Application of MNX1-AS1 inhibitor in preparation of product for enhancing breast cancer radiotherapy sensitivity and preparation method of sensitizer

Abstract

The invention relates to the technical field of tumor molecular biology, and provides application of an MNX1-AS1 inhibitor in preparation of a product for enhancing breast cancer radiotherapy sensitivity and a preparation method of a sensitizer. The invention also provides application of the long-chain non-coding RNAMNX1-AS1 detection reagent in preparation of products for predicting and diagnosing breast cancer cell radiotherapy resistance and / or prognosing breast cancer cell radiotherapy curative effect. The method has the advantages that (1) the long-chain non-coding RNA MNX1-AS1 detection is beneficial for timely judging whether breast cancer cells of a patient have the tendency of radiotherapy resistance or easy-to-acquire radiotherapy resistance or not, and a reference basis for the effective degree of radiotherapy is provided, so that a proper treatment strategy is timely adjusted and given, and the lifetime of the patient is prolonged; (2) the novel breast cancer radiotherapy nano sensitizer provided by the invention can be used for inhibiting two main signal transduction molecules causing radiotherapy resistance and tumor metastasis while breaking the abnormal oxidation-reduction state of cells in a three-in-one manner, and is beneficial to enhancing the radiotherapy sensitivity of breast cancer cells and improving the radiotherapy curative effect.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor molecular biology, and more specifically, to an application of an MNX1-AS1 inhibitor in preparing a product for enhancing the sensitivity of breast cancer radiotherapy and a method for preparing a sensitizer. Background Art

[0002] Breast cancer is the most common malignant tumor in women worldwide. The 2024 Cancer Statistics Report shows that breast cancer ranks second among all cancers in incidence. Radiotherapy is an integral component of comprehensive breast cancer treatment. Radiotherapy for early-stage breast cancer patients following breast-conserving surgery has become the standard treatment modality and is widely used clinically. This treatment approach maximizes the preservation of organ function. For patients undergoing mastectomy, postoperative radiotherapy to the chest wall ± the lymph node drainage area is recommended if there is at least one axillary lymph node metastasis or the primary tumor is larger than 5 cm. Studies have shown that postoperative radiotherapy can significantly improve disease-free and overall survival. However, while radiotherapy can improve local breast cancer control, there is still a certain risk of local recurrence or distant metastasis after radiotherapy, suggesting the existence of radioresistance. Therefore, addressing radioresistance is a crucial prerequisite for improving local breast cancer control and overall survival.

[0003] The specific molecular mechanisms underlying radioresistance in breast cancer remain incompletely elucidated, but studies suggest a correlation with abnormally elevated intracellular GSH levels and the expression of several signaling molecules. Currently, there are no proven, reliable diagnostic tools available for early prognosis of a breast cancer patient's sensitivity to radiotherapy and the likelihood of developing acquired radioresistance during radiotherapy. This is often only possible after prolonged radiotherapy, when the patient's sensitivity and disease outcomes are assessed based on treatment efficacy. Currently, physicians typically only detect and recognize radioresistance in a patient's tumor when radioresistance significantly interferes with treatment efficacy. For patients with radioresistant breast cancer, continued conventional radiotherapy, without prior knowledge of this condition, often fails to achieve the desired therapeutic outcome. This can increase the patient's burden, hindering treatment goals and exposing them to unnecessary side effects. More importantly, it can also lead to missed opportunities for other effective treatment strategies, potentially preventing patients from achieving their desired survival goals. Therefore, timely confirmation of breast cancer radiation resistance is crucial for the implementation of radiation therapy for breast cancer patients and the adjustment of the overall treatment plan, and is of great significance for improving the prognosis and survival time of patients with such refractory breast cancer. Therefore, the prior art urgently needs a product related to identifying radiation-resistant breast cancer, so as to identify radiation-resistant breast cancer earlier, provide more effective treatment strategies for radiation therapy of breast cancer, and prolong patient survival. In addition, when breast cancer patients develop radiation resistance, there is a lack of effective treatment methods. Therefore, the prior art urgently needs an effective prevention / treatment method and drug for radiation-resistant breast cancer. Summary of the Invention

[0004] The present invention aims to overcome at least one of the deficiencies of the above-mentioned prior art, and provides a new strategy for treating breast cancer radiotherapy resistance by using an MNX1-AS1 inhibitor in the preparation of a product that increases the sensitivity of breast cancer to radiotherapy and a method for preparing a sensitizer.

[0005] Specifically provide the following invention:

[0006] One object of the present invention is to provide a sensitizer for increasing the radiosensitivity of breast cancer, including an MNX1-AS1 inhibitor.

[0007] In some embodiments, the MNX1-AS1 inhibitor is siRNA.

[0008] In some embodiments, the MNX1-AS1 inhibitor is siRNA, the sense strand sequence of the siRNA is 5′-GAACAACGCAGACAACATA-3′ (SEQ ID NO: 4), and the antisense strand sequence of the siRNA is 5′-TATGTTGTCTGCGTT GTTC-3′ (SEQ ID NO: 5).

[0009] In some embodiments, the MNX1-AS1 inhibitor is siRNA, the sense strand sequence of the siRNA is 5′-CTGCCTGCATGCTTTACCA-3′ (SEQ ID NO: 6), and the antisense strand sequence of the siRNA is 5′-TGGTAAAGCATGCAG GCAG-3′ (SEQ ID NO: 7).

[0010] Furthermore, the sensitizer for enhancing the radiotherapy sensitivity of breast cancer also includes a GSH depleting agent and / or a TROP2 inhibitor.

