Application of FIBCD1 marker

Abstract

The invention relates to the technical field of molecular biology diagnosis, in particular to application of an FIBCD1 marker. The invention provides an application of a substance for inhibiting expression of an FIBCD1 marker in preparation of a medicine for preventing and / or treating breast cancer complicated with diabetes mellitus. The invention also discloses application of a reagent for detecting expression of the FIBCD1 marker in preparation of a kit for diagnosis and / or prognosis evaluation of breast cancer combined with diabetes mellitus. According to the application, a novel cancer-promoting regulation molecule FIBCD1 is found, and through experimental analysis, the FIBCD1 is abnormally highly expressed in focus tissues of a BC-DM patient and is positively correlated with the fasting blood-glucose value of the patient, so that a novel target is provided for precise prevention and treatment and targeted intervention of the unique breast cancer subtype BC-DM.

Description

Technical Field

[0001] The present application belongs to the field of molecular biological diagnosis technology, and in particular relates to an application of a FIBCD1 marker. Background Art

[0002] Breast Cancer (BC) has become one of the most common malignant tumors. Diabetes Mellitus (DM) is an increasingly common chronic metabolic disease. A large number of epidemiological studies have found that there is a close relationship between diabetes and breast cancer. Diabetes can induce and increase the risk of breast cancer and is one of the risk factors for increased breast cancer mortality. It is reported that women with diabetes have a significantly increased risk of breast cancer, and diabetes is significantly associated with an increased risk of all-cause mortality in breast cancer patients. With the changes in people's lifestyles and the increasing prominence of the aging phenomenon, the incidence of breast cancer combined with diabetes (BC-DM) is increasing. Clinical studies have found that BC-DM has the characteristics of late pathological stage, easy metastasis and recurrence, worse prognosis, and high mortality, and is an independent prognostic risk factor for breast cancer metastasis and recurrence.

[0003] Although a close association between breast cancer and diabetes has been found clinically, the molecular mechanism of the association between the two is still unclear. At present, there is still a lack of clear understanding of the complication of breast cancer and diabetes (BC-DM), and the relevant clinical prevention and treatment strategies have not yet been established, which seriously affects the malignant outcome of the disease and the prognosis of patients. On the one hand, since both diseases are invisible chronic diseases, there are no significant symptoms in the early stage and they are not easy to detect. In addition to routine examinations such as fasting blood sugar, urine routine and glycosylated hemoglobin, there is currently a lack of characteristic monitoring and prevention for BC-DM patients. On the other hand, the interaction between diabetes and breast cancer is intricate, and diabetes may affect the occurrence and prognosis of breast cancer through hyperglycemia, hyperinsulinemia, immune escape, etc. Therefore, exploring the molecular mechanism of the association between breast cancer and diabetes, developing effective BC-DM early warning markers, strengthening the precise classification diagnosis and treatment of BC-DM patients, and scientific prognosis monitoring and intervention management are of great clinical significance for improving the survival rate of this group of patients.

[0004] Hyperglycemia is an important pathological feature of diabetes. Some studies have shown that hyperglycemia is associated with an increased risk of breast cancer and a poor prognosis. Other studies have found that hypoglycemic drugs such as metformin can reduce the risk of recurrence, metastasis and death in BC-DM patients, and the better the blood sugar control, the better the prognosis. In recent years, some literature has reported that hyperglycemia can promote breast cancer proliferation and metastasis by impairing the expression of angiotensinogen; other studies have reported that diabetic hyperglycemia promotes breast tumor progression by glycosylation of the extracellular matrix; and other studies have shown that hyperglycemia promotes tumor progression in breast cancer by inducing overexpression of neuregulin 1. All of the above suggest that hyperglycemia plays an important role in the malignant outcome of breast cancer lesions, providing new clues for finding characteristic biomarkers and intervention targets for breast cancer with diabetes (BC-DM). However, there are currently very few related molecules that can be used for the diagnosis and / or treatment and / or prognostic evaluation of BC-DM, limited by the fact that the molecular link between hyperglycemia and breast cancer risk remains largely unknown. Summary of the invention

[0005] The purpose of this application is to provide an application of a FIBCD1 marker, aiming to solve the technical problem of providing a new molecular marker related to breast cancer combined with diabetes.

[0006] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] In a first aspect, the present application provides a use of a substance that inhibits the expression of a FIBCD1 marker in the preparation of a drug for preventing and / or treating breast cancer complicated with diabetes.

[0008] In a second aspect, the present application provides a use of a reagent for detecting the expression of a FIBCD1 marker in the preparation of a kit for diagnosing and / or prognosticating breast cancer complicated with diabetes.

