Application of DTX3 or agents that promote DTX3 expression in the preparation of products that reverse radioresistance in nasopharyngeal carcinoma.

Abstract

This invention relates to the application of DTX3 or reagents that promote DTX3 expression in the preparation of products reversing radioresistance in nasopharyngeal carcinoma (NPC), belonging to the field of biomedical technology. The invention found that, compared to parental NPC cells, Deltex E3 ubiquitin ligase 3 (DTX3) expression is significantly reduced in radioresistant NPC. Overexpression of DTX3 can reverse radioresistance in NPC cells and inhibit homologous recombination repair after radiotherapy in NPC, thus allowing DTX3 to be used to reverse radioresistance in NPC. This invention provides a new biomarker for the diagnosis of radiotherapy response in NPC, an effective method for reversing radioresistance in NPC, and a new treatment option for NPC.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of DTX3 or agents that promote DTX3 expression in the preparation of products that reverse radiotherapy resistance in nasopharyngeal carcinoma. Background Technology

[0002] Radiotherapy resistance is a significant cause of treatment failure in nasopharyngeal carcinoma (NPC). Exploring its molecular mechanisms and therapeutic targets is crucial for improving radiotherapy efficacy. A deeper understanding of these mechanisms can provide a theoretical basis for reversing radiotherapy resistance and optimizing radiotherapy strategies. However, currently, there is a lack of effective predictive indicators for radiotherapy resistance in NPC. Physicians typically assess patients based on factors such as age, TNM stage, and comorbidities when selecting radiotherapy regimens, but these factors cannot predict the intrinsic radiotherapy resistance determined by tumor cell genes, limiting the implementation of personalized radiotherapy. Ubiquitination is a multifunctional signaling mechanism that regulates various biological processes, including protein degradation and activity regulation, DNA damage repair, cell cycle, apoptosis, and immune regulation. By affecting the stability and activity of oncogenes or tumor suppressor genes, ubiquitination can promote or inhibit tumor development. Studies have shown that ubiquitination is involved in the regulation of radiosensitivity, and targeting ubiquitination or deubiquitination pathways may provide a theoretical basis and new clinical strategies for improving the radiosensitivity of tumor cells. E3 ubiquitin ligase DTX3 has a dual role in tumors. For example, it acts as a tumor suppressor molecule in esophageal cancer, triple-negative breast cancer, colorectal cancer, papillary thyroid carcinoma, and bladder cancer, while acting as a tumor promoter molecule in highly proliferative luminal breast cancer, gastric cancer, and ovarian cancer. Currently, the function and mechanism of DTX3 in enhancing radiosensitization in nasopharyngeal carcinoma are unclear.

[0003] The mechanism of radiotherapy resistance in nasopharyngeal carcinoma remains unclear, and there is currently a lack of effective molecular markers, related reagent kits, or detection methods for predicting radiotherapy resistance in nasopharyngeal carcinoma in clinical practice. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide the use of Deltex E3 ubiquitin ligase 3 (DTX3) or reagents that promote the expression of Deltex E3 ubiquitin ligase 3 (DTX3) in the preparation of products that reverse radiotherapy resistance in nasopharyngeal carcinoma.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the use of Deltex E3 ubiquitin ligase 3 (DTX3) or a reagent that promotes the expression of Deltex E3 ubiquitin ligase 3 (DTX3) in the preparation of a product that reverses radiotherapy resistance in nasopharyngeal carcinoma, wherein the coding sequence of the Deltex E3 ubiquitin ligase 3 is shown in SEQ ID NO.1; and the amino acid sequence of the Deltex E3 ubiquitin ligase 3 is shown in SEQ ID NO.2.

[0006] This invention has found that overexpression of DTX3 can reverse radiotherapy resistance in nasopharyngeal carcinoma cells, and DTX3 or agents that promote DTX3 expression can be used to reverse radiotherapy resistance in nasopharyngeal carcinoma.

[0007] DTX3 is a subtype of the Deltex family, located on chromosome 12q13.3. The human Deltex family consists of five subtypes: DTX1, DTX2, DTX3, DTX3L, and DTX4. Deltex proteins contain three distinct functional domains: an N-terminal WWE tandem module, a central Pro-rich region, and a C-terminal RFD domain. Unlike other Deltex family subtypes, DTX3 retains only a central Pro-rich region and a C-terminal RFD domain. The DTX3 protein consists of 350 amino acids and is primarily expressed in the cell nucleus.

[0008] As a preferred embodiment of the application described in this invention, the reagent for promoting the expression of Deltex E3 ubiquitin ligase 3 includes a plasmid carrying the coding sequence of Deltex E3 ubiquitin ligase 3.

