CN115025226A - Application of PTGES expression inhibitor in preparation of medicine for improving sensitivity of tumor cells to chemotherapeutic medicine - Google Patents

Abstract

The invention discloses an application of a PTGES expression inhibitor in preparing a medicament for improving the sensitivity of tumor cells to chemotherapeutic medicaments. The invention discovers that the drug resistance of osteosarcoma to chemotherapeutic drugs, especially lobaplatin, can be weakened by knocking out or inhibiting the expression of PTGES, and suggests that the drug resistance of osteosarcoma to chemotherapeutic drugs can be overcome when an inhibitor or a knock-out reagent for synthesizing PTGES is combined with lobaplatin for application, so that the drug resistance of osteosarcoma to chemotherapeutic drugs can be overcome when the osteosarcoma is treated, and the PTGES has important guiding significance and broad prospects in clinical application.

Description

Application of PTGES expression inhibitor in preparation of medicine for improving sensitivity of tumor cells to chemotherapeutic medicine

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a PTGES expression inhibitor in preparation of a drug for improving sensitivity of tumor cells to chemotherapeutic drugs.

Background

Osteosarcoma is the most common primary malignant bone tumor, which is better in juvenile and has poor prognosis. The current treatment for osteosarcoma is a combination of active surgical resection with neoadjuvant chemotherapy. The platinum chemotherapeutic medicine is a medicine commonly used in osteosarcoma clinic and comprises cisplatin, oxaliplatin and lobaplatin. Lobaplatin is one of the third-generation platinum antitumor drugs, and has the advantages of less side effects and good antitumor activity. However, after several courses of treatment, relapse and metastasis remain a problem for the patient. Most patients who die of osteosarcoma exhibit significant chemoresistance.

Lipid metabolism plays a key role in signal transduction for many cellular activities and is of great interest as a key emerging effect in cancer cell behavior. Cancer cells use lipid metabolism to acquire energy and signaling molecules to proliferate, metastasize, and respond to various therapies. Furthermore, blocking cancer-associated adipocyte lipolysis and free fatty acid uptake has a beneficial effect on tumor suppression in preclinical animal models. With respect to chemotherapy resistance, there is increasing evidence that the hypoxic environment of tumors can trigger lipid metabolism and produce high levels of ATP, a key factor for chemotherapy resistance. However, personalized therapeutic guidelines related to lipid metabolism in osteosarcoma remain to be explored.

PTGES is a key enzyme in the arachidonic acid metabolic pathway to synthesize PGE2, catalyzes the conversion of PGH2 to PGE2, is significantly up-regulated in inflammatory tissues and tumors, and has been shown to be critical for tumorigenicity, migration and metastasis of non-small cell lung cancer cells. PTGES is associated with immunosuppression and pulmonary tumorigenesis in a Gprc5a knockout mouse model. PTGES/PGE2 signaling is currently believed to be critical for cell dryness, including expression of ABCG2 and EMT-related markers, and may induce expression of PD-L1 in macrophages and MDSCs, inhibiting tumor immunity. However, the role of PTGES in tumor chemotherapy resistance is not clear. Therefore, exploring the role of PTGES in platinum drug chemotherapy of osteosarcoma and developing drugs for sensitizing osteosarcoma chemotherapy are of great significance in treating patients with osteosarcoma.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the application of the PTGES expression inhibitor in preparing the medicament for improving the sensitivity of tumor cells to chemotherapeutic medicaments, and the response rate of osteosarcoma patients to lobaplatin can be improved and the generation of medicament resistance can be reduced by inhibiting or knocking out PTGES genes.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

the PTGES is used as a target point to prepare a medicament for improving the sensitivity of tumor cells to chemotherapeutic medicaments.

In addition, the application of the PTGES expression inhibitor in preparing the medicament for improving the sensitivity of tumor cells to chemotherapeutic medicaments is also provided.

Further, the drug improves the sensitivity of tumor cells to chemotherapeutic drugs by inhibiting the expression of PTGES.

Further, the tumor includes osteosarcoma.