[0009] This application proves a new cancer-promoting regulatory molecule FIBCD1 through experimental analysis, thus providing a new target for the precise prevention and targeted intervention of BC-DM, a unique subtype of breast cancer. FIBCD1 is abnormally highly expressed in the lesion tissue of BC-DM patients and is positively correlated with the patient's fasting blood glucose value. The results show that high sugar can upregulate the expression of FIBCD1 in breast cancer cells; hypoglycemic drugs can downregulate the expression of FIBCD1 in breast cancer cells; under high sugar conditions, FIBCD1 has the ability to significantly promote the proliferation, migration and invasion of breast cancer cells; inhibiting the expression of FIBCD1 can significantly inhibit the proliferation, migration and invasion of breast cancer cells; its regulatory mechanism is related to the expression of the cell proliferation-related factor MCM5; experiments have shown that interfering with FIBCD1 expression has a significant tumor-suppressing effect on the BC-DM mouse model. Therefore, substances that inhibit the expression of the FIBCD1 marker can be used to prepare drugs for the prevention and / or treatment of BC-DM; at the same time, reagents for detecting the FIBCD1 marker can be used to prepare kits for the diagnosis and / or prognosis evaluation of BC-DM. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0011] Figure 1 This is a graph of differentially expressed genes in tumor tissues of BC-DM patients and BC-ND patients based on RNA sequencing in the examples of this application;

[0012] Figure 2 This is a graph showing the difference in FIBCD1 expression levels in tumor tissues of BC-DM patients and BC-ND patients based on qRT-PCR detection in the examples of the present application;

[0013] Figure 3 This is a graph showing the correlation analysis results between the FIBCD1 expression level and the fasting blood glucose (FBG) value of BC patients in the embodiment of the present application;

[0014] Figure 4 This is a diagram showing changes in the gene level of FIBCD1 in breast cancer cells under different sugar concentration culture conditions in the examples of this application;

[0015] Figure 51 is a diagram showing the overexpression of FIBCD1 in breast cancer cells MDA-MB-231 in the examples of the present application; wherein A is the overexpression of FIBCD1 mRNA level detected by qRT-PCR, and B is the overexpression of FIBCD1 protein level detected by Western blot;

[0016] Figure 6 This is a graph showing the experimental results of promoting the proliferation of breast cancer cells MDA-MB-231 under different sugar concentration culture conditions in the examples of the present application;

[0017] Figure 7 This is a graph showing the experimental results of FIBCD1 overexpression promoting the migration of breast cancer cells MDA-MB-231 under different sugar concentration culture conditions in the examples of the present application;

[0018] Figure 8 This is a graph showing the experimental results of FIBCD1 overexpression promoting the invasion of breast cancer cells MDA-MB-231 under different sugar concentration culture conditions in the examples of the present application;

[0019] Fig. 9 1 is a diagram showing the low expression of FIBCD1 in breast cancer cells MDA-MB-231 in the examples of the present application; wherein A is the low expression of FIBCD1 mRNA level detected by qRT-PCR; B is the low expression of FIBCD1 protein level detected by Western blot;

[0020] Fig.10 This is a graph showing the experimental results of the inhibition of proliferation of breast cancer cells MDA-MB-231 by low expression of FIBCD1 under different sugar concentration culture conditions in the examples of the present application;

[0021] Fig.11 This is a graph showing the experimental results of the inhibition of breast cancer cell MDA-MB-231 migration by low expression of FIBCD1 under different sugar concentration culture conditions in the examples of the present application;

[0022] Fig.12 This is a graph showing the experimental results of the inhibition of breast cancer cell MDA-MB-231 invasion by low expression of FIBCD1 under culture conditions of different sugar concentrations in the examples of the present application;

[0023] Fig.13 This is a graph showing the down-regulation of FIBCD1 expression in breast cancer cells MDA-MB-231 under metformin treatment based on Western blot detection in the examples of the present application;

[0024] Fig.14 This is a result diagram of FIBCD1 regulating the expression of cell proliferation-related factor MCM5 in the embodiment of the present application;

[0025] Fig.15 This is a graph showing the results of low expression of FIBCD1 inhibiting tumor growth in breast cancer and diabetic model mice in the examples of the present application. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0027] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0028] In the present application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0029] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0030] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0031] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0032] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0033] The BC-DM-related characteristic differential gene FIBCD1 in the embodiments of the present application includes wild type, mutant type or fragments thereof. A representative FIBCD1 gene sequence is shown in the genome database (Ensemble) (ENSG00000130720), and the FIBCD1 nucleotide full-length sequence or fragments thereof of the present application can usually be obtained by PCR amplification, recombination or artificial synthesis.

[0034] The gene expression levels in the examples of the present application are detected using a variety of detection techniques known to those of ordinary skill in the art, including but not limited to techniques for detecting gene or protein expression levels.

[0035] "Detecting the expression level of a gene" refers to determining the presence of mRNA of a target gene in a biological sample and its expression level in order to screen or predict the prognosis of BC-DM and can be achieved by measuring the amount of mRNA. The analytical methods used for this purpose include but are not limited to RT-PCR, real-time quantitative PCR (qRT-PCR), DNA microarray chips, etc. "Detecting the expression level of a protein" refers to determining the presence of a protein expressed by a target gene in a biological sample and its expression level in order to screen or predict the prognosis of BC-DM, and the amount of the protein can be determined by using antibodies that specifically bind to the protein expressed in the above-mentioned gene. The analytical methods used for this purpose include but are not limited to Western blot, ELISA (enzyme-linked immunosorbent assay), tissue immunostaining, flow cytometry, protein chips, etc. The non-amplified or amplified nucleic acids or proteins in the embodiments of the present application can be detected by any conventional means.

