Application of TRO gene as target spot in screening and / or preparing preparation for improving sensitivity of tumor cell chemotherapeutic drugs

Abstract

The invention discloses application of a TRO gene as a target spot in screening and / or preparing a preparation for improving the sensitivity of a tumor cell chemotherapy drug, and belongs to the technical field of biology. By inhibiting or knocking out the TRO gene, the response rate of osteosarcoma patients to cis-platinum can be improved, and the drug resistance generated by cells can be reduced. The invention finds that the drug resistance of osteosarcoma to chemotherapeutic drugs, especially cis-platinum, can be weakened by knocking out or inhibiting the expression of TRO, and prompts that the combined application of an inhibitor or a knockout reagent for synthesizing TRO and cis-platinum can overcome the drug resistance of osteosarcoma to chemotherapeutic drugs when being used for treating osteosarcoma; the method has important guiding significance in clinical application and has a wide prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the use of TRO genes as targets in screening and / or preparing preparations for enhancing the sensitivity of tumor cells to chemotherapy drugs. Background Art

[0002] Osteosarcoma, a malignant bone tumor originating from mesenchymal cells, seriously affects the health of adolescents and the elderly. Between 2016 and 2020, the incidence of osteosarcoma in the United States was highest in children aged 10 to 19 years. Untreated osteosarcoma patients generally have a poor prognosis, with a low five-year survival rate and often accompanied by distant metastasis. Currently, the main treatment for osteosarcoma includes a combination of surgery and chemotherapy. Among them, cisplatin is the first-line chemotherapy drug for the treatment of osteosarcoma, which has greatly improved the long-term survival rate of osteosarcoma patients. However, the ensuing problem of chemotherapy resistance has become a major challenge in clinical practice.

[0003] The adhesion ability of tumor cells plays a dual and dynamic role in the process of tumor metastasis and invasion. On the one hand, weakened adhesion ability allows cells to detach from the primary lesion and enter the circulatory system; on the other hand, enhanced adhesion ability makes it easier for tumor cells to colonize in distal tissues. This contradictory phenomenon reflects the differentiated requirements for adhesion properties at different stages of tumor progression. Tumor cells lose epithelial markers (such as E-cadherin) through epithelial-mesenchymal transition and acquire mesenchymal characteristics, resulting in weakened intercellular adhesion and detachment from the primary lesion. Epithelial-mesenchymal transition can promote carcinogenesis by inducing oxidative stress and DNA repair defects, increasing mutation accumulation; on the other hand, epithelial-mesenchymal transition causes cells to highly express ABC transporter pumps, thereby increasing drug efflux, activating survival pathways, and actively participating in tumor resistance to chemotherapy.

[0004] Trophinin (TRO) is an adhesion molecule that plays a key role in embryonic implantation. Recent studies have found that it is highly expressed in various malignant tumors and is closely associated with tumor invasion, metastasis, and prognosis. TRO promotes invasion and metastasis by enhancing the adhesion ability of tumor cells. This process is similar to the invasive behavior of trophoblast cells during embryonic implantation and may involve similar molecular mechanisms. In pancreatic cancer tissue, high trophinin expression is significantly associated with clinical stage (with increasing expression from stages I to III), poor tumor differentiation, and neural invasion. Trophinin-related proteins are expressed at significantly higher levels in lung adenocarcinoma than in normal tissue and are associated with disease stage, distant metastasis, and high Ki-67 expression, suggesting that their role in promoting tumor progression is universal across cancer types. Therefore, exploring the role of TRO in platinum-based chemotherapy for osteosarcoma and developing drugs to enhance chemotherapy sensitivity in osteosarcoma are of great significance for the treatment of osteosarcoma patients. Summary of the Invention

[0005] In response to the above-mentioned deficiencies in the prior art, the present invention provides a method for using the TRO gene as a target in screening and / or preparing a preparation for enhancing the sensitivity of tumor cells to chemotherapy drugs. By inhibiting or knocking out the TRO gene, the response rate of osteosarcoma patients to cisplatin can be increased and the development of drug resistance can be reduced.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0007] The purpose of the present invention is to provide the use of TRO gene as a target in screening and / or preparing a preparation for enhancing the sensitivity of tumor cells to chemotherapy drugs.

