CN115025226B

CN115025226B - Application of PTGES expression inhibitor in preparation of medicine for improving sensitivity of tumor cells to chemotherapeutic drugs

Info

Publication number: CN115025226B
Application number: CN202210760058.0A
Authority: CN
Inventors: 马琼; 孙瑾; 王欢; 李晨宇; 吴永虹
Original assignee: Air Force Medical University of PLA
Current assignee: Air Force Medical University of PLA
Priority date: 2022-06-30
Filing date: 2022-06-30
Publication date: 2023-05-23
Anticipated expiration: 2042-06-30
Also published as: CN115025226A

Abstract

The invention discloses an application of a PTGES expression inhibitor in preparing a medicament for improving sensitivity of tumor cells to chemotherapeutic drugs. The invention discovers that the drug resistance of osteosarcoma to a chemotherapeutic drug, in particular to lobaplatin, can be weakened by knocking out or inhibiting the expression of PTGES, and indicates that the inhibitor for synthesizing PTGES or the knocking-out reagent and lobaplatin are combined for application, can overcome the drug resistance of osteosarcoma to the chemotherapeutic drug when treating osteosarcoma, and has important guiding significance and broad prospect in clinical application.

Description

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

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 medicament for improving sensitivity of tumor cells to chemotherapeutic drugs.

Background

Osteosarcoma is the most common primary malignant bone tumor, which is good for teenagers and has poor prognosis. Current treatments for osteosarcoma are a combination of active surgical resection with neoadjuvant chemotherapy. Platinum-based chemotherapeutic agents are the agents commonly used in osteosarcoma clinic, including cisplatin, oxaliplatin and lobaplatin. Lobaplatin is one of the third-generation platinum antitumor drugs, and has few side effects and good antitumor activity. However, after several courses of treatment, recurrence and metastasis remain problems facing the patient. Most patients dying from osteosarcoma exhibit significant chemical resistance.

Lipid metabolism plays a key role in the signal transduction of many cellular activities and is of great interest as a new emerging key effect in cancer cell behavior. Cancer cells utilize lipid metabolism to acquire energy and signaling molecules to proliferate, metastasize, and respond to various treatments. Furthermore, blocking cancer-associated lipolysis of adipocytes 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 in chemotherapy resistance. However, personalized therapeutic guidelines associated with lipid metabolism in osteosarcoma remain to be explored.

PTGES is a key enzyme in the metabolic pathway of arachidonic acid for the synthesis of PGE2, and can catalyze the conversion of PGH2 into PGE2, and the expression in inflammatory tissues and tumors is significantly up-regulated, and has been demonstrated to be important for tumorigenicity, migration and metastasis of non-small cell lung cancer cells. In the Gprc5a knockout mouse model, PTGES was associated with immunosuppression and pulmonary tumorigenesis. It is currently believed that PTGES/PGE2 signaling is critical for cell stem performance, including expression of ABCG2 and EMT related markers, and can induce expression of PD-L1 in macrophages and MDSCs, inhibiting tumor immunity. However, the role of PTGES in tumor chemoresistance is not yet clear. Therefore, the research on the role of PTGES in osteosarcoma platinum drug chemotherapy and the development of drugs for chemosensitization of osteosarcoma are significant for the treatment of osteosarcoma patients.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides application of a PTGES expression inhibitor in preparing a medicament for improving the sensitivity of tumor cells to chemotherapeutic drugs, and the PTGES gene is inhibited or knocked out, so that the response rate of patients with osteosarcoma to lobaplatin can be improved, and the generation of drug resistance can be reduced.

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

application of PTGES as target in preparing medicine for improving sensitivity of tumor cells to chemotherapeutic drugs.

In addition, the application of the PTGES expression inhibitor in preparing medicines for improving the sensitivity of tumor cells to chemotherapeutic medicines 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.

Further, the chemotherapeutic drug is a platinum chemotherapeutic drug.

Further, the chemotherapeutic agent is lobaplatin.

Further, the PTGES expression inhibitor includes shRNA, siRNA, small molecule compound or mab.

A combination chemotherapeutic comprising an inhibitor of PTGES expression and a platinum-based chemotherapeutic.

