CN115944617A

CN115944617A - Application of natural phenethylisothiocyanate in cruciferous vegetables in preparation of anti-endometrial cancer drug

Info

Publication number: CN115944617A
Application number: CN202211575619.6A
Authority: CN
Inventors: 胡丽丹; 陈祥军; 余威
Original assignee: Childrens Hospital of Zhejiang University School of Medicine
Current assignee: Childrens Hospital of Zhejiang University School of Medicine
Priority date: 2022-12-07
Filing date: 2022-12-07
Publication date: 2023-04-11

Abstract

The invention discloses application of a naturally-occurring phenethyl isothiocyanate in cruciferous vegetables to an anti-endometrial cancer medicament, wherein the phenethyl isothiocyanate can play a role in resisting endometrial cancer through targeting SF3A3, can inhibit survival of endometrial cancer cells, inhibit proliferation capacity of the endometrial cancer cells, can induce cycle retardation and apoptosis of the endometrial cancer cells, and has a good application prospect in preparation of anti-tumor medicaments.

Description

Application of natural phenethylisothiocyanate in cruciferous vegetables in preparation of anti-endometrial cancer drug

Technical Field

The invention relates to a new application of a medicament, in particular to an anti-endometrial cancer medicament application of phenethylisothiocyanate naturally existing in cruciferous vegetables, and the anti-endometrial cancer medicament application plays an anti-tumor role aiming at a new target SF3A 3.

Background

Cancer is a disease with extremely high mortality in the world, the morbidity is continuously increased, and the latest global cancer statistical report shows that 1910 ten thousands of new cancer cases and 870 ten thousands of new cancer cases occur in 2018. At present, the treatment methods for cancer mainly include chemical drug therapy, radiotherapy, surgical resection and the like, and the treatment methods usually cause damage to normal cells while killing tumor cells, so that the side effects are great. In recent years, a targeted therapy is gradually developed, and the therapy treats specific sites closely related to tumors, so that tumor cells die specifically without affecting normal tissue cells around the tumors, toxic and side effects are effectively reduced, and the survival rate of cancer patients is greatly improved. Endometrial Cancer (EC) is one of the most common female diseases as Endometrial cancer, and is also the fourth most common cancer in women. Although the majority of women with endometrial cancer are diagnosed with early stage disease and the prognosis is good. However, there is still a poor prognosis in 15-20% of patients and a high risk of distant metastasis. Currently, the primary treatment modalities for endometrial cancer are surgery, including total abdominal or laparoscopic hysterectomies and bilateral salpingo-oophorectomy. Specific biomarkers for endometrial cancer have not been identified. In addition, the combination of cisplatin, doxorubicin, and paclitaxel is the most effective chemotherapeutic regimen reported in advanced or recurrent endometrial cancer. However, chemotherapeutic drugs often have major toxic side effects. Thus, a thorough understanding of the molecular mechanisms underlying the development and progression of endometrial cancer may help to develop new therapies to improve treatment outcome and prognosis in patients with endometrial cancer. The study of the molecular mechanisms of endometrial cancer has helped to improve target specific therapy. Among them, SF3A3 has been demonstrated to exert an antitumor effect in various cancers as an important target related to tumors, and therefore, drugs designed for SF3A3 as a target are expected to exert great potential in the development of antitumor drugs.

According to the report of the world health organization, the tumor is the second leading cause of death in the world, the development of the anti-tumor drug is rapid since the forty years in the 20 th century, the number of the anti-tumor drugs in the current domestic and foreign clinical experiments is up to 100, and although the anti-tumor drug has been developed in a long time after the development of nearly 70 years, the problems of cytotoxicity, drug resistance of the tumor and the like still exist. The anticancer drugs mainly comprise cytotoxic micromolecular drugs, natural product anticancer drugs and molecular targeted drugs, paclitaxel is taken as a representative, compounds derived from natural products occupy an important position in the research and development of the antitumor drugs, abundant traditional Chinese medicinal materials are treasury of the antitumor drugs, and the discovery of antitumor natural active substances by the traditional Chinese medicinal materials is also an important subject of the research of modern traditional Chinese medicines.