[0008] Furthermore, the chemotherapy sensitivity of tumor cells can be improved by inhibiting TRO gene expression.

[0009] Another object of the present invention is to provide a use of a TRO expression inhibitor in the preparation of a drug for enhancing the sensitivity of tumor cells to chemotherapy drugs.

[0010] Furthermore, the tumor is osteosarcoma.

[0011] Furthermore, the chemotherapy drug is a platinum chemotherapy drug.

[0012] Furthermore, the chemotherapy drug is cisplatin.

[0013] Furthermore, the TRO expression inhibitor includes shRNA, siRNA, small molecule compounds or monoclonal antibodies.

[0014] Another object of the present invention is to provide a pharmaceutical composition for chemotherapy, which comprises the above-mentioned TRO expression inhibitor and a platinum chemotherapy drug.

[0015] Furthermore, the platinum chemotherapy drug is cisplatin.

[0016] Beneficial effects of the present invention:

[0017] The present invention found that by knocking out or inhibiting the expression of TRO, the resistance of osteosarcoma to chemotherapy drugs, especially cisplatin, can be weakened. This suggests that the combined use of synthetic TRO inhibitors or knockout reagents and cisplatin can overcome the resistance of osteosarcoma to chemotherapy drugs in the treatment of osteosarcoma. This has important guiding significance in clinical application and has broad prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 IHC staining was used to detect the expression of TRO in cisplatin-sensitive and -resistant osteosarcoma tissues; scale bar: 100 μm (magnification, 400x); the bar graph shows significant differences, ***P < 0.001;

[0019] Figure 2The expression levels of TRO mRNA and protein in osteosarcoma cells after TRO knockdown by lentivirus; *P < 0.05, ***P < 0.001;

[0020] Figure 3 The expression levels of activated caspase-3 and PARP in osteosarcoma cells transfected with shTRO and treated with cisplatin; **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0021] Figure 4 Flow cytometry was used to detect the apoptosis rate of osteosarcoma cells in different treatment groups after 24 h of cisplatin treatment; *P < 0.05, ***P < 0.001, ****P < 0.0001;

[0022] Figure 5 Scratch assay to detect the migration of TRO-knockdown osteosarcoma cells after treatment with cisplatin; **P < 0.01, ***P < 0.001;

[0023] Figure 6 The proliferation ability of osteosarcoma cells transfected with shTRO and treated with cisplatin was detected by clone formation assay; ***P < 0.001, ****P < 0.0001;

[0024] Figure 7 CCK8 assay was used to detect the viability of osteosarcoma cells transfected with shTRO and then treated with cisplatin;

[0025] Figure 8 The sensitivity of osteosarcoma to cisplatin after TRO knockdown was detected in nude mouse subcutaneous xenograft tumor experiments. DETAILED DESCRIPTION

[0026] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0027] Example 1 TRO and drug resistance of osteosarcoma

[0028] Immunohistochemical staining was performed on tumor samples from patients who underwent surgery for osteosarcoma in the Department of Orthopedics, Second Affiliated Hospital of Air Force Medical University from 2022 to 2025. The specific process is as follows:

[0029] Tumor tissue was fixed with 10% formalin solution at room temperature overnight, dehydrated with gradient ethanol (70%, 80%, 95% and 100%), and then embedded in paraffin and cut into 2-3 μm sections. Paraffin sections were kept at a constant temperature of 60°C overnight and dewaxed to water: xylene 2×15 min, 100% ethanol 2×5 min, 95% ethanol 2×5 min, 70% ethanol 2×5 min, ddH2O 2×5 min. Microwave antigen retrieval was performed and the sections were allowed to cool to room temperature; the sections were completely immersed in 3% hydrogen peroxide solution and incubated at room temperature for 15 min; the sections were washed with PBS for 2×5 min; the sections were dried and sufficient blocking solution (PBS containing 5% goat serum) was added to the tissue, gently shaken to completely cover the tissue, and placed in a humidified chamber for incubation at room temperature for 15 min; 50 μL of diluted TRO primary antibody (Novus, 1:100) was added to the tissue to completely cover the tissue, and placed in Incubate overnight at 4°C in a humidified chamber; wash sections with PBS for 3×5 min; add 50 μL of rabbit and mouse universal ready-to-use secondary antibody to the tissue and incubate in a humidified chamber at 37°C for 1 h; wash sections with PBS for 3×5 min; add 50 μL of freshly prepared DAB color development substrate solution to the tissue and incubate at room temperature for 5-10 min; when the color development signal-to-noise ratio reaches the optimal value, rinse the sections slowly under running water for 1 min; stain with hematoxylin, differentiate with hydrochloric acid and alcohol, dehydrate and seal the sections, dry in a fume hood, and then examine under a microscope.

[0030] The staining results were independently evaluated under an upright microscope (Olympus BX51). Five complementary fields of view were selected for each specimen, and the score was calculated based on the area of positive cells and the intensity of positive cell staining in each field of view.

[0031] Scoring criteria:

[0032] a. Positive area scoring: 0 points if the number of positive cells is ≤5%; 1 point if the number of positive cells is 6%-20%; 2 points if the number of positive cells is 21%-50%; 3 points if the number of positive cells is 51%-70%; 4 points if the number of positive cells is ≥71%;

[0033] b. Staining intensity score: yellow, 1 point; brownish yellow, 2 points; tan, 3 points.

[0034] Comprehensive score = positive area score × staining intensity score.

[0035] like Figure 1 As shown in the data, the expression of TRO in samples of patients resistant to cisplatin treatment was enhanced compared with that in samples sensitive to treatment, and the difference was significant.

[0036] Example 2 Construction of TRO-knockdown osteosarcoma cell line

[0037] 1. Culture of human osteosarcoma cells

[0038] Human osteosarcoma cell lines MG63 and U2OS were cultured with modified Eagle's medium and McCoy's 5A medium containing 10% fetal bovine serum, respectively, and cultured in a cell culture incubator at 37° C. and 5% CO 2 .

[0039] 2. Lentivirus infection

[0040] The shTRO knockdown lentiviral particles and random control lentiviral particles were provided by Shanghai Genegene Co., Ltd. 1×10 5 Osteosarcoma cells were seeded in 6-well culture plates and infected at an MOI (multiplicity of infection) of 100. The amount of virus added per well (μL) was calculated as follows: MOI × number of cells / titer × 1000. MG63 and U2OS cells were then selected for infection in the presence of 25 μg / mL and 2 μg / mL puromycin (Thermo Fisher Scientific), respectively, for 5 days and maintained in a medium containing half the amount of puromycin to establish stably infected cells, i.e., TRO-knockdown osteosarcoma cell lines.

[0041] 3. Real-time fluorescence quantitative PCR analysis

[0042] Total cellular RNA was extracted using the GeneJET RNA purification kit (Thermo Scientific). RNA purity, concentration, and integrity were assessed using a Nanodrop spectrophotometer and agarose gel electrophoresis. cDNA was synthesized using a reverse transcription kit (QIAGEN Biotechnology) with 1 μg of total RNA as a template. Quantitative real-time PCR was performed using the Rotor-Gene Q system (QIAGEN). cDNA samples were diluted 10-fold and subjected to real-time PCR using 5× SmArt RT Master Mix (QIAGEN Biotechnology).