The invention has the beneficial effects that:

the invention discovers that the drug resistance of osteosarcoma to a chemotherapeutic drug, in particular to lobaplatin, can be weakened by knocking out or inhibiting the expression of PTGES, and indicates that the inhibitor for synthesizing PTGES or the knocking-out reagent and lobaplatin are combined for application, can overcome the drug resistance of osteosarcoma to the chemotherapeutic drug when treating osteosarcoma, and has important guiding significance and broad prospect in clinical application.

Drawings

FIG. 1 is a chart showing IHC staining to detect PTGES expression in lobaplatin sensitive and drug resistant osteosarcoma tissue; scale bar: 100 μm (magnification, 200 times), scale bar: 50 μm (magnification, 400); the violin map shows that the differences are significant.

FIG. 2 shows the expression levels of PTGES mRNA and protein in osteosarcoma cells after lentiviral knockdown of PTGES; * P < 0.001;

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

FIG. 4 shows flow cytometry to detect apoptosis rate of lobaplatin treated for 48 hours in various treated osteosarcoma cells; p <0.05, P < 0.001;

FIG. 5 shows TUNEL staining to detect apoptosis of PTGES knocked down osteosarcoma cells after lobaplatin treatment; scale bar: 100 μm;

FIG. 6 is a graph showing the proliferation potency of clonogenic assays to detect transfected shPTGES and loblatin treated osteosarcoma cells; * P < 0.001;

fig. 7 is a graph showing the viability of lobaplatin-treated osteosarcoma cells after CCK8 assay to detect shPTGES transfection.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate 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 all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.

EXAMPLE 1 resistance of PTGES to osteosarcoma

Immunohistochemical staining was performed on tumor samples of patients who were operated after osteosarcoma in 2019 to 2022 in Tang Dou hospital, and the specific procedure was as follows:

tumor tissue was fixed overnight at room temperature with 10% formalin solution, gradient ethanol (70%, 80%, 95% and 100%) dehydrated, and after paraffin embedding the tissue was cut into 2-3 μm sections. Paraffin sections were kept at 60 ℃ overnight, dewaxed to water: xylene 2X 15min,100% ethanol 2X 5min,95% ethanol 2X 5min, 70% ethanol 2X 5min, ddH ₂ O2X 5min. Repairing antigen by microwave method, standing and cooling to room temperature; immersing the slices in 3% hydrogen peroxide solution completely, and incubating for 15min at room temperature; washing the slices with PBS for 2X 5min; spin-drying the sections, dripping a sufficient amount of blocking solution (PBS containing 5% goat serum) onto the tissues, lightly shaking to completely cover the tissues, and placing the tissues in a wet box for incubation at room temperature for 15min; 50. Mu.L of diluted PTGES primary antibody (Cayman, 1:50) was added dropwise to the tissue to cover the tissue completely and incubated overnight at 4℃in a wet box; washing the slices with PBS for 3X 5min; dripping 50 mu L of universal rabbit and mouse secondary antibody into the tissue, and incubating for 1h at 37 ℃ in a wet box; washing the slices with PBS for 3X 5min; dropwise adding 50 mu L of freshly prepared DAB chromogenic substrate solution into the tissue, and incubating for 5-10min at room temperature; when the color signal to noise ratio reaches the best, taking the slice under a tap, and slowly flushing with running water for 1min; hematoxylin lining, hydrochloric acid alcohol differentiation, dehydration, sealing, drying in a fume hood and microscopic examination.

Staining results were independently assessed under a positive microscope (Olympus BX 51), and 5 complementary fields of view were selected for each specimen, and scored according to positive cell area and positive cell staining intensity in each field. Scoring criteria: a. positive area score: the number of positive cells is less than or equal to 5 percent, and 0 percent is calculated; 6-20%, counting 1 minute; 21% -50%, count 2; 51% -70%, counting 3 minutes; 4 minutes in the proportion of more than or equal to 71 percent; b. staining intensity score: yellow, 1 minute; brown yellow, 2 minutes; tan, 3 minutes. Composite score = positive area score x staining intensity score.

The left side of fig. 1 shows the results of immunohistochemical detection, which show the expression of PTGES in the lobaplatin-treated susceptible group and lobaplatin-treated resistant group osteosarcoma specimens, respectively, with the upper panel at 200-fold magnification and the lower panel at 400-fold magnification; the statistical result is on the right side, and the difference of the violin graph display is significant.

