Application of fh quantitative reagent in triple-negative breast cancer

Abstract

The application discloses application of FH quantitative reagent in triple-negative breast cancer. The inventors have found that, by interfering with the expression of FH by using shRNA, or by treating triple-negative breast cancer cell lines with an inhibitor of FH, regular spheroids can be formed in three-dimensional culture, and the expression levels of breast differentiation protein markers GATA3, KRT8, KRT18 and CDH1 are up-regulated, and the expression level of VIM is down-regulated. Clinical data analysis shows that FH is highly expressed in triple-negative breast cancer and is related to a shorter survival period, indicating that FH is a key gene leading to dedifferentiation of triple-negative breast cancer. Therefore, by detecting the content of FH in a sample, triple-negative breast cancer can be better diagnosed or assisted in diagnosis, and the prognosis of triple-negative breast cancer can also be better determined. This application is contrary to the results in other tumors; by further combining the expression amounts of GATA3, KRT8, KRT18, CDH1 and VIM, triple-negative breast cancer can be better diagnosed or assisted in diagnosis, and the prognosis of triple negative breast cancer can also be better determined.

Description

Technical Field

[0001] This invention belongs to the field of diagnostics, specifically relating to the application of FH quantitative reagent in triple-negative breast cancer. Background Technology

[0002] Breast cancer is one of the most common malignant tumors in women. According to the 2015 China Cancer Statistics, the incidence of breast cancer ranks first among malignant tumors in women in my country, and there is a trend of younger age of onset. It is the leading cause of death for female cancer patients under the age of 45.

[0003] Triple-negative breast cancer (TNBC) is a high-risk type of breast cancer characterized by negative expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (Her-2). Therefore, it cannot benefit from endocrine therapy targeting hormone receptors or Her-2-targeted therapy. Thus, although surgical resection and targeted therapy have brought significant benefits to some patients, effective treatments remain lacking for patients with advanced or triple-negative breast cancer. Therefore, identifying therapeutic targets for breast cancer, especially triple-negative breast cancer, and exploring new treatment strategies has significant theoretical and practical value.

[0004] Fumarate hydratase (FH) is also known as fumarate enzyme. FH participates in the citric acid cycle, and studies have shown that FH deficiency or mutation is associated with renal cell carcinoma and multiple leiomyomas of the uterus. In other tumors, FH has not shown valuable applications or there are no relevant reports (Schmidt C, Sciacovelli M, Frezza C. Fumaratehydratase in cancer: A multifaceted tumor suppressor[C] / / Seminars in cell & developmental biology. Academic Press, 2020, 98: 15-25., King A, Selak MA, Gottlieb E. Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer[J]. Oncogene, 2006, 25(34): 4675-4682.). Summary of the Invention

[0005] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide the application of FH quantitative reagent in triple-negative breast cancer.

[0006] The technical solution adopted in this invention is: The first aspect of the present invention provides: Application of FH protein or FH mRNA quantitative reagents in the preparation of reagents for the diagnosis, auxiliary diagnosis or prognosis of triple-negative breast cancer.

[0007] In some applications, FH protein quantification reagents are selected from ELISA reagents and immunohistochemical analysis reagents; FH mRNA quantification reagents are selected from PCR reagents.

[0008] In some applications, the quantitative reagent is used to detect tumor tissue samples.

[0009] In some applications, the triple-negative breast cancer is undifferentiated triple-negative breast cancer.

[0010] A second aspect of the present invention provides: The application of a quantitative reagent kit in the preparation of reagents for the diagnosis, auxiliary diagnosis, or prognosis of triple-negative breast cancer, wherein the quantitative reagent kit contains the following quantitative reagents: Quantitative reagents for FH protein or FH mRNA; and A reagent for quantifying at least one of GATA3, KRT8, KRT18, CDH1, and VIM.

[0011] In some application examples, the quantitative reagent kit contains reagents for quantifying GATA3, KRT8, KRT18, CDH1, and VIM.