[0036] In the present application, breast cancer complicated with diabetes (BC-DM) refers to patients with both breast cancer and diabetes, and the corresponding patients are breast cancer patients with diabetes; breast cancer without diabetes (BC-ND) refers to patients with breast cancer but without diabetes, and the corresponding patients are breast cancer patients without diabetes. For breast cancer and diabetes, clinical symptoms commonly used in medicine can be used for identification.

[0037] Based on the fact that there are very few related molecules that can be used for diagnosis and / or treatment and / or prognosis evaluation of BC-DM, the molecular connection between hyperglycemia and breast cancer risk is still largely unknown. In the embodiment of the present application, whole transcriptome sequencing (RNA-seq) and comparative analysis were performed on the lesion tissues of BC-DM patients and BC-ND patients (control group), and a differential gene highly expressed in BC-DM patients, namely fibrinogen C-containing domain 1 (FIBCD1) gene, was provided, which can be used for diagnosis and / or prognosis evaluation and prevention and / or treatment of BC-DM, thus providing a new intervention target. The specific scheme is as follows.

[0038] In a first aspect, the present application provides an application. Specifically, the present application provides an application of a substance that inhibits the expression of a FIBCD1 marker in the preparation of a drug for preventing and / or treating breast cancer combined with diabetes.

[0039] The embodiments of the present application have undergone extensive and in-depth research, and performed whole transcriptome sequencing (RNA-seq) and comparative analysis on tumor tissues of BC-DM patients and BC-ND patients to discover differentially expressed genes and explore their role in the association between diabetes and breast cancer, in order to find better ways and methods for accurate classification and treatment of BC-DM patients, scientific prognosis monitoring and intervention management, and targeted treatment.

[0040] Specifically, the application examples found that FIBCD1 was abnormally highly expressed in the lesion tissues of BC-DM patients and was positively correlated with the fasting blood glucose values ​​of the patients. Experiments have shown that a high sugar environment can enhance the expression level of FIBCD1 in breast cancer cells, and under high sugar conditions, FIBCD1 overexpression can promote the proliferation, migration, and invasion of breast cancer cells. Conversely, by reducing the expression level of FIBCD1, the proliferation, migration, and invasion of breast cancer cells can be effectively inhibited, and the tumor growth of breast cancer and diabetes model mice can be significantly inhibited. Interfering with FIBCD1 gene expression can become a new way to prevent or treat BC-DM.

[0041] In some embodiments, the gene sequence information and encoded protein information of the FIBCD1 marker are as follows:

[0042] >NC_000009.12:c130940709-130902440FIBCD1[organism=Homo sapiens][GeneID=84929][chromosome=9].

[0043] sp|Q8N539|FBCD1_HUMAN Fibrinogen C domain-containing protein 1OS=Homo sapiens OX=9606GN=FIBCD1 PE=1SV=2.

[0044] SEQ ID No.1:

[0045] MVNDRWKTMGGAAQLEDRPRDKPQRPSCGYVLCTVLLALAVLLAVAVTGAVLFLNHAHAPGTAPPPVVSTGAASANSALVTVERADSSHLSILIDPRCPDLTDSFARLESAQASV LQALTEHQAQPRLVGDQEQELLDTLADQLPRLLARASELQTECMGLRKGHGTLGQGLSALQSEQGRLIQLLSESQGHMAHLVNSVSDILDALQRDRGLGRPRNKADLQRAPARGTR PRGCATGSRPRDCLDVLLSGQQDDGVYSVFPTHYPAGFQVYCDMRTDGGGWTVFQRREDGSVNFFRGWDAYRDGFGRLTGEHWLGLKRIHALTTQAAYELHVDLEDFENGTAYAR YGSFGVGLFSVDPEEDGYPLTVADYSGTAGDSLLKHSGMRFTTKDRDSDHSENNCAAFYRGAWWYRNCHTSNLNGQYLRGAHASYADGVEWSSWTGWQYSLKFSEMKIRPVREDR.

[0046] In some embodiments, the substance that inhibits the expression of the FIBCD1 marker includes at least one of a protein, a polypeptide, and a small molecule compound that inhibits the activity of the protein encoding the FIBCD1 marker. The protein may be an antibody, a proteolytic enzyme, and the small molecule may be a ligand, a protein binding molecule, etc. These substances can bind to the activity of the protein encoding the FIBCD1 marker whose amino acid sequence is shown in SEQ ID No. 1, and can degrade or reduce its activity, thereby significantly inhibiting the tumor growth of breast cancer combined with diabetes.

[0047] In some embodiments, the substance that inhibits the expression of the FIBCD1 marker includes a substance that knocks down the gene of the FIBCD1 marker or a substance that knocks out the gene of the FIBCD1 marker.

[0048] The substance that knocks down the gene of the FIBCD1 marker can be any substance that makes the gene of the FIBCD1 marker difficult to express, such as a substance that silences the gene. For example, the substance that knocks down the gene of the FIBCD1 marker includes at least one of miRNA, siRNA, dsRNA, and shRNA that silences the gene of the FIBCD1 marker.