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

EXAMPLE 2 construction of PTGES knocked-down osteosarcoma cell line

1. Culture of human osteosarcoma cells

Human osteosarcoma cell line MG63, SOSP-9607 was cultured with modified Eagle medium and RPMI-1640 medium containing 10% fetal bovine serum, respectively, and placed at 37℃in 5% CO ₂ Is cultured in a cell culture incubator.

2. Lentiviral infection

shPTGES lentiviral particles with knockdown PTGES and random control lentiviral particles were supplied by Shanghai Ji Kai Gene Co. Will be 1X 10 ⁵ Osteosarcoma cells were plated in 6-well plates and infected at a ratio of MOI (multiplicity of infection) =100, with viral load per well (μl) =moi×cell number/titer×1000. Infected cells were then screened for 5 days in the presence of 4 μg/mL puromycin (Thermo Fisher Scientific) and maintained in medium containing 2 μg/mL puromycin to obtain a stable PTGES knockdown osteosarcoma cell line.

Will be 1X 10 ⁵ Osteosarcoma cells were seeded in 6-well plates and infected at a MOI of 50% at 50% confluency with lentiviral particles. The infected cells were then selected in the presence of 4 μg/mL puromycin for 14 days and maintained in medium containing 2 μg/mL puromycin to construct a stable infected cell, 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 the purity, concentration and integrity of the RNA was assessed by Nanodrop spectrophotometry, agarose gel electrophoresis. cDNA was synthesized using reverse transcription kit (YEASEN) with 1. Mu.g total RNA as template. Quantitative real-time PCR was performed using the Rotor-Gene Q system (QIAGEN). The cDNA samples were diluted 10-fold and real-time PCR reactions were performed using Hieff UNICON Universal Blue Qpcr SYBR Green Master Mix (YEASEN). The amplification conditions were: 95 ℃ for 2 minutes; 95℃for 10 seconds, a total of 35 cycles, then 60℃for 30 seconds. GAPDH served as a housekeeping gene to normalize all samples. The relative expression level of PTGES mRNA was calculated using the 2- ΔΔCt method. Details of the primer sequences of the PTGES are shown below.

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

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

GAPDH upstream primer: 5'-CTCCTCCACCTTTGACGCTG-3' the number of the individual pieces of the plastic,

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

4. Western blot analysis

Cell lysates were collected with a protein lysis buffer containing 1% protease inhibitor and protein was quantified using BCA method. 20. Mu.g of total protein was placed in a 10% SDS-PAGE gel, and after 80V electrophoresis to isolate the gel, 120V electrophoresis was performed until bromophenol blue was run out of the gel, and the electrophoresis was terminated. After appropriate labeling, a transfer system was prepared from negative to positive by carefully peeling off the gel, followed by sponge, filter paper, gel, 0.22 μm PVDF film, filter paper and sponge. And (3) transferring the film for 1.5 hours under the condition of constant pressure of 100V, and carrying out ice bath in the whole process. After the film transfer is finished, marking the direction and the front and back of the PVDF film, cutting off the positions of the target strips and the internal references, and sealing for 1h at 37 ℃ by using 5% skimmed milk powder (TBST dilution); PTGES primary antibody (Cayman, 1:200) was incubated overnight at 4 ℃. Washing the membrane for 10min X2 times on a TBST (Tunnel boring machine) in the next morning, washing the membrane for 10min on a TBS, adding HRP (horse radish peroxidase) labeled secondary antibody, and combining for 1h at room temperature; after 10min×2 times of TBST washing, the strips were immersed in TBS solution. Dripping a luminescent working solution on a PVDF film, covering with a preservative film, carrying out luminescent development by a BIO-RAD chemiluminescent instrument, quantifying the signal intensity of each wave band by using Image-Pro-Plus software, and calculating the expression abundance of the protein according to the ratio of each band to the beta-actin gray level.

FIG. 2 shows the levels of PTGES mRNA and protein in osteosarcoma cells after lentiviral knockdown of PTGES, wherein A is the real-time quantitative PCR detection of PTGES mRNA expression; panel B shows western blot detection of PTGES protein expression.