[0009] Secondly, the present invention provides the use of Deltex E3 ubiquitin ligase 3 (DTX3) or a reagent that promotes the expression of Deltex E3 ubiquitin ligase 3 (DTX3) in the preparation of a product that inhibits homologous recombination repair after radiotherapy for nasopharyngeal carcinoma, wherein the coding sequence of the Deltex E3 ubiquitin ligase 3 is shown in SEQ ID NO.1; and the amino acid sequence of the Deltex E3 ubiquitin ligase 3 is shown in SEQ ID NO.2.

[0010] This invention has found that overexpression of DTX3 can inhibit homologous recombination repair after radiotherapy for nasopharyngeal carcinoma. DTX3 or agents that promote DTX3 expression can be used to inhibit homologous recombination repair after radiotherapy for nasopharyngeal carcinoma.

[0011] As a preferred embodiment of the application described in this invention, the reagent for promoting the expression of Deltex E3 ubiquitin ligase 3 includes a plasmid carrying the coding sequence of Deltex E3 ubiquitin ligase 3.

[0012] Thirdly, the present invention provides the application of a reagent for detecting the expression level of Deltex E3 ubiquitin ligase 3 (DTX3) in the preparation of products for diagnosing radiotherapy responsiveness and prognosis of nasopharyngeal carcinoma; the coding sequence of the Deltex E3 ubiquitin ligase 3 is shown in SEQ ID NO.1; the amino acid sequence of the Deltex E3 ubiquitin ligase 3 is shown in SEQ ID NO.2.

[0013] This study found that DTX3 expression was significantly increased in primitive nasopharyngeal carcinoma cell lines and significantly decreased in radioresistant cell lines. By detecting DTX3 expression levels, it is possible to determine whether nasopharyngeal carcinoma has developed radioresistance.

[0014] As a preferred embodiment of the application described in this invention, the reagent for detecting the expression level of Deltex E3 ubiquitin ligase 3 (DTX3) includes primers that specifically amplify Deltex E3 ubiquitin ligase 3 or antibodies that specifically bind to Deltex E3 ubiquitin ligase 3.

[0015] As a preferred embodiment of the application described in this invention, the sequences of the primers for specifically amplifying Deltex E3 ubiquitin ligase 3 are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0016] In a preferred embodiment of the application described in this invention, the radiotherapy reactivity includes radiotherapy resistance and radiotherapy sensitivity.

[0017] As a preferred embodiment of the application described in this invention, the product includes a chip, a reagent kit, or a test strip.

[0018] Fourthly, the present invention provides a kit for diagnosing radiotherapy responsiveness and prognosis of nasopharyngeal carcinoma, comprising a reagent for detecting the expression level of Deltex E3 ubiquitin ligase 3, wherein the coding sequence of Deltex E3 ubiquitin ligase 3 is shown in SEQ ID NO.1; and the amino acid sequence of Deltex E3 ubiquitin ligase 3 is shown in SEQ ID NO.2.

[0019] As a preferred embodiment of the kit described in this invention, the reagent for detecting the expression level of Deltex E3 ubiquitin ligase 3 (DTX3) includes primers that specifically amplify Deltex E3 ubiquitin ligase 3 or antibodies that specifically bind to Deltex E3 ubiquitin ligase 3. The sequences of the primers for specifically amplifying Deltex E3 ubiquitin ligase 3 are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention reveals that DTX3 expression is significantly increased in parental nasopharyngeal carcinoma cells and significantly decreased in radioresistant nasopharyngeal carcinoma cells. Overexpression of DTX3 can reverse radioresistance in nasopharyngeal carcinoma cells and inhibit homologous recombination repair after radiotherapy, suggesting that DTX3 can be used to reverse radioresistance in nasopharyngeal carcinoma. This invention provides a new biomarker for the diagnosis of radiotherapy response in nasopharyngeal carcinoma, an effective method for reversing radioresistance in nasopharyngeal carcinoma, and a new treatment option for nasopharyngeal carcinoma. Attached Figure Description