Phenylisothiocyanate (PEITC) is naturally occurring phenylisothiocyanate in vegetables of the family Brassicaceae, and is a chemopreventive agent. There is currently no study on this compound for the treatment of endometrial cancer.

Disclosure of Invention

The invention aims to: aiming at the existing problems, the invention aims to provide an anti-endometrial cancer drug application of phenethylisothiocyanate naturally existing in cruciferous vegetables, wherein the phenethylisothiocyanate plays an anti-tumor role through SF3A 3.

The technical scheme is as follows: the invention provides application of phenethyl isothiocyanate in preparing an anti-endometrial cancer medicament, wherein the structural formula of the phenethyl isothiocyanate is shown as a formula I:

the application is that the isothiocyanic acid phenethyl ester can inhibit the activity of SF3A3 in a targeted way.

The application of the phenethylisothiocyanate inhibits survival of tumor cells, inhibits cloning formation of the tumor cells, inhibits proliferation capacity of the tumor cells, and induces apoptosis of the tumor cells.

Application of phenethylisothiocyanate as the only active ingredient in preparing anti-endometrial cancer drugs.

A pharmaceutical composition is prepared from phenethylisothiocyanate as the only active component and pharmaceutically acceptable adjuvants.

The application of the pharmaceutical composition in preparing anti-endometrial cancer drugs.

Application of phenethylisothiocyanate in preparing medicines for treating SF3A3 gene related diseases is provided.

The SF3A3 gene related disease is endometrial cancer.

The above-mentioned isothiocyanatobenzene ethyl ester has a molecular formula of C ₆ H ₅ CH ₂ CH ₂ NCS, code PEITC, chemical name: 2-phenylethyl isothiocyanate.

The PEITC has a cytotoxic effect on endometrial cancer cells, and further research finds that the PEITC has the effects of growth inhibition, cycle blocking and tumor cell apoptosis induction on endometrial cancer cell lines at different concentrations.

PEITC can inhibit tumor progression by preventing the cell cycle and inducing apoptosis in various human cancer cell models. Currently, there are several ongoing clinical trials exploring the role of PEITC in various tumors, either as a single drug or in combination with chemotherapy, radiotherapy, targeted drugs or other immunotherapeutic drugs. In addition, the search for related drugs for treating endometrial cancer based on related biomarkers also has certain clinical significance. Based on bioinformatics analysis, the inventor finds that Phenethylisothiocyanate (PEITC) can be a potential SF3A3 targeting drug and is expected to have a certain therapeutic effect on endometrial cancer. In addition, the compound has been verified in various models, and shows a certain clinical prospect.

Has the advantages that: the PEITC is a natural product from cruciferous vegetables, has a good anti-tumor effect in vivo, can play an anti-tumor role through an anti-tumor key target SF3A3, kills endometrial cancer tumor cells, and has research and development values of anti-tumor drugs.

Drawings

FIG. 1 is a graph showing that SF3A3 has a prognostic adverse effect in endometrial cancer;

fig. 2 is an effect of SF3A3 survival in endometrial cancer;

FIG. 3 is the effect of PEITC against endometrial cancer cells;

FIG. 4 is a graph to investigate the effect of PEITC on SF3A3 gene and protein;

FIG. 5 is a graph of the anti-tumor effect of PEITC on ISHIKAWA and KLE endometrial cancer cells after si-SF3A3 stem administration;

FIG. 6; the in vivo effect of SF3A3 and PEITC on ISHIKAWA and KLE endometrial cancer cells was explored.

Detailed Description

The present invention will be described in detail below with reference to examples.

The source of the drug is as follows: purchased from sigma;

cell line:

IAHIKAWA cell: human endometrial cancer cell lines, which grow adherently, were purchased from Shanghai cell bank of Chinese academy of sciences;

KLE cells: human endometrial cancer cell lines, growing adherently, were purchased from Shanghai cell bank, chinese academy of sciences.

Animals: nude mice, 18-22g,4-6 weeks, purchased from slaike, were housed in huajia chi school district, university, zhejiang, under the animal care center regulations, and the experimental protocol for mice was approved by the animal ethics committee, university, zhejiang.

