CN112646886B - Application of FOXD1 in invasive breast cancer - Google Patents

Application of FOXD1 in invasive breast cancer Download PDF

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CN112646886B
CN112646886B CN202011557892.7A CN202011557892A CN112646886B CN 112646886 B CN112646886 B CN 112646886B CN 202011557892 A CN202011557892 A CN 202011557892A CN 112646886 B CN112646886 B CN 112646886B
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任栋
杨春潇
张鑫
王斌
姜春林
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Jiangmen Central Hospital
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Abstract

The invention makes clear for the first time that FOXD1 is significantly up-regulated in multiple molecular subtypes of invasive breast cancer, especially in the BasL subtype. Furthermore, over-expression of FOXD1 specifically predicts bone metastasis in patients with multiple molecular subtypes of invasive breast cancer. Importantly, silencing FOXD1 inhibited invasive breast cancer bone metastasis in vivo, but had no effect on liver, brain and lung metastasis. Therefore, the invention provides an application scheme of FOXD1 in invasive breast cancer, and provides a new path for improving the survival quality of breast cancer patients.

Description

Application of FOXD1 in invasive breast cancer
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to application of FOXD1 in invasive breast cancer.
Background
Breast cancer (Breast cancer, BCa) is the most common malignancy in women. Although the treatment of primary breast cancer has made major progress in recent decades with the progress of precision medicine, cancer metastasis remains the cause of most cancer deaths. The most common metastatic organs of breast cancer include bone, brain, liver, lung, and 15 years follow-up studies found that as many as 51.5% of breast cancer patients develop metastasis. Bone is the main site of metastasis of breast cancer, and the subtypes that develop bone metastasis are mainly LumA, lumB and HER2. Once bone metastasis of breast cancer occurs, a series of bone-related complications can develop, including fractures, intractable pain, hypercalcemia, or nerve compression syndrome, which seriously affect the quality of life and survival time of breast cancer patients.
FOXD1 (Forkhead Box D1, HGNC: 3802) belongs to the Forkhead Box (Fox) transcription factor family, is characterized by a unique Forkhead domain, and has been reported to play an important role in retinal and renal development, and to regulate inflammatory responses and prevent autoimmunity.
The present invention has been made in view of the above background, in conjunction with the research results that the present invention has been made for the first time.
Disclosure of Invention
The invention provides at least one of the following technical schemes:
the invention relates to an application of a reagent for detecting FOXD1 expression level in preparing a product for diagnosing whether a subject has invasive breast cancer.
The invention also relates to application of the reagent for detecting the FOXD1 expression level in preparing a product for predicting the survival prognosis of an invasive breast cancer subject.
The invention also relates to application of the reagent for detecting the expression level of FOXD1 in preparing a product for predicting bone metastasis tendency of an invasive breast cancer subject.
The invention also relates to application of the FOXD1 inhibitory reagent in preparing a medicine for preventing, inhibiting and/or treating invasive breast cancer bone metastasis.
The beneficial effects obtained by the invention comprise at least any one of the following:
the invention makes clear for the first time that FOXD1 is significantly upregulated in multiple molecular subtypes of invasive breast cancer, especially in the BasL subtype. Furthermore, over-expression of FOXD1 specifically predicts bone metastasis in patients with multiple molecular subtypes of invasive breast cancer. Importantly, silencing FOXD1 inhibited invasive breast cancer bone metastasis in vivo, but had no effect on liver, brain and lung metastasis. Therefore, the invention provides an application scheme of FOXD1 in invasive breast cancer, and provides a new path for improving the survival quality of breast cancer patients.
