Application of GRHL1 in the diagnosis and prognosis of HER2-breast cancer

Abstract

The present invention discloses the application of GRHL1 in the diagnosis and prognosis evaluation of HER2− breast cancer. The present invention provides the use of a GRHL1 gene, mRNA, cDNA, protein or its detection reagent for preparing a reagent or composition for detecting breast cancer or the risk of breast cancer, or for the prognosis evaluation of breast cancer patients. GRHL1 provided by the present invention is highly expressed in breast cancer samples, and HER2− breast cancer patients with high expression of GRHL1 have a worse prognosis. GRHL1 promotes the proliferation of breast cancer cells. At the same time, the GRHL1 protein provided by the present invention can promote the proliferation rate of HER2− breast cancer cells, resulting in a poor prognosis, opening up a new direction for the personalized treatment of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of cancer diagnosis, and specifically relates to the application of GRHL1 in the diagnosis and prognosis evaluation of HER2- (human epidermal growth factor receptor 2 negative) breast cancer. Background Art

[0002] Breast cancer is one of the most common malignant tumors that endanger women's health. The genetic susceptibility of breast cancer plays an extremely important guiding role in the entire medical intervention process of breast cancer prevention, diagnosis, treatment and prognosis. The continued search for genes related to breast cancer susceptibility will greatly improve the efficiency of breast cancer risk prediction, family genetic risk assessment and early screening, and will greatly promote the development of individual precision treatment of breast cancer.

[0003] The GRHL1 (Grainyhead-like 1) protein belongs to the transcription factor family, which means it is involved in regulating gene expression. GRHL1 is widely expressed in animals, especially playing an important role in embryonic development and tissue formation. It plays an important role in the morphogenesis of epithelial cells, the differentiation of the epithelial layer, and the maintenance of cell polarity. GRHL1 affects cell adhesion, cell migration, cell proliferation, and the determination of cell fate by binding to DNA and regulating gene expression. Summary of the invention

[0004] The first purpose of the present invention is to address the deficiencies of the prior art and provide new risk monitoring markers and treatment methods for breast cancer diagnosis and treatment, and specifically provide an application of GRHL1 in breast cancer diagnosis and prognosis assessment.

[0005] A use of a GRHL1 gene, mRNA, cDNA, protein or a detection reagent thereof, for preparing a reagent or composition, wherein the reagent or composition is used for one or more uses selected from the following group:

[0006] (1) Used to detect breast cancer or the risk of breast cancer;

[0007] (2) Used for prognostic assessment of breast cancer patients.

[0008] Preferably, the prognosis evaluation is a prognosis evaluation of breast cancer patients after endocrine therapy or chemotherapy; further, the prognosis evaluation includes predicting the survival time of breast cancer patients after treatment.

[0009] Preferably, the protein comprises a full-length protein or a protein fragment.

[0010] Preferably, the detection reagent includes a GRHL1-specific antibody, a GRHL1-specific binding molecule, a specific amplification primer, a probe, a nucleic acid chip (such as a DNA chip) or a protein chip.

[0011] Preferably, the detection reagent is coupled with or carries a detectable label; further, the detectable label is selected from the following group: a chromophore, a chemiluminescent group, a fluorophore, an isotope or an enzyme.

[0012] Preferably, the antibody is a monoclonal antibody or a polyclonal antibody.

[0013] Preferably, the nucleic acid chip comprises a substrate and specific oligonucleotide probes spotted on the substrate, and the specific oligonucleotide probes comprise probes that specifically bind to GRHL1 polynucleotides (mRNA or DNA).

[0014] Preferably, the protein chip comprises a substrate and specific antibodies spotted on the substrate, and the specific antibodies comprise specific antibodies against GRHL1.

[0015] Preferably, the detection is for in vitro samples; further, the in vitro samples include: tissue samples, body fluid samples, cell samples, blood samples, or a combination thereof; further, the cell samples include: tumor cell samples, paracancerous cell samples, and normal breast cell samples.

[0016] Preferably, the breast cancer includes: Luminal-A breast cancer, Luminal-B breast cancer, HER2-positive breast cancer, HER2-negative breast cancer, triple-negative breast cancer, and Claudin-low breast cancer.

[0017] The second object of the present invention is to provide a kit for detecting breast cancer or breast cancer risk, wherein the kit comprises a detection reagent for detecting GRHL1 gene, mRNA, cDNA, protein or a combination thereof.

[0018] Preferably, the kit further contains GRHL1 gene, mRNA, cDNA, and / or protein as a reference substance or quality control substance.

[0019] Preferably, the detection of breast cancer or breast cancer risk refers to detecting whether breast cancer has occurred, the location of occurrence, and / or determining the possibility (susceptibility) of breast cancer; further, the determination includes pre-determination (prediction).