[0043] Amplification conditions were: 95°C for 30 seconds; 95°C for 10 seconds, 55°C for 10 seconds, and 72°C for 30 seconds, for a total of 40 cycles. GAPDH was used as a housekeeping gene for normalization across all samples. The relative expression of TRO mRNA was calculated using the 2-ΔΔCt method. Detailed information on the primer sequences for TRO is shown below.

[0044] TRO upstream primer: 5′-gAgCAgACgCCTTCTggATT-3′;

[0045] TRO downstream primer: 5′-gAgggCCCTgAAATAgAggC-3′;

[0046] GAPDH upstream primer: 5′-CTCCTCCACCTTTgACgCTg-3′;

[0047] GAPDH downstream primer: 5′-TCCTCTTgTgCTCTTgCTgg-3′.

[0048] 4. Western Blot Analysis

[0049] Cell lysates were collected using protein lysis buffer containing 1% protease inhibitors, and protein was quantified using the BCA assay. 20 μg of total protein was placed on a 10% SDS-PAGE gel and electrophoresed at 80 V until the separation gel was reached, followed by 120 V until bromophenol blue ran out of the gel. Carefully peel the gel and, after appropriate labeling, prepare the transfer system from negative to positive pole, following the order of sponge, filter paper, gel, 0.22 μm PVDF membrane, filter paper, and sponge. Transfer was performed at a constant voltage of 100 V for 1.5 hours, with an ice bath throughout. After transfer, the PVDF membrane was marked for direction and positive and negative, and the target band and internal control were cut open. Block the membrane with 5% skim milk powder (diluted in TBST) at 37°C for 1 hour. Incubate with the TRO primary antibody (Novus, 1:1000) at 4°C overnight. The next morning, the membrane was washed twice with TBST for 10 minutes on a decolorizing shaker. After a 10-minute wash with TBS, HRP-conjugated secondary antibody was added and allowed to bind at room temperature for 1 hour. After a 10-minute wash with TBST, the membrane strips were immersed in TBS. Luminescence working solution was added dropwise to the PVDF membrane, covered with plastic wrap, and developed using a BIO-RAD chemiluminescence instrument. The signal intensity of each band was quantified using Image-Pro-Plus software, and protein expression abundance was calculated as the ratio of the grayscale of each band to that of GAPDH.

[0050] Figure 2 Figure 5. TRO mRNA and protein levels in osteosarcoma cells after lentiviral knockdown of TRO, with GAPDH as an internal reference (**P < 0.01, ***P < 0.001, ****P < 0.0001). Figure A shows the results of real-time fluorescence quantitative polymerase chain reaction analysis, and Figure B shows Western blot analysis.

[0051] according to Figure 2 The detection results showed that the expression of TRO in osteosarcoma cells was significantly reduced in the TRO knockdown osteosarcoma cells infected by lentivirus.

[0052] Example 3: Enhanced sensitivity to cisplatin after TRO knockdown in osteosarcoma

[0053] 1. Western blotting

[0054] TRO knockdown osteosarcoma cells were treated with 5 μg / mL cisplatin for 24 hours. Total proteins were extracted and subjected to SDS-PAGE electrophoresis. The primary antibodies were Cleaved-Caspase 3 (Affinity, 1:1000) and Cleaved-PARP (CST, 1:3000), respectively. GAPDH was used as an internal control to detect the sensitivity of osteosarcoma cells to cisplatin treatment after TRO knockdown.

[0055] like Figure 3 As shown in the figure, in TRO knockdown osteosarcoma cells, after treatment with 5 μg / mL cisplatin, the protein levels of activated caspase 3 (Cleaved-Caspase 3) and activated PARP (Cleaved-PARP) increased compared with osteosarcoma cells without TRO knockdown ( Figure 3 A).