[0011] Furthermore, the GSH depleting agent is reduction-responsive polydisulfide amide (PDSA); the TROP2 inhibitor is an anti-TROP2 monoclonal antibody, i.e., an anti-human trophoblast cell surface antigen 2 (TROP2) monoclonal antibody. In some embodiments, the anti-TROP2 monoclonal antibody is a monoclonal antibody against the human TROP2 molecule.

[0012] Another object of the present invention is to provide a long non-coding RNA MNX1-AS1 detection reagent for use in preparing a product for predicting and diagnosing breast cancer cell radiotherapy resistance and / or prognosing breast cancer cell radiotherapy efficacy.

[0013] Furthermore, the long non-coding RNA MNX1-AS1 detection reagent includes primers and / or probes for detecting long non-coding RNA MNX1-AS1 in breast cancer samples.

[0014] Another object of the present invention is to provide a use of the above-mentioned sensitizer in the preparation of a product for enhancing the sensitivity of breast cancer to radiotherapy.

[0015] Another object of the present invention is to provide a method for preparing any of the above-mentioned sensitizers, comprising:

[0016] S1, mixing an aqueous solution of siRNA for knocking down lncRNA MNX1-AS1 and a DMF solution of G0-C14 to form a mixture A;

[0017] S2, DMF solution of PDSA polymer, DSPE-mPEG3 000 DMF solution, DSPE-PEG3 000 -NHS DMF solution to form mixture B;

[0018] S3. Mix the mixture A and the mixture B to form a mixture C, add the mixture C dropwise into enzyme-free water, stir, and ultrafilter to obtain a concentrated solution as the sensitizer.

[0019] In this scheme, DMF (N,N-Dimethylformamide) is N,N-dimethylformamide, CAS number 68-12-2;

[0020] PDSA is a reduction-reactive polydisulfide. Its chemical formula is: (C18H30N2O6S2);

[0021] DSPE-mPEG 3000 (1,2-Distaroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyle-ne glycol)- 3000 ]) is distearoylphosphatidylethanolamine-polyethylene glycol 3000, CAS No. 147867-65-0;

[0022] DSPE-PEG 3000 -NHS(1,2-distaroyl-sn-glycero-3-phosphoethanolamine-N-[succinimidyl-(polyethylene glycol)- 3000 ]) is 1,2-distearoyl-SN-glycerol-3-phosphoethanolamine-polyethylene glycol 3000-N-hydroxysuccinimide;

[0023] G0-C14 is a cationic lipid compound alkyl-modified polyaminoamine (PAMAM) dendrimer with a CAS number of 1510653-27-6.

[0024] In some embodiments, in step S2, an anti-human trophoblast cell surface antigen 2 (TROP2) monoclonal antibody is further added. The abbreviation of the anti-TROP2 monoclonal antibody is αTrop2.

[0025] In some embodiments of the present invention, the sensitizer is prepared by the following method:

[0026] Dissolve 0.8-1.2 nmol of Cy5-labeled siMNX1-AS1 in water to prepare an aqueous solution with a concentration of 0.5-1.5 nmol / μL.

[0027] Mix 45-55 μL of the DMF solution of G0-C14 and 8-12 μL of the above aqueous solution to form a mixture A.

[0028] Take 160-240 μL of PDSA polymer DMF solution (concentration is 18-22 mg / mL DMF), 135-145 μL of DSPE-mPEG 3000 DMF solution, 18-22 μL DSPE-PEG 3000 The DMF solution of -NHS was mixed with 50 μL of αTrop2 to form mixture B.

[0029] Mixture A and mixture B are mixed to form mixture C, and mixture C is added dropwise to 4 to 6 mL of enzyme-free water while stirring at a stirring speed of 1000 to 2000 rpm. After the addition is completed, stirring is continued at room temperature for 25 to 35 minutes. The organic reagent and free compounds are removed using an ultrafiltration tube, and the final concentrated solution obtained is the sensitizer TREARA.

[0030] The novel breast cancer radiotherapy nanosensitizer (TREARA) prepared by the present invention achieves efficient targeting and large-scale deposition in breast cancer tissue in vivo through its unique nanodrug structure. Furthermore, while silencing the expression of lncMNX1-AS1, an important target for regulating breast cancer radiotherapy sensitivity, it also inhibits the cancer-promoting signals transduced by trophoblast surface antigen 2 (TROP2) and depletes abnormally elevated glutathione (GSH) in radiotherapy-resistant breast cancer cells. This "three-in-one" approach not only breaks the abnormal cellular redox state but also inhibits the two major signal transduction molecules that lead to radiotherapy resistance and tumor metastasis, thereby enhancing the radiotherapy sensitivity of breast cancer cells and improving the efficacy of radiotherapy.

[0031] Another object of the present invention is to provide a long non-coding RNA MNX1-AS1 detection reagent for use in preparing a product for predicting and diagnosing breast cancer cell radiotherapy resistance and / or prognosing breast cancer cell radiotherapy efficacy.

[0032]

[0033] In some embodiments, a reagent for detecting long noncoding RNA MNX1-AS1 includes primers and / or probes for detecting long noncoding RNA MNX1-AS1 in breast cancer samples. In one or more embodiments of the present invention, the forward primer sequence is 5'-CAGCCTCCACGGAAAATGTG-3' (SEQ ID NO: 8), and the reverse primer sequence is 5'-TCTCCTCCTCCCACTAGGGT-3' (SEQ ID NO: 9).

[0034] Another object of the present invention is to provide a use of an MNX1-AS1 inhibitor in the preparation of a product for increasing the sensitivity of breast cancer to radiotherapy.

