Application of MEIS1 in preparation of endometriosis therapeutic agent and therapeutic agent prepared from MEIS1

Abstract

The invention relates to application of MEIS1 in preparation of an endometriosis therapeutic agent and the therapeutic agent prepared by the MEIS1. According to the invention, the influence of MEIS1 on an endometrial stromal cell apoptosis pathway is studied, endometrial stromal cell apoptosis is induced by overexpression of MEIS1 in endometrial stromal cells, and an MEIS1 lentivirus overexpression vector is used for administration to an endometriosis mouse model, so that the size and weight of a mouse endometriosis focus are remarkably reduced, and the application of MEIS1 in the endometriosis mouse model is realized. Liver and kidney biochemical indexes show that the LV-MEIS1 does not damage main organ functions and brings a new potential hope for treatment of endometriosis.

Description

Technical Field

[0001] The present invention belongs to the field of endometriosis treatment, and specifically relates to application of MEIS1 in preparing an endometriosis therapeutic agent and the prepared therapeutic agent. Background Art

[0002] Endometriosis is an estrogen-dependent benign gynecological disease, which refers to the appearance of endometrial tissue outside the uterine cavity. It is estimated that 6% to 10% of women of childbearing age suffer from endometriosis, and its main symptoms include pelvic pain, dysmenorrhea and infertility, which may lead to poor quality of life for 35% to 50% of women of childbearing age and bring a heavy economic burden. The common types of endometriosis are superficial (or peritoneal) endometriosis, ovarian endometriosis (OMA) and deep infiltrating endometriosis, of which OMA accounts for about 44%. The pathogenesis of endometriosis is still unclear, and the widely accepted retrograde menstrual theory cannot explain the mechanism of all phenotypes. However, the dysregulation and resistance of apoptosis enable ectopic endometrial tissue to escape immune clearance, and various apoptotic pathways may play a vital role in this process.

[0003] Apoptosis activation can be triggered by different stimuli, including physiological and pathological factors. Apoptosis promoters in endometriosis include progesterone, Fas and Fas ligand (Fas / FasL), DNA fragmentation factor, and P53. Apoptosis inhibitors include estradiol, NF-κβ, serine-threonine kinase (AKT), B-cell lymphoma-2 (BCL-2), and survivin. Strether et al. found that abnormal regulation of the Fas / FasL system prevented ectopic endometrial cells from growing in ectopic implants through the Fas / FasL-mediated apoptotic pathway. Other researchers have found that NF-κB can upregulate BCL-2 expression by activating the AKT / PI3K pathway, thereby increasing the expression of X-linked inhibitor of apoptosis protein and ultimately reducing apoptosis in patients with endometriosis.

[0004] Therefore, finding suitable drugs to activate the apoptosis pathway of endometriosis cells and induce apoptosis of related cells can effectively treat endometriosis. Summary of the invention

[0005] During our study, we found that both RNA and protein levels of MEIS1 were reduced in eutopic and endometriotic cells of endometriosis compared with normal endometrium. Higher levels of MEIS1 in endometriosis induced apoptosis of endometrial stromal cells, where MEIS1 activated TNFR1 transcriptional activity to induce apoptosis through the caspase pathway and prevented the formation of endometriotic lesions in mice.

[0006] Based on the above research, the present invention provides the use of meis homeobox I in preparing an agent for treating endometriosis.

[0007] In a specific embodiment, the amino acid sequence of the meis homeobox I is shown in SEQ ID NO:1.

[0008] The present invention also provides an agent for treating endometriosis, comprising an expression vector containing a meis homeobox I expression frame.

[0009] In a specific embodiment, the amino acid sequence of the meis homeobox I is shown in SEQ ID NO:1.

[0010] In a specific embodiment, the expression vector is a lentivirus-based expression vector.

[0011] The present invention also provides a non-therapeutic method for promoting cell apoptosis, comprising the step of increasing the expression level of meis homeobox I in the cells.

[0012] In a specific embodiment, the amino acid sequence of the meis homeobox I is shown in SEQ ID NO:1.

[0013] In a specific embodiment, the cells are endometrial stromal cells.