Furthermore, the chemotherapy drug is a platinum chemotherapy drug.

Further, the chemotherapeutic agent is lobaplatin.

Further, PTGES expression inhibitors include shRNA, siRNA, small molecule compounds or monoclonal antibodies.

A combined chemotherapy medicine comprises PTGES expression inhibitor and platinum chemotherapy medicine.

The invention has the beneficial effects that:

the invention discovers that the drug resistance of osteosarcoma to chemotherapeutic drugs, particularly lobaplatin, can be weakened by knocking out or inhibiting the expression of PTGES, and suggests that the inhibitor or knock-out reagent for synthesizing PTGES can be combined with lobaplatin for application, so that the drug resistance of osteosarcoma to chemotherapeutic drugs can be overcome when the osteosarcoma is treated, and the invention has important guiding significance and wide prospect in clinical application.

Drawings

FIG. 1 is an IHC stain to detect expression of PTGES in lobaplatin-sensitive and drug-resistant osteosarcoma tissue; scale bar: 100 μm (magnification, 200 times), scale bar: 50 μm (magnification, 400 times); the violin plots show that the differences are significant.

FIG. 2 is a graph of the expression levels of PTGES mRNA and protein in osteosarcoma cells following lentiviral knockdown of PTGES; p < 0.001;

FIG. 3 shows the expression levels of activated cysteine protease 9, cysteine protease 3 and PARP after treatment of osteosarcoma cells transfected with shPTGES with lobaplatin; p <0.05, P < 0.01;

FIG. 4 is a graph showing the detection of apoptosis rate of osteosarcoma cells in different treatment groups treated with lobaplatin for 48 hours by flow cytometry; p <0.05, P < 0.001;

FIG. 5 shows TUNEL staining to detect apoptosis in knockdown PTGES-treated osteosarcoma cells treated with lobaplatin; scale bar: 100 μm;

FIG. 6 is a clone formation assay to examine the proliferative capacity of shPTGES-transfected and lobaplatin-treated osteosarcoma cells; p < 0.001;

FIG. 7 shows the activity of osteosarcoma cells treated with lobaplatin after transfection of shPTGES in CCK8 assay.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

Example 1 resistance of PTGES to osteosarcoma

Immunohistochemical staining is carried out on tumor samples of patients subjected to operations after osteosarcoma in 2019 to 2022 years in orthopedics department of Tangdu hospital, and the specific process is as follows:

tumor tissues were fixed with 10% formalin overnight at room temperature, dehydrated with graded ethanol (70%, 80%, 95% and 100%), and paraffin embedded and the tissues were cut into 2-3 μm sections. Paraffin sections were incubated overnight at 60 ℃ and deparaffinized to water: xylene 2 × 15min, 100% ethanol 2 × 5min, 95% ethanol 2 × 5min, 70% ethanol 2 × 5min, ddH ₂ O2X 5 min. Performing antigen retrieval by a microwave method, standing and cooling to room temperature; completely immersing the slices in a 3% hydrogen peroxide solution, and incubating for 15min at room temperature; PBS wash section, 2X 5 min; spin-drying, adding a sufficient amount of blocking solution (5% goat serum in PBS) to the tissue, and gently shaking to remove the blocking solutionFully covering the tissues, and incubating at room temperature for 15min in a wet box; 50 μ L of diluted PTGES primary antibody (Cayman, 1:50) was added dropwise to the tissue to completely cover the tissue and incubated overnight at 4 ℃ in a wet box; PBS wash section, 3X 5 min; dripping 50 mu L of universal ready-to-use secondary antibody for rabbit and mouse to the tissue, and incubating for 1h at 37 ℃ in a wet box; PBS wash section, 3X 5 min; dropwise adding 50 mu L of freshly prepared DAB chromogenic substrate liquid into the tissue, and incubating for 5-10min at room temperature; when the color development signal-to-noise ratio reaches the best, the slices are taken to be under a faucet and slowly washed for 1min by running water; hematoxylin lining staining, hydrochloric acid alcohol differentiation, dehydration and mounting, drying in a fume hood and microscopic examination.

