Application of ZNF263 in preparation of medicine for treating breast cancer

Abstract

The invention provides an application of ZNF263 in preparation of a medicine for treating breast cancer. The ZNF263 regulates ferroptosis in breast cancer cells by activating transcription of FOSL2. The ZNF263 disclosed by the invention plays a key role in regulating and controlling ferroptosis and oxidative stress of breast cancer cells, and the ZNF263 indirectly regulates and controls lipid peroxidation, iron metabolism imbalance and autophagic flow abnormality by activating FOSL2 transcription; the ZNF263 and other markers have a synergistic effect in early diagnosis of the breast cancer, a theoretical basis is provided for a multi-marker joint detection strategy, and a technical support is provided for early screening and individualized treatment of the breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to the application of ZNF263 in the preparation of drugs for treating breast cancer. Background Art

[0002] Breast cancer is one of the malignant tumors with the highest incidence and mortality rates among women worldwide. Early diagnosis and treatment are crucial for the survival rate and quality of life of patients. However, current breast cancer detection and diagnosis methods have various limitations. For example, traditional imaging examinations such as mammography X-ray may be difficult to detect small breast lesions in the early stage due to limited sensitivity. As the "gold standard" for diagnosis, tissue biopsy has limitations such as high invasiveness, high cost, and long time consumption. In addition, the detection of a single biomarker is also prone to false negative or false positive results, thus affecting the accuracy of diagnosis and the reliability of clinical decisions. Summary of the Invention

[0003] In view of this, the present invention aims to overcome the defects in the prior art and proposes the application of ZNF263 in the preparation of drugs for treating breast cancer.

[0004] To achieve the above object, the technical solution of the present invention is realized as follows:

[0005] The present invention provides the application of a biomarker in the preparation of drugs for treating breast cancer, and the biomarker is ZNF263.

[0006] Furthermore, ZNF263 regulates ferroptosis in breast cancer cells by activating the transcription of FOSL2.

[0007] Furthermore, knocking out ZNF263 down-regulates the expression of FOSL2; knocking out ZNF263 activates the autophagy of breast cancer cells.

[0008] Furthermore, knocking out ZNF263 increases the level of oxidized ROS and enhances the level of lipid ROS induced by Erastin.

[0009] Furthermore, knocking out ZNF263 enhances the oxidative stress of breast cancer cells induced by erastin; overexpressing ZNF263 promotes the malignant growth of breast cancer cells.

[0010] Furthermore, ZNF263 regulates immune cell subsets in breast cancer.

[0011] Furthermore, overexpressing ZNF263 inhibits the proportions of CD3+ T cells, CD4+ T cells, and CD8+ T cells, and at the same time reduces the proportion of CD19+ B cells; overexpressing ZNF263 promotes the function of CD56+ NK cells.

[0012] Furthermore, ZNF263 weakens humoral immunity by inhibiting B cell differentiation and T cell function, while activating NK cells to reshape the immunosuppressive microenvironment; ZNF263 targets key genes of B cells or inhibits the function of B cells by regulating the IL-6 / STAT3 pathway; the key gene of B cells is PAX5 or BCL6.

[0013] The breast cancer is Luminal subtype breast cancer.

[0014] The present invention also provides an application of a biomarker in the preparation of a reagent for detecting breast cancer, and the biomarker is ZNF263.

[0015] Furthermore, the application of the ZNF263 in the preparation of a breast cancer auxiliary diagnosis detection kit.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] The ZNF263 described in the present invention plays a key role in regulating ferroptosis and oxidative stress of breast cancer cells. ZNF263 indirectly regulates lipid peroxidation, iron metabolism imbalance and autophagy flux abnormality by activating FOSL2 transcription; ZNF263 and other markers have a synergistic effect in the early diagnosis of breast cancer, providing a theoretical basis for the multi-marker combined detection strategy and technical support for the early screening and individualized treatment of breast cancer.