[0035] The inventors evaluated the effect of si-MNX1-AS1 (siRNA that specifically knocks down the expression of long non-coding RNA MNX1-AS1, referred to as si-MNX1-AS1) on the radiosensitivity of breast cancer radiotherapy-resistant cells MDA-MB-231R through a single-click multi-target experiment. The results showed that after using si-MNX1-AS1 to knock down the expression of long non-coding RNA MNX1-AS1 (hereinafter referred to as lncMNX1-AS1), the radiosensitivity of breast cancer radiotherapy-resistant cells was significantly enhanced, and their resistance to radiotherapy was significantly reduced. At the same time, the effect of exogenous expression of lncMNX1-AS1 on the radiosensitivity of breast cancer cells was also evaluated through a single-click multi-target experiment. The results showed that after exogenous expression of lncMNX1-AS1 using the lncMNX1-AS1 overexpression plasmid, the radiosensitivity of breast cancer cells was significantly decreased, and radiotherapy resistance was significantly enhanced. Therefore, inhibiting lncMNX1-AS1 expression can enhance the radiosensitivity of breast cancer cells and greatly improve the treatment effect.

[0036] In some embodiments, the radiation used in the radiotherapy is photon radiation.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The present invention provides the use of long non-coding RNA MNX1-AS1 as a marker of breast cancer radiotherapy resistance. Detection of its expression level facilitates timely diagnosis of radiotherapy resistance during breast cancer radiotherapy, facilitates timely determination of whether a breast cancer patient's tumor cells have potential or existing radiotherapy resistance, and provides a reference for the effectiveness of radiotherapy treatment methods, so as to timely adjust and administer appropriate treatment strategies, avoid ineffective or low-efficacy radiotherapy treatment methods and unnecessary radiotherapy side effects, and reduce patient discomfort during treatment. This facilitates timely adjustment and adoption of other effective treatment strategies, thereby prolonging patient survival.

[0039] (2) The present invention provides the use of long non-coding RNA MNX1-AS1 as a target for radiotherapy resistance in breast cancer, and provides interfering RNA that inhibits its expression, thereby increasing the radiotherapy sensitivity of breast cancer and greatly improving the effect of breast cancer radiotherapy.

[0040] (3) The sensitizer provided by the present invention is a nanoformulation based on the co-delivery of a long non-coding RNA MNX1-AS1 inhibitor, a GSH depletor, and a TROP2 inhibitor. The sensitizer has strong breast cancer targeting based on active TROP2 targeting, high long non-coding RNA MNX1-AS1 silencing efficiency, and efficient GSH consumption. While breaking the abnormal redox state of cells and improving the intracellular microenvironment of radiotherapy-resistant cells, it inhibits the two major signal transduction molecules, long non-coding RNA MNX1-AS1 and TROP2, which lead to radiotherapy resistance and tumor metastasis, thereby enhancing the radiotherapy sensitivity of breast cancer cells and improving the radiotherapy efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Display: 1A: The difference in expression between triple-negative breast cancer and non-triple-negative breast cancer is 2 5 Heat map of lncRNAs with expression differences of 2 in normal breast tissue and breast cancer tissue. 5 Heat map of lncRNAs with fold increase. 1C: lncRNAs with fold increase of at least 2 in triple-negative breast cancer and breast cancer samples. 5 1D: qRT-PCR validation of lncMNX1-AS1 expression in breast cancer cell lines and normal breast epithelial cell lines (*P < 0.05, **P < 0.01, and ***P < 0.001).

[0042] Figure 2 Shown: 2A, 2B, 2C: Verification of the radiotherapy-resistant phenotype of the MDA-MB-231 cell line: MDA-MB-231-Resistant (MDA-MB-231R) cells have a higher survival fraction and higher D0 and Dq values under different doses of irradiation compared with their parental cells MDA-MB-231-Parenta (MDA-MB-231P).

[0043] Figure 3 Shown: Intracellular localization of lncMNX1-AS1. 3A: FISH results; 3B: Nuclear-cytoplasmic fractionation results.

[0044] Figure 4 Shown: Validation of the efficiency of siMNX1-AS1 knockdown of lncMNX1-AS1 in MDA-MB-231 cells.

[0045] Figure 5 Shown: 5A: After knocking down lncMNX1-AS1 in MDA-MB-231 cells, the proliferation ability of breast cancer cells was weakened (detected by MTS method); 5B and 5C: After knocking down lncMNX1-AS1 in MDA-MB-231 cells, the clone formation ability of breast cancer cells was weakened; 5D: After knocking down lncMNX1-AS1 in MDA-MB-231 cells, the apoptosis rate of breast cancer cells was increased (**P<0.01).

[0046] Figure 6 Display: q-PCR results show the expression level of lncMNX1-AS1 in MDA-MB-231 and MDA-MB-231R.

[0047] Figure 7 Shown: Validation of the efficiency of siMNX1-AS1 knockdown of lncMNX1-AS1 in MDA-MB-231R cells.

[0048] Figure 8 Figures 8A and 8B show that knockdown of lncMNX1-AS1 in MDA-MB-231R cells attenuated the colony-forming ability of breast cancer cells. Figures 8C and 8D show that knockdown of lncMNX1-AS1 in MDA-MB-231R cells attenuated the migration ability of breast cancer cells. Figures 8E and 8F show that knockdown of lncMNX1-AS1 in MDA-MB-231R cells attenuated the invasion ability of breast cancer cells (**P<0.01 and ***P<0.001).

[0049] Figure 9 Shown: 9A: After knocking down lncMNX1-AS1 in MDA-MB-231R, the survival rate of breast cancer cells after radiotherapy was significantly decreased; 9B: After knocking down lncMNX1-AS1, the D0 and Dq of the MDA-MB-231R cell line were reduced.