Staining results were evaluated independently under an upright microscope (Olympus BX51), and 5 complementary fields were selected for each specimen and scored for the area of positive cell regions and the intensity of positive cell staining in each field. The scoring standard is as follows: a. positive area scoring: the number of positive cells is less than or equal to 5 percent, and the score is 0; 6% -20%, accounting for 1 minute; 21% -50%, 2 points; 51% -70%, 3 points; not less than 71 percent, and 4 minutes; b. staining intensity scoring: yellow, 1 point; brown yellow, 2 points; tan, 3 points. Composite score-positive area score x staining intensity score.

In fig. 1, the left side shows immunohistochemical detection results, which are the expression of PTGES in osteosarcoma specimens of the lobaplatin-treatment sensitive group and the lobaplatin-treatment drug-resistant group, respectively, and the upper part is a picture at 200-fold magnification, and the lower part is a picture at 400-fold magnification; the statistical results are shown on the right side, and the violin graph shows that the differences are significant.

As shown in figure 1, PTGES expression was enhanced in samples tolerated by the lobaplatin-treated patients compared to treatment-sensitive samples, with significant differences.

Example 2 construction of an osteosarcoma cell line with knockdown of PTGES

1. Culture of human osteosarcoma cells

Human osteosarcoma cell lines MG63 and SOSP-9607 were cultured in modified Eagle medium containing 10% fetal calf serum and RPMI-1640 medium respectively at 37 deg.C and 5% CO ₂ Cultured in a cell culture box.

2. Lentivirus infection

PTGES-knocked-down shpts lentiviral particles and random control lentiviral particles were supplied by shanghai gekhae gene ltd. Will be 1 × 10 ⁵ Osteosarcoma cells were seeded in 6-well culture plates and infected at a ratio of MOI (multiplicity of infection) to 100, with a virus dose (μ L) per well of MOI × number of cells/titer × 1000. Infected cells were then screened for 5 days in the presence of 4. mu.g/mL puromycin (Thermo Fisher Scientific) and maintained in medium containing 2. mu.g/mL puromycin to obtain a stable PTGES knockdown osteosarcoma cell line.

Will be 1 × 10 ⁵ Osteosarcoma cells were seeded in 6-well culture plates and infected at 50% confluence with lentiviral particles at 50% MOI. Infected cells were then selected in the presence of 4. mu.g/mL puromycin for 14 days and maintained in medium containing 2. mu.g/mL puromycin to construct stable infected cells, i.e., PTGES knockdown osteosarcoma cell line.

3. Real-time fluorescent quantitative PCR analysis

Total cellular RNA was extracted using the GeneJET RNA purification kit (Thermo Scientific) and RNA purity, concentration and integrity were assessed by Nanodrop spectrophotometer, agarose gel electrophoresis. cDNA was synthesized using a reverse transcription kit (YEASEN) using 1. mu.g of total RNA as a template. Quantitative real-time PCR was performed using the Rotor-Gene Q system (QIAGEN). The cDNA samples were diluted 10-fold and subjected to real-time PCR reaction using a Hieff UNICON Universal Blue Qpcr SYBR Green Master Mix (YEASEN). The amplification conditions were: at 95 ℃ for 2 minutes; 35 cycles of 95 ℃ for 10 seconds, then 60 ℃ for 30 seconds. GAPDH was used as a housekeeping gene to normalize all samples. The relative expression of PTGES mRNA was calculated by the 2- Δ Δ Ct method. Details of the primer sequences for PTGES are shown below.

PTGES upstream primer: 5'-CCCAAGGTTTGAGTCCCTCC-3', respectively;

PTGES downstream primer: 5'-CCCATCAAGGGGACATTTGC-3', respectively;

GAPDH upstream primer: 5'-CTCCTCCACCTTTGACGCTG-3' the flow of the air in the air conditioner,

GAPDH downstream primer: 5'-TCCTCTTGTGCTCTTGCTGG-3' are provided.

