Cancer therapeutic drug

Abstract

The application relates to a cancer treatment drug and belongs to the technical field of cancer treatment drugs. The application scheme clarifies the correlation between BHLHE40 expression and TNBC prognosis, determines the possibility of tumor hypoxia-induced BHLHE40 as a TNBC treatment target, clarifies the mechanism of rapid induction of BHLHE40 mRNA and protein levels by tumor hypoxia from two aspects of hypoxia-mediated transcriptional regulation and mitochondrial ROS production and regulation, and clarifies the mechanism of BHLHE40 in tumor hypoxia-mediated TNBC poor prognosis, thereby providing sufficient theoretical basis for optimizing the existing radiotherapy method of TNBC.

Description

Technical Field

[0001] This application relates to cancer treatment drugs and belongs to the field of cancer treatment drug technology. Background Technology

[0002] Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer cases. Due to the simultaneous lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) as therapeutic targets, TNBC patients typically face higher recurrence rates, a greater likelihood of metastasis, and shorter overall survival compared to other breast cancer subtypes. Therefore, identifying novel targets and mechanisms in TNBC and optimizing existing treatment methods has become an urgent clinical challenge.

[0003] Intratumoral hypoxia, caused by the imbalance between the oxygen demand of cancer cells and stromal cells and the oxygen supply from dysfunctional microvessels within the tumor, has been shown to be a key characteristic of tumor-associated neoplasia (TNBC) and may be associated with poor prognosis. However, the underlying mechanisms by which tumor hypoxia affects the prognosis of TNBC have not yet been fully elucidated. Notably, to adapt to hypoxia, tumor cells can initiate a wide range of biological responses to maintain their survival and proliferation. These key responses include: 1) induction of hypoxia-inducible factors (HIFs) mediating transcriptional regulation; and 2) induction of mitochondrial reactive oxygen species (ROS) mediating redox regulation.

[0004] On one hand, cells regulate ROS levels and maintain cellular redox balance through antioxidant enzyme systems. Superoxide dismutase (SOD) regulates ROS levels by dismutating highly reactive superoxide anion free radicals into H2O2 and O2. Peroxiredoxin (PRDX) is another highly conserved antioxidant enzyme in cells; it reduces intracellular ROS levels by catalyzing the reduction of H2O2 to H2O. Glutathione peroxidase (GPX) utilizes glutathione as a reducing agent to reduce H2O2 and other peroxides to H2O, thereby reducing ROS levels and protecting cells from oxidative stress damage. Catalase (CAT) is another peroxidase; unlike GPX, CAT is an iron-dependent redox enzyme that also reduces ROS levels by reducing H2O2 to H2O. Glutaredoxin (GLRX) plays a key role in the cellular antioxidant enzyme system by catalyzing the reversible reduction of glutathione-protein disulfide.

[0005] In addition to the antioxidant enzyme systems mentioned above, cells can also directly utilize small-molecule antioxidants to reduce ROS. For example, vitamin C is a potent antioxidant that can directly reduce ROS. - It undergoes redox reactions with substances like H2O2 to generate H2O, thereby reducing ROS levels. Furthermore, it can reduce oxidized glutathione (GSSG) to reduced glutathione (GSH), enhancing the antioxidant capacity of cells. Human cells cannot synthesize vitamin C themselves and primarily absorb it through the sodium-dependent vitamin C transporter (SVCT). Therefore, the expression level of SVCT can indirectly reflect the antioxidant capacity of cells.

[0006] Furthermore, reduced glutathione (GSH) is one of the most important intracellular antioxidants. GSH is a tripeptide composed of glutamate, cysteine, and glycine. Glutamate and cysteine ​​first combine under the action of γ-glutamylcysteine ​​synthase (γ-GSC) to form γ-glutamylcysteine, which is then further combined with glycine under the action of glutathione synthase (GS) to finally form glutathione. Notably, cysteine, used in GSH synthesis, is itself an antioxidant. Studies have also shown that tumor cells primarily rely on the cysteine ​​transporter SLC7A11 on the cell membrane to obtain cysteine ​​from the extracellular environment. This transport system includes the active protein subunit SLC7A11 and the chaperone protein subunit SLC3A2, which can export intracellular glutamate and import extracellular cysteine ​​in a 1:1 ratio. One molecule of cysteine ​​can be further converted into two molecules of cysteine, providing raw materials for GSH synthesis and cellular antioxidant responses.

[0007] The aforementioned responses influence cellular biological functions by directly regulating mRNA and protein levels, helping cells adapt to the harsh hypoxic environment of tumors and ultimately affecting cancer treatment outcomes. Therefore, these hypoxia-driven biological responses are not only key mechanisms for cellular adaptation to tumor hypoxia but also highly attractive targets for tumor therapy. However, existing research indicates that the effects of HIF-transcribed target genes and ROS-redox-regulated target proteins on tumor cell resistance to treatment are controversial, necessitating in-depth analysis targeting specific mechanisms.

[0008] E40 (basic helix-loop-helix family member E40) is a widely reported transcription factor. Its gene promoter region contains conserved sequences that can bind to various transcription factors; therefore, current research primarily focuses on changes in BHLHE40 mRNA levels, with very few reports on direct effects on BHLHE40 protein levels. Furthermore, although existing studies have reported that HIF-1 can transcribe and activate BHLHE40 gene expression after 6 hours or longer of hypoxia, research on how early hypoxia affects BHLHE40 expression remains lacking. Functionally, BHLHE40 protein typically functions as a transcriptional repressor, but limited research suggests that it can also transcribe and activate certain genes. Additionally, the role of BHLHE40 in tumor cell fate remains controversial, thus requiring further investigation into the precise mechanisms by which BHLHE40 influences cancer treatment efficacy, in conjunction with specific therapeutic approaches.

[0009] Current research mainly focuses on how BHLHE40 mRNA changes and how BHLHE40 functions as a transcriptional repressor, while reports on direct regulation of BHLHE40 protein levels and BHLHE40’s non-transcriptional factor functions are few and far between.

[0010] Therefore, the following questions still need to be explored and studied: 1) How early hypoxia (2 hours or earlier) affects BHLHE40 expression has not been elucidated; 2) BHLHE40 may play a potential transcriptional activation function or a non-transcription factor function; 3) Existing research on the impact of BHLHE40 on tumor cell fate is controversial, so it is necessary to further explore the exact mechanism by which BHLHE40 affects tumor progression and treatment efficacy in conjunction with specific treatment methods; 4) The current changes and exact role of BHLHE40 in TNBC are still unclear, and it is necessary to explore in depth the specific molecular mechanism of BHLHE40 in TNBC and its possibility as a potential therapeutic target. Summary of the Invention

[0011] To address the aforementioned issues, this application, through experiments and research, clarified the correlation between BHLHE40 expression and TNBC prognosis, and determined the potential of tumor hypoxia-induced BHLHE40 as a therapeutic target for TNBC. Furthermore, it elucidated the mechanism by which tumor hypoxia rapidly induces BHLHE40 mRNA and protein levels from two aspects: hypoxia-mediated transcriptional regulation and mitochondrial ROS production and its regulation. Finally, it clarified the mechanism of action of BHLHE40 in the poor prognosis of tumor hypoxia-mediated TNBC, providing sufficient theoretical basis for optimizing existing radiotherapy methods for TNBC.

[0012] This application provides an isolated nucleotide fragment comprising a sense strand as shown in SEQ ID No. 1 and an antisense strand as shown in SEQ ID No. 2; or, The nucleotide fragments include a sense strand as shown in SEQ ID No. 3 and an antisense strand as shown in SEQ ID No. 4.

[0013] This application provides the use of the above-mentioned nucleotide fragments in the preparation of drugs for treating and / or alleviating tumor occurrence and growth.

[0014] Optionally, the tumor may be lung cancer, breast cancer, colon cancer, or cervical cancer.

[0015] Optionally, the breast cancer is triple-negative breast cancer.

[0016] This application provides a tumor treatment drug comprising the isolated nucleotide fragments described above.

[0017] Optionally, the tumor treatment drug can reduce the activity of SLC7A11 in tumor tissue.

[0018] Optionally, the tumor treatment drug can inhibit the transcription of SLC7A11, thereby reducing the activity of SLC7A11 in tumor tissue.

[0019] This application provides a cancer treatment drug that can target mitochondria, and the cancer treatment drug further includes a component that can break disulfide bonds.

[0020] Optionally, the cancer treatment drug includes a) a mitochondrial localization signal peptide and b) a reducing agent capable of breaking disulfide bonds.

[0021] This application provides a drug that reduces poor prognosis after chemotherapy for breast cancer, the drug being able to reduce the activity of BHLHE40 in cancer tissue.

[0022] The beneficial effects of this application include, but are not limited to: 1. Based on the cancer treatment drug of this application, it was first discovered that TNBC can induce increased BHLHE40 expression in the early stage of hypoxia (2 hours or earlier), and the dual mechanism of rapid induction of BHLHE40 expression by tumor hypoxia was elucidated in detail: through HIFs-mediated increase in BHLHE40 mRNA expression and ROS-mediated decrease in BHLHE40 protein degradation, thereby rapidly inducing high expression of BHLHE40 in TNBC.

[0023] 2. According to the cancer treatment drug of this application, the BHLHE40 protein, which has been previously reported as a transcription factor, is found to be distributed in mitochondria for the first time. At the same time, the level of mitochondrial BHLHE40 protein can be induced to increase by hypoxia. The mitochondrial distribution of BHLHE40 protein overcomes the compartmentalization restriction of redox reactions with mitochondrial ROS generated by hypoxia and the problem of competitive reaction with a large number of intracellular ROS scavenging enzymes.

[0024] 3. Based on the cancer treatment drug of this application, BHLHE40-mediated hypoxia-induced radioresistance in TNBC was discovered for the first time, and the intrinsic mechanism of this biological action was elucidated in detail: BHLHE40 upregulated by hypoxia increases the expression of a series of antioxidant genes and the level of intracellular antioxidants, thereby negatively regulating ROS levels, highlighting the important role and core position of BHLHE40 in maintaining cellular redox homeostasis under tumor hypoxic conditions.

