CN118006779B

CN118006779B - Application of targeting ZNF468/PRMT1/VEGF-C signal shaft in early diagnosis and treatment of esophageal cancer lymph node metastasis

Info

Publication number: CN118006779B
Application number: CN202410183397.6A
Authority: CN
Inventors: 朱金容; 张荣信; 邱湘喻; 欧胜明; 聂小雅; 金鑫; 唐久人; 李岩
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2024-02-19
Filing date: 2024-02-19
Publication date: 2024-11-15
Anticipated expiration: 2044-02-19
Also published as: CN118006779A

Abstract

The present invention relates to an application of a targeted ZNF468/VEGF-C signaling axis in the treatment, prognosis and lymph node metastasis of tumors. The present invention finds that when ZNF468 is overexpressed, the body significantly promotes lymphangiogenesis and lymph node metastasis of tumor patients (such as esophageal squamous cell carcinoma) by recruiting and binding to PRMT1 to enhance the transcriptional activity of VEGF-C; the present invention combines the use of an agent that inhibits PRMT1 expression to significantly inhibit the lymphangiogenesis mechanism of esophageal squamous cell carcinoma with overexpression of ZNF468. The important role of ZNF468 in lymphangiogenesis and lymph node metastasis in patients with esophageal squamous cell carcinoma can be used as a new treatment strategy for ZNF468-overexpressing tumors.

Description

Application of targeting ZNF468/PRMT1/VEGF-C signal shaft in early diagnosis and treatment of esophageal cancer lymph node metastasis

Technical Field

The invention relates to the technical field of biology, in particular to application of a targeting ZNF468/PRMT1/VEGF-C signal shaft in treatment of esophageal cancer lymph node metastasis and early diagnosis.

Background

Esophageal cancer is a major global health challenge, the sixth leading cause of global cancer-related death in 2020, with 5-year survival rates of less than 20%, while Esophageal Squamous Cell Carcinoma (ESCC) is the leading cause of cancer death, accounting for about 90% of esophageal cancer cases.

Diagnosis of esophageal cancer often occurs in advanced stages, the primary treatment for esophageal cancer is esophageal resection, however, overall prognosis remains poor.

Studies show that ESCC patients generate lymphangiogenesis in early stage and cause lymph node metastasis, and that poor prognosis of ESCC is related to lymphangiogenesis and lymph node metastasis, so that molecular mechanisms and identification of ESCC lymphangiogenesis and lymph node metastasis are urgently needed to determine a new therapeutic target to improve ESCC prognosis.

Vascular endothelial growth factor family (VEGF, e.g., VEGFA, VEGFB, VEGF-C, VEGFD) and placental growth factor (PLGF) are involved in lymphangiogenesis, where VEGF-C (vascular endothelial growth factor C) is a stimulator of lymphatic endothelial cells, VEGF-C and VEGFR3 play a major role in lymphangiogenesis.

Furthermore, lymph node metastasis, poor prognosis, lymphatic infiltration and distant metastasis are associated with increased expression of VEGF-C in tumors, with expression levels of VEGF-C associated with lower 5-year survival in ESCC.

However, selective inhibitors against VEGF-C receptor VEGFR3 are currently under preclinical investigation, such as EVT801 and SAR131675, and therefore, the determination of new therapeutic targets to inhibit tumor-associated lymphangiogenesis and lymph node metastasis may be a more effective strategy for clinically intervening in cancer.

ZNF468 is a member of the zinc finger protein family, the globally largest family of transcription factors with finger domains, playing an important role in a number of biological processes, however, little information remains about the role of ZNF468 in ESCC, and recent studies have established that a variety of zinc finger proteins are key regulators of cancer initiation and progression.

For example, zhang et al report that the expression of the potential cancer suppressor ZNF671 is down-regulated in nasopharyngeal carcinoma, promoting cell proliferation and tumorigenicity.

An and colleagues research shows that ectopic expression of ZNF575 can inhibit colorectal cancer cell proliferation, promote apoptosis and inhibit tumor growth.