[0012] In some application examples, the primers for quantitative detection of FH are: Upstream primer F: 5'-CCGCTGAAGTAAACCAGGATTATG-3' Downstream primer R: 5'-ATCCAGTCTGCCATACCACGAG-3' The primers for quantitative detection of GATA3 are: Upstream primer F: 5'-ACCACAACCACACTCTGGAGGA-3' Downstream primer R: 5'-TCGGTTTCTGGTCTGGATGCCT-3' The primers for quantitative detection of KRT8 are: Upstream primer F: 5'-CAGAAGTCCTACAAGGTGTCCA-3' Downstream primer R: 5'-CTCTGGTTGACCGTAACTGCG-3' The primers for quantitative detection of KRT18 are: Upstream primer F: 5'-GGCATCCAGAACGAGAAGGAG-3' Downstream primer R: 5'-ATTGTCCACAGTATTTGCGAAGA-3' The primers for quantitative detection of CDH1 are: Upstream primer F: 5'-GCCTCCTGAAAAGAGAGTGGAAG-3' Downstream primer R: 5'-TGGCAGTGTCTCTCCAAATCCG-3' The primers for quantitative detection of VIM are: Upstream primer F: 5'-AGGCAAAGCAGGAGTCCACTGA-3' Downstream primer R: 5'-ATCTGGCGTTCCAGGGACTCAT-3'.

[0013] A third aspect of the present invention provides: Systems used for the diagnosis, auxiliary diagnosis, or prognosis of triple-negative breast cancer include: A quantitative device used to determine the expression level of FH protein or FH mRNA in a sample; The analytical device determines triple-negative breast cancer or its prognosis based on the expression levels of FH protein or FH mRNA in the sample. The result output device outputs the analysis results from the analysis device.

[0014] In some system instances, the quantitative device also quantifies the expression level of at least one of GATA3, KRT8, KRT18, CDH1, and VIM in the sample, and determines triple-negative breast cancer or its prognosis based on the expression level of FH protein or FH mRNA in the sample and the expression level of at least one of GATA3, KRT8, KRT18, CDH1, and VIM in the sample.

[0015] In some system instances, the quantification device is a PCR quantification device.

[0016] In some systematic instances, FH is highly expressed in triple-negative breast cancer, which has been identified as a cause of shorter survival.

[0017] The beneficial effects of this invention are: The inventors discovered that interfering with FH expression using shRNA, or treating triple-negative breast cancer cell lines with an FH inhibitor (FHi), resulted in the formation of regular spherical structures in three-dimensional culture. Furthermore, the expression levels of breast differentiation protein markers GATA3, KRT8, KRT18, and CDH1 were upregulated, while VIM expression was downregulated. Clinical data analysis showed that high FH expression in triple-negative breast cancer was associated with shorter survival, indicating that FH is a key gene leading to dedifferentiation in triple-negative breast cancer. Therefore, detecting the FH content in samples can provide a good basis for the diagnosis or auxiliary diagnosis of triple-negative breast cancer, and can also help determine its prognosis. This finding contradicts the results observed in other tumors.

[0018] Some examples of this invention, by further combining the expression levels of GATA3, KRT8, KRT18, CDH1, and VIM, can better diagnose or assist in the diagnosis of triple-negative breast cancer, and can also better determine the prognosis of triple-negative breast cancer. Attached Figure Description

[0019] Figure 1 These are experimental results of rutecarpine-induced differentiation of triple-negative breast cancer cells in a three-dimensional culture model.

[0020] Figure 2 Experimental results showing that rutecarpine inhibits tumor growth and promotes breast cancer cell differentiation in a mouse in vivo model.