[0025] 4. According to the cancer treatment drug of this application, BHLHE40 upregulated by hypoxia enhances tumor hypoxia-induced TNBC radioresistance by promoting the expression of antioxidant genes and reducing intracellular ROS levels. Based on this, the expression level of BHLHE40 is positively correlated with the poor prognosis of TNBC patients. This study shows that BHLHE40 plays an important role in maintaining cellular redox homeostasis under tumor hypoxia, clarifying the feasibility of tumor hypoxia-induced BHLHE40 as a target for TNBC treatment, and providing an important theoretical basis for optimizing existing radiotherapy methods for TNBC.

[0026] 5. According to the cancer treatment drug of this application, the BHLHE40 protein, previously reported as a transcription factor, is distributed in mitochondria. As a "sensor" of hypoxia-induced mitochondrial ROS, it can directly react with ROS to form intermolecular disulfide bonds, mediating the formation of its homodimer. Furthermore, the BHLHE40 homodimer has transcription factor activity, which affects the level of intracellular antioxidants (such as cysteine, reduced glutathione, etc.) by increasing the expression of a series of antioxidant genes, and ultimately negatively regulates ROS, thus exerting its "regulator" function for intracellular ROS levels. Attached Figure Description

[0027] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1a The results of RT-qPCR experiments on the mRNA of FOS, ANGPTL4, ADM, LOX, STBD1, P4HA2, BHLHE40, PFKFB3 and CITED2 in the MDA-MB-231 cell line involved in Example 2 of this application after normoxic / hypoxic treatment for 2 hours are presented.

[0028] Figure 1b The results of RT-qPCR experiments on the mRNA of FOS, ANGPTL4, ADM, LOX, STBD1, P4HA2, BHLHE40, PFKFB3 and CITED2 in the MDA-MB-468 cell line involved in Example 2 of this application after normoxic / hypoxic treatment for 2 hours are presented.

[0029] Figure 1c The results of RT-qPCR experiments on BHLHE40 mRNA were obtained by subjecting the HS278T cell line involved in Example 2 of this application to normoxic / hypoxic treatment for 2 hours.

[0030] Figure 1dThe results of RT-qPCR experiments on BHLHE40 mRNA were obtained by subjecting the SUM159 cell line involved in Example 2 of this application to normoxic / hypoxic treatment for 2 hours.

[0031] Figure 1e The results of RT-qPCR experiments on BHLHE40 mRNA were obtained by subjecting the MCF7 cell line involved in Example 2 of this application to normoxic / hypoxic treatment for 2 hours.

[0032] Figure 2a The results of RT-qPCR experiments on BHLHE40 mRNA were obtained by subjecting the MDA-MB-231 cell line involved in Example 2 of this application to normoxic / hypoxic treatment for 0-72 hours.

[0033] Figure 2b The results of RT-qPCR experiments on BHLHE40 mRNA were obtained by subjecting the MDA-MB-468 cell line involved in Example 2 of this application to normoxic / hypoxic treatment for 0-72 hours.

[0034] Figure 3a The results of Western blot experiments on BHLHE40 and HIF-1α proteins were performed on the breast cancer cell line involved in Example 2 of this application after normoxic / hypoxic treatment for 0-2 hours.

[0035] Figure 3b The results of Western blot experiments on BHLHE40 and HIF-1α proteins were performed on the breast cancer cell line involved in Example 2 of this application after normoxic / hypoxic treatment for 0-72 h.

[0036] Figure 3c The results of Western blot (WB) experiments on BHLHE40 protein were performed on the breast cancer cell line involved in Example 2 of this application after normoxic / hypoxic treatment for 0-24 hours.

[0037] Figure 4a The weight of the 4T1-BALB / c mouse involved in Example 3 of this application.

[0038] Figure 4b The tumor size is that of the 4T1-BALB / c mouse involved in Example 3 of this application.

[0039] Figure 4c This is the original image of the 4T1-BALB / c mouse tumor sample involved in Example 3 of this application.

[0040] Figure 4d The images shown are representative images of immunohistochemically labeled BHLHE40 and HIF-1α proteins from serial sections of tumor samples from 4T1-BALB / c mice involved in Example 3 of this application (blue and red boxes indicate higher magnification, and scale bars are 200 μm and 20 μm).

[0041] Figure 5 The images shown in Example 3 of this application are representative images of immunofluorescence-labeled BHLHE40 and BNIP3 proteins in different regions of human TNBC tumor tissue (blue represents DAPI-labeled cell nuclei; red represents BHLHE40 protein; green represents BNIP3 protein; scale bar is 50 μm).

[0042] Figure 6 The BHLHE40 involved in Embodiment 4 of this application high and BHLHE40 low KM survival analysis results for TNBC patients (HR is the hazard ratio).

[0043] Figure 7 The results of Western blot experiments on BHLHE40, HIF-1β, HIF-1α and BNIP3 proteins were performed on MDA-MB-231 and MDA-MB-468 cells with HIF-1β gene knockdown as described in Example 5 of this application after normoxic / hypoxic treatment for 0, 1, 2 or 24 hours.

[0044] Figure 8a The results of RT-qPCR experiments on BHLHE40 mRNA in MDA-MB-231 cells with HIF-1β gene knockdown as described in Example 5 of this application after normoxic / hypoxic treatment for 0, 1, 2 or 24 hours.

[0045] Figure 8b The results of RT-qPCR experiments on BHLHE40 mRNA in MDA-MB-468 cells with HIF-1β gene knockdown as described in Example 5 of this application after normoxic / hypoxic treatment for 0, 1, 2 or 24 hours.

[0046] Figure 9a The results of the CCK-8 experiment of MDA-MB-231 cells involved in Example 5 of this application under normoxic / hypoxic conditions for 2 hours, with and without NAC / VH298 treatment.

[0047] Figure 9b The results of the CCK-8 experiment of the MDA-MB-468 cells involved in Example 5 of this application under normoxic / hypoxic conditions for 2 hours, with and without NAC / VH298 treatment.

[0048] Figure 9c The results of Western blot experiments on BHLHE40, HIF-1α, HIF-2α, and HIF-1β proteins were performed on MDA-MB-231 and MDA-MB-468 cells involved in Example 5 of this application with and without 0.1 mMVH298 treatment.

[0049] Figure 9d The results of RT-qPCR experiments on BHLHE40 mRNA in MDA-MB-231 cells with and without 0.1 mM VH298 treatment, as described in Example 5 of this application.

[0050] Figure 9e The results of RT-qPCR experiments on BHLHE40 mRNA in MDA-MB-468 cells with and without 0.1 mM VH298 treatment, as described in Example 5 of this application.

[0051] Figure 10a The results of flow cytometry experiments of MDA-MB-231 cells involved in Example 6 of this application under normoxic / hypoxic conditions for 2 hours, with and without NAC treatment (vertical axis is the median value of the total area of ​​DCF fluorescence channels; DCF is 2',7'-dichlorofluorescein, and A is the area).

[0052] Figure 10b The application described in Embodiment 6 involves the following: Figure 10a The statistical results of three biological replicates of the experiment described herein (vertical axis represents the median of the total area of ​​the DCF fluorescence channel; DCF is 2',7'-dichlorofluorescein, and A is the area).

[0053] Figure 10c The results of flow cytometry experiments of MDA-MB-468 cells involved in Example 6 of this application under normoxic / hypoxic conditions for 2 hours, with and without NAC treatment (vertical axis is the median value of the total area of ​​DCF fluorescence channels; DCF is 2',7'-dichlorofluorescein, and A is the area).

[0054] Figure 10d The application described in Embodiment 6 involves the following: Figure 10c The statistical results of three biological replicates of the experiment described herein (vertical axis represents the median of the total area of ​​the DCF fluorescence channel; DCF is 2',7'-dichlorofluorescein, and A is the area).

[0055] Figure 11a The results of Western blot experiments on BHLHE40, HIF-1α, and HIF-1β proteins were performed on MDA-MB-231 and MDA-MB-468 cells involved in Example 6 of this application under normoxic / hypoxic conditions for 2 hours, with / without H2O2 / NAC treatment.

[0056] Figure 11b The results of RT-qPCR experiments on BHLHE40 mRNA in MDA-MB-231 cells involved in Example 6 of this application under normoxic / hypoxic conditions for 2 hours, with and without H2O2 / NAC treatment.

[0057] Figure 11c The results of RT-qPCR experiments on BHLHE40 mRNA in MDA-MB-468 cells involved in Example 6 of this application under normoxic / hypoxic conditions for 2 hours, with and without H2O2 / NAC treatment.

[0058] Figure 12 The results of the Western blot experiment on the BHLHE40 protein were obtained by treating the MDA-MB-231 and MDA-MB-468 cells involved in Example 6 of this application with specified treatment.

[0059] Figure 13 The results of Western blot experiments on BHLHE40 protein in whole cells and cytoplasm of MDA-MB-231 and MDA-MB-468 cells involved in Example 6 of this application under normoxic / hypoxic treatment for 2 or 24 hours are shown (Tubulin is the cytoplasmic protein internal control; Lamin B1 is the nuclear protein internal control; WCL is the whole cell lysis buffer; CYTO is the cytoplasmic lysis buffer).

[0060] Figure 14a Immunoelectron microscopy results of the MDA-MB-468 cells involved in Example 6 of this application after normoxic / hypoxic treatment for 2 / 24 hours (the area circled in dashed is the mitochondria, the red arrow and black colloidal gold particles are BHLHE40 protein, and the scale bar is 1 μm and 500 nm).

[0061] Figure 14b The application described in Embodiment 6 involves the following: Figure 14a The statistical results of three biological replicates of the experiment (vertical axis represents the number of colloidal gold particles, i.e., BHLHE40 protein).

[0062] Figure 14c After the MDA-MB-231 cells involved in Example 6 of this application were subjected to normoxic / hypoxic treatment for 2 / 24 hours, the results of immunofluorescence experiments on BHLHE40 and TOM20 were obtained (red represents BHLHE40, green represents TOM20, blue represents DAPI, white arrows represent co-localization of BHLHE40 and TOM20, and scale bars are 50 μm and 10 μm).

[0063] Figure 14d The application described in Embodiment 6 involves the following: Figure 14c The fluorescence quantitative results of MDA-MB-231 cells subjected to normoxic treatment for 2 hours as described in the experiment (vertical axis is...) Figure 14c (Fluorescence intensity at points on the yellow dashed line in the combined red-green fluorescence graph).

