and ITGAV proteins have a structural basis for forming a stable compound. The specific steps are as follows: The structural files of WDHD1 and ITGAV proteins were obtained from the protein structure website UniProt, with their numbers O75717 and P06756, respectively. The structural files were then uploaded to the rigid docking website ZDock for molecular docking.

[0035] Fluorescent colocalization: Fluorescent colocalization is an important application of fluorescence microscopy, used to study whether there are spatial relationships between two or more molecules within a cell. When two different fluorescent markers emit light in the same region within a cell, it can be inferred that the two markers may be in the same complex or in the same organelle. In this study, cells were treated with primary antibodies against WDHD1 and ITGAV, and then organelles expressing WDHD1 and / or ITGAV proteins were fluoresced in green and / or red using fluorescent secondary antibodies. The distribution of red and green fluorescence was then compared to determine whether the two proteins overlapped in spatial distribution. The specific steps are as follows: After digesting, centrifuging, and resuspending the cells in the culture flask, they were seeded into 24-well plates. When the cell confluence was observed to be 70-80%, the complete culture medium in the wells was discarded, and the cells were washed three times with PBS, 5 min each time. After washing, 4% paraformaldehyde was added, and the cells were incubated at room temperature for 20 min to fix the cells. After washing three times with PBS, 0.5% Triton was added, and the cells were incubated at room temperature for 20 min to disrupt the cell membrane structure. After washing three times with PBS, add 5% goat serum and incubate at room temperature for 50 minutes to reduce non-specific antigen-antibody binding. Wash three times with PBS, add a pre-diluted mixture of WDHD1 and ITGAV primary antibodies according to the manufacturer's instructions, and incubate overnight at 4°C. If the volume of primary antibody added is insufficient to cover the bottom of the wells, gently shake on a shaker. After primary antibody incubation, wash the wells three times with PBS, add diluted fluorescent secondary antibody, and incubate at room temperature in the dark for 50 minutes. After secondary antibody incubation, add DAPI solution, incubate at room temperature in the dark for 20 minutes, then wash three times with PBS and discard any remaining liquid in the wells. The sample preparation is now complete. Place the wells under a fluorescence inverted microscope to observe the spatial distribution of cell nuclei (DAPI field), WDHD1 protein (GFP field), and ITGAV protein (RFP field).

[0036] Co-immunoprecipitation (Co-IP) is a classic method for studying protein-protein interactions based on the specific interaction between antibodies and antigens. It is an effective method for determining the physiological interactions between two proteins within intact cells. When cells are lysed under non-denaturing conditions, many protein-protein interactions present within intact cells are preserved. If protein B is immunoprecipitated with an antibody against protein A, then protein B, which binds to A in vivo, will also precipitate. Co-IP technology can determine whether two proteins can form a protein complex in vivo. This study used the classic Protein A / G immunoprecipitation kit, IP-grade ITGAV primary antibody, and IP-grade WDHD1 primary antibody for co-precipitation. The specific steps are as follows.

[0037] (1) Cell treatment: Remove the culture medium and wash twice with PBS at a ratio of 150 μl per 10⁵ cells. Scrape off the cells with a cell scraper and collect them into a 1.5 ml centrifuge tube. Add lysis / wash buffer at a ratio of 200-300 μl per 10⁶ cells, along with a protease inhibitor. Mix well and incubate on ice for 5-20 min. Then, use an ultrasonic processor to disrupt the cells (39% power, 5 s sonication, 5 s interval, 1 min total). Centrifuge (4℃, 120000g, 10 min). Take a small amount of supernatant to determine the protein concentration. Keep the remaining supernatant on ice for later use.

[0038] (2) Antigen-antibody conjugation: Use a pipette to draw 500 μl of supernatant and add it to a 1.5 ml centrifuge tube. Then add 2-10 μg of antibody (ITGAV antibody concentration is 200 μg / ml, a total of 40 μl is added; WDHD1 antibody concentration is 1 mg / ml, a total of 8 μl is added; IgG antibody concentration is 1 mg / ml, a total of 8 μl is added). Incubate on a centrifuge at room temperature for 1 h to form antigen-antibody complexes.