[0021] Figure 1 This is a comparison of the number of differentially expressed genes (DEGs) between the radiotherapy-resistant cell line HK1-IR and the original cell line HK1 in Example 1 of the present invention, as well as the comparison results of the differentially expressed genes (DEGs) and the list of E3 ubiquitin ligases. Figure 2 This is a heatmap showing some of the differential genes of E3 ubiquitin ligase between the radiotherapy-resistant cell line HK1-IR and the original cell line HK1 in Example 1 of the present invention. Figure 3 This represents the relative expression level of the DTX3 gene in transfected cells in Example 2 of this invention. Figure 4 The results of Western blot analysis of transfected cells in Example 2 of this invention; Figure 5 This is a diagram showing the formation of cell clones after treatment with different irradiation doses in Example 3 of the present invention. A represents a clonal statistics chart, and B represents the clone survival rate. Figure 6 The results of the comet experiment in Example 4 of this invention are shown. A represents the fluorescence diagram of the comet experiment (n=10, scale bar is 50μm), and B represents the statistical diagram of the tail moment of the comet experiment. Figure 7 The results of the homologous recombination reporter and the non-homologous recombination reporter in Example 4 of the present invention are shown. A represents non-homologous end joining (NHEJ) and B represents homologous recombination (HR). Detailed Implementation

[0022] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0023] Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0024] In this invention, DTX3 refers to Deltex E3 ubiquitin ligase 3.

[0025] The nasopharyngeal carcinoma progenitor cells HK1 used in this invention are from Professor Xia Yunfei's research group at Sun Yat-sen University Cancer Center.

[0026] The GFP-HR or GFP-NHEJ reporter plasmids used in this invention are from Professor Xia Yunfei's research group at Sun Yat-sen University Cancer Center.

[0027] Example 1 To identify genes that can reverse radioresistance, this invention irradiated nasopharyngeal carcinoma progenitor cells HK1 and constructed a radioresistant nasopharyngeal carcinoma cell line according to the method described in "Combining decitabine with radiotherapy to enhance nasopharyngeal carcinoma radiosensitivity via the TFAP2C-OTUD1-SLC25A11 axis" DOI: 10.1038 / s41419-025-07858-9.

[0028] Irradiation doses of 2, 4, 6, and 8 Gy were used, with the interval between each irradiation adjusted according to the cell growth stage to ensure that the cells were in the logarithmic growth phase before each irradiation. This process was repeated, with a cumulative dose of 60 Gy over 6 months. Using this method, the radioresistant cell line HK1-IR, capable of stable growth at a 60 Gy irradiation dose, was successfully established.

[0029] RNA from the radiotherapy-resistant cell line HK1-IR and the original cell line HK1 was collected and deep sequenced. Sequencing was performed by Guangzhou Nobelio Biotechnology Co., Ltd. An E3 ubiquitin ligase list was obtained from http: / / ubibrowser.ncpsb.org.cn for comparison. The number of differentially expressed genes (DEGs) between the radiotherapy-resistant cell line HK1-IR and the original cell line HK1, as well as the comparison results between the differentially expressed genes (DEGs) and the E3 ubiquitin ligase list, are shown below. Figure 1 The amino acid sequence of DTX3 is as SEQ ID As shown in NO.2, MPILSSSGSKMAACGGTCKNKVTVSKPVWDFLSKETPARLARLREEHRVSILIDGETSDIYVLQLSPQGPPPAPPNGLYLARKALKGLLKEAEKELKKAQRQGELMGCLALGGGGEHPEMHRAGPPPLRAAPLLPPGARGLPPPPPPLPPPLPPRLREEAEEQESTCPICLGE IQNAKTLEKCRHSFCEGCITRALQVKKACPMCGRFYGQLVGNQPQNGRMLVSKDATLLLPSYEKYGTIVIQYVFPPGVQGAEHPNPGVRYPGTTRVAYLPDCPEGNKVLTLFRKAFDQRLTFTIGTSMTTGRPNVITWNDIHHKTSCTGGPQLFGYPDPTYLTRVQEELRAKGITDD (SEQ ID NO.2).

[0030] A partial differential gene heatmap of E3 ubiquitin ligase between the radiotherapy-resistant cell line HK1-IR and the original cell line HK1 is shown below. Figure 2 As shown in the results, DTX3, TRIM15, and KCTD17 all showed differences between the radiotherapy-resistant cell line HK1-IR and the original cell line HK1, with DTX3 showing the most significant difference.

[0031] Example 2 A cell line overexpressing DTX3 was constructed. First, a recombinant plasmid overexpressing DTX3 was constructed and then transfected into the human nasopharyngeal carcinoma carcinoma line (NPC / HK-1 cells) using the recombinant plasmid to construct a cell line overexpressing DTX3.

[0032] 1. Recombinant plasmids overexpressing DTX3 Using pCDH-CMV-MCS-EF1-Puro as a blank vector (vec), SEQ ID NO.1 from Example 1 was introduced into the blank vector to construct the pCDH-CMV-DTX3-HA-EF1-Puro recombinant plasmid (DTX3). The construction of the recombinant plasmid was completed by Guangzhou Jidan Biotechnology Co., Ltd.

