CN117129690B - Application of GRHL1 in the diagnosis and prognosis of HER2-breast cancer - Google Patents
Application of GRHL1 in the diagnosis and prognosis of HER2-breast cancer Download PDFInfo
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Abstract
本发明公开GRHL1在HER2‑乳腺癌诊断及预后评估中的应用。本发明提供一种GRHL1基因、mRNA、cDNA、蛋白或其检测试剂的用途，用于制备一种试剂或组合物，所述试剂或组合物用于检测患乳腺癌或患乳腺癌风险，或用于乳腺癌患者的预后评估。本发明提供的GRHL1在乳腺癌样本中高表达，且高表达GRHL1的HER2‑乳腺癌患者预后更差，GRHL1促进乳腺癌细胞的增殖。同时，本发明提供的GRHL1蛋白能促进HER2‑乳腺癌细胞的增殖速率，导致较差的预后，为乳腺癌的个性化治疗开辟新的方向。
The present invention discloses the application of GRHL1 in the diagnosis and prognosis evaluation of HER2 - breast cancer. The present invention provides 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 to detect breast cancer or the risk of breast cancer, or for the prognosis evaluation of breast cancer patients. The 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, and 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
The invention belongs to the technical field of cancer diagnosis, and particularly relates to application of GRHL to diagnosis and prognosis evaluation of HER2- (human epidermal growth factor receptor 2 negative) breast cancer.
Background
Breast cancer is one of the most common malignant tumors that endanger the health of women. The gene susceptibility of the breast cancer plays an important guiding role in the whole medical intervention process of the prevention, diagnosis, treatment and prognosis of the breast cancer, and the continuous search of the gene related to the susceptibility of the breast cancer greatly improves the efficiency of the breast cancer risk prediction, family genetic related risk assessment and early screening, and greatly promotes the development of the accurate treatment of the breast cancer individuals.
GRHL1 (GRAINYHEAD-like 1) belongs to the family of transcription factors, which means that it is involved in regulating gene expression. GRHL1 is widely expressed in animals and plays an important role in embryogenesis and histogenesis, among others. It plays an important role in morphogenesis of epithelial cells, differentiation of epithelial layers and maintenance of cell polarity. GRHL1 affects cell adhesion, cell migration, cell proliferation and cell fate decisions by binding to DNA and modulating gene expression.
Disclosure of Invention
The first object of the present invention is to provide a new risk monitoring marker and therapeutic means for diagnosing and treating breast cancer, and in particular to provide an application of GRHL in diagnosing and prognosis evaluation of breast cancer.
Use of GRHL gene, mRNA, cDNA, protein or detection reagents thereof for the preparation of a reagent or composition for one or more uses selected from the group consisting of:
(1) For detecting breast cancer or a risk of breast cancer;
(2) Is used for prognosis evaluation of breast cancer patients.
Preferably, the prognosis evaluation is performed after the breast cancer patient is subjected to endocrine treatment or chemotherapy treatment, and further, the prognosis evaluation comprises the step of predicting the survival time of the breast cancer patient after the breast cancer patient is subjected to treatment.
Preferably, the protein comprises a full-length protein or protein fragment.
Preferably, the detection reagent comprises GRHL specific antibodies, GRHL specific binding molecules, specific amplification primers, probes, nucleic acid chips (such as DNA chips) or protein chips.
Preferably, the detection reagent is coupled to or carries a detectable label, further wherein the detectable label is selected from the group consisting of a chromophore, a chemiluminescent group, a fluorophore, an isotope, or an enzyme.
Preferably, the antibody is a monoclonal antibody or a polyclonal antibody.
Preferably, the nucleic acid chip comprises a substrate and specific oligonucleotide probes spotted on the substrate, wherein the specific oligonucleotide probes comprise probes specifically binding to GRHL polynucleotides (mRNA or DNA).
Preferably, the protein chip comprises a substrate and a specific antibody spotted on the substrate, wherein the specific antibody comprises a specific antibody of GRHL.
Preferably, the detection is for an ex vivo sample, further the ex vivo sample comprises a tissue sample, a body fluid sample, a cell sample, a blood sample, or a combination thereof, further the cell sample comprises a tumor cell sample, a paracancestor cell sample, and a normal breast cell sample.
Preferably, the breast cancer comprises Luminal-A type breast cancer, luminal-B type breast cancer, HER2 positive breast cancer, HER2 negative breast cancer, triple negative breast cancer and Claudin-low type breast cancer.
A second object of the present invention is to provide a kit for detecting breast cancer or a risk of breast cancer, the kit comprising a detection reagent for detecting GRHL gene, mRNA, cDNA, protein or a combination thereof.
Preferably, the kit further contains GRHL genes, mRNA, cDNA and/or protein as a control or quality control.