[0049] siRNA (Small interfering RNA) is a small infectious RNA, also known as short interfering RNA (short interfering RNA) or silencing RNA (silencing RNA), which is a double-stranded RNA of about 20 to 25 nucleotides in length, which regulates gene expression in a specific way. shRNA (short hairpin RNA) is a small hairpin RNA, which is a section of RNA sequence with a tight hairpin loop, and is used to inhibit the expression of specific genes. Therefore, both siRNA and shRNA can effectively inhibit the expression of target genes.

[0050] In some embodiments, the positive strand of the siRNA for silencing the gene of the FIBCD1 marker includes at least one of the sequences of SEQ ID No. 2-3; and the shRNA for silencing the gene of the FIBCD1 marker includes the sequence shown in SEQ ID No. 4.

[0051] SEQ ID No. 2: 5'-GCATGAGGTTCACCACCAA-3'.

[0052] SEQ ID No. 3: 5'-GGCAGTACTCACTCAAGTT-3'.

[0053] SEQ ID No. 4: 5'-GCGAGCAAGCTGCAGTTATGT-3'.

[0054] When screening effective siRNA sequences, the present application designed and synthesized a variety of siRNA sequences through a large number of comparison analyses, and verified them by transfecting breast cancer cells with transfection reagents, selecting the above siRNAs with good interference effects, and verifying them at the cell level. The results show that for cell experiments, the inhibition efficiency is good. The present application designed and synthesized a variety of shRNA sequences, accessed vectors and packaged them with viruses, and then infected breast cancer cells with them for verification, screened out the above shRNAs with good interference effects, and verified them at the animal level. The results show that their tumor inhibition effect is very good.

[0055] Wherein, the material of the gene of knocking out FIBCD1 marker can be a material that realizes that breast cancer combined with diabetic cancer cells do not produce the functional protein product of the gene (as shown in SEQ ID No.1) in any way, and the specific method is such as removing all or part of the coding gene sequence, introducing frameshift mutation so that no functional protein is produced, removing or changing the regulatory component (such as promoter editing) so that the coding gene sequence is not transcribed, and preventing translation by combining with mRNA. Usually, knocking out is carried out at the genomic DNA level so that the offspring of the cell also permanently carry the knockout. For example, the material of the gene of knocking out the FIBCD1 marker includes the CRISPR / Cas9 gene editing system of the gene of knocking out the FIBCD1 marker.

[0056] The drug for preventing and / or treating BC-DM prepared in the present application can be a pharmaceutical composition, comprising a substance capable of inhibiting the expression of FIBCD1, and a pharmaceutically acceptable carrier. For example, the substance capable of inhibiting the expression of FIBCD1 includes nucleic acid inhibitors siRNA and / or shRNA, and the drug can further include a hypoglycemic drug.

[0057] The above-mentioned substance that inhibits the expression of FIBCD1 marker is used to prepare a pharmaceutical composition for inhibiting BC-DM. The substance that inhibits the expression of FIBCD1 marker includes but is not limited to inhibitors, antagonists, blockers, blocking agents, nucleic acid inhibitors, etc. The inhibitor of FIBCD1 gene or protein refers to any substance that can reduce the activity of FIBCD1 protein, reduce the stability of FIBCD1 gene or protein, down-regulate the expression of FIBCD1 protein, or inhibit the transcription and translation of FIBCD1 gene, and these substances can be used to prevent or treat BC-DM. For example, it can be: nucleic acid inhibitor, protein inhibitor, antibody, ligand, proteolytic enzyme, protein binding molecule, as long as it can down-regulate the expression of FIBCD1 at the gene or protein level.

[0058] The pharmaceutical composition for preventing and / or treating BC-DM contains an effective amount of a substance that inhibits the expression of FIBCD1 and a pharmaceutically acceptable carrier. Any of the aforementioned substances that inhibit the expression of FIBCD1 can be used in the preparation of the composition.

[0059] "Effective amount" refers to an amount that can produce functions or activities on humans and / or animals and can be accepted by humans and / or animals. "Pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including (but not limited to) liposome nanoparticles, diluents, excipients, adhesives, disintegrants, surfactants, adsorption carriers, buffers, stabilizers, chelating agents, pH control agents, etc. This term refers to such pharmaceutical carriers: they are not necessary active ingredients themselves and are not overly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. In the composition, the pharmaceutically acceptable carrier may contain a liquid, such as water, saline, buffer. In addition, these carriers may also contain auxiliary substances, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffer substances, etc. The carrier may also contain a cell transfection reagent. Specifically, the inhibitor or its transcription gene, or its pharmaceutical composition can be administered to mammals using a variety of methods well known in the art. Including but not limited to: subcutaneous injection, intramuscular injection, transdermal administration, local administration, implantation, sustained release administration, etc.

[0060] In addition, the embodiments of the present application can use the early warning marker FIBCD1 to screen compounds for preventing or treating BC-DM. For example, in one embodiment, the steps include: in the test group, adding the test compound to the culture system, and observing the expression and / or activity of FIBCD1 in the cells of the test group; in the control group, no test compound is added to the same culture system, and the expression and / or activity of FIBCD1 in the cells of the control group is observed; wherein, if the expression and / or activity of FIBCD1 in the cells of the test group is lower than that of the control group, it indicates that the test compound is a candidate drug for treating BC-DM that has an inhibitory effect on the expression and / or activity of FIBCD1.