[0064] Figure 14e The application described in Embodiment 6 involves the following: Figure 14c The results of quantitative fluorescence analysis of MDA-MB-231 cells subjected to hypoxia treatment for 2 hours as described in the experiment (vertical axis is...). Figure 14c (Fluorescence intensity at points on the yellow dashed line in the combined red-green fluorescence graph).

[0065] Figure 14f The application described in Embodiment 6 involves the following: Figure 14c The results of quantitative fluorescence analysis of MDA-MB-231 cells after 24 hours of hypoxia treatment described in the experiment (vertical axis is...). Figure 14c (Fluorescence intensity at points on the yellow dashed line in the combined red-green fluorescence graph).

[0066] Figure 15a The IP experiment (R is a rabbit-derived antibody, M is a mouse-derived antibody) was performed on the MDA-MB-231 cells involved in Example 6 of this application under normoxic / hypoxic treatment for 2 / 24 hours, with / without NAC treatment.

[0067] Figure 15b The results of the DTNB experiment are for the mitochondrial BHLHE40 protein involved in Example 6 of this application after denaturation and reduction treatment following immunoprecipitation.

[0068] Figure 15c The results of the DTNB experiment are for the mitochondrial BHLHE40 protein after immunoprecipitation and denaturation treatment, as described in Example 6 of this application.

[0069] Figure 15d The content of disulfide bonds in the mitochondrial BHLHE40 protein after immunoprecipitation, as measured by the DTNB experiment, is shown in Example 6 of this application (Mito-BHLHE40 is the mitochondrial BHLHE40 protein).

[0070] Figure 15e The results of non-reducing and reducing Western blot experiments on mitochondrial BHLHE40 protein were performed on MDA-MB-231 and MDA-MB-468 cells involved in Example 6 of this application under normoxic / hypoxic treatment conditions of 2 / 24 hours (VDAC1 is the internal control of mitochondrial protein, red arrows are BHLHE40 homodimers, gray arrows are heterodimers formed by BHLHE40 monomers binding with other proteins via disulfide bonds, black arrows are BHLHE40 monomers, and asterisks are BHLHE40 protein complexes unrelated to disulfide bonds).

[0071] Figure 16a Microscopic images of the radioresistance of MDA-MB-231 and MDA-MB-468 and their wild-type cell lines involved in Example 7 of this application.

[0072] Figure 16bThe Western blot (WB) experiment (scale bar 100 μm) was performed on the radioresistance of MDA-MB-231 and MDA-MB-468 and the BHLHE40 protein of their wild-type cell lines as described in Example 7 of this application.

[0073] Figure 17a This is the result of an RT-qPCR experiment on BHLHE40 mRNA in MDA-MB-231 cells with BHLHE40 gene knockdown, as described in Example 8 of this application.

[0074] Figure 17b This is the result of an RT-qPCR experiment on BHLHE40 mRNA in MDA-MB-468 cells with BHLHE40 gene knockdown, as described in Example 8 of this application.

[0075] Figure 17c The results of the Western blot (WB) experiment on the BHLHE40 protein in MDA-MB-231 and MDA-MB-468 cells with BHLHE40 gene knockdown, as described in Example 8 of this application.

[0076] Figure 18a The results of CCK-8 experiments on the MDA-MB-231 cells involved in Example 8 of this application after 24, 48 and 72 hours of different doses of radiotherapy.

[0077] Figure 18b The results of CCK-8 experiments on the MDA-MB-468 cells involved in Example 8 of this application after 24, 48 and 72 hours of different doses of radiotherapy.

[0078] Figure 18c The results of a plate clone experiment were obtained after the MDA-MB-231 cells involved in Example 8 of this application were treated with different doses of radiotherapy for 14 days.

[0079] Figure 18d The application described in Embodiment 8 involves the following: Figure 18c The statistical results of the experiment were obtained from three biological replicates (vertical axis represents absorbance relative to the 0 Gray group).

[0080] Figure 18e The results of a plate clone experiment were obtained after the MDA-MB-468 cells involved in Example 8 of this application were treated with different doses of radiotherapy for 14 days.

[0081] Figure 18f The application described in Embodiment 8 involves the following: Figure 18e The statistical results of the experiment were obtained from three biological replicates (vertical axis represents absorbance relative to the 0 Gray group).

[0082] Figure 19aThe results of the CCK-8 assay are as follows: MDA-MB-231 cells with BHLHE40 gene knockdown, as described in Example 8 of this application, were treated with normoxic / hypoxic methods for 24 hours, followed by radiotherapy with different doses for 24, 48, and 72 hours.

[0083] Figure 19b The results of a plate clone experiment were obtained after MDA-MB-231 cells with BHLHE40 gene knockdown, as described in Example 8 of this application, were treated with normoxic / hypoxic methods for 24 hours, followed by radiotherapy with different doses for 14 days.

[0084] Figure 19c The results of the CCK-8 assay are as follows: MDA-MB-468 cells with BHLHE40 gene knockdown, as described in Example 8 of this application, were treated with normoxic / hypoxic methods for 24 hours, followed by radiotherapy with different doses for 24, 48, and 72 hours.

[0085] Figure 19d The results of a plate clone experiment were obtained after MDA-MB-468 cells with BHLHE40 gene knockdown, as described in Example 8 of this application, were treated with normoxic / hypoxic methods for 24 hours, followed by radiotherapy with different doses for 14 days.

[0086] Figure 20a The results of flow cytometry experiments on MDA-MB-231 cells with knocked-down / non-knocked-down BHLHE40 as described in Example 9 of this application after normoxic / hypoxic treatment for 24 hours.

[0087] Figure 20b The application described in Embodiment 9 involves the following: Figure 20a The statistical results of three biological replicates of the experiment are presented (vertical axis represents the median of the total area of ​​the DCF fluorescence channel; DCF is 2',7'-dichlorofluorescein).

[0088] Figure 21a The gene expression levels of SLC7A11 and SLC3A2 in MDA-MB-231 cells with BHLHE40 gene knockdown involved in Example 10 of this application after 24 hours of normoxic / hypoxic treatment.

[0089] Figure 21b A Western blot experiment was performed on the SLC7A11 protein in MDA-MB-231 cells with BHLHE40 gene knockdown as described in Example 10 of this application after 24 hours of normoxic / hypoxic treatment.

[0090] Figure 22a The cysteine ​​level of MDA-MB-231 cells with BHLHE40 gene knockdown involved in Example 10 of this application after being treated with normoxic / hypoxic conditions for 24 hours.

[0091] Figure 22b The reduced glutathione level in MDA-MB-231 cells with BHLHE40 gene knockdown as described in Example 10 of this application after 24 hours of normoxic / hypoxic treatment.

[0092] Figure 23a The results of Western blot experiments on BHLHE40, HIF-1α and BNIP3 proteins were performed on MDA-MB-231 cells with BHLHE40 gene knockdown involved in Example 11 of this application after being treated with normoxic / hypoxic conditions for 24 hours.

[0093] Figure 23b This is a schematic diagram of the HIF transcriptional active reporter plasmid designed based on the dual-luciferase reporter enzyme principle involved in Example 11 of this application.

[0094] Figure 23c Dual-luciferase reporter enzyme assay was performed on MDA-MB-231 cells with BHLHE40 gene knockdown as described in Example 11 of this application after normoxic / hypoxic treatment for 24 hours.

[0095] Figure 23d The expression levels of a series of classic HIF target genes were obtained by subjecting the BHLHE40 gene knockdown MDA-MB-231 cells involved in Example 11 of this application to normoxic / hypoxic treatment for 24 hours.

[0096] Figure 24a After treating the MDA-MB-231 cells with knocked-down / non-knocked-down BHLHE40 involved in Example 12 of this application with normoxic / hypoxic treatment for 24 hours, the level of SLC7A11 on the cell membrane surface was measured by flow cytometry.

[0097] Figure 24b The application described in Embodiment 12 involves the following: Figure 24a The statistical results of three biological replicates of the experiment (vertical axis represents the median fluorescence value of the FITC channel (reflecting the SLC7A11 level in the cell membrane)).

[0098] Figure 25 This refers to the direct binding of the hypoxia-enhanced BHLHE40 and SLC7A11 promoter involved in Embodiment 12 of this application.

[0099] Figure 26a This application's embodiment 13 involves the competitive blocking of the binding of BHLHE40 to the SLC7A11 promoter by decoy #1.

[0100] Figure 26b This application's embodiment 13 involves the use of decoy #1 to inhibit SLC7A11 expression.

[0101] Figure 26c The decoy #1 involved in Example 13 of this application specifically reduces SLC7A11 protein without affecting the stability of BHLHE40 protein.

[0102] Figure 27a The results of dsODN-decoy #1 inhibiting cystine uptake are shown in Example 13 of this application.

[0103] Figure 27b The results of dsODN-decoy #1 inhibiting glutathione synthesis are shown in Example 13 of this application.

[0104] Figure 27c The original flow cytometry results for dsODN-decoy #1 promoting intracellular ROS accumulation under hypoxic conditions, as described in Example 13 of this application.

[0105] Figure 27d This is the statistical result of the dsODN-decoy #1 promoting intracellular ROS accumulation under hypoxic conditions, as described in Example 13 of this application.

[0106] Figure 27e The results of dsODN-decoy #1 restoring radiosensitivity of hypoxic tumor cells are shown in Example 13 of this application. Detailed Implementation

[0107] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of the present application are all purchased through commercial channels.

[0108] The present application solution will be described below through specific embodiments.

[0109] Part 1: Increased BHLHE40 expression in triple-negative breast cancer induced by tumor hypoxia. To better understand tumor hypoxia in TNBC and further explore novel targets that influence TNBC prognosis, thereby elucidating the precise mechanism by which tumor hypoxia affects TNBC prognosis, we first constructed a hypoxia-prognosis model of TNBC patients. We discovered that tumor hypoxia induces increased expression of the basic helix-loop-helix family member E40 (BHLHE40), and verified that BHLHE40 expression is positively correlated with poor prognosis of TNBC, thus clarifying the possibility of tumor hypoxia-induced BHLHE40 as a therapeutic target for TNBC.