[0033] 2. Construction of cell lines stably overexpressing DTX3 NPC / HK-1 cells were seeded in 6cm culture dishes. When the cells reached a density of 70%, transient transfection of the plasmid was initiated. 2μg of the recombinant plasmid (or a blank vector as a control group) was added to 100μL of Opti-MEM medium at a ratio of 1:2.5 with Lipofectamine 3000 transfection reagent. The mixture was gently mixed and incubated at room temperature for 15 minutes to form a stable transfection complex. The transfection complex was then slowly added to the 6cm culture dishes containing NPC / HK-1 cells, and the cells were incubated at 37°C for 24-36 hours to obtain transfected cells. Cells were collected, and the expression level of DTX3 was detected using qRT-PCR and Western blot to verify the successful construction of the stable transfected cell line.

[0034] (1) RNA was extracted from transfected cells using a kit and cDNA was obtained by reverse transcription. Using cDNA as a template and DTX3-F and DTX3-R as primers, qRT-PCR was performed. DTX3-F (Forward): 5'-CCAGCGTCTCACCTTCACTATCG-3' (SEQ ID NO.3); DTX3-R (Reverse): 5'-TGGTCTTGTGGTGGATGTCGTTC-3' (SEQ ID NO.4).

[0035] The results of the relative expression level of the DTX3 gene in transfected cells are as follows: Figure 3 As shown, DTX3 refers to the cells transfected with the recombinant plasmid, and vec refers to the cells transfected with the blank vector. The results showed that the relative expression level of the DTX3 gene was significantly increased in the cells transfected with the recombinant plasmid, indicating that the transfected cells could overexpress the DTX3 gene.

[0036] (2) Extract the protein from the transfected cells and detect the expression efficiency of DTX3 protein by Western blot.

[0037] Western blot results of transfected cells are as follows Figure 4 As shown, DTX3 refers to cells transfected with the recombinant plasmid, and vec refers to cells transfected with the blank vector; the results show that DTX3 protein can be overexpressed in cells transfected with the recombinant plasmid.

[0038] Example 3 The transfected cells collected in Example 2 were seeded into 6-well plates, grouped into groups of 200, 800, 2000, 4000, and 10000 cells per well. On the second day, after cell attachment, these cells were irradiated with 0 Gy, 2 Gy, 4 Gy, 6 Gy, and 8 Gy, respectively. After irradiation, the cells were cultured at 37°C for 10-14 days to observe colony formation. Once colonies formed, the original culture medium was discarded, and each well was carefully rinsed three times with 1 mL of PBS to remove residual culture medium. Then, 1 mL of 4% paraformaldehyde solution was added to each well, and the cells were fixed at room temperature for 30-60 minutes to maintain cell morphology and structural stability. After fixation, the cells were carefully rinsed three times again with PBS to remove residual paraformaldehyde. Finally, the cells were stained with 1 mL of crystal violet dye per well for 30 minutes at room temperature. After staining, carefully rinse three times with tap water, taking care to avoid detaching clones. Place in a fume hood and allow to air dry at room temperature. The next day, photograph and record the cells, and count the number of colonies containing more than 50 cells. A linear quadratic model was used: survival fraction = exp(-αD - βD) 2 Fit the cell survival curve.

[0039] Cell clonal formation after treatment with different irradiation doses, as follows Figure 5 As shown in the figure, A represents the clonal statistics and B represents the clonal survival rate. The results showed that, compared with the control group cells transfected with the blank vector, the nasopharyngeal carcinoma cells overexpressing DTX3 formed significantly fewer cell colonies after irradiation, indicating that DTX3 overexpression reversed the radioresistance of nasopharyngeal carcinoma cells.

[0040] Example 4 The effects of DTX3 overexpression on homologous recombination repair after radiotherapy in nasopharyngeal carcinoma were investigated using neutral comet assays and reporter assays.

[0041] 1. Neutral comet experiment The transfected cells collected in Example 2 were seeded in 6-well plates and irradiated with 6 Gy after cell adhesion. Viable cells were collected at 0, 1, 6, and 24 hours after irradiation. A comet electrophoresis DNA damage detection kit (Jiangsu Kaiji Biotechnology Co., Ltd.) was used, and neutral electrophoresis buffer was prepared according to the manufacturer's instructions for comet assays. Images were observed and obtained using an inverted fluorescence microscope at 20x magnification. Quantitative analysis of the images was performed using Caslab-comet Assay software to quantify the tail torque, calculating the tail torque of 10 cells per sample.