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FIG. 1: IHC detects the expression condition of FOXD1 in different tissues; in the figure, benign represents benign hyperplasia of mammary glands tissue, precan represents malignant atypical hyperplasia of mammary glands tissue, in situ represents In situ breast cancer tissue, and invadeve represents Invasive breast cancer tissue; basL represents a BasL type invasive breast cancer tissue, her2 represents a Her2 type invasive breast cancer tissue, lumA represents a LumA type invasive breast cancer tissue, and LumB represents a LumB type invasive breast cancer tissue;
FIG. 2: analysis of FOXD1 expression and different survival periods in BasL subtypes and other subtypes; in the figure, OS represents the total survival period, DMFS represents the survival period without distant metastasis, boMFS represents the survival period without bone metastasis, liMFS represents the survival period without liver metastasis, brMFS represents the survival period without brain metastasis, luMFS represents the survival period without lung metastasis, other represents non-BasL subtype invasive breast cancer tissues and comprises the invasive breast cancer tissues of Her2, lumA and LumB subtypes; cutoff (SI) represents a cutoff value set according to SI, L represents low FOXD1 expression, and H represents high FOXD1 expression;
FIG. 3: constructing a down-regulated expression FOXD1 model in a breast cancer cell line, taking Scr as a reference, and respectively constructing a down-regulated expression model by using two FOXD1 shRNAs for sh #1 and sh # 2;
FIG. 4: down-regulation of FOXD1 effect on bone metastasis in vivo, panel a is a schematic imaging; panel B is a bone X-ray, panel C is a bone H & E staining, panel D is an osteolytic score assessed by X-ray, panel E is tumor area assessed according to H & E, panel F is a survival curve without bone metastasis, panel G is tumor area metastatic to brain, panel H is tumor area metastatic to liver, panel I is tumor area metastatic to lung, and scarmble is a control.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more embodiments of which are described below. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
The invention provides an implementation mode, and an application of a reagent for detecting the expression level of FOXD1 in preparing a product for diagnosing whether a subject has invasive breast cancer. "detection" in the context of the present invention is to be understood in its broadest sense, including both qualitative and quantitative. In some embodiments, the expression level of FOXD1 in invasive breast cancer is significantly higher than that in benign hyperplasia of mammary glands, malignant atypical hyperplasia of mammary glands or in situ breast cancer, so that whether a subject suffers from invasive breast cancer can be effectively judged by detecting the expression level of FOXD 1. For example, if the subject FOXD1 expression level is higher than a set cutoff value, then aggressive breast cancer is diagnosed.
In some embodiments, the invasive breast cancer is selected from the BasL subtype, her2 subtype, the LumA subtype, or the LumB subtype; in preferred embodiments, the invasive breast cancer is the BasL subtype; wherein, the expression level of FOXD1 in the BasL subtype invasive breast cancer is obviously higher than that of a Her2 subtype, a LumA subtype or a LumB subtype, so that whether a subject suffers from the BasL subtype invasive breast cancer or not can be specifically judged by detecting the expression level of FOXD 1. For example, if the subject FOXD1 expression level is higher than the set cutoff value, it is diagnosed as having BasL subtype aggressive breast cancer.
The invention also provides an implementation mode of the application of the reagent for detecting the FOXD1 expression level in preparing a product for predicting the survival prognosis of an invasive breast cancer subject. By "predicting" herein is meant predicting the predisposition or risk of a subject to develop a disease/metastasis before the subject does not develop the disease/metastasis. In some embodiments, where the invasive breast cancer subject is the BasL subtype, the product is used to predict overall survival and/or distant metastasis free survival of the subject. For example, the overall survival of a subject, or the survival without distant metastasis, can be predicted by detecting FOXD1 expression levels in BasL subtype aggressive breast cancer subjects; wherein the shorter overall survival, the greater the risk of death; or the shorter the survival without distant metastasis, the greater the risk of distant metastasis. Predicting a subject to have a poor overall survival (or output a corresponding survival time, or higher risk of death), a poor distant metastasis-free survival (or input a corresponding distant metastasis-free time, or higher risk of distant metastasis) if the FOXD1 expression level of a BasL subtype aggressive breast cancer subject is higher than a set cutoff value; the choice of intervention means may be guided.
In some embodiments, where the invasive breast cancer subject is a non-BasL subtype, the product may predict distant metastasis-free survival of the subject; for example, distant metastasis-free survival of a subject can be predicted by detecting FOXD1 expression levels in non-BasL subtype aggressive breast cancer subjects. Predicting a subject to have poor distant metastasis-free survival (or input a corresponding distant metastasis-free time, or higher risk of distant metastasis) if the FOXD1 expression level of a non-BasL subtype aggressive breast cancer subject is higher than a set cutoff value; the choice of intervention means may be guided. In some embodiments, the non-BasL subtype is selected from the Her2 subtype, the LumA subtype, or the LumB subtype.