In addition, hu and colleagues demonstrated that ZNF668 can inhibit the progression of cancer through the p53 pathway and determined that it is a potential tumor suppressor in breast cancer. He and colleagues found ZNF154 as a tumor suppressor in ESCC and was associated with poor prognosis.

We speculate that ZNF468 may also play a key role in the tumorigenesis and progression of ESCC. Therefore, exploring the potential of ZNF468 as a target for ESCC therapy might help us develop new therapeutic strategies, improving the prognosis of ESCC patients.

Disclosure of Invention

The invention aims at solving the problems existing in the prior art and provides an application of a targeting ZNF468/PRMT1/VEGF-C signal shaft in treatment of esophageal cancer lymph node metastasis and early diagnosis.

In order to achieve the above object, the present invention provides the following technical solutions:

the application of ZNF468 as a target in preparing products for treating tumor, prognosis tumor and predicting the effect of tumor treatment.

Preferably, the tumor treatment, tumor prognosis, or/and product for predicting the effect of tumor treatment comprises a diagnostic kit.

Preferably, the diagnostic kit contains at least one of a reagent for detecting the expression level of ZNF468, a reagent for detecting the expression level of VEGF-C and a reagent for detecting the expression level of PRMT 1.

Preferably, the reagent for detecting ZNF468 comprises at least one of a reagent for detecting whether ZNF468 gene is overexpressed, a reagent for quantitatively detecting RNA transcription level of ZNF468, and a reagent for quantitatively detecting protein expression level of ZNF 468.

An application of an agent for inhibiting ZNF468 protein expression in preparing a medicament for inhibiting lymphangiogenesis and improving tumor treatment effect.

An agent for inhibiting PRMT1 expression, an agent for inhibiting VEGF-C expression, and the application of the agent in preparing a medicament for treating ZNF468 over-expression tumor and/or a medicament for improving the immune treatment effect of the ZNF468 over-expression tumor.

Preferably, the agent that inhibits PRMT1 expression comprises the chemical AMI-1.

An agent for improving the therapeutic effect and prognosis of tumor therapy, which comprises at least one of an agent for inhibiting ZNF468 expression, an agent for inhibiting VEGF-C expression, and an agent for inhibiting PRMT1 expression.

An agent for inhibiting ZNF468 over-expression tumor to treat.

Preferably, the agent for inhibiting the overexpression of ZNF468 has at least one of an agent for inhibiting the expression of ZNF468, an agent for inhibiting the expression of VEGF-C and an agent for inhibiting the expression of PRMT 1.

The invention has the beneficial effects that:

1) The invention discovers that under the condition of the overexpression of ZNF468, the organism obviously promotes the lymphangiogenesis and the lymph node metastasis of tumors (such as esophageal squamous cell carcinoma) by recruiting a path of combining with PRMT1 to enhance the VEGF-C transcriptional activity, and the combined application of the agent for inhibiting the expression of the PRMT1 obviously inhibits the lymphangiogenesis mechanism of the esophageal squamous cell carcinoma of the ZNF 468. These results demonstrate that ZNF468 plays an important role in lymphangiogenesis and lymph node metastasis in patients with esophageal squamous cell carcinoma, and can be used as a novel therapeutic strategy for ZNF468 over-expressed tumors;

2) The invention finds application of the ZNF468 gene in generating lymphangiogenesis and lymph node metastasis in esophageal squamous cell carcinoma. The invention successfully builds an esophageal squamous carcinoma cell model which over-expresses ZNF468 and interferes with the expression of ZNF468, successfully builds a mouse esophageal squamous carcinoma callus lymph node metastasis model, and discovers through experimental analysis such as immunohistochemistry, cell tubule formation and the like that the inhibition of ZNF468 can reduce in-vivo and in-vitro lymphatic vessel generation and lymph node metastasis capacity, thereby inhibiting tumor growth. The invention uses TCGA database analysis to find that the up-regulation of ZNF468 protein expression is closely related to the metastasis survival rate of patients suffering from esophageal squamous cell carcinoma.