Preferably, the detection of breast cancer or breast cancer risk refers to detecting whether or not breast cancer has occurred, the site of occurrence, and/or determining the size of the possibility (susceptibility) of occurrence of breast cancer, and further, the determination includes a preliminary determination (prediction).
Preferably, the kit further comprises a label instruction, wherein the label or the instruction notes that the kit is used for detecting breast cancer or breast cancer risk, and further, when the kit is used for detecting breast cancer or breast cancer risk, the label or the instruction notes that:
If the GRHL expression level of the test subject is significantly higher than the control expression level, the subject is at greater risk of having breast cancer or having breast cancer than normal, or the subject has a higher risk of having metastasis or breast cancer metastasis.
Preferably, the control expression level is GRHL concentrations in the same samples in the normal population, GRHL concentrations in the same samples in the general cancer patient population except for the breast cancer patient.
It is a third object of the present invention to provide GRHL a1 inhibitor which is an interfering fragment of the GRHL gene which is capable of reducing the expression of the GRHL gene.
Preferably, the GRHL inhibitor is a neutralizing antibody, a small molecule compound, an antisense nucleic acid, or a combination thereof.
A fourth object of the present invention is to provide the use of GRHL inhibitors in the manufacture of a medicament for the prevention or treatment of breast cancer.
A fifth object of the present invention is to provide a pharmaceutical composition comprising GRHL a inhibitor and a pharmaceutically acceptable carrier.
Preferably, the pharmaceutical composition is a liquid, solid, or semi-solid, more preferably a liquid composition.
Preferably, the dosage form of the pharmaceutical composition comprises a tablet, a granule, a capsule, an oral liquid or an injection, and more preferably an oral preparation.
Compared with the prior art, the invention has the beneficial effects that:
The GRHL1 provided by the invention is highly expressed in a breast cancer sample, is related to the survival rate of a poor breast cancer patient without distant metastasis HER2 -, the prognosis of the breast cancer patient with high expression GRHL1 is worse, and is related to the prognosis of a breast cancer patient with HER2 - only, and GRHL1 promotes the proliferation of breast cancer cells of HER2 -. Meanwhile, GRHL protein provided by the invention can promote proliferation rate of breast cancer cells, so that poor prognosis is caused, and a new direction is opened for personalized treatment of breast cancer.
Drawings
FIG. 1 is a graph showing GRHL1 expression profiles in individual tumor samples;
FIG. 2 is a graph showing the analysis of GRHL1 expression in each of the typed breast tumor samples;
FIG. 3 shows GRHL that the absence of cells resulted in inhibition of cell growth;
FIG. 4 shows that the absence of GRHL results in a significant reduction in the number of clones of cells, wherein (a) cell growth proliferation was observed by the colony formation assay after transfection of control or siGRHL1 in MCF-7 cells, (b) was a quantitative analysis of the number of clones from three independent replicates, <0.01;
FIG. 5 shows that the in situ tumor growth rate of mammary gland in mice vaccinated with MDA-MB-231 cells stably expressing GRHL shRNA was significantly slower than that of mice vaccinated with control cells, wherein (a) the constructed expression vector or specific shRNA lentiviral-infected MDA-MB-231-Luc-D3H2LN cells were vaccinated in situ on mammary fat pads of 6-week-old female SCID beige mice. In situ tumor size was determined by luminescence imaging of small animals after 7 weeks. (b) For in situ tumor size (n=6) measurement of mice at days 39, 42, 44, 46, 48, 50 after tumor cell inoculation, statistical analysis was performed using two-way ANOVA analysis with P <0.001;
FIG. 6 shows that GRHL.sup.1 high expression correlates with poor distantly located metastasis survival (DMFS), where (a) shows the relationship between GRHL.sup.1 expression and distantly located metastasis survival (DMFS) in breast cancer analyzed by Kaplan-Meier survival software, and (b-e) shows the relationship between GRHL.sup.1 expression and distantly located metastasis survival (DMFS) in different subtypes (HER 2 +、HER2-、ER+、ER-) of breast cancer analyzed by Kaplan-Meier survival software.
Detailed Description
The invention will be better understood from the following examples. However, it will be readily appreciated by those skilled in the art that the description of the embodiments is provided for illustration and explanation of the invention only and is not intended to limit the invention as described in detail in the claims. Unless otherwise indicated, reagents, methods and equipment employed in the present invention are conventional methods and test materials used, unless otherwise indicated, are available from commercial companies.
The invention is further described below with reference to examples and figures.