[0021] Figure 3 This is the experimental result of inhibiting FH to promote the differentiation of triple-negative breast cancer cells in a three-dimensional culture model. Detailed Implementation

[0022] A three-dimensional culture model of breast cancer cells can better simulate the biological characteristics and microenvironment of breast cancer cells in vitro compared to two-dimensional culture. In the three-dimensional culture medium, normal breast cells first proliferate from a single cell into a regular spherical structure, followed by apoptosis of the central cell, ultimately forming a hollow, regular sphere resembling the lumen of a normal breast duct. Well-differentiated breast cancer cells form a relatively regular, solid sphere; while poorly differentiated triple-negative breast cancer cells, such as MDA-MB-231 and BT-549, form an irregular stellate tissue structure with multiple pseudopodia. Based on the different spherical morphologies formed by breast cancer cells at different differentiation levels in three-dimensional culture, we screened more than 100 natural compounds and found that rutecarpine had a significant differentiation-inducing effect. Triple-negative breast cancer cells treated with rutecarpine, such as MDA-MB-231 and BT-549, formed regular spheres in three-dimensional culture. Furthermore, the expression levels of breast differentiation protein markers GATA3, KRT8, KRT18, and CDH1 were upregulated, while the expression level of VIM was downregulated. In animal tumor formation, the tumor volume was significantly reduced, and HE staining and immunofluorescence staining revealed cavitary structures within the tumors resembling normal breast tissue. These results demonstrate that rutecarpine can effectively induce differentiation of triple-negative breast cancer cells.

[0023] Further research using the DARTS method revealed that the target protein of rutecarpine is fumarate hydratase (FH). Interference with FH expression using shRNA, or treatment of triple-negative breast cancer cell lines with an FH inhibitor (FHi), resulted in the formation of regular spherical structures in three-dimensional culture. Furthermore, the expression levels of breast differentiation protein markers GATA3, KRT8, KRT18, and CDH1 were upregulated, while VIM expression was downregulated. Clinical data analysis showed that FH is highly expressed in triple-negative breast cancer and is associated with shorter survival, indicating that FH is a key gene leading to dedifferentiation in triple-negative breast cancer.

[0024] The first aspect of the present invention provides: Application of FH protein or FH mRNA quantitative reagents in the preparation of reagents for the diagnosis, auxiliary diagnosis or prognosis of triple-negative breast cancer.

[0025] In some applications, FH protein quantification reagents are selected from ELISA reagents and immunohistochemical analysis reagents; FH mRNA quantification reagents are selected from PCR reagents.

[0026] In some applications, the quantitative reagent is used to detect tumor tissue samples.

[0027] In some applications, the triple-negative breast cancer is undifferentiated triple-negative breast cancer.

[0028] A second aspect of the present invention provides: The application of a quantitative reagent kit in the preparation of reagents for the diagnosis, auxiliary diagnosis, or prognosis of triple-negative breast cancer, wherein the quantitative reagent kit contains the following quantitative reagents: Quantitative reagents for FH protein or FH mRNA; and A reagent for quantifying at least one of GATA3, KRT8, KRT18, CDH1, and VIM.

[0029] In some application examples, the quantitative reagent kit contains reagents for quantifying GATA3, KRT8, KRT18, CDH1, and VIM.

[0030] In some application examples, the primers for quantitative detection of FH are: Upstream primer F: 5'-CCGCTGAAGTAAACCAGGATTATG-3' Downstream primer R: 5'-ATCCAGTCTGCCATACCACGAG-3' The primers for quantitative detection of GATA3 are: Upstream primer F: 5'-ACCACAACCACACTCTGGAGGA-3' Downstream primer R: 5'-TCGGTTTCTGGTCTGGATGCCT-3' The primers for quantitative detection of KRT8 are: Upstream primer F: 5'-CAGAAGTCCTACAAGGTGTCCA-3' Downstream primer R: 5'-CTCTGGTTGACCGTAACTGCG-3' The primers for quantitative detection of KRT18 are: Upstream primer F: 5'-GGCATCCAGAACGAGAAGGAG-3' Downstream primer R: 5'-ATTGTCCACAGTATTTGCGAAGA-3' The primers for quantitative detection of CDH1 are: Upstream primer F: 5'-GCCTCCTGAAAAGAGAGTGGAAG-3' Downstream primer R: 5'-TGGCAGTGTCTCTCCAAATCCG-3' The primers for quantitative detection of VIM are: Upstream primer F: 5'-AGGCAAAGCAGGAGTCCACTGA-3' Downstream primer R: 5'-ATCTGGCGTTCCAGGGACTCAT-3'.