Example 1 growth inhibition of SF3A3 on endometrial cancer cells

Biological information analysis

The patent discloses TCGA database (https:// www. Cancer. Gov/about-nci/organization/ccg/research/structure-genes/TCGA), human protein gene database (https:// www. Protein atlas. Org.), survival curve analysis database (http:// kmplot. Com), and database analysis, and the results in FIG. 1 show that SF3A3 is associated with poor prognosis of endometrial cancer, and SF3A3 exhibits high expression in endometrial cancer samples compared with paracarcinoma tissues.

Western Blot

(1) Protein sample preparation

Firstly, uniformly spotting cells in a 6-well plate, and adding PEITC (3, 15 and 75 mu M) and a medicament for treatment for 72h after the cells are adhered to the wall; the cell sample was taken out from the carbon dioxide incubator, the cell density state was observed under a microscope, and after the medium was aspirated, the cell sample was washed twice with 4 ℃ precooled 1 × PBS buffer. The suspension cells were centrifuged and then precooled at 4 ℃ in 1 XPBS buffer

And washing twice. Add 100. Mu.L of cell lysate premixed phosphatase and protease inhibitors and place gently on ice for lysis for 20min. After scraping the cells with a clean spatula, the cells were collected in a 1.5mL centrifuge tube, centrifuged for 15min at 12000rpm at 4 ℃ in a high speed refrigerated centrifuge, and the cell lysate was transferred to the 1.5mL centrifuge tube with a gun. The entire operation was performed on ice as much as possible and the volume of the lysate was quantified using a pipette. The suspension was lysed directly in a 1.5mL centrifuge tube. And (4) subpackaging the centrifuged supernatant into centrifuge tubes and preserving at-20 ℃.

(2) Protein quantification (BCA method protein quantification)

A standard of Bovine Serum Albumin (BSA) for gradient dilution was prepared, and a clean 1.5mL centrifuge tube was prepared and numbered. Precisely pipetting 7. Mu.L of the protein sample and the standard, and adding ultrapure water to dilute 10-fold to 70. Mu.L. A BCA working solution (working solution A: working solution B = 50. 20 mu L of diluted protein sample and standard dilution are added into a 96-well plate (3 wells for each sample), 200 mu L of mixed BCA working solution is added, and the mixture is gently shaken up. The 96-well plate was placed in an oven at 37 ℃ and allowed to stand for 15min. And measuring the OD value at the position where the excitation wavelength detected by the full-wavelength microplate reader is 562 nm. A standard curve was established and the protein concentration was calculated. Adding 5 × sample buffer solution of 1/5 volume of the sample, mixing by vortex, heating in metal bath at 100 deg.C for 10min for denaturation, and storing at-20 deg.C or-80 deg.C.

(3) SDS-Polyacrylamide gel electrophoresis

Selecting corresponding separation gel concentration (table 1) according to the molecular weight of the target protein, fixing the washed gel-making glass plate in a gel preparation device, pouring the prepared separation gel according to table 2 after checking and confirming that no liquid leakage exists, slightly adding an ultrapure water protective layer, adding the mixed solution of the concentrated gel according to table 2 after the separation gel is solidified, inserting comb teeth with corresponding specifications, paying attention to no bubbles, and waiting for the solidification of the concentrated gel for later use. And (3) fixing the prepared glue on the electrophoresis clip A, adding a newly configured 1 Xelectrophoresis liquid buffer solution, slightly and vertically pulling out the comb teeth, and adding the sample and the pre-dyed Marker into the corresponding pore channel by using a liquid transfer device. Electrophoresis program: 80V,40min;120V,50min.

