Application of phenylethyl isothiocyanate naturally present in cruciferous vegetables as a drug against endometrial cancer

Abstract

The invention discloses an anti-endometrial cancer drug application of phenethyl isothiocyanate naturally present in cruciferous vegetables. Phenethyl isothiocyanate can exert an anti-endometrial cancer effect by targeting SF3A3, can inhibit the survival of endometrial cancer cells, inhibit the proliferation ability of endometrial cancer cells, and can induce endometrial cancer cell cycle arrest and cell apoptosis, and has good application prospects in the preparation of anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to a new application of drugs, and in particular to an application of phenylethyl isothiocyanate naturally present in cruciferous vegetables as a drug for anti-endometrial cancer, and the drug exerts an anti-tumor effect against a new target SF3A3. Background Art

[0002] Cancer is a disease with a very high mortality rate worldwide, and the incidence rate is still rising. The latest global cancer statistics report shows that in 2018, there were 19.1 million new cases of cancer and 8.7 million new deaths from cancer. At present, the main treatments for cancer include chemotherapy, radiotherapy, and surgical resection. While killing tumor cells, these treatments usually also damage normal cells and have great side effects. In recent years, targeted therapy has gradually emerged. This therapy treats sites that have been clearly identified and are closely related to tumors, causing tumor cells to die specifically without affecting normal tissue cells around the tumor, effectively reducing toxic side effects and greatly improving the survival rate of cancer patients. Endometrial carcinoma (EC) is one of the most common female diseases and the fourth most common cancer in women. Although most women with endometrial cancer are diagnosed with early-stage disease and have a good prognosis. However, 15-20% of patients still have a poor prognosis and a high risk of distant metastasis. At present, the main treatment for endometrial cancer is surgery, including total abdominal or laparoscopic hysterectomy and bilateral salpingo-oophorectomy. Specific biomarkers for endometrial cancer have not yet been identified. In addition, the combination of cisplatin, doxorubicin, and paclitaxel is the most effective chemotherapy regimen reported in advanced or recurrent endometrial cancer. However, chemotherapy drugs generally have significant toxic side effects. Therefore, a deeper understanding of the molecular mechanisms of the occurrence and development of endometrial cancer may help develop new treatments to improve treatment outcomes and the prognosis of patients with endometrial cancer. Research on the molecular mechanisms of endometrial cancer will help improve targeted specific therapies. Among them, SF3A3, as an important tumor-related target, has been shown to play an anti-tumor role in a variety of cancers. Therefore, drugs designed with SF3A3 as a target are expected to play a significant potential in the development of anti-tumor drugs.

[0003] According to the World Health Organization, cancer is the second leading cause of death in the world. Since the 1940s, anti-tumor drugs have developed rapidly. At present, there are as many as 100 anti-tumor drugs in clinical trials at home and abroad. Although anti-tumor drugs have made great progress after nearly 70 years of development, there are still problems such as cytotoxicity and tumor resistance. Among them, anti-cancer drugs are mainly divided into cytotoxic small molecule drugs, natural product anti-cancer drugs and molecular targeted drugs. With paclitaxel as a representative, compounds derived from natural products occupy an important position in the research and development of anti-tumor drugs. The rich Chinese medicinal materials are a treasure trove of anti-tumor drugs. The discovery of natural active substances in Chinese medicinal materials for anti-tumor is also an important theme of contemporary Chinese medicine research.

[0004] Phenyl isothiocyanate (PEITC), a naturally occurring ester of phenylethyl isothiocyanate found in cruciferous vegetables, is a chemopreventive agent. There are no studies on this compound for the treatment of endometrial cancer. Summary of the invention

[0005] Purpose of the invention: In view of the existing problems, the purpose of the present invention is to provide an anti-endometrial cancer drug application of phenethyl isothiocyanate naturally present in cruciferous vegetables, which exerts an anti-tumor effect through SF3A3.

[0006] Technical solution: The present invention provides the use of phenethyl isothiocyanate in the preparation of anti-endometrial cancer drugs. The structural formula of phenethyl isothiocyanate is shown in Formula I:

[0007]

[0008] In the application, phenylethyl isothiocyanate targets and inhibits the activity of SF3A3.

