Method for promoting renal clear cell carcinoma metastasis by VSIG4 through epithelial-mesenchymal transition

Abstract

The invention discloses a method for promoting renal clear cell carcinoma metastasis by VSIG4 through epithelial-mesenchymal transition, which comprises the following steps: S1, clinical data analysis of ccRCC patients: S1.1, data collection: collecting clinical data of ccRCC patients, including two groups of primary metastatic-free tumor patients and metastatic patients, and observing whether there is statistical difference in expression of VSIG4 on renal carcinoma pathological tissues of the two groups of patients; s1.2, immunohistochemical staining and scoring: performing VSIG4 immunohistochemical staining and scoring on pathological section tissues of the ccRCC patients, grouping according to whether the VSIG4 is highly expressed, and observing whether the patients have disease imaging progression and whether the total lifetime is different; and S1.3, expression difference research: determining the effect of the VSIG4 in invasion and metastasis of the kidney cancer according to the expression difference of the VSIG4 among the kidney cancer, the para-carcinoma and the cancerous pseudoenvelop. According to the application disclosed by the invention, the VSIG4 promotes the metastasis of the renal clear cell carcinoma through epithelial-mesenchymal transition, and a potential means is provided for treating metastatic renal carcinoma by taking the VSIG4 as a target.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to a method for promoting renal clear cell carcinoma metastasis by VSIG4 through epithelial-mesenchymal transition. Background Art

[0002] Renal cell carcinoma (RCC) is a malignant renal tumor that originates from the urothelial epithelium of the renal parenchyma. It primarily includes clear cell carcinoma, papillary cell carcinoma, and chromophobe cell carcinoma. Renal cell carcinoma accounts for 2% to 3% of adult malignant tumors, second only to prostate cancer and bladder cancer among genitourinary tumors, but it is the most lethal malignant tumor of the genitourinary system. V-set and immunoglobulin domain containing 4 (VSIG4), also known as CRIg, Z39IG, encodes a protein containing V-set and immunoglobulin domains. This protein is structurally similar to proteins encoded by members of the B7 family of immunomodulatory proteins and belongs to the B7-related family. This protein also serves as a receptor for the complement components C3b and iC3b.

[0003] Approximately 30-40% of renal cancers eventually progress to a metastatic state after surgery, which is difficult to treat and ineffective, seriously endangering the patient's health and survival. Providing better treatment strategies for patients with metastatic renal cancer is a topic we are constantly striving to address.

[0004] During the epithelial-mesenchymal transition (EMT) process, epithelial cells lose their polarity and intercellular connections while acquiring mesenchymal cell characteristics, such as enhanced migration and invasion capabilities. EMT exhibits hallmark therapeutic resistance in glioma cells. Understanding the mechanisms behind these characteristics is crucial for developing effective therapies. Studies have found that VSIG4 protein is upregulated in glioblastoma. Overexpression of VSIG4 induces EMT and significantly promotes the invasion and migration of glioblastoma U-87MG cells. In addition, we found that its overexpression promotes the formation of a glioma stem cell phenotype in U-87MG cells. It has also been reported that VSIG4 promotes the progression of chronic kidney disease by upregulating the NF-kB signaling axis and then regulating EMT.

[0005] To better treat metastatic renal cancer, we proposed a method to promote the metastasis of renal clear cell carcinoma by VSIG4 through epithelial-mesenchymal transition. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for promoting renal clear cell carcinoma metastasis by VSIG4 through epithelial-mesenchymal transition, so as to solve the problems in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for promoting renal clear cell carcinoma metastasis by VSIG4 through epithelial-mesenchymal transition, comprising the following steps:

[0008] S1. Clinical data analysis of ccRCC patients:

[0009] S1.1. Data Collection: Clinical data of ccRCC patients were collected, including patients with primary tumors without metastasis and patients with metastasis. The expression of VSIG4 in renal cancer pathological tissues between the two groups was observed to determine whether there was a statistically significant difference.

[0010] S1.2 Immunohistochemical staining and scoring: ccRCC patients were immunohistochemically stained for VSIG4 and scored. Patients were grouped based on whether VSIG4 was highly expressed to observe whether they had radiographic disease progression and overall survival.