(4) Wet rotor

After electrophoresis is finished, carefully prying the glass plate, taking out gel after electrophoretic separation, cutting off the gel where the needed target protein is located according to the position of a prestained Marker, clamping a wet rotating clamp according to the sequence of positive electrode (white), sponge, filter paper, PVDF (polyvinylidene fluoride) membrane, gel, filter paper, sponge and negative electrode (black), placing the PVDF membrane in methanol for activation for 30s before use, evacuating bubbles between the gel and the PVDF membrane in the process of clamping the wet rotating clamp, then placing the PVDF membrane in a wet rotating tank according to the corresponding directions of the positive electrode and the negative electrode, adding 1 multiplied wet rotating liquid pre-cooled at 4 ℃ and an ice box, carrying out ice bath in the wet rotating tank to maintain a low-temperature state, and rotating the membrane under the condition of constant current of 0.32A. The wet-turn time was determined according to the protein molecular weight at 1 min/kDa. SF3A3 target protein can be used for wet transformation of liquid, the membrane transformation time is 0.64A constant current, and the condition is 60 min.

(5) Blocking and incubation of antibodies

After the wet-spinning was completed, the PVDF membrane protein band was carefully removed, placed in a protein incubation cassette, added to blocking solution (5% BSA), and blocked on a shaker at room temperature for 1 hour. After the blocking, the blocking solution was recovered, primary antibody dilution was added, and the mixture was incubated for 12 hours at 4 ℃ in a shaker. After the primary antibody incubation is finished, recovering the primary antibody diluent, adding 1 XTSST, and placing on a shaker to wash the primary antibody for 5min each time for 3 times; discarding 1 × TBST, adding secondary antibody diluent, incubating for 1h at 4C in a shaking table, recovering the secondary antibody diluent after the secondary antibody incubation is finished, adding 1 × TBST, and removing unbound secondary antibody diluent in a shaking table for 5min each time for 4 times. After the secondary washing resist is finished, exposure is carried out.

(6) Chemiluminescent imaging

And (3) preparing ECL (solution A: solution B = 1: 1) for exposure, carefully taking out a PVDF membrane protein band, placing the PVDF membrane protein band on an imaging plate in a Bio-Rad full-automatic gel imager, opening a program, adjusting parameters such as focal brightness, storing a template, dripping the prepared light-emitting solution, clicking for exposure, and storing data.

TABLE 1 optimum separation Range for separation gels of different concentrations

Adding 100 mu L of 10% ammonium persulfate solution into the separation gel and 10 mu L of TEMED solution into the small gel; the concentrated gel was mixed with 30. Mu.L/gel of 10% ammonium persulfate solution and 5. Mu.L/gel of TEMED solution.

TABLE 2 separation gel/concentrated gel formulations

The results in FIG. 1 show that SF3A3 protein is significantly elevated in cancer samples from clinical samples.

Construction of endometrial cancer cell line with knockdown of over-expressed SF3A3

Cell lines: human endometrial cancer cell line ISHIKAWA cells, human endometrial cancer cell line KLE cells;

the experimental method comprises the following steps: constructing a si-SF3A3 cell strain; SF3A3 knockdown overexpression stable transgenic cell lines; the CCK8 method;

the experimental steps are as follows:

(1) Taking a bottle of cells in exponential growth phase, adding 0.25wt% of trypsin digestive juice, digesting to make adherent cells fall off, counting (2-4) × 10 ⁴ Preparing cell suspension per mL;

(2) Inoculating the cell suspension on a 96-well plate at 90 μ L/well, and placing in constant temperature CO ₂ Culturing in an incubator;

(3) After 24 hours, adding si-SF3A3 (10 mu L/hole), changing the liquid after 6-8 hours, and detecting by adopting a CCK8 kit after culturing for 48-72 hours;

(4) Adding the CCK8 reagent into a 96-well plate, and incubating for 2 hours in an incubator at a concentration of 10 mu L/well;

(5) The absorbance of each well was measured at a wavelength of 450nm using an enzyme linked immunosorbent assay and the corresponding cell inhibition was calculated.

The results of the experiment are shown in FIG. 2, ISHIKAWA and KLE endometrial cancer cells were treated with siRNA. SF3A3 shows obvious effect of inhibiting survival of endometrial cancer cells on the endometrial cancer cells, and shows stable knocking-down or over-expression of SF3A3 protein or gene on two endometrial cancer cells which are stably knocked down or over-expressed.