[0050] Figure 10 Shows that after the lncMNX1-AS1 overexpression plasmid was transfected into the MDA-MB-231 parental cell line, the expression level of MNX1-AS1 increased by more than 20 times

[0051] Figure 11 Shown: 11A: After overexpression of lncMNX1-AS1 in MDA-MB-231, the survival rate of breast cancer cells after radiotherapy was significantly increased; 11B: After overexpression of lncMNX1-AS1, the D0 and Dq of the MDA-MB-231 cell line were increased.

[0052] Figure 12Shown: 12A: Schematic diagram of TREARA. 12B: TEM images of NP-siNC, REARA, and TREARA. 12C: Particle size distribution of NP-siNC, REARA, and TREARA. 12D: Zeta potential of NP-siNC, REARA, and TREARA.

[0053] Figure 13 Shown: Cellular uptake assays of free siMNX1-AS1, REARA, and TREARA.

[0054] Figure 14 Shown: qPCR experiments detected the expression level of lncMNX1-AS1 in MDA-MBA-231R cells under treatment with different siRNA doses of TREARA or REARA.

[0055] Figure 15 Shown: 15A and 15B: TREARA combined with radiotherapy significantly inhibited the colony formation of MDA-MB-231R cells.

[0056] Figure 16 Shows: 16A and 16B: TREARA combined with radiotherapy significantly weakened the migration ability of MDA-MB-231R cells; 16C and 16D: TREARA combined with radiotherapy significantly weakened the invasion ability of MDA-MB-231R cells.

[0057] Figure 17 Figures 17A and 17B show the ROS generation in MDA-MB-231 cells under different treatments and the corresponding statistics. Figures 17C and 17D show the ROS generation in MDA-MB-231R cells under different treatments and the corresponding statistics.

[0058] Figure 18 Shown: 18A: Live / dead dual fluorescence images of MDA-MB-231 and MDA-MB-231R cells after different treatments. Dead cells: red fluorescence of PI; live cells: green fluorescence of Calcein AM. (Magnification, ×100); 18B and 18C: Corresponding statistics of the dead / live cell ratios of MDA-MB-231 and MDA-MB-231R cells after different treatments.

[0059] Figure 19 Shown: 19A, 19C: After treatment with TREARA, the survival rate of MDA-MB-231 cells after radiotherapy was significantly decreased; 19B, 19D: After treatment with TREARA, the survival rate of MDA-MB-231R breast cancer cells after radiotherapy was significantly decreased.

[0060] Figure 20 Shown: 20A, 20B: In vitro fluorescence images and statistical graphs of tumors and major organs in the siMNX1-AS1-Cy5, REARA, and TREARA groups.

[0061] Figure 21 Figures 21A: Tumor inoculation and treatment procedures for MDA-MB-231R tumor-bearing mice (n=3). 21B: In vitro images of mouse tumors on day 30 after treatment. 21C: Mouse tumor growth curve. 21D: Mouse tumor weight statistics. 21E: Flowchart for establishing the mouse MDA-MB-231R-Luc lung metastasis model and treatment. 21F: In vivo bioluminescence images of mice bearing MDA-MB-231R-Luc lung metastases. 21G and 21H: In vitro bioluminescence images of lung tissue from mice bearing MDA-MB-231R-Luc lung metastases and their corresponding statistical graphs. *P<0.05, **P<0.01, ***P<0.001.

[0062] Figure 22 Shown: 22A: H&E staining of heart, liver, spleen, lung, and kidney histological sections of mice in all treatment groups showed no significant abnormalities. 22B: Serum biochemical parameters including ALT, AST, ALP, TP, ALB, GLOB, UREA, and CREA were normal in all treatment groups. *P < 0.05, **P < 0.01, ***P < 0.001. DETAILED DESCRIPTION

[0063] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0064] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0065] The present invention will now be further described in conjunction with specific examples. The following examples are only intended to explain the present invention but do not limit the present invention. The test samples and test procedures used in the following examples include the following (if the specific experimental conditions are not specified in the examples, they are usually based on conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources).

[0066] In the following examples, MDA-MB-231 is a human breast cancer cell line, product number: iCell-h133, obtained from Mirror Qidian (Shanghai) Cell Technology Co., Ltd.); MDA-MB-468 is a human breast cancer cell line, product number: iCell-h138, obtained from Mirror Qidian (Shanghai) Cell Technology Co., Ltd.

[0067] Example 1 (Screening out the key lncMNX1-AS1 involved in regulating breast cancer radiotherapy resistance)

[0068] 1. We analyzed the following from the TCGA dataset: (A) lncRNAs with a 5-fold differential expression between triple-negative breast cancer and non-triple-negative breast cancer; (B) lncRNAs with a 5-fold differential expression between normal breast tissue and breast cancer tissue. The intersection of datasets A and B revealed that lncMNX1-AS1 was the only candidate lncRNA upregulated more than 5-fold. qRT-PCR was then used to verify that lncMNX1-AS1 was significantly upregulated in breast cancer cell lines compared with the normal breast epithelial cell line MCF10A. The greatest upregulation of lncMNX1-AS1 was observed in the triple-negative breast cancer (TNBC) cell lines MDA-MB-231 and MDA-MB-468. Figure 1 ).

[0069] 2. We further constructed MDA-MB-231R. We established a radioresistant cell line by inducing it through dose-escalation hyperfractionation. Specifically, we initially irradiated the cells with high-energy X-rays at 2 Gy / dose, with hyperfractionation (approximately 2-3 times per week). This radiation induction process continued until the cells reached 70% density, then irradiated them again with the same dose. After the cells grew to a density of over 90%, they were passaged. After passage, the cells were stabilized and irradiated again with the same dose. This process was repeated until the cells showed significant radioresistance. The dose was then gradually increased until the radioresistant breast cancer cell line MDA-MB-231R was established with stable radioresistance.