[0009] In the application, the phenethyl isothiocyanate inhibits the survival of tumor cells, inhibits the clone formation of tumor cells, inhibits the proliferation ability of tumor cells, and induces apoptosis of tumor cells.

[0010] Application of phenethyl isothiocyanate as the sole active ingredient in the preparation of anti-endometrial cancer drugs.

[0011] A pharmaceutical composition is prepared by taking phenethyl isothiocyanate as the only active ingredient and adding pharmaceutically acceptable excipients.

[0012] The application of the pharmaceutical composition in preparing an anti-endometrial cancer drug.

[0013] Application of phenethyl isothiocyanate in the preparation of drugs for treating SF3A3 gene-related diseases.

[0014] The SF3A3 gene-related disease is endometrial cancer.

[0015] The above-mentioned phenylethyl isothiocyanate has a molecular formula of C6H5CH2CH2NCS, a code name of PEITC, and a chemical name of 2-phenylethyl isothiocyanate.

[0016] The PEITC has a cytotoxic effect on endometrial cancer cells. Further studies have found that at different concentrations, PEITC has growth inhibition, cycle blocking and tumor cell apoptosis induction effects on endometrial cancer cell lines.

[0017] PEITC can inhibit tumor progression by arresting the cell cycle and inducing apoptosis in various human cancer cell models. Currently, there are several ongoing clinical trials exploring the role of PEITC as a single agent or in combination with chemotherapy, radiotherapy, targeted drugs or other immunotherapy drugs in various tumors. In addition, it is also of certain clinical significance to find relevant drugs for the treatment of endometrial cancer based on relevant biomarkers. Based on bioinformatics analysis, we found that phenethyl isothiocyanate (PEITC) may be a potential drug targeting SF3A3, which is expected to have a certain therapeutic effect on endometrial cancer. In addition, this compound has been verified in multiple models and shows certain clinical prospects.

[0018] Beneficial effects: The PEITC described in the present invention is derived from natural products in cruciferous vegetables, has good anti-tumor effects in vivo, can play an anti-tumor role through the key anti-tumor target SF3A3, kill endometrial cancer tumor cells, and has anti-tumor drug research and development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the discovery of the adverse prognostic role of SF3A3 in endometrial cancer;

[0020] Figure 2 is the impact of SF3A3 on survival in endometrial cancer;

[0021] Figure 3 It is the effect of PEITC against endometrial cancer cells;

[0022] Figure 4 is to explore the effects of PEITC on SF3A3 gene and protein;

[0023] Figure 5 After si-SF3A3 intervention, PEITC exerted antitumor effects on ISHIKAWA and KLE endometrial cancer cells;

[0024] Figure 6 ; To investigate the in vivo effects of SF3A3 and PEITC on ISHIKAWA and KLE endometrial cancer cells. DETAILED DESCRIPTION

[0025] The present invention is described in detail below by way of examples.

[0026] Drug source: purchased from Sigma;

[0027] Cell lines:

[0028] IAHIKAWA cells: human endometrial cancer cell line, adherent growth, purchased from Shanghai Cell Bank, Chinese Academy of Sciences;

[0029] KLE cells: human endometrial cancer cell line, grown adherently, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0030] Animals: Nude mice, 18–22 g, 4–6 weeks old, were purchased from Slack and maintained at the Huajiachi Campus of Zhejiang University in accordance with the regulations of the Animal Management Center of Zhejiang University, and the mouse experimental protocols were approved by the Animal Ethics Committee of Zhejiang University.

[0031] Example 1: Growth inhibition of endometrial cancer cells by SF3A3

[0032] Bioinformatics analysis

[0033] This patent was obtained through TCGA database (https: / / www.cancer.gov / about-nci / organization / ccg / research / structural-genomics / tcga), human protein gene database (https: / / www.proteinatlas.org / ), survival curve analysis library (http: / / kmplot.com), and database analysis. Figure 1 The results showed that SF3A3 was associated with poor prognosis of endometrial cancer, and SF3A3 was highly expressed in endometrial cancer samples compared with adjacent adjacent tissues.