Growth curve experiment

(1) Taking the growth state well in logarithmDigesting and centrifuging the cells in the stage, counting the cells after resuspending the cells, and diluting the cells to 1-2X 10 ⁵ one/mL, the desired volume of cell suspension was taken, resuspended in complete medium, and after pipetting well, at 500. Mu.L/well, 5X 10 per well ⁴ Inoculating the cells in a 24-well plate, and placing in constant temperature CO ₂ Culture in dressing box

(2) After 24h of culture, fresh low serum medium was replaced and randomized into 5 groups: negative control group, administration group (low, medium and high dose), and positive control group, and culturing in dressing box.

(3) Cells were digested and counted once a day for 6 consecutive days.

Results of the experiment shown in FIG. 2, ISHIKAWA and KLE cells were administered siRNA and stably transfected cell lines were constructed for 6 days and counted daily to plot growth curves. The results show that SF3A3 is able to inhibit proliferation of endometrial cancer cells.

As can be seen from fig. 2, SF3A3 can significantly inhibit the growth of endometrial cancer cells in both endometrial cancer cells.

Plate clone formation experiment

(1) And taking the cells which are in good growth state and in logarithmic phase, digesting and centrifuging, counting after cell resuspension, taking the cell suspension with the corresponding volume in a complete culture medium, blowing and uniformly mixing, and adding the cell suspension into a 6-hole plate. 2mL of cell suspension per well, containing 1000 cells. Shaking up and down, left and right to form six orifice plates, placing in constant temperature CO ₂ And (5) applying in a box for culturing.

(2) After 24h of culture, fresh complete medium was replaced and randomly divided into 5 groups: negative control group, administration group (low, medium, high dose), positive control group; the culture is continued in a coating box, the culture medium is changed every 3 to 4 days until the single cell mass contains at least 50 cells, and the culture is stopped.

(3) Taking out a six-hole plate, discarding the supernatant, washing with PBS once, adding 4% paraformaldehyde into each hole, fixing at room temperature for 30min, discarding the fixing solution, adding 1mL of 0.8% crystal violet dye solution into each hole, and dyeing at room temperature in a dark place for 30min; and finally, washing the bottom of the six-hole plate by using tap water with small flow, washing away the crystal violet dye solution, and taking a picture after air drying.

Results of the experiment as shown in fig. 2, in the colony formation experiment, ISHIKAWA cells and KLE cells were administered siRNA, respectively, 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 show that the clonality of endometrial cancer cells is inhibited with SF3A3 knock-down.

As can be seen in fig. 2, SF3A3 significantly inhibited proliferation of endometrial cancer cells.

Example 2 in vitro inhibition ratio IC of PEITC to human endometrial cancer cell line ₅₀

the experimental method comprises the following steps: the CCK8 method;

the experimental steps are as follows:

(3) After 24 hours, the test drug PEITC (10. Mu.L/well) was added and cultured for 72 hours;

(4) Adding the MTT reagent into a 96-well plate, and incubating for 2 hours in an incubator at a concentration of 20 mu L/well;

The results of the experiment are shown in FIG. 3, and ISHIKAWA and KLE endometrial cancer cells were treated with a final concentration of 0.1,0.3,1,3, 10, 30, 100, 300. Mu.M PEITC. The cell survival rate is detected by a CCK8 method, and the result is expressed by half lethal concentration IC ₅₀ Standard deviation (n = 6) indicates that with increasing dose, a clear indication is shown on both endometrial cancer cellsInhibiting the survival of endometrial cancer cells, and in two endometrial cancer cells KLE and ISHKAWA, IC of PEITC ₅₀ 24.78. Mu.M and 58.21. Mu.M, respectively.

Example 3 growth inhibition of endometrial cancer cells by PEITC

The experimental steps are as follows:

plate clone formation experiment

(3) Taking out a six-hole plate, discarding the supernatant, washing with PBS once, adding 4% paraformaldehyde into each hole, fixing at room temperature for 30min, discarding the fixing solution, adding 1mL of 0.8% crystal violet dye solution into each hole, and dyeing at room temperature in a dark place for 30min; and finally, washing the bottom of the six-hole plate by using tap water with small flow, washing off the crystal violet dye solution, and taking a picture after air drying.