[0011] S1.3. Expression differential study: Based on the differential expression of VSIG4 between renal cell carcinoma, adjacent tissues, and pseudocapsule, the role of VSIG4 in renal cell carcinoma invasion and metastasis was clarified.

[0012] S2. Study the mechanism of VSIG4 in promoting invasion and metastasis at the level of renal cell carcinoma;

[0013] S3. In vivo studies in mice showed the role of VSIG4 in promoting the invasion and metastasis of renal cell carcinoma and the synergistic effect of VSIG4 gene knockout on PD-L1 inhibitors.

[0014] Preferably, the step S2 specifically includes the following steps:

[0015] S2.1 Cell experiments: Renal cancer cells were cultured and stably transfected VSIG4 knockout cell lines were constructed. Changes in cell proliferation, colony formation, invasion, and metastasis phenotypes before and after VSIG4 knockout were compared.

[0016] S2.2. Exploration of EMT Mechanisms: Exploring the relationship and mechanism between cell invasion and metastasis and EMT;

[0017] S2.3. Synergistic effect of PD-L1 inhibitors: To explore whether knocking out the VSIG4 gene has a synergistic inhibitory effect on PD-L1 inhibitors against renal cancer cells;

[0018] S2.4. Compound screening: Use the compound library to screen cancer cells and select compounds that have inhibitory function on VSIG4.

[0019] Preferably, the exploration of the EMT mechanism in S2.2 specifically explores whether the effect of VSIG4 on cell invasion and metastasis is related to the epithelial-mesenchymal transition (EMT) process.

[0020] Preferably, the step S3 specifically includes the following steps:

[0021] S3.1. Animal Model Construction: Stably transfected renal cancer cell lines with VSIG4 knockout were injected into mice to establish subcutaneous tumor formation models and tail vein injection lung metastasis models.

[0022] S3.2. Efficacy evaluation: Compare the tumor size between wild-type and VSIG4 knockout cell lines, and evaluate the efficacy and safety of the compound combined with PD-L1 inhibitors.

[0023] S3.3. Metastasis model evaluation: After the successful establishment of a lung metastasis model by tail vein injection of a stably transfected renal cancer cell line with VSIG4 knockout, the sizes of tumors in the lung metastasis model of the wild-type cell line and the stably transfected cell line were compared. The efficacy and toxicity of the compound were evaluated by combining the compound with a PD-L1 inhibitor.

[0024] S3.4. Functional study of macrophages and T lymphocytes in gene-knockout mice: Using VSIG4-knockout mice, we analyzed the functional changes in their immune system, especially macrophages and T lymphocytes.

[0025] Preferably, the efficacy evaluation in S3.2 is specifically as follows: after successfully constructing a subcutaneous tumor model with a stably transformed renal cancer cell line in which VSIG4 is knocked out, the tumor formation ratio and tumor size of the wild-type and stably transformed cell lines are compared to evaluate the effect of VSIG4 in promoting tumor formation; at the same time, the compounds screened at the above-mentioned cell level are combined with PD-L1 inhibitors to treat tumor-bearing mice, and the changes in tumor size and mouse weight are observed to evaluate the efficacy and toxicity of the screened compounds.

[0026] Preferably, after the lung metastasis model of the stably transfected renal cancer cell line with VSIG4 knockout in S3.3 is successfully established by tail vein injection, the screened compound is simultaneously administered in combination with a PD-L1 inhibitor to treat metastases, and the changes in tumor size and mouse weight are observed to evaluate the efficacy and toxicity of the compound.

[0027] Preferably, the VSIG4 gene in the mice to be knocked out in S3.3 is successfully generated, the bone marrow and thymus of the mice are taken, and the cells are sorted to observe the changes in the functions of the mouse macrophages and T lymphocytes, and to evaluate the effects of VSIG4 on macrophages and T lymphocytes.

[0028] Compared with the prior art, the beneficial effects of the present invention are: VSIG4 promotes the metastasis of renal clear cell carcinoma through epithelial-mesenchymal transition, and using VSIG4 as a target provides a potential means for treating metastatic renal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0030] Figure 1 is a flow chart of the present invention;

[0031] Figure 2 It is a functional diagram of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0033] See also Figure 1 and 2 In an embodiment of the present invention, a method for promoting renal clear cell carcinoma metastasis by VSIG4 through epithelial-mesenchymal transition comprises the following steps:

[0034] S1. Clinical data analysis of ccRCC patients:

[0035] S1.1. Data Collection: Clinical data of ccRCC patients were collected, including patients with primary tumors without metastasis and patients with metastasis. The expression of VSIG4 in renal cancer pathological tissues between the two groups was observed to determine whether there was a statistically significant difference.