The invention also provides an implementation mode of the application of the reagent for detecting the expression level of FOXD1 in preparing a product for predicting the bone metastasis tendency of an invasive breast cancer subject. In some embodiments, the invasive breast cancer subject is selected from the BasL subtype, her2 subtype, the LumA subtype, or the LumB subtype. Bone metastasis can be specifically predicted in different typed aggressive breast cancer subjects. For example, the predisposition/risk of a subject to develop bone metastasis can be predicted by detecting FOXD1 expression levels in an aggressive breast cancer subject. For example, when the FOXD1 expression level in an aggressive breast cancer subject is higher than a set cutoff value, the subject may be predicted to have a high propensity/risk for bone metastasis.
"expression" refers to transcription into oligonucleotides, translation into polypeptides or their modifications, splicing, etc., and the level can be expressed in high/low form or in a specific quantitative value. In some embodiments, the expression level comprises mRNA levels and protein levels. In some embodiments, the reagent comprises a primer, probe, antibody, or aptamer that detects the expression level of FOXD 1. Wherein, it is understood that primers or probes are used for detecting the mRNA level of FOXD1, and antibodies or aptamers are used for detecting the protein level of FOXD1, which can be obtained by conventional design/screening in the art or commercially available, and which can also be carried on a solid support. In some embodiments, detecting FOXD1mRNA levels can employ a variety of detection modalities, such as fluorescent quantitative PCR, rolling circle nucleic acid amplification, loop-mediated isothermal amplification, strand displacement amplification, and the like. In some embodiments, the protein level of FOXD1 can be detected by ELISA, chemiluminescence, immunohistochemistry, and the like.
Herein, the cutoff value is also called a cutoff value, and those skilled in the art can set an effective cutoff value according to the specificity, sensitivity, etc. of the prepared diagnosis or prediction product for diagnosing/predicting the risk of the aforementioned diseases or processes, for example, by counting the FOXD1 levels of a large population of target subjects and setting according to the product target, even setting a corresponding cutoff value by using a computer model, or setting a cutoff value by combining with other markers.
The invention also relates to an implementation mode, namely application of the FOXD1 inhibitory reagent in preparing a medicament for preventing, inhibiting and/or treating invasive breast cancer bone metastasis. In some embodiments, the inhibitory agent of FOXD1 is selected from the group consisting of: substances capable of completely or partially inhibiting the expression of the FOXD1 gene, substances capable of completely or partially inhibiting the FOXD1 protein from exerting efficacy; in some embodiments, the inhibitory agent of FOXD1 is selected from the group consisting of: proteins, oligonucleotides, oligonucleotide expression vectors, small molecule compounds. In some embodiments, the inhibitory agent of FOXD1 is, for example, an interfering RNA, an antisense oligonucleotide, or CRISPRi.
The product of the invention is obtained by detecting a sample from a subject, wherein the sample can be an isolated body fluid sample or a tumor sample; body fluids include, but are not limited to, blood, plasma, serum, lymph, cerebrospinal fluid, synovial fluid, urine, saliva, mucus, and the like; forms of tumor samples include, but are not limited to, forms of living tissue, paraffin embedded tissue, frozen tissue, and the like. The subject's sample may also be selected from cells that can be isolated from the subject, such as peripheral blood mononuclear cells, T lymphocytes, B lymphocytes, circulating tumor cells, and the like.
"bone metastasis free" in the medical field means that bone metastasis does not occur, and generally, a collected tumor sample is analyzed and classified as a bone metastasis free tumor sample if the tumor sample does not have bone metastasis, and it should be understood by those skilled in the art that the bone metastasis free tumor sample may be a tumor sample in which no metastasis occurs, and may also be a tumor sample in which other non-bone metastasis (such as liver, brain, lung) occurs. The same understanding is understood as "no lung metastasis", "no liver metastasis" and "no brain metastasis".