3) In the mechanism level, ZNF468 is combined with a VEGF-C promoter through CHIP-PCR, luciferase reporter gene experiments and the like, so that the transcription activity of VEGF-C is increased; through mass spectrometry experiments, co-immunoprecipitation techniques and ChIP experiments, ZNF468 is found to increase the expression of VEGF-C in esophageal squamous cell carcinoma and promote lymphangiogenesis and lymph node metastasis; the specific binding region of ZNF468 and a downstream gene VEGF-C promoter is analyzed, and a novel molecular regulation mechanism is provided for regulating a target gene by using the oncogene ZNF 468; finally, immunohistochemical experiments and analysis are carried out on tumor tissue samples utilized by the invention, and the combined application of PRMT1 inhibitor is found to greatly reduce the expression of VEGF-C protein in tumors. This suggests that the ZNF468 gene can be an important target molecule for promoting lymphangiogenesis and lymph node metastasis in patients with esophageal squamous cell carcinoma. In conclusion, the study finds that ZNF468 is an important promoter for the development of esophageal squamous cell carcinoma, and has important significance in the development process of cancer. This will provide a new therapeutic direction for clinical tumor treatment. The invention provides a new diagnosis and treatment method and a drug screening platform for tumor diseases.

The features and advantages of the present invention will be described in detail in the detailed description that follows.

Drawings

FIG. 1 (A) is a graph showing the expression of ZNF468 in ESCC tissues with or without lymph node metastasis (LN+);

FIG. 1 (B) is a graph showing the immunohistochemical staining analysis of the expression of ZNF468 and Lyve-1 in ESCC tissues;

FIG. 1 (C) is a Kaplan-Meier survival curve;

FIG. 1 (D) is a graph showing mRNA expression of ZNF468 in ESCC tissues with or without lymph node metastasis in TCGA dataset;

FIG. 1 (E) is a graph of a prognosis analysis for survival in TCGA dataset;

FIG. 2 (A) is a graph of a mouse lymph node metastasis model;

FIG. 2 (B) is a lymph node map (left) and a lymph node volume quantification map (right) for each experimental group;

FIG. 2 (C) shows the IHC assay set Lyve-1 expression pattern;

FIG. 2 (D) is a graph showing the expression pattern of ZNF468 in each group (left) and the lymph node metastasis rate of the cells shown (right) in IHC test;

FIG. 3 (A) is a graph of HLECs cell tube formation ability validated for a tubule formation experiment;

FIG. 3 (B) is a graph showing the results of a cell perforation experiment;

FIG. 4 (A) is a map of predicted ZNF468 binding sites using JASPAR database;

FIG. 4 (B) is a graph showing the results of ChIP-PCR using IgG or ZNF468 antibody for detection of two ESCC cells;

FIG. 4 (C) is a graph of relative luciferase activity in cells;

FIG. 4 (D) is a specific map of locations where ZNF468 may be located in the VEGF-C promoter for the ChIP-PCR analysis;

FIG. 5 (A) is a schematic diagram looking for key factors for VEGF-C regulation (left) and a graph of endogenous ZNF468 interactions with endogenous PRMT1 in ESCC cells (right);

FIG. 5 (B) is a Western blot;

FIG. 5 (C) is a graph of H ₄R₃ me2a enrichment in the VEGF-C promoter region;

FIG. 5 (D) is a graph of luciferase activity of PRMT1 affecting the VEGF-C promoter;

FIG. 5 (E) is a graph of the levels of VEGF-C protein in PRMT 1-affected ESCC cells;

FIG. 6 (A) is a diagram showing IHC measurement of expression of ZNF468 in each group and a diagram showing lymph node metastasis rate of cells;

FIG. 6 (B) is a diagram showing the IHC assay of each group Lyve-1;

FIG. 7 is a genetic map of various cancers.

Detailed Description

In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. Examples of which are illustrated in the accompanying drawings. It should be understood that the specific examples described in the following embodiments of the present invention are intended to be illustrative of the specific embodiments of the present invention and are not to be construed as limiting the invention.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein. In the description of the present application, unless otherwise indicated, the meaning of "a", "an", "a plurality" and the like are two or more.