As shown in FIG. 4, in the colony formation experiment, after giving PEITC interventions at doses of 3. Mu.M, 15. Mu.M and 75. Mu.M to ISHIKAWA cells and KLE cells, respectively, for 14 days, the clones were stained with crystal violet. The results show that the clonality of endometrial cancer cells is inhibited with increasing dose.

As shown in figure 3, the compound PEITC can obviously inhibit the proliferation of endometrial cancer cells.

Growth curve experiment

(1) Collecting cells with good growth state and log phase, digesting, centrifuging, re-suspending, counting, and diluting to 1-2 × 10 ⁵ Per mL, taking the desired bodyThe cell suspension was resuspended in complete medium, and after pipetting well, the volume was 5X 10 per well at 500. Mu.L/well ⁴ Inoculating the cells in a 24-well plate, and placing in constant temperature CO ₂ Culture in dressing box

(3) Cells were digested and counted once a day for 6 consecutive days.

Experimental results as shown in figure 5, ISHIKAWA and KLE cells were administered PEITC intervention treatments at 3 μ M, 15 μ M, 75 μ M doses, respectively, for 6 days and counted daily to plot growth curves. The results show that the proliferative capacity of endometrial cancer cells is inhibited with increasing dose.

According to the graph 3, the compound PEITC can obviously inhibit the growth of endometrial cancer cells in two endometrial cancer cells.

Method for detecting apoptosis by Annexin V/PI double staining method

The experimental steps are as follows:

(1) Cell inoculation and administration, and the method is the same as cell cycle detection;

(2) Cell collection: taking 15mL of a centrifuge tube, collecting cell supernatant, washing with 4 ℃ precooled 1 xPBS, collecting supernatant in the same centrifuge tube, digesting cells with EDTA-free pancreatin, stopping digestion with the cell supernatant after digestion (surely noting that excessive digestion is not needed so as to avoid false positive results), blowing down the cells, collecting the cells in the same centrifuge tube, centrifuging at 1000rpm for 5min, discarding the supernatant, adding 1mL of precooled PBS for resuspension, transferring to 1.5mL of the centrifuge tube, centrifuging, discarding the supernatant, repeatedly washing with PBS once, centrifuging, discarding the supernatant (paying attention to discard the supernatant, and sleeving a small gun head with a large gun head);

(3) Dyeing: adding 100 μ L of 1 × binding buffer into each tube, blowing the cells evenly, adding 5 μ L of FITC Annexin V and 5 μ L of PI respectively, vortex mixing evenly, and reacting for 15min in a dark place;

(4) And (3) computer detection: and adding 400 mu L of 1 × binding buffer into each tube, blowing uniformly, sucking into a flow tube, and detecting, wherein the excitation light of annexin V-FITC is green fluorescence, and the excitation light of PI is red fluorescence (whole light-shielding operation).

Experimental results as shown in fig. 6, cells were subjected to PEITC intervention at doses of 3 μ M, 15 μ M, and 75 μ M, respectively, for 48 hours, and analyzed for apoptosis by PI staining and flow cytometry. The results show that the compound can induce apoptosis of human endometrial cancer cells ISHIKAWA and KLE with increasing dosage. As shown in the figure, the compound PEITC can cause human endometrial cancer cells ISHIKAWA and apoptosis in G2/M stage.

According to the graph 3, the compound PEITC can induce the endometrial cancer cells to undergo apoptosis.

Western Blot

As shown in figure 3, compound PEITC was able to significantly increase P53 expression in endometrial cancer and inhibited the apoptosis marker BCL-2 with no significant effect on the expression of the apoptosis-promoting protein BAX.