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever.
The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
Example 1 FOXD1 was highly expressed in invasive breast cancer tissue
Clinical samples were collected from the central hospital in the Jiangmen city, 67 cases of benign hyperplastic breast lesions, 30 cases of malignant atypical hyperplastic breast lesions, 49 cases of in situ breast cancer tissues and 394 cases of invasive breast cancer tissues, wherein 394 cases of invasive breast cancer tissues were typed, 104 cases of BasL type, 96 cases of Her2 type, 95 cases of LumA type and 99 cases of LumB type.
Immunohistochemistry (IHC) staining of the above tissues: firstly, thermally treating a tissue slice at 65 ℃ for 1h, and then dewaxing the tissue slice in xylene for 2 times and 5min each time; hydrating the slices with 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol and pure water in sequence, each time for 1min; taking out, adding 3% hydrogen peroxide dropwise, incubating at room temperature for 10min, and inactivating endogenous peroxidase; placing the slices in a container containing EDTA pH9.0 repairing solution, and repairing under high pressure for 10min; taking out the slices, surrounding the tissues by an immunohistochemical pen, rinsing the tissues for 3min by using PBS buffer solution, dripping goat serum confining liquid (reagent A) after spin-drying, and incubating for 20min at room temperature; removing the reagent A, dropwise adding FOXD1 primary antibody working solution diluted by the antibody diluent, and incubating overnight at 4 ℃; rinsing with PBST buffer solution for 3 times and 5min each time after removing the primary antibody, dripping biotin-labeled secondary antibody working solution (reagent B) after spin-drying, and incubating at room temperature for 20min; discarding the reagent B, rinsing with PBST buffer solution for 3 times and 5min each time, dripping horse radish peroxidase-labeled streptavidin working solution (reagent C) after spin-drying, and incubating at room temperature for 10min; removing the reagent C, rinsing the mixture for 3 times by using PBST buffer solution for 5min each time, dripping DAB developing solution after spin-drying, and incubating the mixture for 1-5 min at room temperature; immersing the slices in tap water to stop color development, re-staining with hematoxylin staining solution for 1min, washing with tap water, and differentiating with hydrochloric acid alcohol for 5s; dehydrating the slices with 75% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol for 3min each time, and drying in a drying oven at 37 deg.C for 30min to obtain environmentally friendly resin sealed tablet. And comprehensively scoring the expression of the protein according to the positive rate and the staining depth of the immunohistochemical tumor cells. First, scoring is performed according to the positive rate of the tumor cells of the section, and the tumor cells without positive are: 0 minute; <10% of tumor cells positive: 1 minute; 10% -35% of the tumor cells are positive: 2 min; 35% -70% of the tumor cells are positive: 3 min; >70% tumor cells are positive: and 4, dividing. Scoring is then performed according to the overall depth of staining of positive tumor cells: no staining signal was 0min; the light yellow is 1 minute; the deep yellow is 2 points; the brown-yellow color is 3 points. Finally, the positive rate score is multiplied by the Staining intensity score to obtain immunohistochemical Staining Scores (SI) of 9 grades of 0, 1, 2, 3, 4, 6, 8, 9 or 12.
As a result, as shown In FIG. 1, FOXD1 was mainly detected In the nucleus (Panel A In FIG. 1), and expression of FOXD1 was significantly increased In Invasive breast cancer tissue (Invasive) as compared with benign hyperplastic breast lesion tissue (benign) and malignant atypical hyperplastic breast lesion tissue (precan.) and In situ breast cancer tissue (In situ) (Panel B In FIG. 1). Therefore, whether a subject suffers from invasive breast cancer can be effectively judged by detecting the expression level of FOXD 1.
The treatment strategies of different molecular subtypes of breast cancer are very different, and the research result shows that FOXD1 has difference in different molecular subtypes of breast cancer. FOCD1 is differentially upregulated in all breast cancer subtypes compared to benign proliferative breast lesions, and is particularly significantly elevated in the BasL subtype (FIG. 1, panels C and D). Therefore, whether the subject suffers from BasL subtype aggressive breast cancer can be specifically judged by detecting the expression level of the FOXD 1.