[0110] Example 1 To better understand the status of tumor hypoxia in TNBC and its relationship with poor prognosis, 4 public datasets including TCGA_TNBC were used to construct a hypoxia-prognosis model for TNBC patients, followed by model validation and performance evaluation, and finally based on this hypoxia-prognosis model, Hypoxia high / low KM survival analysis was performed on TNBC patients.

[0111] First, univariate Cox regression was performed on the hypoxia-related gene expression profile (including Clinical data) of TNBC patients constructed based on the RNA-seq data of TCGA_TNBC and the HALLMARK_HYPOXIA gene set, and 9 hypoxia-prognosis-related genes of TNBC patients were screened out. Further, LASSO regression was performed on the expression profile and survival information of the 9 hypoxia-prognosis-related genes obtained from the above screening, and 7 genes with the optimal Lambda value (Lambda.min=0.0099) were screened out as the construction genes of the hypoxia-prognosis model for TNBC patients. TNBC patients were divided into Hypoxia based on the median hypoxia score high and Hypoxialow groups, and the hypoxia-prognosis model for TNBC patients was successfully constructed. The validation results showed that the hypoxia-prognosis model constructed in the present application was positively correlated with 7 other published hypoxia models (except the Ragnum hypoxia model), suggesting that these hypoxia models can predict each other, that is, the hypoxia-prognosis model for TNBC patients constructed in the present application is reliable.

[0112] The AUC value is an important indicator for evaluating the performance of a binary classification model, which represents the area under the ROC curve (area under the curve). The AUC value ranges from 0 to 1, where AUC=0.5 indicates that the model has no classification ability, equivalent to random guessing; 0.5<AUC<0.7 indicates that the model has certain classification ability but with average effect; 0.7≤AUC<0.9 indicates that the model has good classification ability; AUC≥0.9 indicates that the model has very good classification ability. By calculating the AUC values of the TCGA_TNBC and GSE103091 datasets, the results showed that AUC(TCGA_TNBC)=0.7993 and AUC(GSE103091)=0.7016, both greater than 0.7, suggesting that the hypoxia-prognosis model for TNBC patients constructed in the present application has good classification ability.

[0113] Because the hypoxia-prognostic model for TNBC patients constructed in this application has reliability and good classification ability, in order to better understand the tumor hypoxia in TNBC and its relationship with poor prognosis, the hypoxia-prognostic model constructed in this application was used to perform Hypoxia analysis on the TCGA_TNBC and GSE103091 datasets, respectively. high and Hypoxia low Survival analyses were performed on patients in different groups. Results showed that in two independent datasets, patients with high-level tumor hypoxia had significantly shorter overall survival (OS) compared to those with low-level tumor hypoxia, suggesting that high-level tumor hypoxia is associated with poor prognosis in patients with TNBC.

[0114] Example 2 like Figure 1a , Figure 1b , Figure 1c , Figure 1d and Figure 1e As shown, in order to further explore potential targets that affect the poor prognosis of TNBC due to high-level tumor hypoxia, TCGA_TNBC:Hypoxia was obtained by screening based on Log2(FoldChange)>0.5 and p value<0.01. high vs. Hypoxia low A total of 54 hypoxia-upregulated genes were identified as differentially expressed between populations. Simultaneously, using Log2(FoldChange) > 1.2 and upregulation in at least 5 TNBC cell lines as the selection criterion, 44 hypoxia-upregulated genes were identified in GSE111653: 24 hours of hypoxia treatment vs. 24 hours of normoxic treatment. The intersection of these differentially expressed hypoxia-upregulated genes between populations and cells yielded 9 hypoxia-upregulated genes that were differentially expressed in both populations and cells. These 9 genes are: FOS, ANGPTL4, ADM, LOX, STBD1, P4HA2, BHLHE40, PFKFB3, and CITED2, exhibiting universality of differential expression between populations and cells.

[0115] When the hypoxia treatment time was further shortened, and the expression levels of the above nine genes were measured 2 hours after hypoxia treatment, BHLHE40 was significantly elevated in both TNBC cell lines compared to the other eight genes. RNA was extracted from two other TNBC cell lines and one non-TNBC cell line after 2 hours of hypoxia treatment, and significantly elevated BHLHE40 expression levels were also detected. These results indicate that, compared to the other eight hypoxia-related genes that show generally differential expression among populations and cells, BHLHE40 has a unique characteristic: it can be significantly induced to increase in the early stages of hypoxia (2 hours or earlier).

[0116] like Figure 2a , Figure 2b As shown, to better observe the changes of BHLHE40 during hypoxia treatment, the hypoxia time was extended to 72 hours after 24 hours of hypoxia (0.5-hour intervals), and after 24 hours of hypoxia (24-hour intervals). RNA was extracted after 0.5, 1, 1.5, 2, 24, 48, and 72 hours of hypoxia to detect BHLHE40 mRNA levels. In vitro hypoxia for 1.5 hours significantly induced an increase in BHLHE40 mRNA expression, and this increase within 24 hours was hypoxia-time dependent; after 24 hours, no significant change was observed. RPL13A was used as a negative control gene for hypoxia treatment. This indicates that in vitro hypoxia induces an increase in BHLHE40 mRNA expression in a hypoxia-time dependent manner within 24 hours.

[0117] like Figure 3a , Figure 3b , Figure 3c As shown, proteins were extracted after 0.5, 1, 1.5, 2, 24, 48, and 72 hours of hypoxia to observe changes in BHLHE40 protein expression during hypoxia treatment. Hypoxia in vitro significantly induced an increase in BHLHE40 protein expression starting from 0.5 hours, a phenomenon almost synchronous with the increase in HIF-1α protein expression, a positive marker of hypoxia. However, unlike the changes in BHLHE40 mRNA, the increase in BHLHE40 protein expression within 2 hours of hypoxia was hypoxia-time dependent, with no significant change after 2 hours. Protein extraction from two other TNBC cell lines and one non-TNBC cell line after 2 and 24 hours of hypoxia treatment also showed that 2 hours of hypoxia significantly induced an increase in BHLHE40 protein expression, but no significant change after 2 hours. Therefore, hypoxia induces an increase in BHLHE40 protein expression in vitro in a hypoxia-time dependent manner within 2 hours.

[0118] Example 3 like Figure 4a , Figure 4b , Figure 4c , Figure 4dAs shown, a mouse TNBC disease model was established by in situ injection of 4T1 cells into the mammary fat pads of BALB / c mice. The body weight and tumor size of the TNBC-affected mice were monitored. Tumors exceeding 1000 mm² were observed. 3 Mice were sacrificed and tumor tissue was dissected for IHC experiments. Representative images of serial sections from the IHC experiments showed that in mouse TNBC, regions with high BHLHE40 expression co-localized with HIF-1α-labeled hypoxic regions.

[0119] An IF assay was performed using tumor tissue from a TNBC patient obtained from Qilu Hospital of Shandong University, with BNIP3 used as a positive marker for hypoxia. Figure 5 As shown in the representative images from the IF experiment, in human TNBC, the region of high expression of BHLHE40 co-localizes with the hypoxic region labeled with BNIP3.

[0120] Pearson correlation coefficients were calculated for the hypoxia score of each patient in TCGA_TNBC based on Log2(BHLHE40-TPM) and eight other independent hypoxia features (i.e., hypoxia models) published in an article downloaded from PMID 30643250. The results showed that BHLHE40 expression levels were positively correlated with human TNBC hypoxia scores in all eight independent hypoxia models, suggesting a positive correlation between human TNBC hypoxia levels and BHLHE40 expression levels.

[0121] Example 4 Analysis of the GSE76250 dataset revealed that BHLHE40 expression levels were significantly higher in breast cancer tissue compared to adjacent tissue. Further Western blotting using adjacent and cancerous tissue from six TNBC patients collected from Qilu Hospital of Shandong University also showed significantly higher BHLHE40 protein levels in breast cancer tissue compared to adjacent tissue.

[0122] Analysis of the GSE62931 dataset revealed that BHLHE40 expression was significantly higher in TNBC tumor tissues compared to non-TNBC tumor tissues.

[0123] like Figure 6 As shown, by analyzing BHLHE40 in TCGA_TNBC high and BHLHE40 low Survival analysis was performed on patients in different groups, and it was found that BHLHE40 low Compared to patients, BHLHE40 high The patient presented with a poor prognosis, suggesting that BHLHE40 is associated with poor prognosis in patients with TNBC.

[0124] In this part of the study, to better understand tumor hypoxia in TNBC and further explore novel targets that influence TNBC prognosis, a hypoxia-prognostic model of TNBC patients was constructed using bioinformatics methods. The results showed that high levels of tumor hypoxia were associated with poor prognosis in TNBC. Further in vitro and in vivo RT-qPCR, Western blotting, IHC, and IF assays revealed that tumor hypoxia significantly induces increased BHLHE40 expression in the early stages of hypoxia, and that BHLHE40 expression is associated with poor prognosis in TNBC patients. These results clarify the potential of tumor hypoxia-induced BHLHE40 as a therapeutic target for TNBC.

[0125] Compared to the other eight genes, BHLHE40's hypoxia-induced elevation is unique: it is significantly induced in the early stages of hypoxia (2 hours or earlier). Although a similar increase in PFKFB3 gene expression was observed after 2 hours of hypoxia treatment, its change was not as significant as that of BHLHE40, and therefore it was not further investigated in this section of the study. Besides the aforementioned eight genes, the five well-known target genes of HIF transcriptional regulation also do not exhibit this unique characteristic of BHLHE40 being induced in the early stages of hypoxia. This suggests that early hypoxia-induced increase in BHLHE40 expression may have crucial biological significance for the rapid cellular response to tumor hypoxia.

[0126] During the 0-72 hour hypoxia treatment, it was found that early hypoxia induced an increase in BHLHE40 mRNA expression, which was hypoxia-time dependent over 24 hours, but showed no significant change after 24 hours. Similarly, early hypoxia also induced an increase in BHLHE40 protein expression, but unlike gene expression levels, the increase in BHLHE40 protein was hypoxia-time dependent only over 2 hours, and showed no significant change after 2 hours. Thus, the changes in protein expression are more rapid and dramatic than those in mRNA expression.