[0061] As an embodiment of the present application, it also includes: conducting further cell experiments and / or animal experiments on the obtained candidate compounds to further select and determine substances that can be used to prevent, monitor or treat BC-DM from the candidate compounds.

[0062] In a second aspect, the present application also provides an application. Specifically, the present application provides an application of a reagent for detecting the expression of a FIBCD1 marker in the preparation of a kit for diagnosing and / or evaluating the prognosis of breast cancer complicated with diabetes.

[0063] Based on the abnormally high expression of FIBCD1 in the lesion tissues of BC-DM patients and its positive correlation with the patients' fasting blood glucose levels, it is suggested that the detection of FIBCD1 gene and protein levels can become one of the auxiliary indicators for accurate diagnosis and prognosis monitoring of BC-DM. Therefore, the reagent for detecting the expression of FIBCD1 marker can be used for the diagnosis and / or prognosis evaluation of breast cancer combined with diabetes.

[0064] In some embodiments, the above reagents include a primer pair for amplifying the nucleotide sequence of the FIBCD1 marker, and the nucleotide sequence of the primer pair is shown as SEQ ID No.5 and SEQ ID No.6.

[0065] SEQ ID No.5: F: 5'-TGTCATCCCCACTCCTTGTG-3';

[0066] SEQ ID No. 6: R: 5'-TGCCATCGGGTGTCCTTT-3'.

[0067] The kit for diagnosing and / or prognostic evaluation of breast cancer combined with diabetes can be used to monitor BC-DM. The kit contains reagents for detecting FIBCD1; specifically, it can include probes or primers that specifically recognize the FIBCD1 gene; or antibodies or ligands that specifically bind to the protein encoded by FIBCD1. Accordingly, the primer sequences for specifically amplifying the FIBCD1 gene are shown in SEQ ID No. 1 to 2.

[0068] Because FIBCD1 has significant cancer-promoting activity in the embodiments of the present application, it provides a new approach for the personalized and precise diagnosis and treatment of BC-DM. Accordingly, the above-mentioned kit for diagnosing and / or prognostic evaluation of BC-DM includes a reagent for detecting an effective amount of the FIBCD1 gene or a protein encoded therein, selected from one or more of the following substances: primers, containers, instructions for use, positive controls, negative controls, buffers, adjuvants or solvents. For example, primers for detecting FIBCD1, solutions for suspended or fixed cells, detectable labels or markers, solutions for making nucleic acids easy to hybridize, solutions for lysing cells, or solutions for nucleic acid purification.

[0069] The present application has been tested for many times, and part of the experimental results are now cited as a reference to further describe the present application in detail, and the following is a detailed description in conjunction with specific embodiments.

[0070] Example 1 Discovery of FIBCD1

[0071] 1.1 Selection of research subjects

[0072] Pathologically confirmed breast cancer cases that met the research requirements were collected from the Department of Breast Surgery, Harbin Medical University Cancer Hospital. Patients were divided into the following groups: (1) Breast cancer complicated with diabetes (BC-DM) group, with the inclusion criteria being breast cancer patients with fasting blood glucose greater than 7.0 mmol / L for two consecutive times, or random blood glucose greater than 11.1 mmol / L; (2) Breast cancer without diabetes (BC-ND) group, with the inclusion criteria being breast cancer patients with normal blood glucose and the exclusion of other complications. All patients were required to give informed consent and sign the informed consent form, which was reviewed and approved by the Medical Ethics Committee.

[0073] 1.2 Whole transcriptome sequencing (RNA-seq) and comparative analysis of tumor tissues of research subjects

[0074] 1.2.1 Tissue RNA extraction

[0075] Tissue RNA was extracted using the column-based RNA extraction kit TaKaRaMiniBEST Universal RNA Extraction Kit (Cat.#:9767). Specific operations were referred to the instruction manual.

[0076] 1.2.2 Quality control and quantification of RNA samples

[0077] The total RNA of the extracted samples was tested for integrity by agarose gel electrophoresis, and quantified and further quality checked by NanoDrop ND-100. The concentration was ≥200 ng / μL, and the OD260 / 280 was between 1.8 and 2.2.

[0078] 1.2.3 Library construction

[0079] 1-2 μg of total RNA was selected from each sample for sequencing library construction. Poly(A)mRNA Magnetic Isolation Module (NEB enzyme reagent) was used for mRNA enrichment (or RiboZero Magnetic Gold Kit was used for rRNA removal). The product RNA after treatment was used to construct a library using KAPA stranded RNA-Seq library prep kit (Illumina). The KAPA kit uses a dUTP-based strand-specific library construction method, and the specific operations were performed according to the instructions.

[0080] 1.2.4 Sequencing

[0081] The mixed sequencing libraries of different samples were denatured by 0.1 M NaOH to generate single-stranded DNA, diluted to 8 pM concentration and amplified in situ on TruSeq SR Cluster Kit v3-cBot-HS (#GD-401-3001, Illumina), and the generated fragment ends were sequenced for 150 cycles using an Illumina HiSeq 4000 sequencer.