[0020] Preferably, the kit further includes a label or instructions, wherein the label or instructions indicate that the kit is used to detect breast cancer or breast cancer risk; further, when the kit is used to detect breast cancer or breast cancer risk, the label or instructions indicate the following:

[0021] If the GRHL1 expression level of the test subject is significantly higher than the control expression level, the subject suffers from breast cancer or has a higher risk of suffering from breast cancer than a normal person, or the breast cancer of the subject has metastasized or has a high risk of breast cancer metastasis.

[0022] Preferably, the control expression level is the GRHL1 concentration in the same sample in a normal population, or the GRHL1 concentration in the same sample in a general cancer patient population except breast cancer patients.

[0023] The third object of the present invention is to provide a GRHL1 inhibitor, which is an interference fragment of the GRHL1 gene, and the interference fragment can reduce the expression of the GRHL1 gene.

[0024] Preferably, the GRHL1 inhibitor is a neutralizing antibody, a small molecule compound, an antisense nucleic acid, or a combination thereof.

[0025] The fourth object of the present invention is to provide use of a GRHL1 inhibitor in the preparation of a drug for preventing or treating breast cancer.

[0026] The fifth object of the present invention is to provide a pharmaceutical composition comprising a GRHL1 inhibitor and a pharmaceutically acceptable carrier.

[0027] Preferably, the pharmaceutical composition is liquid, solid, or semisolid; more preferably, it is a liquid composition.

[0028] Preferably, the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral solutions or injections, more preferably oral preparations.

[0029] Compared with the prior art, the beneficial effects of the present invention are at least:

[0030] The GRHL1 provided by the present invention is highly expressed in breast cancer samples, and is different from the poor HER2 without distant metastasis. - The prognosis of breast cancer patients with high expression of GRHL1 is worse, and it is only related to HER2 - GRHL1 promotes HER2 - Proliferation of breast cancer cells. At the same time, the GRHL1 protein provided by the present invention can promote the proliferation rate of breast cancer cells, leading to a poor prognosis, and opening up a new direction for personalized treatment of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the expression profile of GRHL1 in various tumor samples;

[0032] Figure 2 The results of the expression analysis of GRHL1 in breast tumor samples of various types;

[0033] Figure 3 The loss of GRHL1 resulted in the inhibition of cell growth;

[0034] Figure 4 The loss of GRHL1 caused a significant decrease in the number of cell clones, where (a) was the observation of cell growth and proliferation by clone formation experiment after transfection of control or siGRHL1 in MCF-7 cells, and (b) was the quantitative analysis of the number of clones in three independent repeated experiments, **P<0.01;

[0035] Figure 5 The growth rate of mammary orthotopic tumors in mice inoculated with MDA-MB-231 cells stably expressing GRHL1 shRNA was significantly slower than that in mice inoculated with control group cells, where (a) the constructed expression vector or specific shRNA lentivirus-infected MDA-MB-231-Luc-D3H2LN cells were orthotopically inoculated into the mammary fat pad of 6-week-old female SCID beige mice. The size of orthotopic tumors was measured by small animal luminescence imaging 7 weeks later. (b) The size of orthotopic tumors in mice was measured on days 39, 42, 44, 46, 48, and 50 after tumor cell inoculation (n=6), and statistical analysis was performed using two-way ANOVA analysis, ***P<0.001;

[0036] Figure 6 The high expression of GRHL1 is associated with poor distant metastasis-free survival (DMFS), where (a) is the relationship between GRHL1 expression and distant metastasis-free survival (DMFS) in breast cancer analyzed by Kaplan-Meier survival curve software, (b) is the relationship between GRHL1 expression and distant metastasis-free survival (DMFS) in breast cancer analyzed by Kaplan-Meier survival curve software for different subtypes (HER2 + HER2 - , ER + , ER - )To analyze the relationship between GRHL1 expression and distant metastasis-free survival (DMFS) in breast cancer. DETAILED DESCRIPTION

[0037] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the implementation cases are only used to illustrate and explain the present invention, and are not used to limit the present invention described in detail in the claims. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional methods unless otherwise specified, and the test materials used can be obtained from commercial companies unless otherwise specified.

[0038] The present invention is further described below in conjunction with embodiments and drawings.

[0039] Example 1

[0040] The expression profile of GRHL1 in each tumor sample was analyzed using an online tool (http: / / gepia.cancer-pku.cn / ). Figure 1 As shown, the GRHL1 gene is significantly overexpressed in tumors such as BLCA (bladder transitional cell carcinoma), BRCA (breast invasive carcinoma), CESC (cervical squamous cell carcinoma and adenocarcinoma), and GBM (glioblastoma multiforme).