[0036] S1.2 Immunohistochemical staining and scoring: ccRCC patients were immunohistochemically stained for VSIG4 and scored. Patients were grouped based on whether VSIG4 was highly expressed to observe whether they had radiographic disease progression and overall survival.

[0037] S1.3. Expression differential study: Based on the differential expression of VSIG4 between renal cell carcinoma, adjacent tissues, and pseudocapsule, the role of VSIG4 in renal cell carcinoma invasion and metastasis was clarified.

[0038] S2. Study the mechanism of VSIG4 in promoting invasion and metastasis at the level of renal cell carcinoma;

[0039] S3. In vivo studies in mice showed the role of VSIG4 in promoting the invasion and metastasis of renal cell carcinoma and the synergistic effect of VSIG4 gene knockout on PD-L1 inhibitors.

[0040] Preferably, the step S2 specifically includes the following steps:

[0041] S2.1 Cell experiments: Renal cancer cells were cultured and stably transfected VSIG4 knockout cell lines were constructed. Changes in cell proliferation, colony formation, invasion, and metastasis phenotypes before and after VSIG4 knockout were compared.

[0042] S2.2. Exploration of EMT Mechanisms: Exploring the relationship and mechanism between cell invasion and metastasis and EMT;

[0043] S2.3. Synergistic effect of PD-L1 inhibitors: To explore whether knocking out the VSIG4 gene has a synergistic inhibitory effect on PD-L1 inhibitors against renal cancer cells;

[0044] S2.4. Compound screening: Use the compound library to screen cancer cells and select compounds that have inhibitory function on VSIG4.

[0045] Preferably, the exploration of the EMT mechanism in S2.2 specifically explores whether the effect of VSIG4 on cell invasion and metastasis is related to the epithelial-mesenchymal transition (EMT) process.

[0046] Preferably, the step S3 specifically includes the following steps:

[0047] S3.1. Animal Model Construction: Stably transfected renal cancer cell lines with VSIG4 knockout were injected into mice to establish subcutaneous tumor formation models and tail vein injection lung metastasis models.

[0048] S3.2. Efficacy evaluation: Compare the tumor size between wild-type and VSIG4 knockout cell lines, and evaluate the efficacy and safety of the compound combined with PD-L1 inhibitors.

[0049] S3.3. Metastasis model evaluation: After the successful establishment of a lung metastasis model by tail vein injection of a stably transfected renal cancer cell line with VSIG4 knockout, the sizes of tumors in the lung metastasis model of the wild-type cell line and the stably transfected cell line were compared. The efficacy and toxicity of the compound were evaluated by combining the compound with a PD-L1 inhibitor.

[0050] S3.4. Functional study of macrophages and T lymphocytes in gene-knockout mice: Using VSIG4-knockout mice, we analyzed the functional changes in their immune system, especially macrophages and T lymphocytes.

[0051] Preferably, the efficacy evaluation in S3.2 is specifically as follows: after successfully constructing a subcutaneous tumor model with a stably transformed renal cancer cell line in which VSIG4 is knocked out, the tumor formation ratio and tumor size of the wild-type and stably transformed cell lines are compared to evaluate the effect of VSIG4 in promoting tumor formation; at the same time, the compounds screened at the above-mentioned cell level are combined with PD-L1 inhibitors to treat tumor-bearing mice, and the changes in tumor size and mouse weight are observed to evaluate the efficacy and toxicity of the screened compounds.

[0052] Preferably, after the lung metastasis model of the stably transfected renal cancer cell line with VSIG4 knockout in S3.3 is successfully established by tail vein injection, the screened compound is simultaneously administered in combination with a PD-L1 inhibitor to treat metastases, and the changes in tumor size and mouse weight are observed to evaluate the efficacy and toxicity of the compound.