[0018] The ZNF263 described in the present invention is significantly highly expressed in breast cancer tissues. Knockout of ZNF263 can enhance Erastin-induced ferroptosis of breast cancer cells, with a significant increase in the LC3II / LC3I ratio and a significant decrease in p62 expression, indicating that knockout of ZNF263 promotes ferroptosis of breast cancer cells by activating autophagy, providing a theoretical basis for the development of new tumor markers. Description of the Drawings

[0019] Figure 1 It is the significance of ZNF263 for the early diagnosis of breast cancer described in the embodiments of the present invention;

[0020] Figure 2 It is the effect of intervening ZNF263 on the viability and apoptosis of breast cancer cells described in the embodiments of the present invention;

[0021] Figure 3 It is the effect of knocking out ZNF263 on the levels of oxidized ROS, lipid ROS, oxidative stress and Fe 2+ content;

[0022] Figure 4 It is the effect of knocking out ZNF263 on ferroptosis-related proteins described in the embodiments of the present invention;

[0023] Figure 5 Effect of knocking out ZNF263 on the expression of LC3II / LC3I and p62 as described in the embodiments of the present invention;

[0024] Figure 6 Potential binding sites of ZNF263 in the promoter region of FOSL2 as described in the embodiments of the present invention;

[0025] Figure 7 Effect and expression of ZNF263 on the promoter region of FOSL2 as described in the embodiments of the present invention;

[0026] Figure 8 Effect of ZNF263 on the expression of FOSL2 as described in the embodiments of the present invention;

[0027] Figure 9 ZNF263 regulates immune cell subsets in breast cancer immune regulation as described in the embodiments of the present invention. Detailed implementation manners

[0028] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. The test reagents used in the following embodiments are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0029] The present invention will be described in detail below in conjunction with the embodiments.

[0030] Example 1 Experimental method

[0031] 1. Isolation and culture of breast cancer organoids

[0032] The surgically resected tumor tissues, adjacent tissues and normal tissues are quickly placed in the transport fluid and transported to the laboratory on ice. Subsequently, the tumor tissues are divided into 3 parts according to experimental requirements, and are respectively used for the extraction of organoids, sequencing of frozen tissues in liquid nitrogen and pathological detection after fixation with 4% paraformaldehyde. The fresh tissue blocks are removed of non-pathological parts on ice, and then washed thoroughly with precooled HBSS containing 1% double antibiotics. Cut into pieces in a 10 cm culture dish, digest with digestive solution, pipette repeatedly, add HBSS solution to dilute and terminate digestion after sufficient dissociation, filter with a 100 μm cell filter, centrifuge, take the precipitate, and wash 2-3 times. Resuspend the precipitate in precooled Matrigel and inoculate it in the center of a 24-well plate to form a Dome shape. Place the culture plate in a humidified incubator at 37°C and 5% CO2. After the Matrigel has completely solidified, add the self-prepared complete medium for breast cancer organoids of different molecular subtypes, and change the medium every 3 days.

[0033] 2. Expression of ZNF263 in breast cancer

[0034] Fifty cases of cancer and adjacent tissue specimens of breast cancer patients pathologically diagnosed after surgery in our hospital were collected to establish a patient-derived organoid (PDOs) model, and immunohistochemistry was used to detect the expression of ZNF236 in breast cancer. According to the results of immunohistochemistry, the staining of ZNF236 in breast cancer was divided into a low-expression group and a high-expression group. Then, statistical software was used to analyze the correlation between the expression of ZNF236 in breast cancer patients and clinicopathological characteristic data (age, tumor size, lymph node metastasis, vascular invasion, neural invasion, clinical stage, menopause status, histological grade). The number of ER receptors, PR receptors, HER-2 receptor negatives and positives, as well as the numbers of the four molecular subtypes, Luminal A type, Luminal B type, HER-2 overexpression type, and triple-negative, were statistically analyzed in patients with high and low expression of ZNF236. The breast cancer prognosis spectrum was downloaded from the biomedical data platform to find the best cut-off value for grouping breast cancer patients, and they were divided into breast cancer patients with high expression of ZNF263 and breast cancer patients with low expression of ZNF263. The Kaplan-Meier curve was plotted and Cox survival analysis was performed.