4. Western blot analysis

Cell lysates were collected with protein lysis buffer containing 1% protease inhibitor and protein was quantitated by BCA method. 20 μ g of total protein was placed on a 10% SDS-PAGE gel, electrophoresed at 80V to gel separation and then at 120V to bromophenol blue run out of the gel, ending the electrophoresis. The gel was carefully peeled off, appropriately labeled, and then a membrane transfer system was prepared from the negative electrode to the positive electrode in the order of sponge, filter paper, gel, 0.22 μm PVDF membrane, filter paper, and sponge. Under the condition of constant pressure of 100V, the film is rotated for 1.5h, and the whole process is ice-cooled. After the film transfer is finished, marking the PVDF film in the direction and the front and back directions, cutting off the positions of a target strip and an internal reference, and sealing for 1h at 37 ℃ by using 5% of skimmed milk powder (TBST for dilution); PTGES primary antibody (Cayman, 1:200) was incubated overnight at 4 ℃. The next morning, washing membrane with TBST on a decolouring shaker for 10min × 2 times, washing membrane with TBS for 10min, adding secondary antibody labeled with HRP, and combining at room temperature for 1 h; after washing membranes for 10min × 2 times with TBST, the membrane strips were soaked in TBS solution. And dripping a luminous working solution on the PVDF film, covering the PVDF film with a preservative film, carrying out luminous development by a BIO-RAD chemiluminescence apparatus, quantifying the signal intensity of each waveband by using Image-Pro-Plus software, and calculating the expression abundance of the protein according to the ratio of each waveband to the gray level of beta-actin.

FIG. 2 is a graph of the levels of PTGES mRNA and protein in osteosarcoma cells following lentivirus knockdown of PTGES, wherein A is real-time quantitative PCR detection of PTGES mRNA expression; panel B shows Western blot detection of PTGES protein expression.

As can be seen from figure 2, PTGES expression was significantly reduced in osteosarcoma cells, indicating successful knockdown of PTGES in osteosarcoma cells.

Example 3 increase in sensitivity to lobaplatin following osteosarcoma knockdown of PTGES

1. Flow cytometry

Will be 3X 10 ⁵ Osteosarcoma cells stably knockdown PTGES infected with lentivirus were seeded in 6-well plates and 24 hours later the cells were treated with 20 μ g/mL lobaplatin. After 24 hours of culture, cells were harvested, washed with PBS and resuspended in complete medium. Apoptosis was assessed using the FITC Annexin V apoptosis detection kit (RUO), the PE Annexin V apoptosis detection kit (RUO) and the Coulter EPICS XL flow cytometer (Beckman). Detecting each sample at least three times, and obtaining apoptosis by using EXP032 ADC analysis softwareAnd (6) data.

In fig. 3, the top two are western blot pictures of two cells, and the bottom two are bar charts obtained by statistically analyzing the gray level of western blots by Image J.

As shown in FIG. 3, after two osteosarcoma cells stably knockdown PTGES, the protein levels of activated cysteine protease 9 (cleared-Caspase 9), activated cysteine protease 3 (cleared-Caspase 3) and activated PARP (cleared-PARP) in the cells were significantly reduced after 24 hours of 20. mu.g/mL lobaplatin treatment.

As shown in fig. 4, it was found by flow cytometry that the proportion of apoptotic cells was significantly increased in PTGES knockdown osteosarcoma cells treated with 20 μ g/mL lobaplatin, i.e., the sensitivity to lobaplatin was increased.

2. TUNEL staining

Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (Tunel) assays were performed using the apoptosis detection kit (Roche, Germany). The osteosarcoma cells stably infected by lentivirus are expressed according to the 2 x 10 ⁴ Spreading each cell/well on a sterile cover glass in a 24-well plate, washing with 1 XPBS for 5 minutes after 24 hours, fixing cells with 4% paraformaldehyde for 15 minutes, washing with PBS for 5 minutes × 3 times, breaking membranes with 0.1% Triton X-100 for 10 minutes, washing with PBS for 5 minutes × 3 times, adding 50 μ L TUNEL into 450 μ L reaction buffer, mixing uniformly and adding into each well of the 24-well plate; incubation at 37 ℃ in the dark for 60 minutes, washing with PBS for 5 minutes × 3 times; sealing the plate with DAPI sealing agent, and drying in the dark. Tunel positive cells were observed and counted under a fluorescence microscope. The percentage of Tunel positive cells was calculated from five random fields in three wells.