[0039] (3) Pretreatment of magnetic beads: Gently pipette the Protein A / G magnetic beads to fully suspend them. Take 30 μl of the magnetic bead suspension and place it in a 1.5 ml centrifuge tube. Add 500 μl of lysis / wash buffer, gently pipette the resuspended magnetic beads, and then let it stand on a magnetic rack for 1 min. After the magnetic beads are adsorbed onto the side wall of the centrifuge tube, discard the supernatant. Repeat this step to wash the magnetic beads 2-3 times.

[0040] (4) Binding of magnetic beads to antigen-antibody complex: The obtained antigen-antibody complex was added to the pretreated magnetic beads and incubated at room temperature for 1 hour on a centrifuge mixer. Then, the mixture was allowed to stand on a magnetic rack for 1 minute until the magnetic beads adhered to the sidewall of the centrifuge tube. The supernatant was then discarded, leaving the antigen-antibody-magnetic bead complex in the centrifuge tube. 500 μl of lysis / wash buffer was added to the antigen-antibody-magnetic bead complex, and the magnetic beads were gently resuspended by pipetting. The mixture was then allowed to stand on a magnetic rack for 1 minute until the magnetic beads adhered to the sidewall of the centrifuge tube. The supernatant was then discarded. This step was repeated twice.

[0041] (5) Denaturation elution: Add 80-100 μl of SDS-page loading buffer to the antigen-antibody magnetic bead complex and mix well. Heat in a metal bath at 100°C for 10 min. After cooling, place the centrifuge tube on a magnetic rack for 1 min. After the magnetic beads are adsorbed onto the side wall of the centrifuge tube, collect the supernatant and perform SDS-page gel electrophoresis.

[0042] 1.2.2 Construction of si-ITGAV cell line

[0043] Transient transfection was performed using RNA TransMate and siRNA. After transfection, silencing efficiency was verified using RT-qPCR and Western blot. To ensure silencing effect, all Si-ITGAV cells used subsequently should be collected within 72 hours after transfection. The specific operation steps are as follows.

[0044] (1) Preparation of RNA TransMate-siRNA complex: Dissolve 10D siRNA in 125μl DEPC water to prepare a 20μM stock solution. Upon receiving the siRNA sample, since the siRNA powder adheres to the tube wall and is easily lost upon opening, centrifugation should be performed before opening the tube to allow the sample to settle to the bottom of the tube wall before adding DEPC water to dissolve it. Take two sterile, enzyme-free centrifuge tubes and label them tube A and tube B, respectively. Add 1ml RPIM1640 basal medium and 7.5μl RNA to tube A, and add 1ml RPIM1640 basal medium and 10μl RNA TransMate to tube B. Gently shake tubes A and B, then mix the liquids in both tubes and incubate at room temperature for 10min to form the RNA TransMate-siRNA complex.

[0045] (2) Cell transfection: The complex was added to a T25 culture flask with a cell confluence of 60%-70%, followed by 2 ml of complete culture medium containing 20% ​​fetal bovine serum. The culture flask was gently shaken back and forth, and the cells were then cultured in a 5% CO2 incubator at 37°C for 24 h. Cells treated with the RNA TransMate-siRNA complex were named si-ITGAV group cells, cells treated with siRNA / NC were named CON group cells, and untreated cells were designated as blank group cells.

[0046] (3) RT-qPCR and Western blot were used to detect the silencing effect of three different siRNA sequences on ITAGV. RNA and protein were extracted from cells in the si-ITGAV group, CON group and blank group, and RT-qPCR and Western blot were used to verify the expression of ITAGV at the transcriptional and translational levels.

[0047] 1.2.3 Western blot analysis of the expression correlation between WDHD1 and ITGAV

[0048] (1) RT-qPCR detection of WDHD1 mRNA expression level

[0049] The expression of ITGAV in OE-WDHD1 and sh-WDHD1 cell lines was detected by RT-qPCR and Western blot, and the expression of WDHD1 in si-ITGAV cell line was detected by RT-qPCR and Western blot (the specific experimental steps of RT-qPCR and Western blot are the same as those in Part 1, 2.2.2 and 2.2.3) to assess the correlation and upstream and downstream relationship of the two genes.