Example 1

Upregulation of ZNF468 was associated with lymphatic metastasis and lymphangiogenesis.

The method comprises the following steps: the relationship between the expression of ZNF468 and lymphatic metastasis and lymphangiogenesis in tumor and normal tissues was evaluated by Immunohistochemical (IHC) results of clinical samples and TCGA database, and the results are shown in fig. 1 (a) to (E).

Results: as shown in FIGS. 1 (A) - (E), IHC results show that the expression level of ZNF468 is significantly increased in tumor tissues, and FIG. 1 (C) compares ESCC patients with higher expression levels of ZNF468 and Lyve-1 or lower expression levels of ZNF468 and Lyve-1; at the same time, the ability of lymph node metastasis and lymphangiogenesis is significantly improved. Under the condition that the TCGA database analysis shows that ZNF468 is expressed lowly, the survival time without transfer is longer.

Conclusion: ZNF468 is highly expressed in tumor tissue, and expression levels of ZNF468 are associated with lymphatic metastasis and lymphangiogenesis.

Example 2

High expression of ZNF468 may promote lymph node metastasis and lymphangiogenesis in vivo.

The method comprises the following steps: a mouse foot pad experimental model is established, a certain amount of esophageal cancer (Eca 109) tumor cells are injected into the sole of a C57 mouse, wherein the tumor cells comprise an Eca109-vector group, an Eca109-ZNF468 group, an Eca109-Control group and an Eca109-shRNA group, and the tumors are subjected to Immunohistochemical (IHC) staining for further analysis, and the results are shown in the figures 2 (A) - (D).

Results: inguinal lymph node analysis of the mouse footpad model found (FIG. 2 (A)), that the lymph node volume of the Eca109-ZNF468 group overexpressed ZNF468 was greater than that of the other groups, while the inguinal lymph node volume of the Eca109 group silenced ZNF468 was significantly reduced. IHC staining results show that the ability of the Eca109-ZNF468 group to produce lymphatic vessels and metastasize lymph nodes is significantly enhanced.

Conclusion: ZNF468 can promote lymph node metastasis and lymphangiogenesis in mice in vivo.

Example 3

Expression of ZNF468 may induce lymphangiogenesis and migration capacity in esophageal cancer cells in vitro.

The method comprises the following steps: two kinds of cells, namely Eca109 and KYSE30, are selected, vector, ZNF468, control, ZNF468-RNA#1 and ZNF468-RNA#2 are respectively transfected, and a tubule formation experiment and a cell migration experiment are carried out by using the transfected cells, and the results are shown in FIG. 3 (A) and FIG. 3 (B).

Results: in the experimental group with high expression of ZNF468 in esophageal cancer cells, the tube formation and migration capacity of lymphatic endothelial cells are obviously enhanced, while the opposite result appears in inhibiting ZNF 468.

Conclusion: expression of ZNF468 promotes lymphangiogenesis and migration in esophageal cancer cells in vitro.

Example 4

ZNF468 binds to the VEGF-C promoter and regulates the transcriptional activity of VEGF-C.

The method comprises the following steps: the potential binding site for ZNF468 in the VEGF-C promoter was first predicted using JASPAR database, and then the relationship between ZNF468 and VEGF-C was verified by chromatin co-immunoprecipitation (CHIP) and luciferase reporter gene detection, and the correlation of ZNF468 in the VEGF-C promoter region was analyzed using CHIP, and the results are shown in fig. 4 (a) to (D).

Results: the JASPAR database predicts three potential ZNF468 binding sites in the VEGF-C promoter. ChIP detection showed that ZNF468 has a significant association with the VEGF-C promoter in esophageal cancer. Luciferase reporter gene detection showed that in esophageal cancer, ZNF468 over-expression promoted luciferase activity of VEGF-C promoter, while silencing ZNF468 inhibited luciferase activity of the promoter. In addition, chromatin immunoprecipitation assays showed that ZNF468 binds to the F5 region of the VEGF-C promoter region.

Conclusion: ZNF468 up-regulates VEGF-C expression in esophageal cancer by targeting VEGF-C promoter transcription.