[0056] Image J software was used to perform statistical analysis on the grayscale values of the protein blot bands. The results showed that after cisplatin treatment, the expression of the above two proteins in osteosarcoma cells with stable TRO knockdown was significantly increased compared with the control group, and the level of cell apoptosis was increased, indicating that the cells were more sensitive to cisplatin treatment ( Figure 3 B).

[0057] 2. Flow cytometry

[0058] 2×10 5 Osteosarcoma cells with stable knockdown of TRO infected with lentivirus were seeded in 6-well plates and treated with 5μg / mL cisplatin after 24 hours. After 24 hours of culture, the cells were collected, washed by centrifugation with pre-cooled PBS, and resuspended in complete culture medium. A blank group, a single-stained Annexin V group, and a single-stained PI group were set as controls, and the experimental group was double-stained with Annexin V and PI. After gentle vortex mixing, incubate at room temperature in the dark for 5 minutes, and evaluate cell apoptosis using a Beckman Coulter-XL flow cytometer. Each sample was tested three times, and the apoptosis data were obtained using EXP032 ADC analysis software. The results are shown in Figure 4 .

[0059] like Figure 4 As shown, flow cytometry detection found that the proportion of apoptotic cells in TRO knockdown osteosarcoma cells treated with 5 μg / mL cisplatin was significantly increased, indicating that the cells were more sensitive to cisplatin.

[0060] 3. Scratch test

[0061] U2OS cells and MG63 cells were divided into empty vector control group, shTRO#1 group and shTRO#2 group, respectively. 5Cells were seeded into 6-well plates at a density of 10 cells / well and cultured at 37°C, 5% CO2 until fully confluent. After confluence, 2 μg / mL and 5 μg / mL of cisplatin were added to each well of U2OS cells and MG63 cells, respectively. A 200 μL sterile pipette tip was used to scratch the plate perpendicularly. After washing with PBS, the plate was replaced with serum-free culture medium. Images were collected using an inverted microscope at 0, 24, and 48 hours, and the scratch area was quantified using Image J software. The results are shown in Figure 2. Figure 5 The mobility calculation formula is as follows:

[0062] [1-(area at time t / area at time t0)]×100%

[0063] The experiment was repeated three times independently, and the data were expressed as mean ± standard deviation. The differences between the groups were evaluated by one-way analysis of variance (p < 0.05 was considered significant).

[0064] like Figure 5 As shown in the figure, the scratch test found that over time, the migration rate of osteosarcoma cells with TRO knockdown was significantly slowed down after cisplatin treatment.

[0065] 4. Clone Formation Assay

[0066] 1000 lentiviral-infected osteosarcoma cells with stable knockdown of TRO and osteosarcoma cells infected with control vector were inoculated in six-well plates, and three biological replicates were performed for each sample. After 5 days, 5μg / mL cisplatin was given, and the cells were cultured for 10-14 days. The cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15-20 minutes, the fixative was aspirated, and the cells were stained with Giemsa stain for 20 minutes. The staining solution was slowly washed away with running water, dried in the air, and photographed and counted under an inverted microscope with the number of clones larger than 10 cells. The clone formation rate was calculated as follows: clone formation rate = (number of clones / number of inoculated cells) × 100%. The results are shown in Figure 6 Figure A shows the clone formation of two osteosarcoma cell lines in different treatment groups after administration of cisplatin, and Figure B shows the clone formation rate of different treatment groups after Student's t-test statistical analysis.

[0067] like Figure 6 As shown in the figure, the clone formation test showed that the clone efficiency of the osteosarcoma cell lines MG63 and U2OS with TRO knockdown was significantly decreased after treatment with cisplatin (5μg / mL) (P<0.01).