[0070] After the radiation-resistant strain was successfully constructed, it was irradiated with X-rays every three generations to maintain its radiation resistance. The corresponding parent cell line MDA-MB-231 was synchronously subcultured with the radiation-resistant strain.

[0071] We used qRT-PCR to verify the expression of lncMNX1-AS1 in MDA-MB-231 and MDA-MB-231R cells. The results showed that the expression of lncMNX1-AS1 in MDA-MB-231R cells was significantly higher than that in MDA-MB-231 cells ( Figure 6 ).

[0072] 3. Current research suggests that radioresistance is common in triple-negative breast cancer, and that lncRNAs may be involved in regulating tumor radioresistance. These experimental results suggest that lncMNX1-AS1 is associated with breast cancer progression and is expressed at higher levels in radioresistant breast cancer lines. This suggests that lncAMNX1-AS1 may be involved in radioresistance. Further experimental verification is needed in triple-negative breast cancer lines with radioresistance.

[0073] Example 2 (Design of siRNA to Knockdown lncMNX1-AS1 and Verification of Its Knockdown Efficiency)

[0074] 1. Nuclear and cytoplasmic RNA was isolated from breast cancer cells using a nuclear-cytoplasmic isolation kit. qRT-PCR was then performed to determine the expression of lncMNX1-AS1 in the nucleus and cytoplasm, respectively. This study clarified the subcellular localization of lncMNX1-AS1 in breast cancer cells and radioresistant breast cancer cells. The results showed that lncMNX1-AS1 was primarily located in the cytoplasm of radioresistant breast cancer cells.

[0075] 2. After confirming that lncMNX1-AS1 is localized in the cytoplasm, RNA interference technology (RNAi) was used to construct siRNA to knock down the expression of lncMNX1-AS1 in breast cancer cell lines. Figure 3 )

[0076] 3. Use RNA interference technology (RNAi) to construct siRNA to knock down lncMNX1-AS1 expression:

[0077] si-1: sense strand sequence: 5′-GAACA ACGCAGACAACATA-3′ (SEQ ID NO: 4);

[0078] Antisense strand sequence: 5′-TATGTTGTCTGCGTTGTTC-3′ (SEQ ID NO: 5)

[0079] si-2: sense strand sequence: 5′-CTGCCTGCATGCTTTACCA-3′ (SEQ ID NO: 6);

[0080] Antisense strand sequence: 5′-TGGTAAAGCATGCAGGCAG-3′ (SEQ ID NO: 7).

[0081] qRT-PCR verified that the efficiency of siMNX1-AS1 knockdown of lncMNX1-AS1 was over 50%.

[0082] Example 3 (Cell experiment)

[0083] 1. Evaluate the effect of siMNX1-AS1 on breast cancer cell proliferation

[0084] MTS cell proliferation assay and plate clone formation assay were used to evaluate the effect of siMNX1-AS1 on the proliferation ability of breast cancer cells. The experimental results showed that siMNX1-AS1 could weaken the proliferation and clone formation ability of breast cancer cells ( Figure 5 ).

[0085] 2. qRT-PCR was used to verify the expression level of lncMNX1-AS1 in MDA-MB-231 and MDA-MB-231R cells: the expression level of lncMNX1-AS1 in MDA-MB-231R cells was significantly higher than that in MDA-MB-231 cells ( Figure 6 ).

[0086] Example 4 (Cellular Experiments in Radiotherapy-Resistant Cells)

[0087] 1. Verify the efficiency of siRNA knockdown of lncMNX1-AS1

[0088] qRT-PCR results confirmed that the knockdown efficiency of lncMNX1-AS1 by siMNX1-AS1 exceeded 50% in MDA-MB-231R cells. Figure 7 )

[0089] 2. Evaluate the effect of siMNX1-AS1 on the proliferation, migration, and invasion of radiotherapy-resistant breast cancer cells

[0090] The effects of siMNX1-AS1 on the clone formation ability and migration / invasion ability of breast cancer cells were evaluated by clone formation assay and transwell migration / invasion assay. The experimental results showed that siMNX1-AS1 could weaken the clone formation ability and migration / invasion ability of breast cancer radiotherapy-resistant cells. Figure 8 ).

[0091] 3. Single-click multi-target assay to evaluate the effect of siMNX1-AS1 on radioresistance of breast cancer cells

[0092] A) Six six-well plates were labeled with 0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy, respectively; breast cancer cells were collected 148 hours after transfection with siMNX1-AS;

[0093] B) Six-well plates were seeded with the appropriate number of cells according to the radiation dose (higher doses require more cells to ensure colony formation). At each radiation dose, cells were divided into an untreated group and a siMNX1-AS1 transfection group. They were then irradiated with 0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy of photon radiation. The cell culture medium was changed on the day after radiation therapy.

[0094] C) Place the six-well plate in a 37°C incubator for 14 days, regularly observing the colony formation status in the six-well plate. Follow the same steps as for the plate colony formation experiment until the number of colonies in the six-well plate is counted.

[0095] D) The number of colonies counted was divided by the total number of cells seeded to obtain the colony formation rate. Finally, the colony formation rate at each dose point was normalized to the value at 0 Gy irradiation.

[0096] E) A single-click multi-target model was used to generate survival curves, and radiobiological parameters (D0 and Dq values) were statistically analyzed to compare the effects of siMNX1-AS1 transfection on the radioresistance of breast cancer cells.