[0014] The present invention studies the effect of MEIS1 on the apoptosis pathway of endometrial stromal cells, achieves overexpression of MEIS1 in endometrial stromal cells to induce apoptosis of endometrial stromal cells, and administers the MEIS1 lentiviral overexpression vector to an endometriosis mouse model, which significantly reduces the size and weight of endometriosis lesions in mice. In addition, biochemical indicators of the liver and kidneys show that LV-MEIS1 does not damage the function of major organs, bringing new potential hope for the treatment of endometriosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a study of OMA susceptibility genes. A is a gene overlap map based on pooled GWAS, RNA sequencing, and public datasets; B is the expression statistics of mRNA in different tissues.

[0016] Figure 2 The expression of MEIS1 protein in women with and without endometriosis. A and C are photos of immunoblotting and immunohistochemistry, respectively, and B and D are quantitative statistical graphs based on A and C, respectively.

[0017] Figure 3MEIS1 regulates the proliferation and apoptosis of endometrial stromal cells. A and B are the detection of MEIS1 mRNA and protein expression in NESCs after treatment with MEIS1 siRNA and its control, with β-actin as the control gene; C is the quantitative statistics of B; D and E are the detection of MEIS1 mRNA and protein expression in EESCs after treatment with MEIS1 overexpression lentivirus and its control virus, with β-actin as the control gene; F is the quantitative statistics of E; G and H are the changes in the proliferation activity of NESCs after silencing MEIS1 by CCK8 and EdU after treatment with MEIS1 siRNA and its control in NESCs; H is the detection of the changes in EESCs apoptosis after overexpression of MEIS1 by flow cytometry after treatment with MEIS1 overexpression lentivirus and its control virus in EESCs.

[0018] Figure 4 The figure shows that MEIS1 activates the TNFR1-caspase8 apoptosis pathway in ectopic endometrial tissue. A is a scan of the total protein detected by human apoptosis antibody chip after EESCs were treated with MEIS1 overexpression lentivirus and control virus; B is a quantitative analysis of A; C is a western blot method to detect the changes in MEIS1, TNFR1, caspase8, cleaved-caspase8, IGF-Ⅱ and β-actin proteins after NESCs and EESCs were treated with MSEI1 siRNA and overexpression lentivirus respectively; D is the effect of MEIS1 lentiviral vector on the luciferase activity of TNFR1 promoter vector in 293T cells. Renilla luciferase expression plasmid was used to normalize the transfection efficiency.

[0019] Figure 5 MEIS1 inhibits the progression of endometriosis lesions. A is the animal research flow chart for the establishment of the nude mouse abdominal endometriosis model and the lentiviral treatment of endometriosis lesions; B is the change in the size of endometriosis lesions before and after MEIS1 lentiviral treatment (marked by red arrows); C is the gross image of endometriosis lesions (above) and lesion weight statistics (below) after 15 days of treatment with MEIS1 lentivirus and its control virus; D is the immunohistochemical detection of MEIS1 and TNFR1 in nude mouse endometriosis lesion tissues, and representative pictures of HE staining and TUNEL staining. DETAILED DESCRIPTION

[0020] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0021] 1. Reduced expression of MEIS1 in eutopic endometrium and OMA tissues of patients with endometriosis

[0022] For OMA susceptibility genes, we analyzed genes from pooled GWAS, RNA sequencing, and public datasets. GSE7305 used normal endometrium and endometriosis tissues. MEIS1 was identified as a gene of interest and is an important transcription factor in cell proliferation and apoptosis ( Figure 1 A). The mRNA expression of MEIS1 in endometriotic tissues was lower than that in normal endometrium at different stages of the menstrual cycle, but there was no difference between eutopic endometrium and ectopic endometrium, and it was not related to the menstrual cycle.

[0023] Immunoblotting and immunohistochemistry were used to examine the expression of MEIS1 protein in women with and without endometriosis. Figure 2 As shown, compared with eutopic endometrium and ectopic endometrial tissue of endometriosis, MEIS1 was overexpressed in both the nuclei and cytoplasm of epithelial cells and stromal cells of normal endometrium, especially in stromal cells.

[0024] These results suggest that abnormal MEIS1 expression may be involved in the pathogenesis of endometriosis.