[0127] Furthermore, the increase in BHLHE40 protein and the increase in HIF-1α protein were found to be almost synchronous. Although HIF-1 can mediate a wide range of transcriptional regulation, current research shows that it takes at least 30 minutes from the initiation of transcription to the completion of translation in different cells. If we hypothesize based on experimental results and existing literature that 0.5 hours of hypoxia significantly increases HIF-1 protein expression levels, initiating transcriptional regulation of BHLHE40, then a significant change in BHLHE40 protein levels should not be observed until at least 1 hour of hypoxia. However, in MDA-MB-231 cells, 0.5 hours of hypoxia significantly induced BHLHE40 protein levels. The only explanation is that there is regulation other than transcriptional regulation that causes rapid and dramatic changes in BHLHE40 protein levels, and this regulatory pathway should be independent of HIF-1.

[0128] Part Two: Mechanism Study of BHLHE40 Expression Induced by Tumor Hypoxia in Triple-Negative Breast Cancer Example 5: HIFs-mediated increase in BHLHE40 mRNA expression induced by tumor hypoxia To investigate how HIFs participate in the biological process of tumor hypoxia-induced BHLHE40, a stable cell line with shRNA-mediated HIF-1β gene knockdown was constructed. This is because the HIF-1β gene encodes a constitutively expressed protein subunit among the transcriptionally active heterodimeric transcription factors HIFs; therefore, knocking down the HIF-1β gene can directly achieve the experimental objective of interfering with HIFs. After adding an appropriate amount of puromycin to the culture medium to screen and amplify the stable cell line with HIF-1β gene knockdown for 15 days, the knockdown efficiency was identified by RT-qPCR. The results showed that in both TNBC cell lines, two different shRNA sequences could achieve a HIF-1β gene knockdown efficiency of over 50%, indicating that the stable cell line with shRNA-mediated HIF-1β gene knockdown was successfully constructed.

[0129] Proteins were further extracted after 1, 2, and 24 hours of hypoxia to observe the effect of HIF-1β knockdown on hypoxia-induced increase in BHLHE40 protein expression. Figure 7The results showed that HIF-1β knockdown significantly inhibited the increase in BHLHE40 protein expression induced by 2 hours of hypoxia, and this inhibitory effect persisted even after 24 hours of hypoxia. It was also observed that HIF-1β knockdown directly led to a decrease in cellular BHLHE40 protein levels, possibly because BHLHE40 is a direct target of HIF-1. Furthermore, compared to the scramble group, the expression of HIF-1β and the hypoxia-positive marker BNIP3 was significantly reduced in the stable HIF-1β knockdown cell line, also indicating the successful construction of the stable HIF-1β knockdown cell line mediated by shRNA; while the increase in HIF-1α protein expression with hypoxia treatment indicated the successful construction of the hypoxia model. These results suggest that HIFs mediate the early hypoxia-induced increase in BHLHE40 protein expression.

[0130] like Figure 8a , Figure 8b As shown, RNA was extracted after 1, 2, and 24 hours of hypoxia to further investigate whether HIF-1β knockdown inhibited the increase in BHLHE40 protein expression induced by early hypoxia through transcriptional regulation by HIFs. HIF-1β knockdown significantly inhibited the increase in BHLHE40 mRNA expression induced by 2 hours of hypoxia, and this inhibitory effect persisted after 24 hours of hypoxia. It was also observed that HIF-1β knockdown directly led to a decrease in cellular BHLHE40 mRNA levels, which may also be due to BHLHE40 being a direct target of HIF-1.

[0131] The above results suggest that HIFs mediate the increase in BHLHE40 mRNA expression induced by early hypoxia through their transcriptional regulation, which ultimately leads to an increase in BHLHE40 protein expression.

[0132] To further confirm the intrinsic mechanism by which HIFs mediate the early hypoxia-induced increase in BHLHE40 expression, the inhibitor VH298, which specifically inhibits the binding of the HIF-α subunit to the VHL complex, was used to inhibit the proteasomal degradation of the HIF-α subunit under normoxic conditions. This aimed to stabilize the HIF-α subunit under normoxic conditions and eliminate the possible influence of hypoxia-induced biological processes other than HIF transcriptional regulation on BHLHE40. CCK-8 assay results showed that treatment with 0.1 mM VH298 for 2 hours did not significantly affect cell viability; therefore, this concentration of VH298 was used for subsequent experiments.

[0133] like Figure 9a , Figure 9b , Figure 9c , Figure 9d , Figure 9eAs shown, Western blot results indicated that treatment with 0.1 mM VH298 for 2 hours effectively stabilized HIF-1α and HIF-2α proteins under normoxic conditions, while having no significant effect on HIF-1β protein expression. Furthermore, treatment with 0.1 mM VH298 for 2 hours significantly increased BHLHE40 protein expression under normoxic conditions. RT-qPCR results showed that treatment with 0.1 mM VH298 for 2 hours significantly increased BHLHE40 mRNA expression under normoxic conditions.

[0134] In summary, the above results suggest that in the early stages of hypoxia, HIFs mediate increased BHLHE40 mRNA expression induced by tumor hypoxia, which ultimately leads to increased BHLHE40 protein expression.

[0135] Example 6: Reduced ROS-mediated tumor hypoxia-induced degradation of BHLHE40 protein Previous studies have shown that hypoxia can lead to increased ROS levels, but whether 2 hours of hypoxia treatment can induce ROS and whether ROS is involved in the biological process of early BHLHE40 induction by tumor hypoxia remains unclear. Therefore, we first used flow cytometry to measure cellular ROS levels after 2 hours of hypoxia treatment, such as... Figure 10a , Figure 10b , Figure 10c , Figure 10d As shown, compared with the normoxic group, the ROS level of TNBC cells in the hypoxia-treated group for 2 hours was significantly increased; and when TNBC cells were pretreated with the ROS inhibitor NAC, 0.5 mM NAC could effectively inhibit the increase in cell ROS level induced by hypoxia for 2 hours.

[0136] like Figure 11a , Figure 11b , Figure 11c As shown, further Western blotting (WB) and RT-qPCR experiments were used to observe whether ROS was involved in the early hypoxia-induced increase in BHLHE40 protein and mRNA expression. Here, H2O2 directly induced an increase in cellular ROS levels. WB results showed that NAC significantly inhibited hypoxia-induced increases in BHLHE40 protein expression, but did not affect hypoxia-induced increases in HIF-1α protein expression; while under normoxic conditions, H2O2 significantly induced an increase in BHLHE40 protein expression, and this induction could be effectively blocked by NAC.

[0137] However, when BHLHE40 mRNA levels were further examined under the same treatment conditions, the results showed that NAC did not inhibit hypoxia-induced increases in BHLHE40 mRNA expression, and H2O2 also failed to induce increases in BHLHE40 mRNA expression under normoxic conditions. These results suggest that ROS participates in the early hypoxia-induced increase in BHLHE40 protein expression, but not in the early hypoxia-induced increase in BHLHE40 mRNA expression. Therefore, further investigation is needed to determine the reasons for ROS-induced increases in BHLHE40 protein levels.

[0138] On the one hand, since the ROS-induced increase in BHLHE40 protein levels is not due to increased mRNA expression, it is hypothesized that ROS may mediate the early hypoxia-induced increase in BHLHE40 protein expression by inhibiting BHLHE40 protein degradation. On the other hand, previous studies have shown that ROS can stabilize target protein levels by forming disulfide bonds through reactions with cysteine ​​sulfhydryl groups of other target proteins, so it is hypothesized that ROS may inhibit BHLHE40 protein degradation by inducing disulfide bond formation in BHLHE40 protein. Here, MG-132 can effectively inhibit the proteasome complex's proteolytic activity, while DTT is a strong reducing agent that can effectively open disulfide bonds in most proteins under living cell conditions.

[0139] like Figure 12 The results showed that the addition of DTT significantly reduced the H2O2-induced BHLHE40 protein level, while the addition of the proteasome inhibitor MG-132 hindered the inhibitory effect of DTT on H2O2-induced BHLHE40 protein. These results suggest that ROS induces the formation of disulfide bonds in BHLHE40 protein, thereby inhibiting its proteasome degradation. When a strong reducing agent is used to break the ROS-induced disulfide bonds in BHLHE40 protein, the proteasome degradation process against BHLHE40 can proceed smoothly again, and the BHLHE40 protein level decreases. Furthermore, when MG-132 is used to further inhibit proteasome activity, the ROS-induced elevated BHLHE40 protein level remains stable.

[0140] However, two key issues need to be addressed regarding the redox reaction between ROS and the cysteine ​​sulfhydryl groups of the BHLHE40 protein: First, hypoxia-induced ROS is mainly produced by mitochondrial complex III, while the BHLHE40 protein, previously widely reported as a transcription factor, is primarily distributed in the nucleus. The compartmentalized distribution of ROS and BHLHE40 protein within the cell directly affects their redox reactions. Second, to overcome the aforementioned mitochondrial compartmentalization limitation, it is hypothesized that ROS can diffuse intracellularly to the vicinity of the BHLHE40 protein and then undergo a redox reaction. However, this raises a new problem: the cytoplasm contains highly abundant and efficient ROS-scavenging enzymes, and ROS may have already reacted with these enzymes before reaching the vicinity of the BHLHE40 protein, thus preventing a redox reaction. Therefore, it is speculated that the BHLHE40 protein may be located near the source of hypoxia-induced ROS, meaning that BHLHE40 may have a mitochondrial distribution. If this hypothesis is true, it would not only solve the problem of mitochondrial compartmentalization that restricts the redox reactions between the two, but also explain why the high concentration of ROS in mitochondria can also explain the competitive reaction between BHLHE40 protein and ROS scavenging enzymes with ROS.

[0141] like Figure 13 Western blot results showed that BHLHE40 protein was present in the cytoplasm and was induced to increase in the early stages of hypoxia.

[0142] Further immunoelectron microscopy was used to observe the distribution of BHLHE40 protein in cells after 2 and 24 hours of hypoxia treatment. Figure 14a , Figure 14b , Figure 14c , Figure 14d , Figure 14e , Figure 14f As shown, BHLHE40 is distributed in mitochondria, and the mitochondrial BHLHE40 protein level is induced to rise after 2 hours of hypoxia, with no significant change after 2 hours. This trend is similar to the trend of whole-cell BHLHE40 protein changes. Immunofluorescence experiments performed under the same treatment conditions showed that, compared with the normoxic group, the co-localization of BHLHE40 and TOM20 fluorescence was significantly increased in the hypoxic treatment group; the latter is often used as a biomarker of the mitochondrial outer membrane.