[0097] The results showed that knocking down the expression of lncMNX1-AS1 using siMNX1-AS1 significantly weakened the radioresistance of breast cancer cells ( Figure 9 ).

[0098] 4. Verify the overexpression efficiency of the lncMNX1-AS1 overexpression plasmid in the MDA-MB-231 parental cell line

[0099] To further verify the effect of lncMNX1-AS1 on radiotherapy resistance, the expression level of lncMNX1-AS1 increased by more than 20 times after the MDA-MB-231 parental cell line was transfected with the lncMNX1-AS1 overexpression plasmid ( Figure 10 ).

[0100] 5. Single-click multi-target assay to evaluate the effect of overexpression of MNX1-AS1 on radiotherapy of breast cancer cells

[0101] A) Six six-well plates were labeled with 0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy, respectively; breast cancer cells were collected 48 hours after transfection with the lncMNX1-AS1 overexpression plasmid;

[0102] B) Six-well plates were seeded with the appropriate number of cells according to the irradiation dose (higher doses require more cells to ensure colony formation). Cells irradiated at each dose were divided equally into a vector group and a lncMNX1-AS1 group. They were then irradiated with 0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy of photon radiation. The cell culture medium was changed on the day after radiotherapy.

[0103] C) Place the six-well plate in a 37°C incubator for approximately 14 days, regularly observing the formation of colonies in the six-well plate. Follow the same steps as for the plate colony formation assay until the number of colonies in the six-well plate is counted.

[0104] D) The number of colonies counted was divided by the total number of cells seeded to obtain the colony formation rate. Finally, the colony formation rate at each dose point was normalized to the value at 0 Gy irradiation.

[0105] E) A single-click multi-target model was used to generate survival curves, and radiobiological parameters (D0 and Dq values) were statistically analyzed to compare the effect of overexpression of lncMNX1-AS1 on the radioresistance of breast cancer cells.

[0106] The results showed that exogenous expression of lncMNX1-AS1 using the lncMNX1-AS1 overexpression plasmid significantly enhanced the radioresistance of breast cancer cells. Figure 11 )

[0107] Example 5 (Preparation and Characterization of Nanosensitizer TREARA)

[0108] 1. After demonstrating the inhibitory function of siMNX1-AS1 in MDA-MB-231R cells, we constructed a multifunctional RNAi nanoplatform, TREARA, to deliver siMNX1-AS1 in a targeted manner. The preparation method for TREARA is as follows:

[0109] Dissolve 1 nmol of Cy5-labeled siMNX1-AS1 in water to prepare an aqueous solution with a concentration of 0.1 nmol / μL.

[0110] 50 μL of a DMF solution of G0-C14 (concentration: 5 mg / mL) was mixed with 10 μL of the above aqueous solution to form a mixture A.

[0111] Take 200 μL of PDSA polymer DMF solution (concentration is 20 mg / mL DMF), 140 μL of DSPE-mPEG 3000 A DMF solution (concentration of 20 mg / mL), 20 μL of a DMF solution of DSPE-PEG3000-NHS (concentration of 20 mg / mL), and 50 μL of αTrop2 (CST) were mixed to form a mixture B.

[0112] Mixture A and mixture B were mixed to form mixture C, which was then added dropwise to 5 mL of enzyme-free water while stirring at a stirring speed of 1400 rpm. After the addition was completed, stirring was continued at room temperature for 30 minutes. The organic reagent and free compounds were removed using an ultrafiltration tube. The final concentrated solution was a sensitizer, which was named TREARA.

[0113] The obtained TREARA was dispersed in 1 mL of PBS buffer (pH 7.4).

[0114] Cy5-labeled siMNX1-AS1 nanoparticles (REARA) were prepared in the same manner as TREARA, but they were not conjugated to αTrop2;

[0115] The difference between Cy5-labeled negative control (NC) siRNA nanoparticles (NP-siNC) and REARA is that the siRNA used is a control siRNA (siCTL), the sequence of which is as follows:

[0116] Sense strand sequence: 5′-CATGACCAAC TGATGG-3′ (SEQ ID NO: 2);

[0117] Antisense strand sequence: 5′-CCATCAGTTGGTCATG-3′ (SEQ ID NO: 3).

[0118] The results showed that the obtained nanoparticles NP-siNC, REARA, and TREARA were all dispersed in a clear spherical shape. The average water content and particle size were further measured by a Malvern particle size analyzer to be 330.0±0.6nm, 274.2±5.0nm, and 257.2±7.0nm, respectively. The zeta potential of the three nanoparticles was negative, which helps to avoid degradation by circulating nucleases. Figure 12 )

[0119] In this embodiment, DMF (N,N-Dimethylformamide) is N,N-dimethylformamide, CAS number is 68-12-2;

[0120] PDSA is a reductive polydisulfide. The synthesis method of PDSA is specifically described in the following literature:

[0121] Xu

[0122] More specifically, the synthesis method of PDSA can be found in the Supplementary Information of the above-mentioned document.