[0031] Studies have shown that these primers have relatively better amplification effects.

[0032] A third aspect of the present invention provides: Systems used for the diagnosis, auxiliary diagnosis, or prognosis of triple-negative breast cancer include: A quantitative device used to determine the expression level of FH protein or FH mRNA in a sample; The analytical device determines triple-negative breast cancer or its prognosis based on the expression levels of FH protein or FH mRNA in the sample. The result output device outputs the analysis results from the analysis device.

[0033] In some system instances, the quantitative device also quantifies the expression level of at least one of GATA3, KRT8, KRT18, CDH1, and VIM in the sample, and determines triple-negative breast cancer or its prognosis based on the expression level of FH protein or FH mRNA in the sample and the expression level of at least one of GATA3, KRT8, KRT18, CDH1, and VIM in the sample.

[0034] In some system instances, the quantification device is a PCR quantification device.

[0035] In some systematic instances, FH is highly expressed in triple-negative breast cancer, which has been identified as a cause of shorter survival. High expression refers to expression levels above the median in validated sequences with available results.

[0036] English-Chinese bilingual edition: Fetal bovine serum (FBS), fumarate hydratase-IN-1 inhibitor.

[0037] Main reagents Fetal bovine serum was purchased from Invitrogen; DMEM basal medium was purchased from Invitrogen; Evodia alkaloid was purchased from MCE; and fumarate hydratase inhibitor was purchased from MCE.

[0038] Cell lines and culture media The examples involve the culture of three cell types. The culture media and formulation methods used are described below.

[0039] 1) MDA-MB-231 cells (human breast cancer cell line) Culture medium: DMEM supplemented with 10% fetal bovine serum (FBS).

[0040] Culture at 37℃ with 5% CO2, and subculture every 2-3 days.

[0041] 2) BT-549 cells (human breast cancer cell line) Culture medium: DMEM supplemented with 10% fetal bovine serum (FBS).

[0042] Culture at 37℃ with 5% CO2, and subculture every 2-3 days.

[0043] 3) 4T1 cells (mouse breast cancer cell line) Culture medium: DMEM supplemented with 10% fetal bovine serum (FBS).

[0044] Culture at 37℃ with 5% CO2, and subculture every 2-3 days.

[0045] mice 4-6 week old BALB / c mice, female.

[0046] The present invention will be further described below with reference to embodiments, but is not limited thereto.

[0047] I. The Differentiation-Inducing Effect of Evodiaein on Triple-Negative Breast Cancer Cells in a Three-Dimensional Model MDA-MB-231 and BT-549 cells were seeded in a three-dimensional matrix gel and treated with DMSO (control) and evodiamine (10 uM), respectively. The culture medium was changed every 2 days. After 7 days, the cells were photographed, and the roundness of the cell spheroids was assessed. The expression level of KRT8 was detected by immunofluorescence staining, and the expression levels of mammary differentiation markers GATA3, KRT8, KRT18, CDH1, and VIM were detected by qPCR. Results are shown below. Figure 1 The control group formed an irregular star-shaped tissue structure, while the cells treated with evodiamine formed a regular spherical structure, and the diameter of the tumor spheres was significantly smaller than that of the control group. Evodiamine significantly upregulated the expression levels of differentiation markers GATA3, KRT8, KRT18 and CDH1, and downregulated the expression level of VIM, indicating that evodiamine effectively inhibits the growth of breast cancer cells and induces the differentiation of breast cancer cells.