Example 1
The expression profile of GRHL A in each tumor sample was analyzed using an online tool (http:// gepia. Cancer-pku. Cn /), as shown in FIG. 1, the GRHL1 gene was significantly highly expressed in tumors such as BLCA (transitional cell carcinoma of the bladder), BRCA (invasive carcinoma of the breast), CESC (squamous carcinoma of the cervix and adenocarcinoma), GBM (glioblastoma multiforme), and the like.
Further, the expression of GRHL1 in each of the typed breast tumor samples was obtained from the TCGA database and analyzed, and the results are shown in fig. 2, wherein GRHL1 is highly expressed in various types of breast tumors, especially in Basal-like breast cancer.
Example 2
SiCTR (negative control) and siGRHL1#1, siGRHL1#2 used in the experiments are interfering fragments chemically synthesized by Shanghai Ji Ma company. For transfectionRNAiMAX。
The MCF-7 cell transfection control or GRHL siRNA is used for detecting cell proliferation by using a CCK kit, and the number of living cells is detected by the following specific steps:
the transfected cells were cultured for 72h, and were subjected to cell counting by digestion with 0.25% pancreatin, and the cells were inoculated in 96-well plates at a concentration of 1X 10 4/ml, 100. Mu.l each well, 10. Mu.l of CCK-8 reagent was added to each well of the 96-well plates under light-shielding conditions, the plates were shaken for 3min, and then placed in an incubator for continuous culture, and after 2h, removed, and OD values were measured at a wavelength of 450nm in an ELISA meter.
The results showed that the deletion of GRHL1 resulted in inhibition of cell growth (FIG. 3), and the clone formation experiments also showed that the deletion of GRHL resulted in a significant reduction in the number of cell clones (FIG. 4). The results of these examples demonstrate that inhibition of GRHL1 expression significantly inhibits proliferation of breast cancer cells.
Example 3
Control and lentivirus carrying GRHL shRNA were constructed to infect MDA-MB-231 cells stably expressing firefly luciferase, and these cells were then implanted in situ at the abdominal mammary fat pad (n=6) of 6 week old female immunodeficiency mice (SCID Beige), and tumor growth was monitored periodically. After 7 weeks, the size of the tumor in the mice was detected by a small animal in vivo imaging technique, and after dissecting the mice, the tumor was removed and compared with the size of the tumor in the mice. As a result, it was found that the in situ tumor growth rate of mammary glands in mice inoculated with the MDA-MB-231 cell line stably expressing GRHL shRNA was significantly slower than that of mice inoculated with the control cell line (FIG. 5), i.e., the deletion of GRHL1 inhibited the growth of the primary tumor.
Example 4
The relationship between GRHL expression levels and survival of breast cancer patients was analyzed by the website of Kaplan-Meier survival analysis (http:// kmpilot. Com/analysis /). The results showed that high expression of GRHL1 was associated with poor distant metastasis-free survival (DMFS) (fig. 6 a), which was only present in HER2 - breast cancer patients and not in HER2 +、ER+、ER- subtype patients (fig. 6 b-e).
Because HER2 - breast cancer patients do not express HER2, they cannot be targeted using monoclonal antibodies to HER2, antibody-coupled drugs to HER2, specific or multi-kinase small molecule inhibitors that inhibit HER2 activity. Meanwhile, as GRHL can promote proliferation of breast cancer cells in vitro and promote growth of in-situ breast cancer of mice in vivo, GRHL1 high expression is related to the survival rate of poor patients without distant metastasis HER2 - breast cancer, GRHL1 may be another meaningful index for predicting the survival rate of the patients without distant metastasis, and designing an inhibitor aiming at GRHL1 may have important significance for treating the patients with HER2 - breast cancer without distant metastasis.
Claims (6)
1. Use of a GRHL gene, mRNA, cDNA, protein detection reagent in the preparation of a kit, characterized in that the kit is used for one or more uses selected from the group consisting of:
(1) For detecting breast cancer or a risk of breast cancer;
(2) Prognosis evaluation for breast cancer patients;
the breast cancer is HER2 - breast cancer.
2. The use according to claim 1, wherein the prognostic evaluation is a prognostic evaluation of a breast cancer patient after endocrine treatment or after chemotherapy treatment.
3. The use according to claim 1, wherein the detection reagent comprises a specific binding molecule of GRHL a.
4. The method of claim 3, wherein the detection reagent comprises GRHL antibodies specific for the nucleic acid, specific amplification primers, probes, nucleic acid chips, or protein chips.
5. The method of claim 4, wherein the nucleic acid chip comprises a substrate and a specific oligonucleotide probe spotted on the substrate, and wherein the specific oligonucleotide probe comprises a probe that specifically binds to GRHL polynucleotides.
6. The method of claim 4, wherein the protein chip comprises a substrate and a specific antibody spotted on the substrate, and wherein the specific antibody comprises a specific antibody against GRHL.
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