[0034] Western Blot

[0035] (1) Protein sample preparation

[0036] First, cells were evenly spotted in a 6-well plate. After the cells adhered to the wall, PEITC (3, 15, 75 μM) and drug treatment were added for 72 h. The cell samples were taken out of the carbon dioxide incubator, and the cell density was observed under a microscope. After the culture medium was discarded, the cells were washed twice with 1× PBS buffer precooled at 4°C. The suspended cells were first centrifuged and then washed with 1× PBS buffer precooled at 4°C.

[0037] Wash twice. Add 100 μL of cell lysis solution premixed with phosphatase inhibitors and protease inhibitors, and place it on ice for 20 minutes. Scrape the cells with a clean scraper, collect them in a 1.5 mL centrifuge tube, centrifuge them in a high-speed refrigerated centrifuge at 12000 rpm and 4°C for 15 minutes, and then use a gun to transfer the cell lysate to a 1.5 mL centrifuge tube. Try to perform the entire operation on ice, and quantify the volume of the lysate with a pipette. If suspended, lyse directly in a 1.5 mL centrifuge tube. Divide the supernatant after centrifugation into centrifuge tubes and store at -20°C.

[0038] (2) Protein quantification (BCA protein quantification)

[0039] Prepare gradient dilutions of bovine serum albumin (BSA) standards, prepare clean 1.5mL centrifuge tubes and number them. Accurately pipette 7μL of protein sample and standard into ultrapure water and dilute 10 times to 70μL. Prepare BCA working solution (working solution A: working solution B = 50:1) as needed, mix well and set aside. Add 20μL of diluted protein sample and standard dilution solution to a 96-well plate (3 replicate wells for each sample), then add 200μL of mixed BCA working solution and shake gently. Place the 96-well plate in a 37℃ oven for 15min. Measure the OD value at an excitation wavelength of 562nm using a full-wavelength microplate reader. Establish a standard curve and calculate the protein concentration. Add 1 / 5 volume of 5* loading buffer to the sample, vortex and mix well, heat and denature in a 100℃ metal bath for 10min, and store the denatured sample at -20℃ or -80℃.

[0040] (3) SDS-polyacrylamide gel electrophoresis

[0041] Select the corresponding separation gel concentration (Table 1) according to the molecular weight of the target protein, fix the cleaned gel glass plate in the gel preparation device, check and confirm that there is no leakage, and then refer to Table 2 to fill the prepared separation gel, gently add the ultrapure water protective layer, and after the separation gel solidifies, add the concentrated gel mixture in Table 2, and insert the comb teeth of the corresponding specifications. Be careful not to generate bubbles, and wait for the concentrated gel to solidify before use. Fix the prepared gel on the A electrophoresis clamp, add the newly configured 1× electrophoresis buffer, gently pull out the comb teeth vertically, and use a pipette to add the sample and pre-stained Marker to the corresponding channel. Electrophoresis program: 80V, 40min; 120V, 50min.

[0042] (4) Wet transfer

[0043] After the electrophoresis is finished, carefully pry open the glass plate, take out the gel separated by electrophoresis, cut the gel where the desired target protein is located according to the position of the pre-stained Marker, and clamp the wet transfer clip in the order of positive electrode (white)-sponge-filter paper-PVDF membrane-gel-filter paper-sponge-negative electrode (black). The PVDF membrane needs to be activated in methanol for 30s before use. During the wet transfer clip, pay attention to emptying the bubbles between the gel and the PVDF membrane, and then put it in the wet transfer tank according to the direction corresponding to the positive and negative electrodes, add 1× wet transfer solution and ice box precooled at 4℃ in advance, and ice bath the wet transfer tank to maintain a low temperature state, and transfer the membrane under 0.32A constant current conditions. The wet transfer time is determined according to the protein molecular weight according to 1min / kDa. The SF3A3 target protein uses Tianneng wet transfer solution, and the transfer time is 0.64A constant current, 60min conditions.