[0082] 1.2.5 Whole transcriptome sequencing data analysis

[0083] Gene expression quantitative analysis is performed at two levels: Gene Level and Transcript Level. The software StringTie compares the results to the known transcriptome, calculates the transcript abundance, and then calculates it through Ballgown. The unit of expression is expressed in FPKM (Fragments Per Kilobase of gene / transcript model per Million mapped fragments). The sequencing results were analyzed and processed by bioinformatics. The screening criteria for differentially expressed genes were: Fold Change ≥ 1.5, p-value < 0.05, and the mean FPKM within the group ≥ 0.5.

[0084] 1.2.6 Sequencing results

[0085] The RNA-seq results showed that there were significant differences between the BC-DM and BC-ND groups. Figure 1 Among them, FIBCD1 showed significant differential expression in tumor tissues of BC-DM patients and BC-ND patients, Fold Change = 4.8, p = 0.01.

[0086] 1.2.7 qRT-PCR method to measure FIBCD1 expression in tissue samples

[0087] (1) According to the results of RNA sequencing, the expression level of FIBCD1 in tumor tissues of BC-DM patients was significantly upregulated compared with that of BC-ND patients. Therefore, the qRT-PCR method was used to further verify the difference in FIBCD1 expression between BC-DM and BC-ND patients.

[0088] (2) The specific steps of RNA extraction were carried out as described in 1.2.1.

[0089] (3) Preparation of cDNA: Using SuperScript TM III Reverse Transcriptase (Invitrogen) kit to prepare cDNA mixed reaction solution. The specific operation is as follows:

[0090] (a) Prepare the annealing mixture, see Table 1.

[0091] Table 1

[0092] RNA 2μg <![CDATA[Oligo(dT) 18 (50μM)]]> 0.8μL N9 (50μM) 0.5μL dNTPs Mix (2.5 mM) 1.6μL <![CDATA[Add RNase-free H 2 O]]> To 13.5 μL (total volume)

[0093] The mixture was incubated in a 65°C water bath for 5 minutes and placed on ice for 2 minutes.

[0094] (b) After a brief centrifugation, add RT reaction solution into the centrifuge tube in sequence, see Table 2.

[0095] Table 2

[0096] 5X First-Strand Buffer 4μL 0.1M DTT 1μL RNase inhibitor 0.5μL SuperScript III RT 1μL

[0097] After mixing, keep the mixture at 37℃ for 1 minute. Then, use a pipette to gently pipette several times to mix evenly; incubate at 50℃ for 60 minutes; incubate at 70℃ for 15 minutes to inactivate the enzyme; place the synthesized cDNA in an ice bath for later use or store it at -20℃.

[0098] (4) qRT-PCR reaction:

[0099] All cDNA samples were configured with Real time PCR reaction system. The system configuration is as follows: 2×MasterMix (5μL); 10μM PCR specific primer F (0.5μL); 10μM PCR specific primer R (0.5μL); add water to a total volume of 8μL. Briefly centrifuge at 5000rpm. Sample addition: a. Add 8μL of the mixture to each corresponding well of the 384-PCR plate; b. Add the corresponding 2μL cDNA; c. Carefully stick the Sealing Film sealing film and briefly centrifuge to mix; d. Place the prepared PCR plate on ice before setting the PCR program; Place the above 384-PCR plate on the Real time PCR instrument for PCR reaction. The PCR reaction program is as follows: 95℃, 10min; 40 PCR cycles (95℃, 10 seconds; 60℃, 60 seconds (collect fluorescence)). The target gene and internal reference ACTB of each sample were subjected to Real time PCR reaction respectively; the data were obtained using 2 -△△CT Method for analysis.

[0100] The primer sequences are as follows:

[0101] Primer sequences for FIBCD1 gene:

[0102] SEQ ID No.5: F: 5'-TGTCATCCCCACTCCTTGTG-3';

[0103] SEQ ID No. 6: R: 5'-TGCCATCGGGTGTCCTTT-3'.

[0104] Internal reference ACTB gene primer sequence:

[0105] SEQ ID No.7: F: 5'-GTGGCCGAGGACTTTGATTG-3';

[0106] SEQ ID No. 8: R: 5'-CCTGTAACAACGCATCTCATATT-3'.

[0107] (5) qRT-PCR results Figure 2 As shown, the results showed that there were significant differences in the expression levels of FIBCD1 in the tumor tissues of BC-DM patients and BC-ND patients. Specifically, the expression level of FIBCD1 in the tumor tissues of BC-DM patients was significantly higher than that in the tumor tissues of BC-DM patients.

[0108] 1.2.8 Correlation between FIBCD1 expression level and fasting blood glucose (FBG) in BC patients

[0109] The expression level (FPKM) of FIBCD1 in tumor tissues of BC patients obtained by whole transcriptome sequencing data and the fasting blood glucose (FBG) value of BC patients were correlated with each other using GraphPad Prism software. The results are as follows: Figure 3 As shown, the expression level of FIBCD1 was significantly positively correlated with the fasting blood glucose (FBG) value of BC patients (p=0.04).