[0053] Preferably, the VSIG4 gene in the mice to be knocked out in S3.3 is successfully generated, the bone marrow and thymus of the mice are taken, and the cells are sorted to observe the changes in the functions of the mouse macrophages and T lymphocytes, and to evaluate the effects of VSIG4 on macrophages and T lymphocytes.

[0054] The working principle of the present invention is: 1. Analyze clinical data of ccRCC patients to find out the impact of differential expression of VSIG4 in renal cancer tissue on patient prognosis:

[0055] 1.1 Collect clinical data from patients with ccRCC, including those with primary tumors without metastasis and those with metastasis, and observe whether there is a statistically significant difference in VSIG4 expression in renal cancer pathological tissue between the two groups;

[0056] 1.2 Pathological sections of ccRCC patients were immunohistochemically stained for VSIG4 and scored. Patients were grouped based on whether VSIG4 was highly expressed and followed up to observe whether the patients had radiographic disease progression and overall survival.

[0057] 1.3 Observe the differences in VSIG4 expression between renal cell carcinoma, adjacent tissues, and pseudocapsule, and clarify the role of VSIG4 in the invasion and metastasis of renal cell carcinoma.

[0058] 2. Study the mechanism of VSIG4 in promoting invasion and metastasis at the renal cancer cell line level:

[0059] 2.1 Culture renal cancer cells and establish a stable VSIG4 knockout cell line to compare changes in cell proliferation, colony formation, invasion, and metastasis before and after VSIG4 knockout;

[0060] 2.2 Explore the relationship and mechanism between changes in cell invasion and metastasis and EMT;

[0061] 2.3 Explore whether knocking out the VSIG4 gene has a synergistic inhibitory effect on PD-L1 inhibitors against renal cancer cells;

[0062] 2.4 Use the purchased compound library to screen cancer cells and select compounds that have inhibitory function on VSIG4.

[0063] 3. In vivo studies in mice: VSIG4 promotes invasion and metastasis of renal cell carcinoma and the synergistic effect of VSIG4 knockout on PD-L1 inhibitors:

[0064] 3.1 Stably transfected renal cancer cell lines with VSIG4 knockout were injected into mice to establish a subcutaneous tumor model and a lung metastasis model via tail vein injection;

[0065] 3.2 After successfully establishing a subcutaneous tumor model with a stably transfected renal cancer cell line that has knocked out VSIG4, compare the proportion and size of tumors formed between wild-type and stably transfected cell lines to evaluate the role of VSIG4 in promoting tumor formation. Simultaneously, compounds screened at the cell level described above can be combined with PD-L1 inhibitors to treat mice with tumors, observing changes in tumor size and mouse weight to evaluate the efficacy and toxicity of the screened compounds.

[0066] 3.3 After the successful establishment of a lung metastasis model by tail vein injection of a stably transfected renal cancer cell line with VSIG4 knockout, the sizes of tumors in the lung metastasis model of the wild-type cell line and the stably transfected cell line were compared. The screened compounds were then combined with PD-L1 inhibitors to treat metastases. The changes in tumor size and mouse weight were observed to evaluate the efficacy and toxicity of the compounds.

[0067] 3.4 Use VSIG4 knockout mice to observe the effects of VSIG4 on macrophages and T lymphocytes. After successful VSIG4 knockout mice, obtain the mouse bone marrow and thymus, sort the cells, observe the changes in the mouse macrophage and T lymphocyte function, and evaluate the effect of VSIG4 on macrophages and T lymphocytes.

[0068] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for promoting renal clear cell carcinoma metastasis by VSIG4 through epithelial-mesenchymal transition, characterized in that: The following steps are involved: S1. Clinical data analysis of ccRCC patients: S1.

1. Data Collection: Clinical data of ccRCC patients were collected, including patients with primary tumors without metastasis and patients with metastasis. The expression of VSIG4 in renal cancer pathological tissues between the two groups was observed to determine whether there was a statistically significant difference. S1.2 Immunohistochemical Staining and Scoring: ccRCC patients were immunohistochemically stained for VSIG4 and scored. Patients were grouped based on whether VSIG4 was highly expressed to observe whether they had radiographic disease progression and overall survival. S1.