[0044] (5) Blocking and antibody incubation

[0045] After wet transfer, carefully remove the PVDF membrane protein band, place it in a protein incubation box, add blocking solution (5% BSA), and place it on a shaker for blocking at room temperature for 1 hour. After blocking, recover the blocking solution, add the primary antibody diluent, and place it on a shaker at 4°C for incubation for 12 hours. After the primary antibody incubation, recover the primary antibody diluent, add 1×TBST, and place it on a shaker to wash the primary antibody, 5 minutes each time, for a total of 3 times; discard 1×TBST, add the secondary antibody diluent, and place it on a shaker at 4C for incubation for 1 hour. After the secondary antibody incubation, recover the secondary antibody diluent, add 1×TBST, and place it on a shaker to remove the unbound secondary antibody diluent, 5 minutes each time, for a total of 4 times. After washing the secondary antibody, expose it.

[0046] (6) Chemiluminescence imaging

[0047] Prepare ECL luminescent liquid (A liquid: B liquid = 1:1) for exposure, carefully take out the PVDF membrane protein band, place it on the imaging plate in the Bio-Rad automatic gel imager, open the program, adjust the focus brightness and other parameters, save the template, add the luminescent liquid prepared in advance, click exposure, and save the data.

[0048] Table 1 Optimal separation range of different separation gel concentrations

[0049]

[0050]

[0051] Add 10% ammonium persulfate solution 100 μL / small gel and TEMED solution 10 μL / small gel to the separation gel; add 10% ammonium persulfate solution 30 μL / small gel and TEMED solution 5 μL / small gel to the concentration gel.

[0052] Table 2 Separation gel / concentration gel formula

[0053]

[0054] Figure 1 The results showed that among clinical samples, SF3A3 protein was significantly elevated in cancer samples.

[0055] Construction of endometrial cancer cell lines with knockdown and overexpression of SF3A3

[0056] Cell lines: ISHIKAWA cells, KLE cells;

[0057] Experimental methods: Construction of si-SF3A3 cell lines; SF3A3 knockdown and overexpression stable cell lines; CCK8 method;

[0058] Experimental steps:

[0059] (1) Take a bottle of cells in good exponential growth phase, add 0.25wt% trypsin digestion solution, digest to make the adherent cells fall off, and count (2-4)×10 4 / mL, and made into cell suspension;

[0060] (2) The cell suspension was inoculated into a 96-well plate at 90 μL / well and cultured in a constant temperature CO2 incubator;

[0061] (3) After 24 hours, si-SF3A3 (10 μL / well) was added, and the medium was changed after 6-8 hours. After 48-72 hours of culture, the CCK8 kit was used for detection;

[0062] (4) Add CCK8 reagent to a 96-well plate, 10 μL / well, and incubate in an incubator for 2 hours;

[0063] (5) Use an enzyme-linked immunosorbent assay (ELISA) to measure the absorbance of each well at a wavelength of 450 nm and calculate the corresponding cell inhibition rate.

[0064]

[0065] The experimental results are shown in Figure 2 ISHIKAWA and KLE endometrial cancer cells were treated with siRNA. SF3A3 showed a significant inhibitory effect on the survival of endometrial cancer cells, and both endometrial cancer cells showed stable knockdown or overexpression of SF3A3 protein or gene.

[0066] Growth curve experiment

[0067] (1) Take cells that are in good growth state and in the logarithmic phase, digest and centrifuge them, count them after resuspending them, and dilute them into 1-2×10 5Take the required volume of cell suspension, resuspend in complete medium, pipette evenly, and add 500 μL / well, 5×10 per well. 4 Cells were inoculated into 24-well plates and cultured in a constant temperature CO2 incubator.

[0068] (2) After 24 h of culture, fresh low-serum culture medium was replaced and the cells were randomly divided into five groups: negative control group, drug-treated group (low, medium, and high doses), and positive control group, and cultured in the incubator.

[0069] (3) Digest the cells and count them once a day for 6 consecutive days.

[0070] The experimental results are as follows Figure 2 As shown, ISHIKAWA and KLE cells were given siRNA and stably transfected cell lines for 6 days, and counted every day to draw growth curves. The results showed that SF3A3 could inhibit the proliferation of endometrial cancer cells.

[0071] according to Figure 2 It can be seen that in the two endometrial cancer cells, SF3A3 can significantly inhibit the growth of endometrial cancer cells.