[0035] 3. The role of ZNF263 in the growth and metastasis of breast cancer

[0036] For the PDOs model, ZNF263 gene intervention was carried out in groups, and the ZNF263 interference sequence or overexpression plasmid was transfected respectively. The ZNF263 gene intervention efficiency was detected, CCK8 was used to detect cell viability, Edu was used to detect cell proliferation, flow cytometry was used to detect the apoptosis rate, scratch assay was used to detect cell migration, and Transwell was used to detect cell invasion. The PDOs cells after the above ZNF263 gene intervention were subcutaneously injected into Balb / c nude mice to establish a nude mouse xenograft tumor model. The tumor size, volume, and mass were detected, and immunohistochemistry was used to detect the expression of KI-67. Balb / c nude mice were injected via the tail vein to establish a nude mouse breast cancer lung metastasis model. The lung tissue was collected, the number of metastatic tumors was counted, and the tumor metastasis index was calculated. HE staining was used to analyze the pathological changes of the lung tissue.

[0037] 4. Knocking out ZNF263 induces ferroptosis in breast cancer by activating autophagy

[0038] The PDOs were treated in groups. While knocking out ZNF263, the ferroptosis activator Erastin was used for treatment. Transmission electron microscopy was used to observe the mitochondrial morphology, C11-BODIPY581 / 591 staining was used to detect lipid ROS, kits were used to detect MDA and GSH, and FerrOrange staining was used to detect Fe. 2+。Transmission electron microscopy was used to observe autophagosomes; immunofluorescence was used to detect LC3; Western blotting was used to detect the expression of LC3-I, LC3-II, Beclin1, and p62. Autophagy inhibitor (3-MA) was used to treat cells while knocking out ZNF263, and lipid ROS, MDA, GSH, and Fe were detected 2+ 。

[0039] 5. ZNF236 promotes breast cancer progression by promoting FOSL2 transcription

[0040] ZNF263 gene intervention was performed on PDOs, that is, ZNF263 interference sequence and control sequence were transfected. After transfection, cells were collected for RNA-seq analysis. Differentially expressed genes after knocking out ZNF263 were screened, and GO, KEGG, and GSEA enrichment analyses were performed to understand the functions of differentially expressed genes after ZNF263 intervention. RT-PCR and immunofluorescence were used to detect the expression of FOSL2 in cells. ChIP-PCR experiment was used to detect the enrichment of ZNF263 on the FOSL2 promoter in PDOs cells. Luciferase reporter gene was used to detect the activity of the FOSL2 promoter in PDOs cells with overexpressed ZNF263. A rescue experiment of ZNF263 and FOSL2 gene intervention was set up in PDOs cells, that is, FOSL2 was overexpressed while knocking out ZNF263. Related indicators of cell autophagy and ferroptosis, cell viability, proliferation, migration, invasion, and apoptosis were detected

[0041] 6. Regulation of immune function of breast cancer by ZNF263

[0042] Organoids were collected from the culture medium, washed twice with pre-cooled PBS, and resuspended in an appropriate amount of 0.25% Trypsin-EDTA digestive enzyme, and incubated at 37 °C for 10 - 15 minutes. Subsequently, the cells were dispersed into a single-cell suspension by pipetting, and complete medium was added to terminate digestion and mix well. Centrifuge at 3000 rpm for 5 minutes, discard the supernatant, resuspend and wash with PBS 1 - 2 times to obtain a single-cell suspension. A cell counting plate and trypan blue staining were used to evaluate cell quantity and viability to ensure the quality of the experiment. Cell samples were collected using an EDTA anticoagulant tube, the sample tubes and flow cytometry tubes were placed well, and corresponding numbers were marked. Take 20 μl of CD3 / CD16 + 56 / CD45 / CD4 / CD19 / CD8 antibodies and add them to the bottom of the labeled flow cytometry tube. After vortexing the single-cell suspension for 3 seconds or manually inverting it 10 times, take 50 μl of the cell suspension and add it to the bottom of the flow cytometry tube using the reverse loading method, and gently mix on the vortex mixer for 3 seconds. Each sample was incubated at room temperature in the dark for 15 min. The hemolytic agent for blood cell analysis was diluted to 1× (1 volume of stock solution: 9 volumes of distilled water), 450 μl of the diluted hemolytic agent was added to each sample tube, and it was gently mixed on the vortex mixer for 3 seconds again. After incubating at room temperature in the dark for 15 min, an Agilent flow cytometer was used for on-machine detection