[0123] DSPE-mPEG 3000 (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyle-ne glycol)-3000]) is distearoylphosphatidylethanolamine-polyethylene glycol 3000, CAS number 147867-65-0; Brand: Ruixibio, Product number: R-1028-2K

[0124] DSPE-PEG 3000 -NHS (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinimidyl-(polyethylene glycol)-3000]) is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 3000-N-hydroxysuccinimide; Brand: Aladdin, Product Number: D163641-100mg

[0125] G0-C14 is a cationic lipid compound alkyl-modified polyaminoamine (PAMAM) dendrimer with CAS number 1510653-27-6. Brand: (GLPBIO), Product number: G0-C14

[0126] Example 6 (Partial Cell Experiment of Nanosensitizer)

[0127] 1. Evaluating TREAR Internalization Effects

[0128] To evaluate the cellular internalization ability of TREARA, MDA-MB-231R cells were cultured overnight in 6-well plates and then treated with PBS, siMNX1-AS1, REARA, and TREARA. After 4 hours of incubation, cells were collected and analyzed using flow cytometry. The results showed that compared with the siMNX1-AS1 and REARA groups, TREARA had the highest cellular uptake efficiency, indicating that the αTrop2-modified nanodelivery system enhanced cellular uptake efficiency. Figure 13 )

[0129] 2. Evaluation of the Silencing Efficiency of REARA and TREARA

[0130] qRT-PCR was used to evaluate the RNA silencing efficiency of REARA and TRERA in MDA-MB-231R cells. The results showed that TRERA silenced approximately 65.4% of lncMNX1-AS1 expression at a 30nM siRNA dose, significantly higher than the 49.7% silencing efficiency of REARA against lncMNX1-AS1 at the same siRNA dose. These results indicate that αTrop2 coupling effectively enhances the silencing efficiency of lncMNX1-AS1 by enhancing the cellular uptake of TRERA. Figure 14 3. Evaluate the effect of TREARA on the proliferation of breast cancer cells

[0131] The plate clone formation experiment evaluated the effect of TREAR combined with radiotherapy on the proliferation ability of breast cancer cells. The experimental results showed that TREAR combined with radiotherapy could weaken the proliferation and clone formation ability of breast cancer cells. Figure 15 )

[0132] 4. Evaluating the Effect of TREARA on Migration and Invasion of Breast Cancer Cells

[0133] Transwell migration / invasion assay was used to evaluate the effect of TREAR combined with radiotherapy on cell migration / invasion ability. The experimental results showed that TREAR combined with radiotherapy significantly inhibited the migration / invasion ability of MDA-MB-231R cells. Figure 16 )

[0134] 5. Evaluating the Effect of TREARA on ROS

[0135] To test whether TREARA could effectively synergize with radiotherapy to enhance ROS production, cells were treated with PBS, NP-siNCs, or TREARA and cultured for 6 hours. The cells were then irradiated at a dose of 0 or 4 Gy. After irradiation, the cells were cultured for an additional 8 hours. The DCF-DA probe was then added and incubated at 37°C in the dark for 30 minutes. Finally, the DCF signal was observed using an inverted fluorescence microscope. The results showed that radiotherapy induced ROS production in some MDA-MB-231 cells, and that combination with NP-siNCs induced even greater ROS production. Furthermore, TREARA combined with radiotherapy significantly increased intracellular ROS production.

[0136] ( Figure 17 )

[0137] 6. Evaluate the ability to kill breast cancer cells

[0138] After verifying the effective stimulation of ROS production by TREARA combined with radiotherapy, we further used live / dead dual fluorescence staining analysis to verify the cell-killing ability of this treatment strategy. The cells were incubated with PBS, NP-siNC or TREARA for 6 hours, and then irradiated with X-rays at a dose of 0 or 4 Gy. After the cells were cultured for 8 hours, calcein AM and propidium iodide (PI) were added to the culture medium and incubated in the dark for 30 minutes, and the staining results were observed under a fluorescence microscope. Calcein AM can dye living cells green, while dead cells will be stained red by PI. The results showed that compared with other treatments, TREARA combined with radiotherapy had the highest killing efficiency for MDA-MB-231 and MDA-MB-231R cells. And this result is consistent with the ROS detection, suggesting that TREARA can improve the efficiency of radiotherapy by promoting ROS generation. ( Figure 18 )

[0139] 7. Single-click multi-target model to evaluate the effect on breast cancer radioresistance

[0140] A) MDA-MB-231 and MDA-MB-231R cells were incubated with naked siNC, Lipo3k / siMNX1-AS1, NP-siNC, or TREARA for 6 hours, followed by replacement with fresh complete medium. Cells were harvested after an additional 48 hours of culture.

[0141] B) Design a gradient of cell number to be seeded based on the irradiation dose (the higher the dose, the more cells to seed to ensure colony formation). The cells collected in step A) are seeded into corresponding six-well plates, cultured overnight, and then irradiated at doses of 0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, and 10 Gy. The cell culture medium is replaced on the day after radiotherapy.

[0142] C) Continue culturing the cells for 14 days, regularly observing the formation of colonies in the six-well plate. Follow the same steps as for the plate colony formation experiment until the number of colonies in the six-well plate is counted.

[0143] D) The clone formation rate of cells in each treatment group was calculated and normalized to the value at 0 Gy irradiation.

[0144] E) A single-target multi-target model was used to generate survival curves, and radiobiological parameters (D0 and Dq values) were statistically analyzed to compare the effects of each treatment group on the radioresistance of breast cancer cells.

[0145] The results showed that TREARA could significantly enhance the radiosensitivity of MDA-MB-231 and MDA-MB-231R breast cancer cells.