Example 4 Effect of PEITC on SF3A3

The experimental method comprises the following steps: RT-PCR

The experimental steps are as follows: (1) Total RNA extraction

Carefully discarding the six-well plate culture medium, slowly adding precooled PBS along the side wall of the six-well plate for washing twice, discarding the residual PBS, adding 1 mL/well Trizol lysate, carrying out ice bath for 10min, carefully blowing the cell fragments after lysis down by using a pipettor, sucking the lysate, adding the lysate into a previously prepared EP tube without enzyme, and preserving at-80 ℃ for later use. Taking out lysate from-80 deg.C, adding 200 μ L chloroform into each tube, vortex shaking for 30s to make the mixed solution into emulsion, and standing on ice for 5min; centrifuging for 15min at a preset temperature of 4 ℃ by using a centrifuge at 12000g/min, gently taking out the EP tube, and obviously layering the solution in the tube: the upper layer is colorless RNA extract, the middle layer is white DNA precipitate, the lower layer is pink protein extract, carefully absorb about 500 muL of supernatant, add isovolume isopropanol, reverse and mix evenly for 10 times, and stand for 10min at room temperature; centrifuging at 4 deg.C at 12000g/min for 10min, wherein white precipitate is usually observed at the bottom of EP tube, and discarding the supernatant; adding 1mL of DEPC water to prepare 75% ethanol, slightly inverting, washing the precipitate to enable the precipitate to float without dispersing, then carefully absorbing and discarding the 75% ethanol, drying the EP tube at room temperature for 20min until the precipitate at the bottom is transparent, then adding 20 mu L of DEPC treated water, dissolving for 10min in a metal bath at 50 ℃, fully mixing and detecting the RNA concentration.

(2) Reverse transcription of RNA

After the total RNA was dissolved uniformly, the RNA concentration was determined using NanoDrop 2000 and diluted to 0.5. Mu.g/. Mu.L with DEPC-treated water. Adding PCR eight-connecting tube according to DEPC treated water 10 μ L, RNA diluent 2 μ L and 4 XgDNA 4 μ L system, sealing, instantly centrifuging, mixing, and reverse transcribing in Bio-Rad My Cycler PCR instrument according to reaction program of 25-10min, 42-30min, 85-5 min.

(3) Real-time fluorescent quantitative PCR

20 mu L of cDNA obtained by reverse transcription is diluted to 250 mu L by DEPC water to prepare cDNA diluent and mixed evenly for later use. Taking an enzyme-free PCR plate, adding 11.4 mu L of SYBR Green Master Mix (premixed ROX Reference Dye 1), 1 mu L of primer (with the concentration of 5 mu M) and 8.6 mu L of cNDA diluent into each hole to prepare a polymerase chain reaction system with the volume of 20 mu L each hole, and adding three times of holes into each sample; and (3) sealing the multiple holes by using a fluorescent quantitative PCR optical sealing plate membrane, and performing instantaneous centrifugation on the mixed system. The PCR reaction program was set up as in Table 3, and the samples were tested by machine reaction.

TABLE 3 PCR reaction procedure

The results in FIG. 4 show that PEITC does not affect the expression of SF3A 3.

Western Blot

The results in FIG. 4 show that endometrial cancer SF3A3 protein is not significantly changed after PEITC treatment.

Growth curve experiment

(1) Collecting cells with good growth state and log phase, digesting, centrifuging, re-suspending, counting, and diluting to 1-2 × 10 ⁵ one/mL, the desired volume of cell suspension was taken, resuspended in complete medium, and after pipetting well, at 2000. Mu.L/well, 20X 10 per well ⁴ Cells inoculated toPlacing constant temperature CO in 6-hole plate ₂ Culture in dressing box

(2) After 24h of incubation, fresh low serum medium was replaced.

The results are shown in FIG. 5, and the ISHIKAWA and KLE cells were administered siRNA and stably transfected cell lines for 3 days, plated in 96-well plates at 2000 cells per well, and after attachment, treated with PEITC (75 μ M), and their growth viability was measured 72 hours later using CCK8 kit. The results of fig. 5 show that SF3A3 is able to inhibit proliferation of endometrial cancer cells, and that after knockdown of SF3A3, the inhibitory effect of PEITC on endometrial cancer is abolished, indicating that PEITC exerts an anti-tumor effect through SF3 A3.

Example 4 in vivo Effect of PEITC on SF3A3

Construction of nude mouse xenograft tumor model

(1) The molding and administration method comprises the following steps:

KLE cells in the vigorous growth stage were inoculated to 2.0X 10 cells per mouse ⁶ A100. Mu.L cell suspension was inoculated subcutaneously into the right axilla of nude mice under sterile conditions. Measuring the diameter of the transplanted tumor with vernier caliper when the tumor grows to 100mm ³ Animals were then randomized into groups. The mice are divided into a si-NC group, a si-SF3A3 group and a PEITC group, small interfering agents of the si-NC and the si-SF3A3 are respectively injected into tumors, the PEITC group is perfused with PEITC 100 mu mol/kg, the number of times of tumor volume measurement is 3 times per week, and the weight of the mice needs to be weighed simultaneously when each measurement is carried out.