Example 5

ZNF468 is involved in VEGF-C-promoted lymphangiogenesis mechanisms by interaction with PRMT 1.

The method comprises the following steps: the mechanism by which ZNF468 transduces activated VEGF-C in esophageal cancer was investigated by biotinylated inactivated Cas9 (dCas 9) capture assay, combined with immunoprecipitation Co-IP and mass spectrometry analysis. Furthermore, among the esophageal cancer cells, PRMT1 activity was inhibited by using a PRMT1 inhibitor (AMI-1) and the esophageal cancer cells were treated with siPRMT, and the results are shown in FIGS. 5 (A) to (E).

As a result, IP/MS experimental analysis shows that ZNF468 and PRMT1 are involved in transcriptional regulation of VEGF-C in esophageal cancer cells. Co-immunoprecipitation (co-IP) and western blot analysis showed that ZNF468 interacted with PRMT1 in esophageal cancer cells. Silencing ZNF468 or PRMT1 significantly reduced the level of H4R3me2a on the VEGF-C promoter, and the expression level of VEGF-C was significantly reduced after treatment of esophageal cancer cells with PRMT1 inhibitor AMI-1 and with siPRMT.

Conclusion: PRMT1 is involved in ZNF 468-induced transcriptional activation of VEGF-C, and upregulates VEGF-C transcriptional levels through methylation modification of H ₄R₃. Demonstrating that ZNF468 is involved in VEGF-C-promoted lymphangiogenesis mechanisms are achieved by interaction with PRMT 1.

Example 6:

Effect of AMI-1 and VEGF-C-RNAi on lymph node metastasis and lymphangiogenesis ability of ZNF468 overexpressing esophageal cancer cells.

The method comprises the following steps: a mouse foot pad model is established, eca109 with high expression of ZNF468 is injected, PRMT1 inhibitor AMI-1 and VEGF-C-RNAi treatment are given to the mice, and a control group Vehicle is set. The tumors of the mice were subsequently subjected to Immunohistochemical (IHC) staining and analyzed, and the results are shown in FIG. 6 (A) and FIG. 6 (B).

Results: in vivo experiments showed that AIM-1 treated mice and VEGF-C-RNAi treated mice had significantly lower lymph node metastasis than control mice, with AMI-1 treated mice being more effective. Also, AIM-1 and VEGF-C-RNAi treated mouse tumor tissue showed a significant decrease in Lyve-1 lymphatic vessels compared to control tumor tissue.

Conclusion: the data show that PRMT1 participates in VEGF-C transcription process induced by ZNF468, and after PRMT1 inhibitor AMI-1 and VEGF-C-RNAi treatment are administered, lymphatic generation and lymph node metastasis of esophageal cancer overexpressed by ZNF468 are obviously inhibited, and ZNF468 and PRMT1 are proved to be involved in regulating VEGF-C.

Example 7

Clinical significance of expression of ZNF468 and VEGF-C in a variety of cancers.

The method comprises the following steps: the results are shown in FIG. 7 by analysis of the GSE and TCGA clinical databases.

Results: binding clinical information demonstrated that ZNF468 expression was positively correlated with VEGF-C expression in numerous cancers, revealing the prevalence of ZNF468 as a tumor biomarker in a variety of tumors.

Conclusion: expression of ZNF468 and VEGF-C was associated with a variety of cancers involving digestion, demonstrating that expression of ZNF468 is positively correlated with expression of VEGF-C in a number of cancers, revealing the prevalence of ZNF468 as a tumor biomarker in a variety of tumors.

It should be noted that the specific parameters or some reagents in the above embodiments are specific embodiments or preferred embodiments under the concept of the present invention, and are not limited thereto; and can be adaptively adjusted by those skilled in the art within the concept and the protection scope of the invention.

Claims

1. Use of a reagent for detecting ZNF468 in the preparation of a diagnostic kit for esophageal cancer lymph node metastasis and lymphangiogenesis, characterized in that the reagent for detecting ZNF468 is at least one of a reagent for detecting whether the ZNF468 gene is overexpressed and a reagent for quantitatively detecting the RNA transcription level of ZNF468.