New application of FOXA1 as target in EMPD treatment

Abstract

The invention discloses a new application of FOXA1 as a target spot in preparation of a product for treating EMPD for the first time, and the FOXA1 is an FOXA1 gene or an FOXA1 protein. The FOXA1 is used as a treatment target of the EMPD, the pathogenic effect of the FOXA1 on the EMPD is proved from multiple angles, and inhibition of the FOXA1 can be used as a novel effective strategy for treating the EMPD.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a new use of FOXA1 as a target in the treatment of EMPD. Background Art

[0002] Paget's disease (PD) is a rare intraepithelial skin malignancy characterized by large adenocarcinoma cells that contain a large amount of mucin. The disease is divided into mammary Paget's disease (MPD) and extramammary Paget's disease (EMPD). The MPD type arises in the nipple-areola complex, while EMPD mainly affects apocrine gland-rich areas such as the skin of the anogenital area. EMPD accounts for 21% of primary scrotal cancers, and the incidence is on the rise.

[0003] Currently, surgery is the main treatment for EMPD. Other treatments include radiotherapy, chemotherapy, photodynamic therapy, laser therapy, imiquimod and 5-fluorouracil.

[0004] Due to the jumpy growth of EMPD tumor cells, surgical resection may not be enough to completely eliminate tumor cells, resulting in a high postoperative recurrence rate (27%-62%). For metastatic EMPD, including extensive lymph node metastasis, systemic chemotherapy cannot improve the patient's survival rate. Because most patients are elderly, the choice of systemic chemotherapy is usually limited.

[0005] Although there are many treatment options, such as radiotherapy, chemotherapy, targeted therapy, etc., choosing the most suitable treatment plan requires comprehensive consideration of the patient's condition and individual differences, which increases the complexity and difficulty of treatment. At present, there is a lack of sufficient clinical research support for the effectiveness and long-term efficacy of certain treatment methods, which limits the optimization and further improvement of treatment plans.

[0006] Therefore, there is an urgent need to explore new therapeutic methods for the prevention and treatment of EMPD, especially precise treatment based on etiology. Summary of the invention

[0007] In order to overcome the problems of the prior art, the present invention proposes a new use of FOXA1 as a target in the treatment of EMPD, which provides a new target for the treatment of EMPD and a new strategy and direction for precision treatment.

[0008] The object of the present invention is achieved in that:

[0009] The first aspect of the present invention provides a new use of FOXA1 as a target in the preparation of a product for treating EMPD.

[0010] Furthermore, the FOXA1 is the FOXA1 gene or the FOXA1 protein.

[0011] Furthermore, the product for treating EMPD is used to reduce the expression of FOXA1 gene or FOXA1 protein.

[0012] A second aspect of the present invention provides use of a reagent for detecting a biomarker in preparing a product for diagnosing EMPD, wherein the biomarker is FOXA1.

[0013] Furthermore, the FOXA1 is the FOXA1 gene or the FOXA1 protein.

[0014] The third aspect of the present invention provides the use of an agent for inhibiting the expression of FOXA1 gene in the preparation of a drug for treating EMPD.

[0015] The advantages and beneficial effects of the present invention are:

[0016] Large sample transcriptome sequencing analysis and mouse model results confirmed that FOXA1 expression can induce the occurrence of EMPD phenotype; thus, the present invention proposes to use FOXA1 as a new therapeutic target for EMPD, providing a new idea for EMPD treatment and drug screening, and inhibiting FOXA1 can be used as a new and effective strategy for treating EMPD. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0018] Figure 1 The volcano plot shows that FOXA1 is significantly overexpressed in EMPD tumor tissues;

[0019] Figure 2 It shows that FOXA1 is a driving transcription factor, which is significantly highly expressed in multiple tumor lesions; the middle is a petal Venn diagram, the upper middle Venn diagram represents the common highly expressed genes, and the lower middle Venn diagram represents the common low expressed genes; the right side is a heat map, the upper right is a heat map of highly expressed genes, and the lower right is a heat map of low expressed genes;

[0020] Figure 3 Representative images of immunohistochemical staining are shown;

[0021] Figure 4 is a schematic diagram of the mouse model construction process;

[0022] Figure 5 This is the Foxa1 immunofluorescence image of abdominal skin tissue of conditional Foxa1 overexpressing mice and wild-type C57 mice 120 hours after smearing doxycycline in Example 2;

[0023] Figure 6After high expression of FOXA1, the mouse skin expressed EMPD tumor cell markers KRT7 and AGR2;

[0024] Figure 7 This is a picture of the abdominal skin phenotype of conditional Foxa1 overexpressing mice and wild-type C57 mice 120 hours after smearing doxycycline in Example 2;

[0025] Figure 8 This is the immunofluorescence image of Krt7+Muc5ac in the abdominal skin tissue of conditional Foxa1 overexpressing mice and wild-type C57 mice in Example 2 120 hours after doxycycline was applied. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