[0043] Data analysis of Example 2

[0044] Breast cancer tumor tissues confirmed by clinical pathology were selected ( Figure 1 A), and a patient-derived organoids (PDOs) model of breast cancer patients was successfully constructed ( Figure 1 B). The expression level of transcription factor ZNF263 was low in normal breast tissues, but significantly up-regulated in breast cancer tissues ( Figure 1 C), suggesting that it may play an important role in the occurrence and development of breast cancer. To further explore the function of ZNF263, we constructed a breast cancer organoid model with ZNF263 knockdown using CRISPR / Cas9 technology. The experimental results showed that after knocking down ZNF263, the growth rate of organoids was significantly slowed down, and at the same time, the proportion of apoptotic positive cells increased significantly ( Figure 1 D- Figure 1 E), indicating that the deletion of ZNF263 can induce apoptosis of breast cancer cells. Bioinformatics analysis showed ( Figure 1 F) that ZNF263 was up-regulated in multiple cancers, especially highly expressed in breast cancer, and had the highest expression in the Luminal subtype. It may participate in tumor progression by regulating the estrogen signaling pathway and immune microenvironment, and is closely related to poor prognosis.

[0045] The ZNF263-targeted interference sequence (si-ZNF263) was transfected into the human breast ductal carcinoma cell line BT-549. si-ZNF263 exerted significant interference, that is, significantly reduced the expression of ZNF263 in cells ( Figure 2 A). In addition, the CCK8 detection results showed that the cell viability of the cells transfected with si-ZNF263 was significantly reduced ( Figure 2 B), while the proportion of cell apoptosis increased significantly ( Figure 2 C). The above experiments showed that ZNF263 highly expressed in breast cancer may be related to the development process of breast cancer. ZNF263 highly expressed in breast cancer may play a role in promoting the malignant growth of breast cancer cells.

[0046] In the organoid model, the C11-BODIPY581 / 591 staining results showed that knocking out ZNF263 significantly increased the level of oxidized ROS ( Figure 3 A), and significantly enhanced the level of lipid ROS induced by Erastin. Detection of oxidative stress-related molecules showed that knocking out ZNF263 led to an up-regulation of MDA expression and a down-regulation of GSH level, significantly increased the level of cellular oxidative stress, and further enhanced the oxidative stress effect induced by Erastin ( Figure 3 B- Figure 3 C). In addition, Fe2+ Content analysis showed that knocking out ZNF263 significantly increased the intracellular Fe 2+ content and synergistically enhanced the Fe 2+ level with Erastin ([[]] Figure 3 D). The Western blot results further revealed that knocking out ZNF263 significantly upregulated the expression of ferroptosis-related proteins COX2, ACSL4, and NOX1, while decreasing the expression of GPX4 and FTH1. Erastin treatment also showed a similar trend, and knocking out ZNF263 significantly enhanced the regulatory effect of Erastin on these proteins ([[]] Figure 4 ), indicating that knocking out ZNF263 enhanced Erastin-induced ferroptosis in breast cancer cells. Meanwhile, the LC3II / LC3I ratio ([[]] Figure 5 A) was significantly increased and the p62 expression was significantly decreased ([[]] Figure 5 B), suggesting that knocking out ZNF263 could further promote ferroptosis in breast cancer cells by activating autophagy flux. In summary, ZNF263 may regulate the lipid peroxidation level by regulating the antioxidant systems GSH and GPX4 and the iron metabolism-related genes FPN1 and TFRC, thus affecting the occurrence of ferroptosis. It plays a key negative regulatory role in the ferroptosis of breast cancer cells, and its deletion may provide a new target for ferroptosis-mediated therapy.