Example 2 over-expression of FOXD1 specifically predicts bone metastasis from various molecular subtypes of breast cancer
In this embodiment, kaplan-Meier survival analysis is adopted, and different cutoff values are set for analysis according to different sample objects, the cutoff values are set according to the median level of the SI sample to be analyzed, the median level is higher expression FOXD1 and the median level is lower expression FOXD 1.
As shown in fig. 2, FOXD1 levels in invasive breast cancer tissues of the BasL subtype correlated with Overall Survival (OS) and distant metastasis-free survival (DMFS), while FOXD1 levels in invasive breast cancer tissues of other subtypes (including luminel a, luminel B, and HER 2) correlated only with distant metastasis-free survival (DMFS) (a-D panels in fig. 2). Therefore, the overall survival or distant metastasis-free survival of the subject can be predicted by detecting the FOXD1 expression level of the BasL subtype invasive breast cancer subject, and the distant metastasis-free survival of the subject can also be predicted by detecting the FOXD1 expression level of the non-BasL subtype invasive breast cancer subject.
Breast cancer has a high propensity for metastasis to bone, lung, liver and brain and is often highly metastatic to bone depending on molecular markers, such as the LumA, lumB and Her2 subtypes; basL is transferred to solid organs such as lung and brain. Surprisingly, it was found from the results of the study that overexpression of FOXD1, whether of the BasL subtype or of other subtypes (including luminel a, luminel B and HER 2), was only associated with bone metastasis free survival (BoMFS) and not with brain metastasis free survival (BrMFS), liver metastasis free survival (LiMFS) and lung metastasis free survival (LuMFS). It is noteworthy that even in the BasL subtype with high potential for solid organ metastasis, overexpression of FOXD1 predicted only BoMFS specifically (E-L panel in FIG. 2). Therefore, the proneness/risk of bone metastasis of a subject can be predicted by detecting the expression level of FOXD1 in the aggressive breast cancer subject.
Example 3 Down-regulation of FOXD1 specifically inhibits breast cancer bone metastasis in vivo
Selecting BasL type breast cancer cells MDA-MB-231 and MDA-MB-468 to construct a cell model for down-regulating FOXD1, wherein cell lines are purchased from a cell bank of Chinese academy of sciences, cells grow in 1640 culture medium (Hyclone), 10% fetal calf serum, 500mg/ml glutamine, 250mg/ml chloramphenicol and 200mg/ml penicillin are added at the same time, and the cells are cultured in a cell culture box with the temperature of 37 ℃ and the carbon dioxide concentration of 5%; according to the state and density of cells to be observed, the culture medium is changed every 2 to 3 days, and the cells are preferably used in an experiment when the cell density is as high as 70 to 80%.
Establishment of a FOXD1 cell model under down-regulation: the method is characterized in that a retrovirus vector (pSuper-retro-puro) is adopted to construct human FOXD1shRNA plasmid (RNA interference plasmid for short) for stably expressing luciferase, FOXD1sh #1 and FOXD1sh #2 are respectively constructed, and the method is consigned to the Sharpo biotechnology limited company in Guangzhou City. The FOXD1shRNA plasmid and a control plasmid (scr.) thereof are respectively infected with MAD-MB-231 and MDA-MB-468, and the FOXD1 expression condition is detected by adopting fluorescent quantitative PCR and Western blotting.
Fluorescent quantitative PCR: grinding 100mg of tissue/cell to be detected in liquid nitrogen with mortar, adding TRizol, cracking at room temperature for 10min, adding 0.2ml chloroform into 1ml TRizol cracking solution, shaking vigorously for 30s, and standing for 5min; centrifuging at 4 deg.C for 15min at 12,000g, sucking supernatant into a new centrifuge tube, adding equal volume of isopropanol, beating uniformly, standing for 10min, centrifuging at 4 deg.C for 5min at 12,000g, washing RNA precipitate with 75% ethanol-containing DEPC water solution for 3 times, drying slightly, and dissolving RNA with DEPC water. After the concentration is measured, reverse transcription is carried out according to the instruction of PrimeScript RT reagent Kit: detecting and analyzing by using a 2ddCt relative quantitative method on a 7500 real-time quantitative PCR system according to the specification of SYBR Premix Ex Taq II Kit, wherein an internal reference gene is GAPDH, and calculating the relative mRNA expression quantity of a target gene; primers for detecting FOXD1mRNA and its reference gene were designed and synthesized by Ruibo Biotech, inc., guangzhou.