[0143] The above results suggest that BHLHE40 is distributed in mitochondria, and that the level of mitochondrial BHLHE40 protein can be induced to increase by hypoxia.

[0144] Since it is hypothesized that BHLHE40 is distributed in mitochondria and thus undergoes redox reactions with hypoxia-induced ROS, mitochondrial proteins were extracted and immunoprecipitated to further analyze mitochondrial BHLHE40 protein in order to observe whether these redox reactions occur within mitochondria. Figure 15a , Figure 15b , Figure 15c , Figure 15d , Figure 15e As shown, hypoxia induced an increase in mitochondrial BHLHE40 protein expression, while pretreatment with the ROS inhibitor NAC blocked the induction of hypoxia on mitochondrial BHLHE40 protein expression, suggesting that ROS mediates hypoxia-induced increase in mitochondrial BHLHE40 protein expression. Further DTNB assays were used to examine the effect of hypoxia on cysteine ​​sulfhydryl groups in mitochondrial BHLHE40 protein. The results showed that hypoxia significantly increased the levels of total cysteine ​​sulfhydryl groups and total free sulfhydryl groups in mitochondrial BHLHE40 protein, while NAC pretreatment blocked the induction of hypoxia on the levels of total cysteine ​​sulfhydryl groups and total free sulfhydryl groups in mitochondrial BHLHE40 protein. Further calculations revealed that hypoxia significantly increased the level of disulfide bonds in mitochondrial BHLHE40 protein, while NAC pretreatment blocked the induction of hypoxia on the disulfide bond content in mitochondrial BHLHE40 protein. These results suggest that hypoxia induces a redox reaction between ROS and cysteine ​​sulfhydryl groups in mitochondrial BHLHE40 protein to form disulfide bonds.

[0145] Western blot (WB) results showed that under non-reducing conditions, hypoxia induced an increase in mitochondrial BHLHE40 homodimer expression, while this phenomenon completely disappeared under reducing conditions. These results indicate that in mitochondria, hypoxia induces a redox reaction between ROS and the cysteine ​​sulfhydryl groups of BHLHE40 protein, forming intermolecular disulfide bonds, which mediates the increase in BHLHE40 homodimer expression; simultaneously, it inhibits the proteasome degradation of BHLHE40, ultimately resulting in increased whole-cell BHLHE40 protein expression.

[0146] To further investigate the underlying mechanisms by which tumor hypoxia rapidly (i.e., in the early stages of hypoxia) induces an increase in BHLHE40 expression, this section employs two main methods. First, it uses shRNA to construct stable HIF-1β gene knockdown cell lines and employs VH298, a specific inhibitor of the HIF-α subunit binding to the VHL complex, to interfere with HIFs. This reveals that HIFs mediate the increase in BHLHE40 mRNA expression induced by tumor hypoxia through transcriptional regulation, leading to elevated BHLHE40 protein levels. Second, building upon the finding that 2 hours of hypoxia induces an increase in cellular ROS levels, small chemical molecules are used to interfere with ROS levels. This reveals that ROS participates in the increase in BHLHE40 protein expression induced by hypoxia, but does not participate in the increase in BHLHE40 mRNA expression induced by hypoxia. Furthermore, using the proteasome inhibitor MG-132 and the strong reducing agent DTT, it is found that ROS oxidation of BHLHE40 to form disulfide bonds mediates a reduction in BHLHE40 protein degradation induced by tumor hypoxia. The direct redox reaction between BHLHE40 and ROS is attributed to its mitochondrial distribution. In mitochondria, ROS can oxidize the cysteine ​​sulfhydryl groups of BHLHE40 protein to form intermolecular disulfide bonds, leading to increased expression of BHLHE40 homodimers and simultaneously inhibiting the degradation of BHLHE40 protein, ultimately resulting in increased expression of BHLHE40 protein throughout the cell.

[0147] Part III: Study on the Enhancement of Radioresistance in Triple-Negative Breast Cancer by BHLHE40 and its Mechanism In Parts 1 and 2, we found that BHLHE40 expression is associated with poor prognosis in TNBC and elucidated the dual mechanism by which tumor hypoxia rapidly induces BHLHE40 expression. Therefore, this part attempts to determine the exact reasons why BHLHE40 affects the poor prognosis of TNBC.

[0148] As a widely used treatment for TNBC patients, radiotherapy has been proven to significantly reduce local recurrence rates and improve patient survival. The high-energy rays (such as X-rays and gamma rays) produced by radiotherapy can excite water molecules to generate free radicals. Oxygen (O2), acting as a radiosensitizer, further reacts with these free radicals to generate large amounts of reactive oxygen species (ROS). Extremely high levels of ROS can induce DNA damage in tumor cells, ultimately leading to tumor cell apoptosis or necrosis. Therefore, tumor hypoxia and cellular antioxidant responses can directly reduce the effectiveness of radiotherapy.

[0149] In this section, we first construct Hypoxia high / lowGene expression profiles of radiation sensitivity signatures (RSS) in TNBC patients revealed a positive correlation between tumor hypoxia and TNBC radioresistance. Subsequently, it was found that BHLHE40 expression was significantly increased in TNBC radioresistance cell lines, and BHLHE40 mediated tumor hypoxia-induced TNBC radioresistance. Mechanistically, tumor hypoxia-induced BHLHE40 homodimers transcribedly activate the expression of a series of antioxidant genes, increase intracellular cysteine ​​and reduced glutathione levels, enhance cellular antioxidant responses, reduce ROS levels, and ultimately enhance tumor hypoxia-induced TNBC radioresistance.

[0150] Example 7 Tumor hypoxia is positively correlated with TNBC radioresistance: by constructing Hypoxia high / low The RSS gene expression profiles of TNBC patients revealed that, compared with Hypoxialow TNBC patients, Hypoxiahigh TNBC patients had higher expression levels of 23 genes positively correlated with radioresistance and lower expression levels of 28 genes negatively correlated with radioresistance, suggesting a positive correlation between tumor hypoxia and radioresistance in TNBC.

[0151] Increased expression of BHLHE40 in TNBC radioresistant cell lines: Subsequently, TNBC radioresistant cell lines were constructed by continuous irradiation of the TNBC cell line using an X-ray irradiator. Figure 16a , Figure 16b As shown, microscopic observation revealed that the cell morphology of the radiation-resistant cell line was significantly altered compared to the wild-type cell line; meanwhile, Western blot results showed that BHLHE40 expression was significantly increased in the TNBC radiation-resistant cell line.

[0152] Example 8: BHLHE40-mediated tumor hypoxia-induced TNBC radioresistance Construction of stable BHLHE40 gene knockdown cell lines mediated by shRNA: To investigate whether BHLHE40 mediates hypoxia-induced radioresistance in TNBC, stable BHLHE40 gene knockdown cell lines were constructed using shRNA-mediated knockdown. After selecting and amplifying stable BHLHE40 gene knockdown cell lines for 15 days with an appropriate amount of puromycin added to the culture medium, the knockdown efficiency of the BHLHE40 gene was assessed by RT-qPCR and Western blotting. Figure 17a , Figure 17b , Figure 17c As shown, in both TNBC cell lines, two different shRNA sequences could achieve BHLHE40 gene knockdown, indicating that the stable transgenic cells with BHLHE40 gene knockdown mediated by shRNA were successfully constructed.

[0153] BHLHE40 knockdown suppressed hypoxia-induced radioresistance in TNBC: CCK-8 assays were performed on two TNBC cell lines after different doses of radiotherapy, followed by 24, 48, and 72 hours of culture, to determine the optimal killing dose for different cell types. Figure 18a , Figure 18b , Figure 18c , Figure 18d , Figure 18e , Figure 18f As shown, 72 hours after radiotherapy, compared with the control group that did not receive radiotherapy, a 2 Gy dose of radiotherapy significantly reduced the survival rate of MDA-MB-231 cells, while a 4 Gy dose significantly reduced the survival rate of MDA-MB-468 cells. In plate cloning experiments conducted 14 days after different doses of radiotherapy on the two TNBC cell lines, compared with the control group that did not receive radiotherapy, a 2 Gy dose of radiotherapy significantly reduced the survival rate of MDA-MB-231 cells, while a 4 Gy dose significantly reduced the survival rate of MDA-MB-468 cells. Therefore, in subsequent experiments, MDA-MB-231 cells were treated with a 2 Gy dose of radiotherapy, while MDA-MB-468 cells were treated with a 4 Gy dose of radiotherapy.

[0154] Further, the BHLHE40 gene knockdown stable cell line constructed using shRNA was subjected to normoxic / hypoxic treatment for 24 hours followed by radiotherapy, and then cultured for 24, 48, and 72 hours before CCK-8 assays were performed. Figure 19a , Figure 19b , Figure 19c , Figure 19d As shown, hypoxia induces radiotherapy resistance in TNBC cells, while BHLHE40 knockdown inhibits hypoxia-induced radioresistance. Plate colony experiments conducted after 14 days of cell culture under the same treatment conditions showed that, compared to the normoxic group, hypoxia increased cell proliferation after radiotherapy treatment, while BHLHE40 knockdown inhibited the hypoxia-induced enhanced cell proliferation. These results suggest that BHLHE40 mediates tumor hypoxia-induced radioresistance in TNBC.

[0155] Example 9: BHLHE40 reduces reactive oxygen species levels BHLHE40 knockdown increased hypoxia-induced cellular ROS levels: ROS levels in stable BHLHE40 knockdown cell lines constructed using shRNA were measured by flow cytometry after 24 hours of normoxic / hypoxic treatment. Figure 20a , Figure 20bAs shown, BHLHE40 knockdown significantly increased hypoxia-induced cellular ROS levels, suggesting that BHLHE40 can reduce ROS levels.