[0048] II. Effects of Evodiaein on Tumorigenicity and Differentiation Induction in In Vivo Models 1×10 5 4T1 mammary gland cells were orally inoculated into the fat pads of BALB / c mice until the tumors grew to 3×3 mm. 2 Subsequently, the tumors were treated with intratumoral injections of PBS and evodiamine (10 μM), respectively. Eighteen days later, the tumors were harvested, photographed, and subjected to immunofluorescence staining. Results Figure 2The results showed that evodiamine significantly inhibited tumor growth, and HE staining and immunofluorescence results showed that the tumors treated with evodiamine exhibited cavitary structures similar to normal breast tissue, with upregulated KRT8 expression and downregulated VIM expression, indicating that evodiamine can inhibit the tumorigenic ability of breast cancer cells and effectively induce breast cancer tumor differentiation.

[0049] III. Inhibition of the differentiation-inducing effect of fumarate hydratase on triple-negative breast cancer cells MDA-MB-231 and BT-549 cells were seeded in a three-dimensional matrix gel and treated with DMSO (control) and fumarate hydratase inhibitor (10 uM), respectively. The culture medium was changed every 2 days. After 7 days, the cells were photographed, and the roundness of the cell spheroids was assessed. The expression level of KRT8 was detected by immunofluorescence staining, and the expression levels of mammary gland differentiation markers GATA3, KRT8, KRT18, and CDH1 were detected by qPCR. Results are shown below. Figure 3 In the control group, irregular stellate tissue structures were formed, while cells treated with fumarate hydratase inhibitors formed regular spherical structures, and the diameter of the tumor spheres was significantly smaller than that of the control group. Fumarate hydratase inhibitors significantly upregulated the expression levels of differentiation markers GATA3, KRT8, KRT18, and CDH1. This indicates that inhibiting fumarate hydratase can inhibit breast cancer cell growth and induce breast cancer cell differentiation. Analysis using a public database (TCGA database, totaling 3801 cases) showed that breast cancer patients with high FH expression had a poorer prognosis. In triple-negative breast cancer, FH was highly expressed, while GATA3 and KRT18 were lowly expressed (…). Figure 3 ).

[0050] The above studies indicate that the FH-GATA3 / KRT8 / KRT18 / CDH1 pathway is a key pathway for dedifferentiation in FH-overexpressing triple-negative breast cancer. Detecting the expression of FH and GATA3 / KRT8 / KRT18 / CDH1 using quantitative real-time PCR or immunohistochemistry can effectively define patients with FH-GATA3 / KRT8 / KRT18 / CDH1-induced dedifferentiation in triple-negative breast cancer, providing diagnostic criteria for subsequent differentiation induction therapy.

[0051] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. Application of fumarate hydratase FH protein quantitative reagent or fumarate hydratase FH mRNA quantitative reagent in the preparation of prognostic reagents for triple-negative breast cancer.

2. The application according to claim 1, characterized in that, The quantitative reagent for fumarate hydratase FH protein is an ELISA reagent; the quantitative reagent for FH mRNA is a PCR reagent.

3. The application according to claim 1, characterized in that, The sample used for quantitative reagent testing is a tumor tissue sample.

4. Systems for the prognosis of triple-negative breast cancer, including: A quantitative device used to quantify the expression levels of fumarate hydratase FH protein or fumarate hydratase FH mRNA in a sample. The analytical device determines the prognosis of triple-negative breast cancer based on the expression levels of fumarate hydratase FH protein or fumarate hydratase FH mRNA in the sample. The result output device outputs the analysis results from the analysis device.

5. The system according to claim 4, characterized in that, The quantitative device is a PCR quantitative device.

6. The system according to claim 4, characterized in that, Fumarate hydratase FH is highly expressed in triple-negative breast cancer, which has been identified as a cause of shorter survival.

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