[0072] Plate colony formation assay

[0073] (1) Take cells that are in good growth state and in the logarithmic phase, digest and centrifuge them, count them after resuspending them, take the corresponding volume of cell suspension in complete culture medium, mix them by pipetting, and add the cell suspension to a 6-well plate. Each well has 2 mL of cell suspension, containing 1000 cells. Shake the 6-well plate up and down and left and right, and place it in a constant temperature CO2 incubator for culture.

[0074] (2) After 24 h of culture, replace with fresh complete culture medium and randomly divide the cells into 5 groups: negative control group, drug-treated group (low, medium, and high doses), and positive control group; continue to culture in the incubator, replace the culture medium every 3-4 days, and terminate the culture when a single cell cluster contains at least 50 cells.

[0075] (3) Take out the six-well plate, discard the supernatant, and wash it once with PBS. Add 4% paraformaldehyde to each well and fix it at room temperature for 30 minutes. Discard the fixative and add 1 mL of 0.8% crystal violet stain to each well. Stain it at room temperature in the dark for 30 minutes. Finally, rinse the bottom of the six-well plate with tap water at a low flow rate to wash off the crystal violet stain. After air drying, take photos.

[0076] The experimental results are as follows Figure 2As shown, in the clone formation experiment, ISHIKAWA cells and KLE cells were given siRNA, and the stably transfected cell lines were plated in six-well plates, and twelve days later, the clones were stained with crystal violet. The results showed that the clone formation ability of endometrial cancer cells was inhibited with the knockdown of SF3A3.

[0077] according to Figure 2 It can be seen that SF3A3 can significantly inhibit the proliferation of endometrial cancer cells.

[0078] Example 2 In vitro inhibition rate IC of PEITC on human endometrial cancer cell lines 50

[0079] Cell lines: ISHIKAWA cells, KLE cells;

[0080] Experimental method: CCK8 method;

[0081] Experimental steps:

[0082] (1) Take a bottle of cells in good exponential growth phase, add 0.25wt% trypsin digestion solution, digest to make the adherent cells fall off, and count (2-4)×10 4 / mL, and made into cell suspension;

[0083] (2) The cell suspension was inoculated into a 96-well plate at 90 μL / well and cultured in a constant temperature CO2 incubator;

[0084] (3) 24 hours later, the test drug PEITC (10 μL / well) was added and cultured for 72 hours;

[0085] (4) Add MTT reagent to a 96-well plate, 20 μL / well, and incubate in an incubator for 2 hours;

[0086] (5) Use an enzyme-linked immunosorbent assay (ELISA) to measure the absorbance of each well at a wavelength of 450 nm and calculate the corresponding cell inhibition rate.

[0087]

[0088] The experimental results are shown in Figure 3 ISHIKAWA and KLE endometrial cancer cells were treated with PEITC at final concentrations of 0.1, 0.3, 1, 3, 10, 30, 100, and 300 μM. Cell viability was detected by CCK8 assay, and the results were expressed as the median lethal concentration (IC 50 ± standard deviation (n=6) indicates that with the increase of dose, PEITC showed a significant inhibitory effect on the survival of endometrial cancer cells in both endometrial cancer cells, and in the two endometrial cancer cells KLE and ISHKAWA, the IC50 24.78 μM and 58.21 μM respectively.

[0089] Example 3 Inhibitory effect of PEITC on the growth of endometrial cancer cells

[0090] Experimental steps:

[0091] Plate colony formation assay

[0092] (1) Take cells that are in good growth state and in the logarithmic phase, digest and centrifuge them, count them after resuspending them, take the corresponding volume of cell suspension in complete culture medium, mix them by pipetting, and add the cell suspension to a 6-well plate. Each well has 2 mL of cell suspension, containing 1000 cells. Shake the 6-well plate up and down and left and right, and place it in a constant temperature CO2 incubator for culture.

[0093] (2) After 24 h of culture, replace with fresh complete culture medium and randomly divide the cells into 5 groups: negative control group, drug-treated group (low, medium, and high doses), and positive control group; continue to culture in the incubator, replace the culture medium every 3-4 days, and terminate the culture when a single cell cluster contains at least 50 cells.