[0050] RT-qPCR is a method for measuring the expression level of specific gene mRNA in a sample. It consists of four steps: total RNA extraction, reverse transcription reaction, sample loading, and PCR.

[0051] Step 1: Total RNA Extraction: Discard the complete culture medium in the culture flask and rinse the flask 2-3 times with PBS. Add 1 ml of PBS, scrape the cells from the bottom of the flask using a cell brush, and pipette the PBS containing the cells into a 1.5 ml sterile, enzyme-free, flat-capped thin-walled tube (1.5 ml EP tube). Centrifuge (4℃, 1000g, 5 min), discarding the supernatant to obtain cell clumps. Add the total RNA extraction reagent RNA so Plus to the EP tube, gently pipette and invert the EP tube to ensure full contact between the cells and RNA so Plus. Incubate on ice for 1 min. Add chloroform (1 / 3 the volume of RNA so Plus), gently invert to mix, incubate on ice for 10 min, and then centrifuge (4℃, 12000g, 15 min). After centrifugation, the liquid in the EP tube will separate into three layers: a clear, transparent upper layer; a cloudy, viscous middle layer; and a pink lower layer. Use a pipette to slowly extract the upper layer and transfer it to an unused, sterile, enzyme-free 1.5ml EP tube. Add isopropanol (50%-75% of the volume of RNAso Plus), invert the EP tube to mix, and incubate on ice for 10 minutes before centrifugation (4℃, 12000g, 10min). After centrifugation, a slightly transparent white precipitate will appear at the bottom of the EP tube. Use a pipette to slowly extract the supernatant along the tube wall, add 75% ethanol (same volume as RNAso Plus), invert to mix, and centrifuge (4℃, 7500g, 5min). Extract the supernatant, add anhydrous ethanol, and repeat the previous step. Discard the supernatant and air-dry the EP tube on ice. The precipitate at the bottom of the EP tube is the total RNA. After dissolving the precipitate in enzyme-free water, the purity and concentration of the obtained RNA were determined using a spectrophotometer. The absorbance at wavelengths of 260 nm (OD260) and 280 nm (OD280) was used to assess RNA purity. An OD260 / OD280 value between 1.8 and 2.1 was considered to meet the RNA purity standard. The obtained RNA was diluted with enzyme-free water to 500 μg / μl and stored on ice for later use or in a -80°C freezer for long-term storage.

[0052] Step Two: The specific steps for the reverse transcription reaction are as follows: Aspirate the sample using the PrimeScript reverse transcription kit. TMReverse transcription was performed using the RTreagent Kit. The reverse transcription system contained 2 μl of 5X PrimeScript Buffer (for Real Time), 0.5 μl of PrimeScript RT Enzyme Mix, 0.5 μl of Oligo dT Primer (50 μM)*1, 0.5 μl of Random6mers (100 μM)*1, 2 μl of total RNA, and 4.5 μl of enzyme-free water. After adding the required reagents to a 0.2 ml PCR tube, the PCR tube was placed in a PCR instrument for reverse transcription. The reaction temperature was 37℃ for 15 min, followed by heating at 85℃ for 5 s. After the reaction, the obtained DNA was diluted 2-3 times with enzyme-free water and stored on ice or at -80℃ for long-term storage.

[0053] Step 3: Sample Loading and RT-qPCR: The RT-qPCR reaction system is as follows: 5 μl TB Green Premix ExTaq II, 0.4 μl upstream primer, 0.4 μl downstream primer, 0.2 μl ROX Reference Dye II, 1 μl cDNA, and 3 μl enzyme-free water. To avoid prolonged exposure of reagents to room temperature and for ease of operation, the total amount of each reagent required should be calculated, and a master tube should be prepared. The master tube is divided into... Premix Ex Taq TM The second tube is a mixing tube for the remaining reagents in the system. After preparing the main tube, use a pipette to transfer the corresponding reagents from the main tube into 0.2ml flat-capped eight-row tubes. Tightly cap the tubes and centrifuge for 1 minute. Check the tubes for air bubbles, as bubbles can affect the accuracy of the results. If bubbles are present, gently tap the tube wall to break them, then centrifuge for another 1 minute. Place the prepared 0.2ml flat-capped eight-row tubes in a Gentier 96R automated PCR instrument. Reaction temperatures: 95℃ for 30s pre-denaturation, 95℃ for 10s, 60℃ for 34s (40 cycles). Melting curve: 60℃ for 1 minute, 95℃ for 15s.