[0041] Furthermore, the expression of GRHL1 in breast tumor samples of various types was obtained from the TCGA database and analyzed. The results are as follows: Figure 2 It was shown that GRHL1 was highly expressed in various types of breast tumors, especially in Basal-like breast cancer.

[0042] Example 2

[0043] The siCTR (negative control) and siGRHL1#1 and siGRHL1#2 used in the experiment are interference fragments chemically synthesized by Shanghai Jima Company. RNAiMAX.

[0044] MCF-7 cells were transfected with control or GRHL1 siRNA, and cell proliferation and the number of live cells were detected using the CCK kit. The specific steps are as follows:

[0045] The transfected cells were cultured for 72 h, digested with 0.25% trypsin, and the cells were counted. The cells were seeded in a 96-well plate at a concentration of 1 × 10 4 / ml, add 100μl to each well; under light-proof conditions, add 10μl of CCK-8 reagent to each well of the 96-well plate, shake the culture plate for 3min, then put the culture plate into the incubator for further culture, take it out after 2h, and measure the OD value at a wavelength of 450nm on an enzyme reader.

[0046] The results showed that the loss of GRHL1 resulted in the inhibition of cell growth ( Figure 3 ); clone formation experiments also showed that the loss of GRHL1 caused a significant decrease in the number of cell clones ( Figure 4 ). These example results show that inhibiting the expression of GRHL1 can significantly inhibit the proliferation of breast cancer cells.

[0047] Example 3

[0048] Control and lentivirus carrying GRHL1 shRNA were constructed to infect MDA-MB-231 cells stably expressing firefly luciferase, and then these cells were orthotopically implanted into the abdominal mammary fat pad of 6-week-old female immunodeficient mice (SCID Beige) (n=6), and the growth of tumors was monitored regularly. After 7 weeks, the size of the mouse tumors was detected by small animal live imaging technology, and the mice were dissected and the tumors were removed to compare the size of the tumors in the mice. The results showed that the growth rate of the mammary orthotopic tumors in the mice inoculated with MDA-MB-231 cells stably expressing GRHL1 shRNA was significantly slower than that of the mice inoculated with control cells ( Figure 5 ), indicating that loss of GRHL1 inhibits primary tumor growth.

[0049] Example 4

[0050] The relationship between GRHL1 expression level and breast cancer patient survival rate was analyzed by the Kaplan-Meier survival analysis website (http: / / kmplot.com / analysis / ). The results showed that high expression of GRHL1 was associated with poor distant metastasis-free survival (DMFS). Figure 6 a) This correlation only exists in HER2 - HER2 is not present in breast cancer patients + , ER + , ER - Among patients with subtype Figure 6 be).

[0051] Because HER2 - Breast cancer patients do not express HER2, so it is impossible to use HER2 monoclonal antibodies, HER2 antibody-drug conjugates, specific or multi-kinase small molecule inhibitors that inhibit HER2 activity for targeted therapy. At the same time, because GRHL1 can promote the proliferation of breast cancer cells in vitro and the growth of in situ breast cancer in mice in vivo, high GRHL1 expression is associated with poor HER2 without distant metastasis. - Therefore, GRHL1 may be another meaningful indicator for predicting the survival rate of breast cancer patients without distant metastasis. Designing inhibitors targeting GRHL1 may have an important effect on HER2-positive breast cancer patients without distant metastasis. - The treatment of breast cancer patients is of great significance.

Claims

1. Use of a GRHL1 gene, mRNA, cDNA, protein detection reagent in preparing a kit, It is characterized in that The kit is used for one or more purposes selected from the following groups: (1) Used to detect breast cancer or the risk of breast cancer; (2) Used for prognostic assessment of breast cancer patients; The breast cancer is HER2 - Breast cancer.

2. The use according to claim 1, It is characterized in that The prognostic evaluation is a prognostic evaluation of breast cancer patients after endocrine therapy or chemotherapy.

3. The use according to claim 1, It is characterized in that The detection reagent includes: a specific binding molecule of GRHL1.

4. The use according to claim 3, It is characterized in that The detection reagents include: GRHL1 specific antibodies, specific amplification primers, probes, nucleic acid chips or protein chips.

5. The use according to claim 4, It is characterized in that The nucleic acid chip comprises a substrate and specific oligonucleotide probes spotted on the substrate; the specific oligonucleotide probes comprise probes specifically binding to GRHL1 polynucleotides.

6. The use according to claim 4, It is characterized in that The protein chip comprises a substrate and specific antibodies spotted on the substrate; the specific antibodies comprise specific antibodies against GRHL1.

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