[0156] BHLHE40 participates in the biological process of cellular antioxidant activity: Further RNA-seq analysis was performed on stable BHLHE40 knockdown cell lines constructed using shRNA after 24 hours of normoxic / hypoxic treatment. GO analysis showed that, compared to the scramble group, differentially expressed genes in the BHLHE40 knockdown group were enriched in the "cellular response to oxidative stress" after 24 hours of hypoxia treatment. KEGG analysis showed that, compared to the scramble group, differentially expressed genes in the BHLHE40 knockdown group were enriched in the "chemical carcinogenesis-reactive oxygen species" pathway, which mainly describes the process by which chemical carcinogens induce cancer through ROS. GSEA results showed that, compared to the scramble group, the BHLHE40 knockdown group was negatively correlated with a series of cellular antioxidant processes after 24 hours of hypoxia treatment, suggesting that BHLHE40 participates in biological processes such as cellular antioxidant activity, thereby reducing ROS levels.

[0157] Example 10: BHLHE40 promotes antioxidant gene expression BHLHE40 knockdown led to a decrease in the expression levels of a series of antioxidant genes: To explore the underlying mechanism by which BHLHE40 reduces ROS levels, the gene expression levels of a series of antioxidant enzymes in stable BHLHE40 knockdown cell lines were further analyzed. The results showed that BHLHE40 knockdown significantly reduced the expression levels of a series of antioxidant enzymes with ROS scavenging functions, including SOD2 / 3 (encoding superoxide dismutase), PRDX1 / 2 / 5 (encoding peroxidase), GPX3 / 4 (encoding glutathione peroxidase), GLRX1 / 2 (encoding glutreonin), and CAT (encoding catalase). In addition, BHLHE40 knockdown also reduced the expression level of the SVCT2 gene, which encodes the vitamin C transporter, suggesting that BHLHE40 can promote the expression of antioxidant genes.

[0158] BHLHE40 knockdown leads to a decrease in SLC7A11 expression levels: (e.g.) Figure 21a , Figure 21bAs shown, besides affecting the expression levels of antioxidant genes such as those encoding ROS scavenging enzymes, BHLHE40 gene knockdown also significantly reduced the expression level of the SLC7A11 gene, which encodes the active protein of the cysteine / glutamate antitransporter, but had no significant effect on the expression level of the SLC3A2 gene, which encodes the chaperone protein of the cysteine / glutamate antitransporter. Since the protein encoded by SLC7A11 is mainly responsible for transporting extracellular cysteine ​​into the cell, where cysteine ​​is reduced to cysteine ​​and then used to synthesize glutathione, both of which are important antioxidants involved in cellular antioxidant processes and protecting cells from oxidative stress damage, these results suggest that BHLHE40 may ultimately reduce ROS levels by increasing SLC7A11 expression.

[0159] BHLHE40 knockdown led to decreased cellular levels of cysteine ​​and reduced glutathione: further examination was conducted to determine the levels of two important intracellular antioxidants, cysteine ​​and reduced glutathione. Figure 22a , Figure 22b As shown, BHLHE40 knockdown leads to a decrease in cellular cysteine ​​and reduced glutathione levels.

[0160] Example 11: No feedback regulation effect of BHLHE40 on HIFs Combining the findings from Part I and Part II, we can conclude that "ROS mediates tumor hypoxia-induced increased BHLHE40 expression, and the increased BHLHE40 promotes antioxidant gene expression, negatively regulating ROS levels, and ultimately enhancing TNBC radioresistance." On the other hand, since another important factor mediating tumor hypoxia-induced increased BHLHE40 expression is HIFs, which can mediate a wide range of transcriptional regulation, we attempt to explore whether HIFs, like ROS, are regulated by BHLHE40 through feedback, thereby affecting cellular redox homeostasis through their transcriptional regulation.

[0161] like Figure 23a , Figure 23b , Figure 23c , Figure 23dAs shown, Western blot results indicated that BHLHE40 knockdown did not affect the protein expression levels of HIF-1α and the classic HIF-1 target gene BNIP3. Furthermore, dual-luciferase reporter assays using BHLHE40 shRNA in cells capable of reporting HIF transcriptional activity revealed that BHLHE40 knockdown did not affect HIF transcriptional activity. Additionally, RT-qPCR experiments showed that BHLHE40 knockdown also did not affect the gene expression levels of a series of classic HIF target genes. These results suggest that although HIFs mediate increased BHLHE40 expression induced by tumor hypoxia, BHLHE40 does not provide feedback regulation of HIFs.

[0162] To ultimately elucidate the mechanism of action of BHLHE40 in the poor prognosis of tumor hypoxia-mediated TNBC, this section focuses on two aspects. First, by constructing a radioresistant TNBC cell line, we found that BHLHE40 expression was significantly increased in the TNBC radioresistant cell line. Second, using shRNA, we constructed a stable BHLHE40 gene knockdown cell line. After hypoxia and radiotherapy treatment, we used CCK-8 assays and plate cloning to demonstrate that BHLHE40 mediates tumor hypoxia-induced TNBC radioresistance. Further analysis using flow cytometry and RNA-seq revealed that ROS-induced elevated BHLHE40 can negatively regulate cellular ROS levels; moreover, its regulation of ROS levels is achieved by promoting the expression of antioxidant genes, rather than through feedback regulation of HIFs-mediated transcriptional regulation. Based on these results, this application proposes that tumor hypoxia-induced BHLHE40 serves as a therapeutic target for TNBC and provides important theoretical basis for optimizing existing radiotherapy methods for TNBC.

[0163] As a transcription factor, BHLHE40 not only broadly influences the expression levels of genes encoding a range of antioxidant enzymes, but also affects the expression level of SLC7A11. SLC7A11, as the active subunit of a cysteine ​​transporter, further influences cysteine ​​and reduced glutathione levels by transporting cysteine ​​into the cell, thus participating in the regulation of cellular redox homeostasis. Unlike the widely reported role of BHLHE40 as a transcriptional repressor, this application emphasizes that BHLHE40 may act as a potential transcriptional activator, influencing the expression of a series of downstream genes with antioxidant functions. However, direct evidence is currently lacking to suggest that BHLHE40 mediates transcriptional activation by binding to the promoter regions of these genes. Therefore, future research should focus on elucidating how BHLHE40 exerts its transcriptional activation function.

[0164] Furthermore, the results of this study suggest that tumor hypoxia-induced BHLHE40 plays an important role in regulating cellular ROS levels. Under hypoxic conditions, ROS-induced upregulation of BHLHE40 further negatively regulates intracellular ROS levels; however, combined with previous findings that hypoxia continuously induces increased ROS levels and increased BHLHE40 expression, it is clear that BHLHE40 did not regulate ROS levels to the levels seen in normoxic conditions. Therefore, the role of this negative feedback loop is obviously to maintain redox homeostasis in cells under tumor hypoxia. Combined with the experimental results in Part II of this study, BHLHE40 has a dual role in responding to and adapting to hypoxia-induced ROS; it is not only a "sensor" of hypoxia-induced mitochondrial ROS but also a "regulator" of intracellular ROS levels. Because BHLHE40 has this function of negatively regulating ROS levels, knocking down BHLHE40 can cause cells subjected to hypoxia to lose their ability to maintain redox homeostasis. The loss of this function further leads to the loss of the radioresistance characteristics of hypoxic cells after radiotherapy, as BHLHE40 knockdown inhibits hypoxia-induced TNBC radioresistance.

[0165] In summary, this application clarifies the possibility of using tumor hypoxia-induced BHLHE40 as a therapeutic target for TNBC, and elucidates the mechanism of action of BHLHE40 in tumor hypoxia-mediated radioresistance in TNBC, providing an important theoretical basis for optimizing existing radiotherapy methods for TNBC.

[0166] Part IV: Research on Cancer Therapeutic Drugs Based on Newly Discovered Mechanisms of Action of BHLHE40 and SLC7A11 Example 12: Research Basis for the Transcription Factor Bait Strategy 1) BHLHE40 knockdown downregulates total SLC7A11 protein expression like Figure 21b As shown, Western blot results indicated that in MDA-MB-231 cells, under normoxic conditions, the SLC7A11 protein level in the shBHLHE40 knockdown group (relative quantitative value 0.6) was lower than that in the shScramble control group (1.0). After 24 hours of hypoxia treatment, SLC7A11 protein expression remained stable in the shScramble group (1.0), while it further decreased to 0.5 in the shBHLHE40 knockdown group. TUBULIN, as an internal control protein, showed uniform expression across all groups.

[0167] These results demonstrate that BHLHE40 is essential for maintaining SLC7A11 protein expression under hypoxic conditions.

[0168] 2) BHLHE40 knockdown inhibits SLC7A11 protein expression and cell membrane localization. like Figure 24a , Figure 24b As shown, flow cytometry (non-permeable cells) analysis revealed that under normoxic conditions, the median fluorescence intensity (FITC-A = 55637) of SLC7A11 on the cell surface of the shBHLHE40 group was lower than that of the shScramble group (77084). After 24 hours of hypoxia, SLC7A11 expression remained high in the shScramble group (77955), while it was significantly reduced in the shBHLHE40 group (57735). The statistical bar chart indicated a significant difference between the shBHLHE40 and shScramble groups after 24 hours of hypoxia. This result suggests that BHLHE40 not only regulates the total protein content of SLC7A11 but also affects its membrane localization function.

[0169] 3) BHLHE40 knockdown specifically inhibits SLC7A11 mRNA transcription. like Figure 21a As shown, RT-qPCR results revealed that at the SLC7A11 mRNA level, the expression level in the shBHLHE40 group was significantly lower than that in the shScramble group under normoxic conditions. After 24 h of hypoxia, SLC7A11 mRNA was further upregulated in the shScramble group, while it was significantly suppressed in the shBHLHE40 group. As a control, there was no significant difference in the mRNA level of the SLC7A11 light chain chaperone molecule SLC3A2 among the groups.

[0170] These results demonstrate that BHLHE40 regulates SLC7A11 at the transcriptional level and has good target gene specificity.

[0171] 4) Hypoxia enhances the direct binding of BHLHE40 to the SLC7A11 promoter. like Figure 25 As shown, the ChIP-qPCR results indicated that the SLC7A11 promoter region showed almost no enrichment when anti-IgG was used as a negative control; however, when immunoprecipitated with anti-BHLHE40 antibody, the promoter region showed significant enrichment under normoxic conditions, and the enrichment level further increased after 24 hours of hypoxia.

[0172] This result directly confirms that BHLHE40 undergoes a sequence-dependent and dynamically enhanced physical binding to the SLC7A11 promoter under hypoxic conditions, providing direct molecular evidence for BHLHE40's transcriptional activation of SLC7A11.