[0094] (3) Take out the six-well plate, discard the supernatant, and wash it once with PBS. Add 4% paraformaldehyde to each well and fix it at room temperature for 30 minutes. Discard the fixative and add 1 mL of 0.8% crystal violet stain to each well. Stain it at room temperature in the dark for 30 minutes. Finally, rinse the bottom of the six-well plate with tap water at a low flow rate to wash off the crystal violet stain. After air drying, take photos.

[0095] The experimental results are as follows Figure 4 As shown, in the clone formation experiment, ISHIKAWA cells and KLE cells were treated with 3μM, 15μM, and 75μM PEITC for 14 days, and the clones were stained with crystal violet. The results showed that the clone formation ability of endometrial cancer cells was inhibited as the dose increased.

[0096] As shown in Figure 3, compound PEITC can significantly inhibit the proliferation of endometrial cancer cells.

[0097] Growth curve experiment

[0098] (1) Take cells that are in good growth state and in the logarithmic phase, digest and centrifuge them, count them after resuspending them, and dilute them into 1-2×10 5 Take the required volume of cell suspension, resuspend in complete medium, pipette evenly, and add 500 μL / well, 5×10 per well. 4 Cells were inoculated into 24-well plates and cultured in a constant temperature CO2 incubator.

[0099] (2) After 24 h of culture, fresh low-serum culture medium was replaced and the cells were randomly divided into five groups: negative control group, drug-treated group (low, medium, and high doses), and positive control group, and cultured in the incubator.

[0100] (3) Digest the cells and count them once a day for 6 consecutive days.

[0101] The experimental results are as follows Figure 5 As shown, ISHIKAWA and KLE cells were treated with 3μM, 15μM, and 75μM PEITC intervention for 6 days, and counted every day to draw a growth curve. The results showed that the proliferation ability of endometrial cancer cells was inhibited as the dose increased.

[0102] As shown in Figure 3, compound PEITC can significantly inhibit the growth of endometrial cancer cells in both endometrial cancer cells.

[0103] Annexin V / PI double staining to detect cell apoptosis

[0104] Experimental steps:

[0105] (1) Cell inoculation and drug administration: the method is the same as cell cycle detection;

[0106] (2) Cell collection: Take a 15 mL centrifuge tube, collect the cell supernatant, wash with 1× PBS precooled at 4°C, collect the supernatant in the same centrifuge tube, digest the cells with EDTA-free trypsin, and terminate the digestion with the cell supernatant after digestion (remember not to over-digest to avoid false positive results), blow off the cells and collect them in the same centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL of precooled PBS to resuspend, transfer to a 1.5 mL centrifuge tube, centrifuge, discard the supernatant, repeat the washing with PBS once, centrifuge, and discard the supernatant (be careful to discard it completely, you can cover the small gun tip with the large gun tip);

[0107] (3) Staining: Add 100 μL 1× binding buffer to each tube and blow the cells evenly, then add 5 μL FITCAnnexin V and 5 μL PI respectively, vortex to mix, and react in the dark for 15 min;

[0108] (4) On-line detection: Add 400 μL of 1× binding buffer to each tube, blow evenly, and then aspirate into the flow tube for detection. The excitation light of Annexin V-FITC is green fluorescence, and the excitation light of PI is red fluorescence (keep away from light throughout the process).

[0109] The experimental results are as follows Figure 6As shown, the cells were given 3μM, 15μM, and 75μM doses of PEITC for 48 hours, and cell apoptosis was analyzed by PI staining and flow cytometry. The results showed that the compound was able to induce apoptosis in human endometrial cancer cells ISHIKAWA and KLE with increasing doses. As can be seen from the figure, the compound PEITC can cause G2 / M phase apoptosis in human endometrial cancer cells ISHIKAWA and KLE.

[0110] As shown in Figure 3, compound PEITC can induce apoptosis of endometrial cancer cells.

[0111] Western Blot

[0112] As shown in Figure 3, the compound PEITC can significantly increase the expression of P53 in endometrial cancer, inhibit the apoptosis marker BCL-2, and has no significant effect on the expression of the apoptosis-promoting protein BAX.