[0027] The specific transgenic FOXA1 mouse model used in the examples of the present invention is constructed as follows:

[0028] Example 1

[0029] In order to explore new targets for the treatment of EMPD, the transcriptome sequencing results of 100 cases of EMPD and 16 normal controls (all from male scrotum and penis) were analyzed and the five genes most upregulated in EMPD compared with normal skin were: CYP4Z1, POTEE, FOXA1, CXCL1, FAM3B ( Figure 1 ), and if Figure 2 As shown, FOXA1 is the only pioneer transcription factor (driver transcription factor) that determines the differentiation of glandular cells. Its high expression is a driving factor for the occurrence of prostate cancer and breast cancer. Therefore, it is speculated that FOXA1 expression plays a key role in the tumorigenesis of PD. Therefore, this application focuses on the study of FOXA1.

[0030] Through multi-region sequencing, we found that FOXA1 is also a driving transcription factor in EMPD tumor cells. Figure 3 The immunohistochemical results shown in the figure showed that FOXA1 was not expressed in normal skin tissues, but was specifically highly expressed in 88% of EMPD tumor cells.

[0031] Example 2

[0032] In order to study the functional consequences of FOXA1 overexpression in the epidermis of the skin in vivo, this example uses C57BL / 6 mice to prepare a specific FOXA1 knock-in transgenic mouse model through CRISPR / Cas. FOXA1 is not expressed in transgenic mice under normal circumstances. After the transgenic mice are hybridized with the corresponding tissue-specific expression Krt14Cre mice, the termination sequence is removed in specific tissues after doxycycline is administered, thereby achieving the purpose of specific expression of transgenic Foxa1 in specific tissues where Krt14 is expressed, and finally inducing the generation of Krt14Cre-Foxa1+ / + mice.

[0033] Specifically, Krt14-tet; TRE-Foxa1 double transgenic mice were constructed using CRISPR / Cas technology. Figure 4 As shown, adult (12 weeks) mice were selected, and after removing the abdominal hair of the mice, 10 mg / ml doxycycline solution was applied once a day. After continuous administration of doxycycline (Dox), Krt14 expression in mouse skin was specifically induced. + Keratinocytes highly express Foxa1. Normally, Foxa1 is expressed at low levels in the suprabasal epidermis of wild-type (WT) mice, similar to human skin, but after continuous administration of doxycycline, Foxa1 expression levels increased in the epidermis of transgenic mice. Figure 5 As shown, the immunofluorescence images of Foxa1 in the abdominal skin tissue of conditional Foxa1 overexpressing mice and wild-type C57 mice were painted with doxycycline 120 hours later. It was confirmed that Foxa1 was lowly expressed in the skin of wild-type C57 mice, and by using doxycycline to induce mice to highly express Foxa1, the conditional Foxa1 overexpressing mouse model was successfully established. Figure 6 As shown, after high expression of FOXA1, the mouse skin expressed EMPD tumor cell markers KRT7 and AGR2.

[0034] After 96 hours of continuous administration of doxycycline, the transgenic mice began to show signs of dehydration and lethargy. After 120 hours of continuous administration of doxycycline, the transgenic mice basically showed a hunched phenotype, white secretions in the eyes, and redness of the abdominal skin. Some mice had scabs and desquamation on their skin ( Figure 7 ). The experiment confirmed that applying doxycycline had no effect on the skin of wild-type C57 mice, but the skin of mice with conditional Foxa1 overexpression showed abnormalities.

[0035] Without intervention, all transgenic mice died rapidly within 7 days after the initial administration of doxycycline. Immunofluorescence was performed on the skin of the lower abdomen and perineum of the mice to observe the expression of Krt7 and Muc5ac, which confirmed that there were no Krt7+Muc5ac+ skin cells with glandular cell characteristics in the skin of wild-type C57 mice. At the same time, Krt7+ and Muc5ac+ epidermal cells were found in the epidermis of transgenic mice, indicating that some squamous epithelial cells were transdifferentiated and had certain glandular cell characteristics ( Figure 8 ).

[0036] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. New use of FOXA1 as a target in the preparation of products for the treatment of EMPD.

2. The new use according to claim 1, characterized in that: The FOXA1 is the FOXA1 gene or the FOXA1 protein.

3. The new use according to claim 2, characterized in that: The product for treating EMPD is used to reduce the expression of FOXA1 gene or FOXA1 protein.

4. Use of a reagent for detecting a biomarker in the preparation of a product for diagnosing EMPD, characterized in that: The biomarker is FOXA1.

5. The use according to claim 1, characterized in that: The FOXA1 is the FOXA1 gene or the FOXA1 protein.

6. Use of an agent that inhibits FOXA1 gene expression in the preparation of a drug for treating EMPD.

Images