[0054] (2) Western blot detection of WDHD1 protein expression level

[0055] Western blot is a imaging technique for semi-quantitative analysis of a protein from a protein mixture. Its basic procedure is protein extraction, SDS-page gel electrophoresis, membrane transfer, specific primary antibody binding, secondary antibody development, and infrared fluorescence imaging. The specific operation steps are as follows.

[0056] Step 1: Protein Extraction: Discard the complete culture medium in the culture flask and wash twice with PBS. After digestion with trypsin, collect the cells by centrifugation or scraping with a cell brush. Place the flask on ice. Excessive digestion with trypsin can damage the cell membrane and affect the expression and activity of membrane proteins. If the target protein is a membrane protein, it is best to collect the suspension by scraping with a cell brush. Place the suspension in a 1.5ml EP tube and centrifuge (room temperature, 1000g, 5min). After discarding the supernatant, add a mixture of high-strength RIPA cell lysis buffer, PMSF, and protease inhibitor to the cell clump. The volume of RIPA cell lysis buffer is 100μl per 106 cells. The ratio of RIPA cell lysis buffer:PMSF:protease inhibitor mixture is 100:1:1. Mix with a pipette and incubate on ice for 10min. Cells were disrupted using an ultrasonic processor (39% power, 5s sonication, 5s interval, 1min total). After standing on ice for 10min, the cells were centrifuged (4℃, 12000g, 15min). A small amount of the supernatant was taken and the protein concentration was determined using a BCA kit. 5× protein loading buffer (1 / 4 the volume of the supernatant) was added to the remaining supernatant. After mixing, the mixture was heated in a metal bath or water bath at 100℃ for 10min. The protein loading preparation was now complete. The mixture was aliquoted into 1.5ml EP tubes. For immediate use, the cells can be temporarily stored in a -20°C freezer. For long-term storage, the cells should be placed in a -80°C freezer or liquid nitrogen.

[0057] Step 2: Preparing the Page Gel: Use the Page gel rapid preparation kit to prepare the electrophoresis gel. One gel requires 2.5 ml of upper gel solution, 2.5 ml of upper gel buffer, 0.75 ml of lower gel solution, 0.75 ml of upper gel solution, and 0.75 ml of upper gel buffer. Before preparing the gel, mix the required upper gel solution and upper gel buffer thoroughly, and mix the lower gel solution and lower gel buffer thoroughly to prepare the mixture. Let the mixture stand at room temperature for 10 minutes to ensure thorough mixing. The concentration of the lower gel solution depends on the molecular weight of the target protein. In this study, since the protein molecular weight is mostly 50-130 kDa, a 10% lower gel solution was selected. Align the bottom ends of the long and short glass plates, with the concave side of the long plate facing inward, and place them on the gel casting holder, securing the clips. Then, add a coagulant (1 / 50th the volume of the mixture) to the lower gel mixture. Use a pipette to transfer the lower gel mixture into the gap between the long and short plates. Add 1 ml of pure water or anhydrous ethanol to remove air bubbles, and let it stand at room temperature for 15 minutes to allow the lower gel mixture to solidify. Discard the pure water or anhydrous ethanol used for pressing the gel. Add another coagulant (1 / 50th the volume of the mixture) to the lower gel mixture, then add the mixture into the gap, insert the 10-well electrophoresis comb, and let it stand at room temperature for 20 minutes to allow the upper gel mixture to solidify. The Page gel preparation is now complete.