[0047] Prediction through the JASPAR database found that there were two potential binding sites of ZNF263 in the promoter region of FOSL2 ([[]] Figure 6 ). Experiments showed that ZNF263 could directly act on the promoter region of FOSL2 to regulate its transcriptional expression ([[]] Figure 7 A- Figure 7 B). Immunofluorescence experiments further showed that overexpressing ZNF263 significantly upregulated the expression of FOSL2, while knocking out ZNF263 significantly downregulated the expression of FOSL2 ([[]] Figure 8 ), indicating that ZNF263 is an important regulator of FOSL2 transcription. After ZNF263 upregulated the expression of FOSL2, it enhanced the transcriptional factor activity of Beclin1 and promoted the expression of lipid metabolism-related genes ACSL4 and LOX, thus increasing the intracellular lipid peroxidation level. At the same time, ZNF263 regulated the content of intracellular Fe 2+ by regulating the expression of the iron transporters FPN1 and TFRC, affecting the iron metabolism balance. In addition, ZNF263 could also activate autophagy flux by regulating the expression of autophagy-related genes LC3 and p62, further promoting the occurrence of ferroptosis. The regulation of autophagy flux by the ZNF263-FOSL2 axis may be achieved by affecting the maturation or degradation efficiency of autophagosomes, and the specific mechanism remains to be further explored.

[0048] ZNF263 plays an important role in breast cancer immune regulation by regulating the proportions of various immune cell subsets. As Figure 9 shown, overexpression of ZNF263 significantly inhibits the proportions of CD3+ T cells (total T cells), CD4+ T cells (helper T cells), and CD8+ T cells (cytotoxic T cells), and at the same time reduces the proportion of CD19+ B cells (B cells). However, overexpression of ZNF263 promotes the function of CD56+ NK cells (natural killer cells), suggesting that ZNF263 may weaken humoral immunity by inhibiting B cell differentiation and T cell function, and at the same time activate NK cells to reshape the immunosuppressive microenvironment. On the contrary, knockdown of ZNF263 can relieve its inhibition of lymphocyte infiltration. ZNF263 may directly target key genes of B cells (such as PAX5, BCL6) or inhibit B cell function by regulating the IL-6 / STAT3 pathway. Humoral immunity mediated by B cells is related to the production of anti-tumor antibodies and the formation of tertiary lymphoid structures in breast cancer, and its inhibition may promote tumor progression, while high expression of ZNF263 may be a biomarker for poor prognosis and a potential target for combined immunotherapy. Therefore, in breast cancer, ZNF263 not only participates in the immune escape process, but may also become a key target for regulating the immune microenvironment, providing an important basis for the development of novel immunotherapies.

[0049] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a biomarker in the preparation of a drug for treating breast cancer, characterized in that: The biomarker described above is ZNF263.

2. The application according to claim 1, wherein: ZNF263 regulates ferroptosis in breast cancer cells by activating the transcription of FOSL2.

3. The application according to claim 2, characterized in that: Knockout of ZNF263 downregulates the expression of FOSL2; knockout of ZNF263 activates autophagy in breast cancer cells.

4. The application according to claim 2, wherein: Knockout of ZNF263 increases the level of oxidized ROS and enhances the level of lipid ROS induced by Erastin.

5. The application according to claim 1, characterized in that: Knockout of ZNF263 enhances erastin-induced oxidative stress in breast cancer cells; overexpression of ZNF263 promotes the malignant growth of breast cancer cells.

6. The application according to claim 1, wherein: ZNF263 regulates immune cell subsets in breast cancer.

7. The application according to claim 6, wherein: Overexpression of ZNF263 inhibits the proportions of CD3+ T cells, CD4+ T cells, and CD8+ T cells, and at the same time reduces the proportion of CD19+ B cells; overexpression of ZNF263 promotes the function of CD56+ NK cells.

8. The application according to claim 7, characterized in that: ZNF263 weakens humoral immunity by inhibiting B cell differentiation and T cell function, and at the same time activates NK cells to reshape the immunosuppressive microenvironment; ZNF263 targets key genes of B cells or inhibits the function of B cells by regulating the IL-6 / STAT3 pathway; the key genes of B cells described above are PAX5 or BCL6.

9. Use of a biomarker in the preparation of a reagent for detecting breast cancer, characterized in that: The biomarker described above is ZNF263.

10. The application according to claim 9, wherein: The application of the said ZNF263 in the preparation of a kit for the auxiliary diagnosis and detection of breast cancer.

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