[0110] Example 2 Effects of high and low sugar culture conditions on FIBCD1 expression

[0111] 2.1 Cell culture:

[0112] Different types of breast cancer cells (MCF-7, T47D, MDA-MB-231, BT-20, BT549) were cultured in DMEM medium containing 10% fetal bovine serum in an atmosphere of 5% CO 2 , cultured at 37°C. By consulting a large number of literature, DMEM medium containing 25mM glucose was used as high glucose medium (High Glucose, HG) to simulate diabetic conditions, and DMEM medium containing 5mM glucose was used as low glucose medium (Low Glucose, LG) to simulate normal physiological conditions. The medium was changed every 2 to 3 days, and when the cells grew to more than 80%, the cells were ready to be collected and extracted for RNA.

[0113] 2.2 Cell RNA extraction:

[0114] Cell RNA was extracted using the column-based RNA extraction kit TaKaRaMiniBEST Universal RNA Extraction Kit (Cat.#:9767). The specific operation was referred to the instruction manual.

[0115] 2.3 Preparation of cDNA: The specific operation steps are as described in 1.2.7(3).

[0116] 2.4 qRT-PCR method to measure the expression level of FIBCD1 under high and low sugar concentration culture conditions: The specific operation steps are carried out as described in 1.2.7(4).

[0117] The results of qRT-PCR assay were as follows Figure 4 As shown, the results showed that compared with low-glucose conditions (control group), the expression of FIBCD1 under high-glucose conditions was significantly upregulated in multiple breast cancer cells.

[0118] Example 3 Construction of FIBCD1 overexpression and FIBCD1 knockdown breast cancer cell lines

[0119] 3.1 Cell culture:

[0120] The commonly used breast cancer cell line MDA-MB-231 was selected to construct FIBCD1 overexpression cell line and FIBCD1 knockdown cell line. The cells were cultured in DMEM medium containing 10% fetal bovine serum and 5% CO 2 , cultured at 37°C.

[0121] 3.2 Cell transfection:

[0122] Observe under a microscope and when the cells are in good condition and in a vigorous growth period, transient cell transfection can be performed. After routine digestion, centrifugation, and resuspending of the cells, cell counting is performed; inoculate in a 6-well plate at 2×10 5 The cells were plated in the wells, and the cell confluence reached 70-80% after 24 hours. The synthesized siRNA or plasmid DNA was taken out, and the siRNA was prepared into a 20μM stock solution with DEPC water; the plasmid DNA was 0.5μg / μL. The cells inoculated the day before were taken out, and 2mL of fresh complete medium was replaced. According to different experimental groups, transfection reagent, first siRNA, second siRNA, and blank plasmid DNA were added respectively, and then placed in the incubator for continued culture.

[0123] The transfection reagent is the FIBCD1 gene recombinant vector in this example, and the plasmid used is pcDNA3.1. Specifically, the human full-length FIBCD1 sequence is amplified using the FIBCD1 gene-specific primers of the sequences shown in SEQ ID No.1 and SEQ ID No.2, and then the FIBCD1 gene fragment is cloned into the expression vector pcDNA3.1 according to the instruction manual of the expression vector pcDNA3.1, and named pcDNA3.1-FIBCD1.

[0124] It should be noted that in this example, the ligation system is configured according to the ratio of target gene to vector volume 3:1 to 8:1. Specifically, the ligation system includes: 1 μL of expression vector pcDNA3.1, 1 μL of PCR amplification recovery product, 0.5 μL of T4 ligase, 1 μL of 10×ligase Buffer, and then ddH 2 0 to 10 μL. The connection conditions were 16°C for 16 hours to complete the connection. The connection product was transformed into E. coli competent cells using the heat shock method, positive colonies were identified by PCR, and first-generation sequencing was performed using sequencing primers. The plasmid was extracted and purified using the Plasmid Maxprep Kit.

[0125] A plurality of small interfering RNA (siRNA) molecules that are specific to FIBCD1 were designed and synthesized, and siRNAs with good interference effects were screened out: the first siRNA and the second siRNA, whose corresponding sequences are shown in SEQ ID No. 2 and SEQ ID No. 3, respectively.

[0126] According to the instruction manual of Lipofectamine 3000, take a 6-well plate as an example to prepare the experimental components for each well. First, take tube A and add lipofectamine 3000 (5μL) and Opti-MEM (125μL) and mix thoroughly. Then take tube B and add siRNA (6μL) / plasmid DNA (5μL) and then add P3000 (5μL, note: siRNA does not add P3000) and Opti-MEM (125μL) and mix thoroughly. Then, mix tube A and tube B evenly, incubate at room temperature for 10 minutes, add dropwise to the corresponding wells of the 6-well plate, shake to mix, and place in the incubator for continued culture. After transfection for 48 hours, conduct subsequent functional experiments.