FIG. 5 shows the staining results of two osteosarcoma cell lines. As shown in fig. 5, apoptosis was significantly increased in tumor cells transfected with lentivirus, as observed under a fluorescent microscope.

3. Clone formation assay

600 lentivirus-infected stably knockdown PTGES osteosarcoma cells and control vector-infected osteosarcoma cells were seeded in 35mm dishes. After 10-14 days of incubation, the cells were fixed with 4% paraformaldehyde for 20 minutes and stained with Giemsa stain for 20 minutes. The staining solution was slowly washed off with running water, dried in air, photographed under an inverted microscope and the number of clones larger than 10 cells was counted. The clone formation rate (number of clones/number of seeded cells) × 100%.

FIG. 6 shows the result of the clone formation experiment, wherein the left image is the clone formation image of two different treatment groups of osteosarcoma cells, and the right image is the clone formation rate of the different treatment groups analyzed by xx statistics. As shown in FIG. 6, the clonogenic assay showed that the cloning efficiency of PTGES-knocked-down osteosarcoma cell lines MG63 and SOSP-9607 was drastically reduced (P <0.05) after treatment with lobaplatin (20. mu.g/mL).

4. CCK8 test for sensitivity of cells to lobaplatin

6000 lentivirus-infected stably knockdown PTGES osteosarcoma cells per well were seeded in 96-well plates and 24 hours later treated with varying concentrations (0. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL, 80. mu.g/mL) of lobaplatin for 24 hours, 10. mu.L of CCK8 reagent was added and the cells were incubated for an additional 2 hours. The absorbance of the cells at 450nm was measured with a full wavelength multifunctional microplate reader (Tecan, Switzerland). Each experimental group contained three biological replicates and the degree of resistance of the cells to lobaplatin was determined at half inhibitory concentrations.

FIG. 7 shows the CCK8 experiment for detecting the cell viability of PTGES stably knocked-down osteosarcoma cells treated with different concentrations of lobaplatin, the left graph shows the experiment result of osteosarcoma MG63 cell line, and the right graph shows the experiment result of osteosarcoma SOSP-9607 cell line. As shown in fig. 7, the IC50 values for lobaplatin were significantly decreased in both PTGES-knockdown osteosarcoma cells, indicating that knockdown of PTGES increased the sensitivity of osteosarcoma cells to lobaplatin.

Therefore, the drug resistance of osteosarcoma to chemotherapeutic drugs, especially lobaplatin, can be weakened by knocking out or inhibiting the expression of PTGES, and the suggestion is that the inhibitor or knocking-out reagent for synthesizing PTGES and lobaplatin are combined for application, so that the drug resistance of osteosarcoma to chemotherapeutic drugs can be overcome when the osteosarcoma is treated, and the medicament has important guiding significance and broad prospects in clinical application.

Claims (6)

1. Use of an inhibitor of PTGES expression in the preparation of a medicament for increasing the sensitivity of tumor cells to chemotherapeutic drugs.
2. 2. The use of claim 1, wherein the tumor comprises osteosarcoma.
3. 3. The use of claim 1, wherein the chemotherapeutic agent is a platinum-based chemotherapeutic agent.
4. 4. The use of claim 3, wherein the chemotherapeutic agent is lobaplatin.
5. 5. The use of claim 1, wherein the PTGES expression inhibitor is a shRNA, siRNA, small molecule compound or a mab.
6. 6. A combined chemotherapy medicament is characterized by comprising a PTGES expression inhibitor and a platinum chemotherapy medicament.

Images