[0113] Example 4 Effect of PEITC on SF3A3

[0114] Experimental method: RT-PCR

[0115] Experimental steps: (1) Total RNA extraction

[0116] Carefully discard the culture medium of the six-well plate, and slowly add pre-cooled PBS along the side wall of the six-well plate to wash twice. After discarding the residual PBS, add 1mL / well Trizol lysis buffer, bathe on ice for 10 minutes, carefully blow off the lysed cell fragments with a pipette, aspirate the lysis buffer and add it to the prepared enzyme-free 1.5mL EP tube, and store it at -80℃ for future use. Take out the lysate from -80℃, add 200μL of chloroform to each tube, vortex and shake for 30s to make the mixed solution into an emulsion, and let it stand on ice for 5min; centrifuge at 12000g / min in a centrifuge with a preset temperature of 4℃ for 15min, gently take out the EP tube, and the solution in the tube will be clearly stratified: the upper layer is a colorless RNA extract, the middle layer is a white DNA precipitate, and the lower layer is a pink protein extract. Carefully aspirate about 500μL of the supernatant, and add an equal volume of isopropanol, mix it upside down 10 times, and let it stand at room temperature for 10min; centrifuge at 12000g / min for 10min at 4℃, and a white precipitate can usually be seen at the bottom of the EP tube. Discard the supernatant; add 1mL of DEPC water to prepare 75% ethanol, gently invert, wash the precipitate, make the precipitate float but do not disperse it, then carefully aspirate and discard the 75% ethanol, dry the EP tube at room temperature for 20min until the bottom precipitate becomes transparent, and then add 20μL Treat the water with DEPC, dissolve in a metal bath at 50°C for 10 min, and test the RNA concentration after thorough mixing.

[0117] (2) RNA reverse transcription

[0118] After the total RNA was dissolved evenly, the RNA concentration was measured using NanoDrop 2000 and diluted to 0.5 μg / μL with DEPC-treated water. 10 μL of DEPC-treated water, 2 μL of RNA diluent and 4 μL of 4×gDNA were added to the PCR eight-tube, sealed, centrifuged and mixed, and then placed in the Bio-Rad My Cycler PCR instrument and reverse transcribed according to the reaction program of 25℃-10min, 42℃-30min, and 85℃-5min.

[0119] (3) Real-time fluorescence quantitative PCR

[0120] Dilute 20 μL of cDNA obtained by reverse transcription with DEPC water to 250 μL and prepare cDNA diluent and mix well for use. Take an enzyme-free PCR plate, add 11.4 μL of SYBR Green Master Mix (premixed with ROX Reference Dye 1), 1 μL of primer (concentration of 5 μM) and 8.6 μL of cDNA diluent to each well, and prepare a polymerase chain reaction system with a volume of 20 μL per well. Each sample is added to three replicate wells; use fluorescent quantitative PCR optical sealing film to seal the replicate wells, and centrifuge the system instantly. Set the PCR reaction program according to Table 3, and load the sample on the machine for reaction detection.

[0121] Table 3 PCR reaction procedure

[0122]

[0123] Figure 4 The results showed that PEITC did not affect the expression of SF3A3.

[0124] Western Blot

[0125] Figure 4 The results showed that there was no significant change in SF3A3 protein in endometrial cancer after PEITC drug treatment.

[0126] Growth curve experiment

[0127] (1) Take cells that are in good growth state and in the logarithmic phase, digest and centrifuge them, count them after resuspending them, and dilute them into 1-2×10 5 Take the required volume of cell suspension, resuspend it in complete medium, pipette evenly, and add 2000 μL / well, 20×10 per well. 4 Cells were inoculated into 6-well plates and cultured in a constant temperature CO2 incubator.

[0128] (2) After 24 h of culture, replace the culture medium with fresh low-serum medium.