[0068] 5. CCK8 detection of cell sensitivity to cisplatin

[0069] 6500 lentiviral-infected osteosarcoma cells with stable TRO knockdown were seeded per well in a 96-well plate. Twenty-four hours later, they were treated with varying concentrations of cisplatin (MG63: 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, and 25 μg / mL; U2OS: 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, and 10 μg / mL) for 24 hours. 10 μL of CCK8 reagent was added, and the cells were incubated for an additional 3 hours. The absorbance of the cells was measured at 450 nm using a full-wavelength multi-function microplate reader (Tecan, Switzerland). Three biological replicates were included for each experimental group, and the 50% inhibitory concentration (CI) was used to determine the degree of cell tolerance to cisplatin.

[0070] like Figure 7 As shown, in two osteosarcoma cell lines with TRO knockdown, the IC 50 The values were significantly decreased, indicating that TRO knockdown enhanced the sensitivity of osteosarcoma cells to lobaplatin.

[0071] 6. Detection of the sensitivity of osteosarcoma to cisplatin using a nude mouse subcutaneous transplant tumor model

[0072] Animal husbandry and related experimental procedures complied with animal welfare and ethical requirements and were approved by the Ethics Committee of Laboratory Animal Science and Technology of the Air Force Medical University of the Chinese People's Liberation Army (20250062). Nude mice aged 4-6 weeks were purchased and acclimated in an SPF-grade nude mouse breeding room for 1 week. They were then randomly divided into a TRO knockdown group and a lentiviral vector control group, with six mice in each group. Lentivirus-constructed TRO knockdown osteosarcoma cell lines and control cell lines were amplified, and 2×10 6 Resuspend the cells in 200 μL of sterile PBS and slowly inject the corresponding cell suspension into the subcutaneous tissue of the right lower limb using a 1 mL syringe. Observe the nude mice daily for tumor formation. Measure and record tumor size with a vernier caliper every three days after tumor formation. Calculate tumor volume using the following formula:

[0073] V=[W×L×(W+L) / 2]×0.5222

[0074] When the tumor volume reaches 0.125 cm 3 At 14:00, nude mice were intraperitoneally injected with cisplatin (3 mg / kg body weight, once every 3 days); when the largest diameter of the tumor in each group was about to reach 1.2 cm, the nude mice were killed by overdose of anesthesia; the animals were dissected, the tumors were removed, photographed, weighed, and divided into small portions and stored at -80°C.

[0075] like Figure 8 As shown in the figure, after cisplatin treatment, the tumor volume of osteosarcoma with TRO knockdown was significantly reduced, and the effect was significant.

[0076] Based on the above test results, it can be seen that by knocking out or inhibiting the expression of TRO, the resistance of osteosarcoma to chemotherapy drugs, especially cisplatin, can be weakened. This suggests that the combined use of synthetic TRO inhibitors or knockout reagents with cisplatin can overcome the resistance of osteosarcoma to chemotherapy drugs in the treatment of osteosarcoma. It has important guiding significance in clinical application and has broad prospects.

[0077] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Use of the TRO gene as a target in screening and / or preparing preparations for enhancing the sensitivity of tumor cells to chemotherapy drugs.

2. The use according to claim 1, characterized in that Improve the chemotherapy sensitivity of tumor cells by inhibiting TRO gene expression.

3. Use of TRO expression inhibitors in the preparation of drugs for enhancing the sensitivity of tumor cells to chemotherapy drugs.

4. The use according to claim 1 or 3, characterized in that The tumor is osteosarcoma.

5. The use according to claim 1 or 3, characterized in that The chemotherapy drug is a platinum chemotherapy drug.

6. The use according to claim 5, characterized in that The chemotherapy drug is cisplatin.

7. The use according to claim 3, characterized in that The TRO expression inhibitor includes shRNA, siRNA, small molecule compounds or monoclonal antibodies.

8. A pharmaceutical composition for chemotherapy, characterized in that: The method comprises the TRO expression inhibitor according to claim 3, and a platinum chemotherapy drug.

9. The pharmaceutical composition for chemotherapy according to claim 8, characterized in that The platinum chemotherapy drug is cisplatin.

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