[0058] Step 3: SDS-Page Gel Electrophoresis: Unlatch the inner electrophoresis tank and place the prepared Page gel into it, ensuring the upper end of the short plate aligns with the edge of the groove. Tighten the inner tank latch and fill it with freshly prepared rapid high-resolution electrophoresis buffer. For the outer tank, recycled rapid high-resolution electrophoresis buffer can be used, filling it to the "2 Gel" or "4 Gel" mark depending on the amount of gel. Gently pull the electrophoresis comb vertically upwards and add 5 μl of pre-stained protein markers to distinguish the lane order and the location of proteins of different molecular weights. Add the pre-prepared sample proteins sequentially, ensuring a consistent loading volume (40-100 μg / well). After loading, replace the electrophoresis tank lid, turn on the power, and set the program to 160V constant voltage for 35 minutes. Proteins are unstable in the gel; immediately discard the electrophoresis buffer after electrophoresis and proceed with the transfer.

[0059] Step 4: Transfer and Blocking: Since the gel does not provide color development, the protein and markers need to be transferred to a PVDF membrane before primary antibody-antigen agglutination. Secondary antibody then binds to the primary antibody for color development. After discarding or recovering the electrophoresis buffer in the inner electrophoresis tank, loosen the clips and remove the page gel. At this point, neatly arranged and clearly spaced markers can be seen. Based on the relative positions of the markers, the approximate locations of the target protein and internal control protein can be determined by referring to the instructions. Immerse the pre-cut PVDF membrane in formaldehyde for 3-5 minutes. Cover the internal control and target protein locations with the polyvinylidene fluoride (PVDF) membrane and place it in the sandwich transfer holder. Pour ice-free rapid transfer buffer into the outer tank, and place the transfer holder into the inner transfer tank, paying attention to the electrode positions and current direction. Transfer conditions: 400mA constant current, 35 minutes. Blocking: Using a rapid blocking solution can make the protein blot more concentrated and reduce stray bands. The principle is that the protein components of the blocking solution bind to the spatial gaps on the membrane surface, reducing the binding of non-specific primary antibodies. The specific steps are as follows: Take the strip that has completed the transfer out of the transfer clamp, immediately put it into the rapid blocking solution, and gently shake it on a shaker for 25 minutes at room temperature.

[0060] Step 5: Binding of the primary antibody to the target protein: The primary antibody, or first antibody, is a highly specific protein that recognizes a specific antigen and binds to it to form an antigen-antibody complex. Simply put, the primary antibody specifically binds to a specific antigenic determinant of the target protein, and the expression of that protein is semi-quantitatively determined by detecting the amount of the antigen-antibody complex. The specific steps are as follows: Dilute the primary antibody with the diluent according to the instructions and place it in an incubation chamber. Remove the blocked PVDF membrane from the rapid blocking solution and wash it three times with Tris-Buffered Saline with Tween (TBST) solution for 5 minutes each time. Place the PVDF membrane in an incubation chamber containing the primary antibody and incubate at 4°C overnight or for 12 hours.

[0061] Step Six: Binding of H&L Fluorescent Secondary Antibody to Primary Antibody: The secondary antibody can bind to the primary antibody, and with the help of its carried luminescent substances or chromogenic genes, the antigen-antibody complex on the PVDF membrane can be visualized as a protein blot under an infrared fluorescence imaging system. Specific operating steps: Following the secondary antibody instructions, dilute the secondary antibody to the appropriate fold using the secondary antibody dilution buffer, and place the diluted secondary antibody in an incubation chamber. After incubation with the primary antibody, wash the PVDF membrane three times with TBST, 5 min each time. After washing, place the PVDF membrane in an incubation chamber containing the secondary antibody and incubate at room temperature for 50 min. Subsequently, wash the PVDF membrane three times with TBST in the incubation chamber, 5 min each time, and scan the protein blot on the PVDF membrane using an infrared fluorescence imaging system.

[0062] 1.2.4 CCK8 assay for detecting si-ITGAV cell proliferation and radiosensitivity

[0063] The si-ITGAV group, CON group, and blank group were prepared into cell suspensions with a density of 10⁴ cells / ml after trypsin digestion. The cell suspensions were gently mixed, and 100 μl of the cell suspension was seeded into each well, with 1000 cells per well and 3 replicates per group. Sterile PBS was added around the perimeter of each well to maintain humidity in the center wells. The 96-well plates with seeded cells were placed in an incubator for culture. After 5-6 hours of culture, the cells were observed under a microscope until they adhered to the well walls; this was recorded as 0h. The original culture medium was discarded, and 100 μl of prepared CCK8 working solution (containing 10 μl CCK8 solution and 90 μl complete culture medium) was added to each well in the dark. The solution was added slowly along the well wall, taking care not to create air bubbles. The 96-well plate was wrapped in aluminum foil and incubated in the dark for 2 hours. The absorbance (OD value) at 450 nm was then measured using a microplate reader. The above procedure was repeated at 8h, 12h, 24h, and 48h, and the OD values ​​were measured. After recording the results by photograph, a proliferation and growth curve for each group of cells was plotted with the detection time point on the x-axis and the OD value on the y-axis. Each group of cells was tested at least three times.