[0042] The results of the comet experiment are as follows Figure 6As shown, A represents the fluorescence spectrum of the comet experiment (n=10, scale bar is 50μm), and B represents the statistical graph of the tail moment of the comet experiment. The results show that, compared with the control group, the tail moment of nasopharyngeal carcinoma cells overexpressing DTX3 was significantly increased after irradiation, indicating that the DNA double-strand breaks of nasopharyngeal carcinoma cells overexpressing DTX3 were more severe after irradiation, and the irradiation damage was more severe. This indicates that DTX3 overexpression reversed the radiotherapy resistance of nasopharyngeal carcinoma cells.

[0043] 2. Homologous recombination reporter and non-homologous recombination reporter experiments Following the method reported in the literature Wang XC, et al. Genome-wide RNAi screening identifies RFC4 as a factor that mediates radioresistance in colorectal cancer by facilitating nonhomologous end joining repair. Clin Cancer Res. 2019;25:4567-4579, the efficiency of two DSB repair pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ), was analyzed using the GFP reporter system.

[0044] The GFP-HR or GFP-NHEJ reporter plasmids were transfected into the transfected cells collected in Example 2. After DSB induction for 48 h with the recombinant plasmid pCMV-NLS-I-SceI of I-SceI enzyme, live cells were collected and the proportion of GFP-positive cells, i.e. recombination repair efficiency, was detected by flow cytometry.

[0045] Experimental results of homologous recombination reporter and non-homologous recombination reporter are as follows Figure 7 As shown, A represents non-homologous end junctions (NHEJ) and B represents homologous recombination (HR). The results showed that the repair efficiency of NHEJ in nasopharyngeal carcinoma cells overexpressing DTX3 was slightly higher than that in the control group, but the difference was not significant. However, the repair efficiency of HR in nasopharyngeal carcinoma cells overexpressing DTX3 was significantly lower than that in the control group. This indicates that DTX3 overexpression has no significant effect on the repair of non-homologous end junctions, but it can inhibit the repair of homologous recombination after radiotherapy in nasopharyngeal carcinoma, thereby reversing radiotherapy resistance in nasopharyngeal carcinoma cells.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of Deltex E3 ubiquitin ligase 3 or an agent promoting expression of Deltex E3 ubiquitin ligase 3 in the preparation of a product for reversing radiotherapy resistance in nasopharyngeal carcinoma, characterized in that, The coding sequence of the Deltex E3 ubiquitin ligase 3 is shown in SEQ ID NO.

1.

2. The application of Deltex E3 ubiquitin ligase 3 or reagents that promote the expression of Deltex E3 ubiquitin ligase 3 in the preparation of products that inhibit homologous recombination repair after radiotherapy for nasopharyngeal carcinoma, characterized in that, The coding sequence of the Deltex E3 ubiquitin ligase 3 is shown in SEQ ID NO.

1.

3. The application as described in claim 1 or 2, characterized in that, The amino acid sequence of the Deltex E3 ubiquitin ligase 3 is shown in SEQ ID NO.

2.

4. The application as described in claim 1 or 2, characterized in that, The reagent that promotes the expression of Deltex E3 ubiquitin ligase 3 includes a plasmid carrying the coding sequence of Deltex E3 ubiquitin ligase 3.

5. The application of a reagent for detecting the expression level of Deltex E3 ubiquitin ligase 3 in the preparation of products for diagnosing radiotherapy responsiveness and prognosis of nasopharyngeal carcinoma, characterized in that... The coding sequence of the Deltex E3 ubiquitin ligase 3 is shown in SEQ ID NO.

1.

6. The application as described in claim 5, characterized in that, The reagents for detecting the expression level of Deltex E3 ubiquitin ligase 3 include primers that specifically amplify Deltex E3 ubiquitin ligase 3 or antibodies that specifically bind to Deltex E3 ubiquitin ligase 3.

7. The application as described in claim 6, characterized in that, The sequences of the primers for specifically amplifying Deltex E3 ubiquitin ligase 3 are shown in SEQ ID NO.3 and SEQ ID NO.

4.

8. The application as described in claim 5, characterized in that, The radiotherapy reactivity includes radiotherapy resistance and radiotherapy sensitivity.

9. The application as described in any one of claims 5 to 8, characterized in that, The products include chips, reagent kits, or test strips.

10. A kit for diagnosing radiotherapy responsiveness and prognosis in nasopharyngeal carcinoma, characterized in that, The reagent includes a reagent for detecting the expression level of Deltex E3 ubiquitin ligase 3, the coding sequence of which is shown in SEQ ID NO.1.

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