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

Example 3 enhancement of lobaplatin sensitivity after osteosarcoma knockdown PTGES

1. Flow cytometry

Will be 3X 10 ⁵ Bone sarcoma cells stably knocked down with PTGES infected with lentivirus were seeded in 6-well plates and after 24 hours the cells were treated with 20. Mu.g/mL lobaplatin. After 24 hours of incubation, cells were collected, washed with PBS and resuspended in complete medium. Apoptosis was assessed using FITC Annexin V apoptosis detection kit (RUO), PE Annexin V apoptosis detection kit (RUO) and Coulter EPICS XL flow cytometer (Beckman). Each sample was tested at least three times and apoptosis data was obtained using EXP032 ADC analysis software.

In fig. 3, the upper two are western blot pictures of two cells, and the lower two are bar graphs after statistical analysis of the grayscale values of western blots by Image J.

As shown in FIG. 3, after stable knock-down of PTGES by two osteosarcoma cells, the protein levels of activated cysteine protease 9 (clear-Caspase 9), activated cysteine protease 3 (clear-Caspase 3) and activated PARP (clear-PARP) in the cells were significantly reduced after 24 hours of treatment with 20. Mu.g/mL lobaplatin.

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

2. TUNEL staining

End deoxynucleotidyl transferase mediated dUTP gap end labeling (Tunel) assay was performed using an apoptosis detection kit (Roche, germany). Bone and meat tumor with stable infection of slow virusThe cells are according to 2X 10 ⁴ The individual cells/wells were spread on sterile coverslips in 24 well plates, washed with 1 XPBS for 5min, 4% paraformaldehyde for 15min, washed with PBS for 5min X3 times, membrane disruption with 0.1% Triton X-100 for 10min, washed with PBS for 5min X3 times, 50. Mu.L TUNEL was added to 450. Mu.L reaction buffer, and mixed well and added to each well of 24 well plates; incubation at 37℃for 60 min protected from light, PBS wash 5min X3 times; sealing tablet containing DAPI, and air drying in dark place. Tunel-positive cells were observed and counted under a fluorescence microscope. The percentage of Tunel positive cells was calculated from five random fields of view in three wells.

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

3. Clone formation assay

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

FIG. 6 shows the results of the test for clonogenic assay, wherein the left panel shows the clonogenic patterns of two different treatment groups of osteosarcoma cells, and the right panel shows the clonogenic rates of different treatment groups analyzed by xx statistics. As shown in FIG. 6, the cloning experiments showed that the osteosarcoma cell lines MG63 and SOSP-9607 knocked down PTGES had a dramatic decrease in cell cloning efficiency (P < 0.05) after lobaplatin (20. Mu.g/mL) treatment.

4. CCK8 detection of sensitivity of cells to lobaplatin

A total of 6000 lentiviral-infected, stable knockdown PTGES bone sarcoma cells per well were seeded in 96-well plates, 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 cultured for an additional 2 hours. The absorbance of the cells at 450nm was measured by a full wavelength multifunctional enzyme-labeled instrument (Tecan, switzerland). Each experimental group contained three biological replicates and the extent of cell resistance to lobaplatin was determined at half the inhibitory concentration.

FIG. 7 shows the experimental results of CCK8 for detecting the cell viability of PTGES stably knocked-down osteosarcoma cells treated with lobaplatin at different concentrations, with the left graph showing osteosarcoma MG63 cell strain and the right graph showing osteosarcoma SOSP-9607 cell strain. As shown in fig. 7, IC50 values of lobaplatin were significantly reduced in both of the bone sarcoma cells in which the PTGES was knocked down, indicating that the knockdown of PTGES enhanced sensitivity of the bone sarcoma cells to lobaplatin.

Therefore, the drug resistance of osteosarcoma to chemotherapeutic drugs, in particular lobaplatin, can be weakened by knocking out or inhibiting the expression of PTGES, and the combination of the synthetic PTGES inhibitor or knocking-out reagent and lobaplatin is suggested, so that the drug resistance of osteosarcoma to chemotherapeutic drugs can be overcome when osteosarcoma is treated, and the composition has important guiding significance and broad prospects in clinical application.

Claims

Use of a PTGES expression inhibitor, which is shRNA or siRNA, for the preparation of a medicament for increasing sensitivity of osteosarcoma cells to lobaplatin.