[0064] When using CCK8 to determine the radiosensitivity of the si-ITGAV cell line, approximately 5000 cells should be seeded into a 96-well plate, with three replicates per group. Except for the control group, the other groups are irradiated with 2 Gy, 4 Gy, 6 Gy, and 8 Gy after 24 hours. After irradiation, the medium is changed, and the cells are cultured for another 60 hours. Then, 100 μl of CCK8 working solution is added, and the cells are incubated in the dark for 2 hours. The OD value at 450 nm wavelength is then measured using a microplate reader, and the cell viability is calculated (cell viability = OD value of irradiated group / OD value of control group × 100%).

[0065] 1.2.5 Colony formation assay to detect radiosensitivity of si-ITGAV cells

[0066] (1) Cell seeding and irradiation: Take C666-1 and CNE-1 cells in the logarithmic growth phase and prepare a cell suspension with a cell concentration of 10⁴ cells / ml. Prepare five 15ml centrifuge tubes and perform serial dilutions of the cell suspension: the total liquid in each 15ml centrifuge tube is 5ml, and add 500, 500, 1000, 1500, and 2000 cells to each tube, respectively. Add 2ml of the prepared cell suspension to a six-well plate. Different irradiation doses correspond to different cell seeding numbers, as follows: 0 Gy group, 1000 cells / well; 2 Gy group, 1000 cells / well; 4 Gy group, 2000 cells / well; 6 Gy group, 3000 cells / well; 8 Gy group, 4000 cells / well. After cell attachment, the cells are irradiated with 0, 2, 4, 6, or 8 Gy 6MV of X-rays and cultured for 10-14 days in a 37°C incubator with 5% CO2.

[0067] (2) Cell fixation and staining: When visible cell colonies appear, stop the culture, discard the original culture medium with a disposable Pasteur pipette, wash the cells 3 times with PBS buffer, fix the cells with 4% paraformaldehyde for 20 minutes, wash the cells 3 times with an appropriate amount of PBS buffer, stain with an appropriate amount of crystal violet for 20 minutes, discard the crystal violet, wash the cells 3 times with PBS buffer, and count the colonies with more than 50 cells after air drying.

[0068] 2. Results

[0069] 2.1WDHD1 interacts with ITGAV protein.

[0070] like Figure 1As shown in Figure A, WDHD1 and ITGAV proteins bind at the molecular level. Molecular docking shows that WDHD1 and ITGAV proteins can form a stable complex. In the complex structure, the two proteins can form six sets of hydrogen bonds to connect them, of which sets b and d are salt bridges. The six sets of hydrogen bonds are: a) ITGAV:ASP-219——WDHD1:HIS-477

[0071] b)ITGAV:ARG-245——WDHD1:GLU-524

[0072] c)ITGAV:PHE-278——WDHD1:SER-521

[0073] d)ITGAV:ARG-339——WDHD1:ASP-520

[0074] e)ITGAV:ARG-339——WDHD1:GLY-560

[0075] f)ITGAV:TYR406——WDHD1:SER521

[0076] like Figure 1 As shown in Figure B, WDHD1 and ITGAV proteins exhibit a high degree of spatial overlap. Immunofluorescence staining of WDHD1 and ITGAV proteins in CNE-1 and C666-1 cell lines was used to determine the spatial distribution of their expression. The results showed that both WDHD1 and ITGAV are nuclear proteins, and their spatial distributions highly overlap, suggesting the existence of binding between WDHD1 and ITGAV proteins.