[0025] 2. Abnormal MEIS1 expression in eutopic endometrium affects the proliferation and apoptosis of endometrial stromal cells

[0026] To investigate the biological function of MEIS1 in the pathogenesis of endometriosis, we downregulated the expression of MEIS1 in normal endometrial stromal cells (NESCs) by siRNA ( Figure 3 A, B, and C). In addition, MEIS1 expression is upregulated in eutopic endometrial stromal cells (EESCs) ( Figure 3 D, E, and F). Cell proliferation assays using CCK8 assay and EuU assay showed that downregulation of MEIS1 increased the proliferation of endometrial stromal cells ( Figure 3 G and H). Flow cytometry analysis showed that the apoptosis rate of EESCs was significantly increased after transfection with LV-MEIS1 ( Figure 3 I).

[0027] These results suggest that downregulation of MEIS1 in EESCs may promote cell proliferation and inhibit apoptosis, thereby contributing to the progression of endometriosis.

[0028] 3. MEIS1 promotes stromal cell apoptosis by activating the TNFR1-mediated caspase pathway

[0029] Since MESI1 can induce apoptosis of endometrial stromal cells, we used Human ApoptosisAntibody Array was used to determine possible apoptotic pathways activated by MEIS1. The results showed that upregulation of MEIS1 in EESCs was positively correlated with caspase cascades induced by TNFR1 and IGF-Ⅱ ( Figure 4 A and B).

[0030] To verify this, we upregulated MEIS1 in EESCs and downregulated MEIS1 in NESCs. Western blot showed that the protein levels of TNFR1 in NESCs and EESCs had a consistent relationship with MEIS1, but there was no such consistency in IGF-Ⅱ ( Figure 4 C). In addition, we also observed that caspase 8 was activated after MEIS1 upregulation ( Figure 4 C).

[0031] Given that MEIS1 is a transcription factor, we hypothesized that MEIS1 might regulate TNFR1 gene expression by enhancing its promoter activity. Figure 4 As shown in D, MEIS1 enhanced the luciferase activity of the TNFR1 promoter plasmid, which supports the above hypothesis. Collectively, these findings suggest that MEIS1 can induce apoptosis by activating the TNFR1-caspase8 cascade in endometriosis.

[0032] 4. MEIS1 treatment inhibits the progression of endometriosis

[0033] The mouse endometriosis model was established by transplanting situ endometrial fragments and suturing them to the peritoneal surface. The model was established 4 weeks after transplantation, and then the lentiviral control LV-CON or lentiviral MEIS1 overexpression vector LV-MEIS1 was intraperitoneally injected every 3 days for 15 days ( Figure 5 A).

[0034] LV-MEIS1 treatment significantly reduced the size and weight of endometriotic lesions in mice ( Figure 5 B and C). Immunostaining showed that MEIS1 and TNFR1 were significantly elevated in endometriotic lesions of MEIS1-treated mice ( Figure 5 D and E). TUNEL staining showed that the apoptosis of the model group treated with MEIS1 increased ( Figure 5 D). Importantly, liver and kidney biochemical parameters indicated that LV-MEIS1 did not impair major organ function. Our data suggest that MEIS1 can reduce endometriotic lesions through activation of apoptotic signaling pathways.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of meis homeobox I in the preparation of therapeutic agents for endometriosis.

2. The use according to claim 1, characterized in that: The amino acid sequence of the meis homeobox I is shown in SEQ ID NO:

1.

3. A therapeutic agent for endometriosis, characterized in that: An expression vector comprising a meis homeobox I expression cassette is included.

4. The therapeutic agent for endometriosis according to claim 2, characterized in that: The amino acid sequence of the meis homeobox I is shown in SEQ ID NO:

1.

5. The therapeutic agent for endometriosis according to claim 2, characterized in that: The expression vector is a lentivirus-based expression vector.

6. A non-therapeutic method for promoting cell apoptosis, characterized in that: The method comprises the step of increasing the expression level of meis homeobox I in the cell.

7. The method according to claim 6, characterized in that The amino acid sequence of the meis homeobox I is shown in SEQ ID NO:

1.

8. The method according to claim 6, characterized in that The cells are endometrial stromal cells.

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