3. Expression differential study: Based on the differential expression of VSIG4 between renal cell carcinoma, adjacent tissues, and pseudocapsule, the role of VSIG4 in renal cell carcinoma invasion and metastasis was clarified. S2. Study the mechanism of VSIG4 in promoting invasion and metastasis at the level of renal cell carcinoma; S3. In vivo studies in mice showed the role of VSIG4 in promoting the invasion and metastasis of renal cell carcinoma and the synergistic effect of VSIG4 gene knockout on PD-L1 inhibitors.

2. The method according to claim 1, wherein VSIG4 promotes renal clear cell carcinoma metastasis through epithelial-mesenchymal transition, The S2 specifically includes the following steps: S2.1 Cell experiments: Renal cancer cells were cultured and stably transfected VSIG4 knockout cell lines were constructed. Changes in cell proliferation, colony formation, invasion, and metastasis phenotypes before and after VSIG4 knockout were compared. S2.

2. Exploration of EMT Mechanisms: Exploring the relationship and mechanism between cell invasion and metastasis and EMT; S2.

3. Synergistic effect of PD-L1 inhibitors: To explore whether knocking out the VSIG4 gene has a synergistic inhibitory effect on PD-L1 inhibitors against renal cancer cells; S2.

4. Compound screening: Use the compound library to screen cancer cells and select compounds that have inhibitory function on VSIG4.

3. The method according to claim 2, wherein VSIG4 promotes renal clear cell carcinoma metastasis through epithelial-mesenchymal transition, The exploration of the EMT mechanism in S2.2 specifically explores whether the effect of VSIG4 on cell invasion and metastasis is related to the epithelial-mesenchymal transition (EMT) process.

4. The method according to claim 1, wherein VSIG4 promotes renal clear cell carcinoma metastasis through epithelial-mesenchymal transition, The S3 specifically includes the following steps: S3.

1. Animal Model Construction: Stably transfected renal cancer cell lines with VSIG4 knockout were injected into mice to establish subcutaneous tumor formation models and tail vein injection lung metastasis models. S3.

2. Efficacy evaluation: Compare the tumor size between wild-type and VSIG4 knockout cell lines, and evaluate the efficacy and safety of the compound combined with PD-L1 inhibitors. S3.

3. Metastasis model evaluation: After the successful establishment of a lung metastasis model by tail vein injection of a stably transfected renal cancer cell line with VSIG4 knockout, the sizes of tumors in the lung metastasis model of the wild-type cell line and the stably transfected cell line were compared. The efficacy and toxicity of the compound were evaluated by combining the compound with a PD-L1 inhibitor. S3.

4. Functional study of macrophages and T lymphocytes in gene-knockout mice: Using VSIG4-knockout mice, we analyzed the functional changes in their immune system, especially macrophages and T lymphocytes.

5. The method according to claim 4, wherein VSIG4 promotes renal clear cell carcinoma metastasis through epithelial-mesenchymal transition, The efficacy evaluation in S3.2 is specifically as follows: after successfully constructing a subcutaneous tumor model with a stably transformed renal cancer cell line in which VSIG4 is knocked out, the tumor formation ratio and tumor size of the wild-type and stably transformed cell lines are compared to evaluate the role of VSIG4 in promoting tumor formation; at the same time, the compounds screened at the above-mentioned cell level are used in combination with PD-L1 inhibitors to treat tumor-bearing mice, and the changes in tumor size and mouse weight are observed to evaluate the efficacy and toxicity of the screened compounds.

6. The method according to claim 4, wherein VSIG4 promotes renal clear cell carcinoma metastasis through epithelial-mesenchymal transition, After the successful establishment of the lung metastasis model by tail vein injection of the stably transfected renal cancer cell line with VSIG4 knockout in S3.3, the screened compounds were simultaneously administered in combination with PD-L1 inhibitors to treat metastases, and the changes in tumor size and mouse weight were observed to evaluate the efficacy and toxicity of the compounds.

7. The method according to claim 4, wherein VSIG4 promotes renal clear cell carcinoma metastasis through epithelial-mesenchymal transition, The VSIG4 gene was successfully knocked out in the S3.3 mice. The bone marrow and thymus of the mice were collected and the cells were sorted to observe the changes in the functions of the mouse macrophages and T lymphocytes and to evaluate the effects of VSIG4 on the macrophages and T lymphocytes.

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