[0077] like Figure 1 As shown in Figure C, o-IP indicates the binding of WDHD1 and ITGAV proteins in nasopharyngeal carcinoma cells. ITGAV and WDHD1 antibodies were used to precipitate the complexes in CNE-1 and C666-1 cell lines, respectively. Western blotting of the complexes revealed that the WDHD1 antibody formed a complex with WDHD1 protein while also precipitating ITGAV protein. Furthermore, after the ITGAV antibody formed a complex with ITGAV protein, WDHD1 protein was also detected in the complex. This suggests that WDHD1 and ITGAV can form protein complexes in the in vivo environment, and that ITGAV is a target gene for the biological function of WDHD1.

[0078] 2.2 The si-ITGAV cell line was successfully constructed.

[0079] like Figure 2As shown, after using three different small interfering RNAs, RT-qPCR analysis revealed that siRNA ITGAV-792 exhibited the highest knockdown efficiency, and Western blot analysis showed that siRNA ITGAV-792 also reduced ITGAV protein expression. Figure 2 A, p < 0.05), while ITGAV expression in the CON group cells was not significantly different from that in the blank group (A, p < 0.05). Figure 2 B (p > 0.05) indicates that the si-ITGAV cell line was successfully constructed.

[0080] 2.3 WDHD1 unidirectionally regulates ITGAV expression

[0081] like Figure 3 As shown, Western blot analysis revealed that ITGAV expression in both sh-WDHD1 and OE-WDHD1 cell lines exhibited the same trend of change, with ITGAV expression positively correlated with WDHD1. Figure 3 ), while WDHD1 expression was not significantly altered in the si-ITGAV cell line. Figure 3 The results suggest that WDHD1 unidirectionally regulates the expression of ITGAV, and the expression of ITGAV is positively correlated with WDHD1, indicating that ITGAV is a downstream target gene of WDHD1.

[0082] 2.4 Silencing ITGAV reduces the proliferative capacity of nasopharyngeal carcinoma cells while increasing their radiosensitivity.

[0083] like Figure 4 As shown, cell viability of si-ITGAV, CON, and control groups was measured using the CCK8 assay. Results showed that in CNE-1 and C666-1 cell lines, the cell proliferation activity of the si-ITGAV group was lower than that of the CON group at 48h and 72h (p < 0.05), suggesting that silencing ITGAV reduces the proliferation capacity of nasopharyngeal carcinoma cells. Cell viability and colony formation ability of the si-ITGAV and CON groups were assessed using CCK8 and plate colony assays after cell irradiation. Results showed that the cell viability and colony formation ability of the si-ITGAV cell line were lower than those of the CON group, suggesting that silencing ITGAV increases the radiosensitivity of nasopharyngeal carcinoma cell lines.

[0084] 2.6 Conclusion

[0085] In this embodiment, molecular docking, immunofluorescence co-localization, and Co-IP technology confirmed the binding of ITGAV to WDHD1. WDHD1 unidirectionally regulates the expression of ITGAV, proving that ITGAV is a downstream target gene of WDHD1 and is unidirectionally regulated by WDHD1. Inhibiting WDHD1 can indirectly inhibit the expression of ITGAV.

[0086] Furthermore, in this embodiment, after silencing ITGAV on nasopharyngeal carcinoma cell lines, when using plate clone assays and CCK8 assays to evaluate the effect of ITGAV on the radiosensitivity of nasopharyngeal carcinoma cells, it was found that the radiosensitivity of nasopharyngeal carcinoma cells increased, that is, the radiosensitivity of nasopharyngeal carcinoma cells increased after silencing ITGAV.

[0087] In summary, ITGAV is a downstream target gene of WDHD1 and is unidirectionally regulated by WDHD1; silencing ITGAV reduces the proliferative activity of nasopharyngeal carcinoma cells and increases their radiosensitivity.

[0088] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the present invention.

Claims

1. A method for inhibiting ITGAV gene expression, characterized in that, It inhibits the expression of WDHD1 in cells.

2. The method for inhibiting ITGAV gene expression according to claim 1, characterized in that, The expression of WDHD1 in cells was suppressed by gene knockdown.

3. The method for inhibiting ITGAV gene expression according to claim 1, characterized in that, Inhibit the expression of WDHD1 in cells by using WDHD1 inhibitors.

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