[0173] The above results form the basis for research on transcription factor bait strategies: First, Figure 25 and Figure 21a This indicates that BHLHE40 activates SLC7A11 transcription through promoter occupancy; Figure 21b and Figure 24a , Figure 24b This indicates that the loss of BHLHE40 not only reduces the total protein content of SLC7A11, but also reduces its membrane surface localization, proving that this transcriptional regulation ultimately affects the physiological function of SLC7A11, namely cystine transport. Figure 25 The results showed that hypoxia significantly enhanced the binding of BHLHE40 to the SLC7A11 promoter, suggesting that hypoxic tumor regions are the main site of action for this regulatory axis, providing a microenvironmental basis for the tumor selectivity of transcription factor bait strategies.

[0174] Based on this, since BHLHE40 drives transcription by directly binding to a specific DNA motif (E-box) on the SLC7A11 promoter, the artificial synthesis of dsODN-decoy containing this motif, and its introduction into MDA-MB-231 cells, can competitively bind a limited amount of BHLHE40 protein to the endogenous SLC7A11 promoter at the molecular level. Because the decoy does not contain promoter active elements, it cannot initiate transcription after binding to BHLHE40, acting only as a molecular sponge to isolate the cells. This mimics or even enhances the phenotype of shBHLHE40, namely, transcriptional repression of SLC7A11, reduction of membrane surface xCT, weakening of antioxidant capacity, and ultimately restoring the radiosensitivity of hypoxic tumor cells.

[0175] Example 13: Development and Results of Cancer Treatment Drug Products Based on previous research, five pairs of double-stranded oligodeoxynucleotide decoys (dsODN-decoy) were designed as shown in Table 1. Before cell transfection, they need to be annealed to form double strands: the 5'→3' strand and the 3'→5' strand are mixed in an equimolar ratio (1:1) in annealing buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA), and then cooled naturally to room temperature after 5 min at 95°C to form dsODN.

[0176] Table 1. Sequences of the sense and antisense strands of 5 pairs of double-stranded oligonucleotide decoys.

[0177] Results of dsODN-decoy on the binding of BHLHE40 to the SLC7A11 promoter and the regulation of target gene expression Figure 26a , Figure 26b , Figure 26c As shown.

[0178] Figure 26aThis study demonstrates that decoy #1 competitively blocks the binding of BHLHE40 to the SLC7A11 promoter. ChIP-qPCR results showed that under hypoxic conditions, the enrichment of the SLC7A11 promoter region precipitated by anti-BHLHE40 antibody (approximately 18-fold higher than the IgG control) was further increased compared to normoxia (approximately 11-fold higher), confirming that hypoxia enhances the physical binding of BHLHE40 to the SLC7A11 promoter. After transfection with decoy #1, hypoxia-induced BHLHE40 enrichment almost completely disappeared (reducing to near IgG background levels); decoy #2 only partially reduced the enrichment (approximately 5-fold); and decoy #3 had no significant effect (approximately 15-fold). These results demonstrate that decoy #1 can competitively block the specific binding of BHLHE40 to the SLC7A11 promoter at the molecular level, and that this blocking effect is strictly sequence-dependent.

[0179] Figure 26b This study demonstrates that decoy #1 inhibits SLC7A11 expression. RT-qPCR results showed that under 24 h hypoxia, the SLC7A11 mRNA expression level (TPMs approximately 34) in the shScramble control group was significantly higher than that in the shBHLHE40 knockdown group (approximately 10). After introducing decoy #1 into the shScramble background, SLC7A11 mRNA expression decreased to a level comparable to shBHLHE40 (approximately 10); decoy #2 partially inhibited SLC7A11 (approximately 20); and decoy #3 showed almost no inhibition (approximately 32). These results are highly consistent with the ChIP-qPCR data, indicating that decoy #1 inhibits SLC7A11 expression by blocking the binding of BHLHE40 to the promoter.

[0180] Figure 26c This study demonstrates that decoy #1 specifically reduces SLC7A11 protein expression without affecting BHLHE40 protein stability. Western blot results showed that under 24 h of hypoxia, both BHLHE40 and SLC7A11 proteins were highly expressed in the shScramble group; both were reduced in the shBHLHE40 group; and in the shScramble+decoy #1 group, the total BHLHE40 protein level was similar to that in the shScramble group, but the SLC7A11 protein level was significantly reduced. These results demonstrate at the protein level that decoy #1 does not affect the stability of BHLHE40 protein (excluding non-specific degradation or toxic effects), but only specifically inhibits the translation of its downstream target gene SLC7A11, consistent with the theoretical expectation of the transcription factor decoy strategy of "competitively occupying the DNA-binding domain."

[0181] Functional validation results of dsODN-decoy #1 on the antioxidant capacity and radiosensitivity of triple-negative breast cancer cells are as follows: Figure 27a , Figure 27b , Figure 27c , Figure 27d , Figure 27e As shown.

[0182] Figure 27a and Figure 27b This study demonstrates that decoy #1 inhibits cystine uptake and glutathione synthesis. Under 24 h of hypoxia, intracellular cystine and reduced glutathione levels in the shScramble control group were significantly higher than those in the shBHLHE40 knockdown group. Cystine and GSH levels in the shScramble+decoy #1 group were reduced to levels comparable to those in the shBHLHE40 group. These results indicate that decoy #1 successfully mimics the antioxidant inhibitory phenotype of BHLHE40 deficiency by inhibiting SLC7A11 transcription, thereby blocking cystine uptake and weakening glutathione synthesis.

[0183] Figure 27c and Figure 27d This study demonstrated that decoy #1 promotes intracellular ROS accumulation under hypoxic conditions. DCFH-DA flow cytometry analysis showed that under 24 h of hypoxia, the intracellular ROS level in the shScramble group (median DCF-A fluorescence intensity approximately 10,412) was low; the shBHLHE40 group showed a significant increase (approximately 68,623); and the ROS level in the shScramble+decoy #1 group (approximately 69,747) was comparable to that in the shBHLHE40 group. Statistical bar charts confirmed that decoy #1 significantly promoted intracellular ROS accumulation under hypoxic conditions, indicating that it disrupts cellular redox homeostasis by inhibiting the SLC7A11-cysteine-GSH axis, thereby impairing the cells' ability to clear radiation-induced ROS.

[0184] Figure 27e This study demonstrates that decoy #1 restores the radiosensitivity of hypoxic tumor cells. CCK-8 assays showed that radiotherapy under normoxic conditions significantly killed cells (relative absorbance approximately 20%), while under hypoxic conditions, cells developed significant radioresistance (survival rate recovered to approximately 70%). Crucially, both shBHLHE40 knockdown and decoy #1 treatment restored cell radiosensitivity under hypoxic conditions (survival rate decreased to approximately 10%, comparable to the normoxic radiotherapy group). These results demonstrate that decoy #1, as a competitive inhibitor of BHLHE40 DNA binding, can effectively overcome hypoxia-mediated radioresistance at a functional level.

[0185] The results indicate that dsODN-decoy #1 isolates BHLHE40 through a "molecular sponge" mechanism, blocking its binding to the SLC7A11 promoter, thereby inhibiting SLC7A11 expression at the transcriptional level. Ultimately, by weakening the cystine-glutathione-ROS clearance axis, it restores the radiosensitivity of hypoxic TNBC cells. This series of experiments successfully validates the scientific validity and therapeutic potential of this novel double-stranded oligonucleotide decoy intervention strategy designed based on a "transcription factor decoy strategy."

[0186] Based on the research results of dsODN-decoy #1, combined with Figure 26a , Figure 26b , Figure 26c The results of dsODN-decoy #2 show that dsODN-decoy #2 can also isolate BHLHE40 through the "molecular sponge" mechanism, block its binding to the SLC7A11 promoter, thereby inhibiting SLC7A11 expression at the transcriptional level, and finally restoring the radiosensitivity of hypoxic TNBC cells by weakening the cystine-glutathione-ROS clearance axis.

[0187] In summary, this application reveals for the first time a novel transcriptional regulatory axis in the hypoxic microenvironment of triple-negative breast cancer (TNBC): in the early stages of hypoxia, reactive oxygen species (ROS)-dependent post-translational modifications (the formation of disulfide-linked homodimers to protect it from proteasome degradation) and hypoxia-inducible factors (HIFs)-dependent transcriptional activation work together to rapidly upregulate the expression of transcription factor BHLHE40; the latter recognizes and binds to the E-box motif on the promoter of the SLC7A11 gene through its basic helix-loop-helix (bHLH) domain, thereby activating SLC7A11 expression at the transcription initiation level, driving cystine uptake and glutathione synthesis, and ultimately mediating tumor radiotherapy resistance.

[0188] Unlike existing technologies that directly target the SLC7A11 protein or function, this application innovatively shifts the intervention target forward to the "transcription factor-promoter" interaction interface. It utilizes double-stranded oligodeoxynucleotide decoys (dsODN-decoy) to competitively occupy the DNA-binding domain of BHLHE40, specifically blocking the binding of BHLHE40 to the SLC7A11 promoter, thereby inhibiting SLC7A11 expression at the transcription initiation level. This strategy offers at least the following advantages: 1) Transcriptional-level blocking, resulting in a more upstream and thorough effect; 2) Utilizing the hypoxia-specific high expression of BHLHE40, it achieves selective intervention in hypoxic tumor regions, potentially reducing off-target toxicity to normoxic normal tissues; 3) It does not interfere with the HIF pathway itself or other non-BHLHE40 target genes, maintaining a relatively independent mechanism; 4) As a nucleic acid drug, it complements the mechanism of small molecule / antibody drugs, providing a novel nucleic acid intervention strategy for overcoming hypoxia-related radiotherapy resistance in TNBC.

[0189] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. An isolated nucleotide fragment, characterized in that, The nucleotide fragment comprises a sense strand as shown in SEQ ID No. 1 and an antisense strand as shown in SEQ ID No. 2; or, The nucleotide fragments include a sense strand as shown in SEQ ID No. 3 and an antisense strand as shown in SEQ ID No.

4.

2. The use of the nucleotide fragment as described in claim 1 in the preparation of a medicament for reducing poor prognosis after radiotherapy for triple-negative breast cancer.

3. A drug, characterized in that, Includes the isolated nucleotide fragments as described in claim 1.

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