Western blotting: taking about 100mg of cells/tissues to be detected, grinding the cells/tissues in liquid nitrogen by using a mortar, adding RIPA lysate to perform ice lysis for 30min, collecting the lysate, centrifuging the lysate for 15min at 4 ℃ under 12,000g, collecting supernatant, performing protein quantification according to the BCA protein quantification kit instruction, adding 5 multiplied by sample loading buffer solution with the volume of 1/4 into the supernatant, performing boiling water bath for 10min, and performing ice bath for 5min. Using SDS PAGE protein electrophoresis system, the sample loading amount is 30 ug total protein, the electrophoresis parameters are 10% separation gel and 5% concentration gel, 100V electrophoresis is performed for 90min until the trypan blue indicator migrates to the bottom, then electrotransfer is performed, and the sample is electrotransferred for 90min by using 0.45 um PVDF membrane and 300mAl ice bath. After the electrotransformation is finished, sealing the membrane by using 5% skimmed milk powder, rinsing by TBST for 3 times and 5min, adding FOXD1 antibody, incubating overnight at 4 ℃, rinsing by TBST for 3 times and 5min, adding secondary antibody diluted by sealing liquid, incubating at room temperature for 1h, rinsing by TBST for 3 times and 5min, adding ECL luminous liquid, placing in a pressing clamp, exposing to X-ray negative for 1-20min, and developing and fixing with alpha-tubulin as a control.
As shown in FIG. 3, the FOXD1 model was successfully constructed in MDA-MB-231 and MDA-MB-468 cell lines.
The FOXD1 cell model is down-regulated by MDA-MB-231 constructed by FOXD1sh #1 to construct a mouse in-vivo model, a nude mouse with the age of 4-5 weeks is taken as a research object, and the density is 1 multiplied by 10 6 one/mL of cells were injected into the left ventricle of mice. Inoculation ofAfter 6 weeks, mice were tested for tumor metastasis (bone, brain, lung and liver) by in vivo imaging system and Xray, and embedded sections were HE stained and observed. Wherein the degree of bone destruction was scored according to Xray for each animal and bone metastasis was scored according to the following criteria: 0: no transfer; 1: covering the width of the bone by the bone injury is less than 1/4;2: covering 1/4-1/2 of the width of the bone for the bone injury; 3: covering 1/2-3/4 of the width of the bone for the bone injury; 4: the width of the bone covered by the bone injury is more than 3/4; the bone metastasis score for each animal was derived from the limb composite score. HE evaluation of tumor area comprehensive limb scores were made based on the area of bone destruction in each animal, expressed as square millimeters.
As shown in FIG. 4, silencing FOXD1 inhibited bone metastasis of breast cancer, significantly reduced the area of bone metastasis foci formation and the extent of bone destruction, and prolonged the life span of mice without bone metastasis (A-F in FIG. 4), while silencing FOXD1 had no effect on breast cancer liver metastasis, lung metastasis, and brain metastasis (G-I in FIG. 4). Therefore, the inhibitory agent of FOXD1 can be used for preventing, inhibiting and/or treating aggressive breast cancer bone metastasis.

Claims (4)

1. Application of a reagent for detecting the expression level of FOXD1 in preparing a product for predicting bone metastasis tendency of a BasL subtype invasive breast cancer subject.
2. Use according to claim 1, characterized in that: the expression levels include mRNA levels and protein levels.
3. Use according to claim 1, characterized in that: the reagent comprises a primer, a probe, an antibody or an aptamer for detecting the expression level of FOXD 1.
The FOXD1 inhibitory reagent is an interfering RNA and can be used for preparing medicines for inhibiting and/or treating BasL subtype invasive breast cancer bone metastasis.
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