[0146] Example 7 (Animal Experiment)

[0147] 1. Biodistribution in vivo

[0148] The in vivo distribution of TREARA was evaluated using the mouse MDA-MB-231R subcutaneous xenograft tumor model. siMNX1-AS1-Cy5, REARA, or TREARA was injected into mice through the tail vein, and the in vivo distribution of the drug was observed 6 hours later using the IVIS Lumina III imaging system. The results showed that the fluorescence signal of TREARA in subcutaneous transplanted tumors of mice was the highest, the signal of REARA lacking αTrop2 modification in the tumor was second, and the signal of the free siMNX1-AS1-Cy5 group was the weakest, indicating that TREARA has efficient in vivo tumor targeting. In addition, compared with the mice in the free siMNX1-AS1-Cy5 group, the mice in the REARA and TREARA groups showed lower renal deposition. ( Figure 20 )

[0149] 2. Animal experiments verify the ability to inhibit tumor growth

[0150] After demonstrating tumor accumulation in vivo, we further investigated the ability of TREARA to inhibit tumor growth under radiotherapy. To this end, mice bearing subcutaneous MDA-MBA-231R xenografts were divided into groups: blank NP group, TREARA group, radiotherapy alone group, or TREARA combined with radiotherapy group. Treatments were administered every two days for three consecutive times, with radiotherapy (10 Gy / mouse) administered the day after the third dose, and a second radiotherapy treatment was given 7 days later. After 30 days of evaluation, tumor size in mice treated with TREARA combined with radiotherapy was reduced by approximately 15%. In contrast, tumor size increased by 797.1%, 485.1%, and 244.6% in mice treated with blank NP, TREARA, and radiotherapy alone, respectively. At day 30, the average tumor weight of mice treated with TREARA combined with radiotherapy was significantly lower than that in mice treated with blank NP, TREARA, and radiotherapy alone. These results strongly demonstrate that TREARA can effectively inhibit the growth of radioresistant tumors in vivo.

[0151] After demonstrating that TREARA can effectively inhibit the growth of radiotherapy-resistant tumors in vivo, a metastatic tumor model established with luciferase (Luc)-expressing MDA-MB-231R cells was used to verify whether TREARA can inhibit the growth of radiotherapy-resistant metastases in combination with radiotherapy. Metastatic tumor growth was monitored by capturing bioluminescent images of the tumor site. Mice with MDA-MBA-231R lung metastases were divided into groups: blank NP group, TREARA group, radiotherapy alone group, or TREARA combined with radiotherapy group, and treated once every 2 days for 3 consecutive times. Radiotherapy (10 Gy / mouse) was given the day after the third dose, and the second radiotherapy was given 7 days later. After treatment, mice treated with TREARA combined with radiotherapy showed the lowest signal at the tumor site compared with the other three treatment groups. Compared with mice treated with radiotherapy alone, the average radiation rate of the ex vivo lung bioluminescence images of mice treated with TREARA combined with radiotherapy was reduced by 48.0±8.1%. These results strongly indicate that the combination of TREARA and radiotherapy can effectively inhibit the growth of radiotherapy-resistant metastases. ( Figure 21 )

[0152] 3. In vivo toxicity evaluation

[0153] To evaluate in vivo toxicity, mice bearing MDA-MB-231R tumors were randomly divided into four groups (n=3) and injected via the tail vein with PBS, naked siMNX1-AS1, REARA, or TREARA. Following treatment, the hearts, livers, spleens, lungs, and kidneys of the mice were histologically sectioned and stained with H&E. In addition, blood samples were collected to assess liver and kidney function. The results showed no significant abnormalities in the hearts, livers, spleens, lungs, or kidneys, and no significant abnormalities in any blood markers were observed.

[0154] ( Figure 22 )。

Claims

1. A sensitizer for enhancing the radiotherapy sensitivity of breast cancer, characterized in that: Including MNX1-AS1 inhibitors.

2. The sensitizer for increasing the radiotherapy sensitivity of breast cancer according to claim 1, characterized in that: The MNX1-AS1 inhibitor is siRNA.

3. The sensitizer for increasing the radiotherapy sensitivity of breast cancer according to claim 2, characterized in that: The sense strand sequence of the siRNA is 5′-GAACA ACGCAGACAACATA-3′, and the antisense strand sequence is 5′-TATGTTGTCTGCGTTGTTC-3′; Alternatively, the sense strand sequence of the siRNA is 5′-CTGCCTGCATGCTTTACCA-3′, and the antisense strand sequence is 5′- TGGTAAAGCATGCAGGCAG-3′.

4. The sensitizer for enhancing the radiotherapy sensitivity of breast cancer according to claim 1, characterized in that Also included are GSH depleting agents and / or TROP2 inhibitors.

5. The sensitizer for enhancing the radiotherapy sensitivity of breast cancer according to claim 3, characterized in that: The GSH depleting agent is reduction-reactive polydisulfide; and the TROP2 inhibitor is an anti-TROP2 monoclonal antibody.

6. Application of long non-coding RNA MNX1-AS1 detection reagent in the preparation of products for predicting and diagnosing breast cancer cell radiotherapy resistance and / or prognosing the efficacy of breast cancer cell radiotherapy.

7. The use according to claim 6, characterized in that The long non-coding RNA MNX1-AS1 detection reagent includes primers and / or probes for detecting long non-coding RNA MNX1-AS1 in breast cancer samples.

8. Use of the sensitizer according to claim 1 in the preparation of a product for enhancing the sensitivity of breast cancer to radiotherapy.

9. A method for preparing the sensitizer according to any one of claims 1 to 5, characterized in that: include: S1, mixing the siRNA aqueous solution and the DMF solution of G0-C14 to form a mixture A; S2, DMF solution of PDSA polymer, DSPE-mPEG 3000 DMF solution, DSPE-PEG 3000 -NHS DMF solution to form mixture B; S3. Mix the mixture A and the mixture B to form a mixture C, add the mixture C dropwise into enzyme-free water, stir, and ultrafilter to obtain a concentrated solution as the sensitizer.

10. The method for preparing a sensitizer according to claim 9, wherein: In step S2, anti-TROP2 monoclonal antibody was also added.

Images