[0129] The experimental results are as follows Figure 5 As shown, ISHIKAWA and KLE cells were given siRNA and stably transfected cell lines for 3 days, and then plated in 96-well plates with 2000 cells per well. After adhesion, they were treated with drug PEITC (75 μM), and their growth activity was measured using a CCK8 kit after 72 hours. Figure 5 The results showed that SF3A3 could inhibit the proliferation of endometrial cancer cells, and after knocking down SF3A3, the inhibitory effect of PEITC on endometrial cancer disappeared, indicating that PEITC exerts its anti-tumor effect through SF3A3.

[0130] Example 4 Effect of PEITC on SF3A3 in vivo

[0131] Construction of nude mouse xenograft tumor model

[0132] (1) Modeling and drug administration methods:

[0133] KLE cells in the vigorous growth stage were taken for inoculation, with 2.0×10 6 / 100μL cell suspension was inoculated subcutaneously in the right axilla of nude mice under sterile conditions. The diameter of the xenograft tumor in nude mice was measured with a vernier caliper. When the tumor grew to 100mm 3 The animals were then randomly divided into si-NC, si-SF3A3 and PEITC groups, which were injected intratumorally with si-NC and si-SF3A3 small interfering reagents, respectively. The PEITC group was gavaged with PEITC 100 μmol / kg. The tumor volume was measured three times a week, and the mouse body weight was also weighed at the same time of each measurement.

[0134] (2) Detection indicators and calculation methods:

[0135] a) Tumor volume (TV), calculated as follows: TV = 1 / 2 × a × b 2 , where a and b represent the length and width respectively.

[0136] b) Relative tumor volume (RTV), calculated as: RTV = T V1 / T V0 Where T V0 is the tumor volume at the time of drug administration (d0), TV t is the tumor volume at each measurement.

[0137] c) Relative tumor proliferation rate T / C (%)

[0138] The calculation formula is: T / C (%) = T RTV / C RTV ×100

[0139] T RTV :RTV in treatment group; C RTV : Negative control group RTV.

[0140] The relative tumor proliferation rate T / C (%) was used as the evaluation index of antitumor activity.

[0141] The experimental results suggest that PEITC can act as an inhibitor of SF3A3 to significantly inhibit tumor growth and has little effect on the weight of mice.

[0142] Construction of SF3A3 nude mouse transplant tumor model:

[0143] Control, OE-SF3A3 cells were inoculated, 2.0×10 per mouse 6 / 100μL cell suspension was inoculated subcutaneously in the right axilla of nude mice under sterile conditions. The diameter of the xenograft tumor in nude mice was measured with a vernier caliper. When the tumor grew to 100mm 3 The animals were then randomly divided into groups and the tumor volume was measured three times a week, and the mice were weighed at the same time for each measurement.

[0144] (2) Detection indicators and calculation methods:

[0145] a) Tumor volume (TV), calculated as follows: TV = 1 / 2 × a × b 2 , where a and b represent the length and width respectively.

[0146] b) Relative tumor volume (RTV), calculated as: RTV = T V1 / T V0 Where T V0 is the tumor volume at the time of drug administration (d0), TV t is the tumor volume at each measurement.

[0147] c) Relative tumor proliferation rate T / C (%)

[0148] The calculation formula is: T / C (%) = T RTV / C RTV ×100

[0149] T RTV :RTV in treatment group; C RTV : Negative control group RTV.

[0150] The relative tumor proliferation rate T / C (%) was used as the evaluation index of antitumor activity.

[0151] The experimental results suggest that overexpression of SF3A3 can significantly promote the growth of endometrial cancer.

[0152] Western Blot

[0153] Figure 6 The results showed that SF3A3 was significantly expressed in the overexpression group.

[0154] In summary, PEITC has a good anti-tumor effect. Further studies have found that PEITC has growth inhibition and induction of tumor cell apoptosis in endometrial cancer. In addition, PEITC can be used as an inhibitor of SF3A3 to play an anti-tumor role in endometrial cancer, and has a good therapeutic effect in vivo with low toxic side effects, suggesting that it may have great potential in the development of targeted drugs.

Claims

1. The application of phenethyl isothiocyanate as the only active ingredient in the preparation of anti-endometrial cancer drugs.

2. The use according to claim 1, characterized in that: Phenyl isothiocyanate targets and inhibits the activity of SF3A3.

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