(2) The detection index and the calculation method comprise the following steps:

a) Tumor Volume (TV) is calculated as: TV =1/2 × a × b ² Wherein a and b represent length and width, respectively.

b) Relative Tumor Volume (RTV), the formula is: RTV = T _V1 /T _V0 . Wherein T is _V0 When the medicine is administered in separate cages (d) ₀ ) Tumor volume, TV _t For the tumor volume at each measurement.

c) Relative tumor proliferation rate T/C (%)

The calculation formula is as follows: T/C (%) = T _RTV /C _RTV ×100

T _RTV : treatment group RTV; c _RTV : negative control group RTV.

The test results used the relative tumor proliferation rate T/C (%) as an index for evaluating antitumor activity.

The experimental result indicates that the PEITC can be used as an inhibitor of SF3A3 to obviously inhibit the growth of tumors and has little influence on the body weight of mice.

The nude mouse transplantation tumor model is constructed by the following steps of SF3A 3:

control, OE-SF3A3 cells were inoculated at 2.0X 10 cells per mouse ⁶ A100. Mu.L cell suspension was inoculated subcutaneously into the right axilla of nude mice under sterile conditions. Measuring the diameter of the transplanted tumor by using a vernier caliper for the nude mouse xenograft tumor until the tumor grows to 100mm ³ Thereafter, the tumor volume was randomly divided into 3 tumor volume measurements per week, and the weight of the mice was also measured for each measurement.

a) Tumor Volume (TV), calculated by the formula: TV =1/2 × a × b ² Wherein a and b represent length and width, respectively.

b) Relative Tumor Volume (RTV), the formula is: RTV = T _V1 /T _V0 . Wherein T is _V0 (d 0) tumor volume, TV, when administered in separate cages _t For the tumor volume at each measurement.

c) Relative tumor proliferation rate T/C (%)

The calculation formula is as follows: T/C (%) = T _RTV /C _RTV ×100

T _RTV : treatment group RTV; c _RTV : negative control group RTV.

The experiment result indicates that the overexpression of SF3A3 can obviously promote the growth of endometrial cancer.

Western Blot

The results in FIG. 6 show that SF3A3 is clearly expressed in the overexpression group.

In conclusion, the PEITC has a good anti-tumor effect, and further research shows that the PEITC has the effects of growth inhibition and tumor cell apoptosis induction in endometrial cancer. In addition, the PEITC can be used as an inhibitor of SF3A3, plays an anti-tumor role in endometrial cancer, has a good treatment effect in vivo, has low toxic and side effects, and suggests that the PEITC has great potential in the development of targeted drugs.

Claims

1. The application of phenethyl isothiocyanate in preparing anti-endometrial cancer drugs is disclosed, wherein the structural formula of the phenethyl isothiocyanate is shown as a formula I:

2. the use of claim 1, wherein the phenethylisothiocyanate is targeted to inhibit SF3A3 activity.

3. The use of claim 1, wherein said phenethylisothiocyanate inhibits tumor cell survival, inhibits clonogenic activity of tumor cells, inhibits proliferative capacity of tumor cells, and induces apoptosis of tumor cells.

4. Application of phenethylisothiocyanate as the only active ingredient in preparing anti-endometrial cancer drugs.

5. A pharmaceutical composition is characterized in that the pharmaceutical composition is prepared by taking phenethylisothiocyanate as the only active ingredient and adding pharmaceutically acceptable auxiliary materials.

6. The use of the pharmaceutical composition of claim 5 in the preparation of an anti-endometrial cancer medicament.

7. Application of phenethylisothiocyanate in preparing medicaments for treating SF3A3 gene-related diseases.

8. The use of claim 7, wherein the SF3A3 gene associated disease is endometrial cancer.