[0127] 3.3 qRT-PCR method to measure the expression of FIBCD1 gene after overexpression and knockdown:

[0128] After transfection, RNA was extracted from the cells using the RNA extraction kit TaKaRaMiniBEST Universal RNA ExtractionKit (Cat.#:9767). For specific operations, refer to the instructions. For preparation of cDNA, the specific steps were as described in 1.2.7(3). The qRT-PCR method was used to measure the expression level of FIBCD1 after overexpression and knockdown. The specific steps were as described in 1.2.7(4). The results of the cell line overexpressing FIBCD1 are shown in Figure 5As shown in A, compared with the control group (OE-NC, blank plasmid), the expression of FIBCD1 in the MDA-MB-231 cell experimental group (OE-FIBCD1) transfected with pcDNA3.1-FIBCD1 plasmid increased significantly. Fig. 9 As shown in A, compared with the control group (si-NC), the expression level of FIBCD1 in MDA-MB-231 cells transfected with siRNA was significantly decreased, and the inhibitory effects of the two siRNAs were both above 65%.

[0129] 3.4 Western blot method to measure the expression of FIBCD1 protein after overexpression and knockdown:

[0130] 3.4.1 Cell protein extraction

[0131] (1) Take out the cells to be extracted from the incubator; (2) Discard the culture medium, wash twice with PBS, and aspirate the residual liquid; (3) Add an appropriate amount of lysis buffer (containing 1× protease phosphatase inhibitor mixture) to the culture dish, shake gently, and place on ice for 5 minutes; (4) Use a cell scraper to collect the cells, collect them into a 1.5mL EP tube, and place them on ice for 30 minutes, shaking once every 5 minutes for a total of 5 to 6 times; (5) Centrifuge at 12,000 rpm for 20 minutes in a 4°C centrifuge. The supernatant is the extracted protein solution and is stored at -80°C for later use.

[0132] 3.4.2 Protein concentration determination

[0133] Dissolve the protein standard on ice and dilute the protein standard to a final concentration of 0.5 mg / mL. Calculate the amount of BCA working solution required according to the number of protein samples, and prepare the BCA working solution according to the ratio of A:B = 50:1. Take 0, 1, 2, 4, 8, 12, 16, and 20 μL of protein standards and add them to the wells of a 96-well plate in sequence, with 2 replicates per group. Add different volumes of standard diluent to each well to make the total volume of each well 20 μL. Add 2 μL of protein sample to each well of the 96-well plate, and then add standard diluent to 20 μL, with 3 replicates per group. Add 200 μL of BCA working solution to each well and incubate in a 37°C oven for 30 minutes. Use an enzyme reader to detect the absorbance of the sample at a wavelength of 562 nm, draw a protein standard curve, and calculate the concentration of each sample.

[0134] 3.4.3 Preparation of protein samples

[0135] (1) Plan to add 20 μg of protein to each well. Calculate the loading volume of the protein sample based on the concentration of the cell protein solution, then add 1× loading to make up to a final volume of 10 μL and vortex to mix; (2) Heat at 95°C to denature the protein for 10 min; (3) Then place the sample on ice or store it at -20°C for subsequent Western blot experiments.

[0136] 3.4.4Western blot

[0137] (1) Glue preparation: Clean the glass plate, assemble it and clamp it on the rack, and use water to check for leaks for 5 minutes. After confirming that there is no leakage, pour out the water and turn it upside down to dry; prepare 10% separation gel according to the instructions, press the gel with water, and let it stand at room temperature for 30-40 minutes; then prepare 5% concentrated gel, add the concentrated gel, gently insert the comb, avoid bubbles, let it stand at room temperature to solidify, and soak it in electrophoresis solution, store at 4℃. (2) Gel electrophoresis: Fix the gel plate to the electrophoresis tank, add electrophoresis solution to the outer tank, and gently pull out the comb; add protein sample to each well, and add 3-5μL protein marker on both sides of the head and tail. Set 90V constant voltage for electrophoresis. After the protein runs out of the concentrated gel, the voltage can be increased to 100-120V. When the target band runs to the appropriate position, turn off the power and stop electrophoresis. (3) Transfer to membrane: Use tweezers to pick up the cut PVDF membrane and soak it in methanol for 15s to activate it. Prepare electrotransfer solution, remove the plate, cut off the upper concentrated gel, and soak the remaining gel in 1× electrotransfer solution. When preparing the electrotransfer sandwich, place the black board down in the order of sponge, filter paper, gel, membrane, filter paper, and sponge, and ensure that there are no bubbles between the membrane and the gel. Insert the sandwich clip into the electrotransfer tank, pour all the electrotransfer solution, add ice packs to the tank, put the entire tank in a foam box, fill it with ice on all sides, and adjust the program to a constant current of 300mA for 90min. (4) Blocking: Determine the position of the target protein and internal reference according to the marker, cut the PVDF membrane, and block the membrane area where the useful band is located with 5% skim milk powder or 5% BSA (prepared in TBST) for 1 to 1.5h. (5) Apply primary antibody: wash the membrane with TBST for 5 min × 3 times, add the diluted antibody, and incubate at 4°C overnight; (6) Apply secondary antibody: take the sample out of the refrigerator, place it at room temperature for 20 min, recover the primary antibody, then wash the membrane with TBST for 5 min × 3 times, take an appropriate amount of secondary antibody and dilute it with TBST (dilution according to the antibody instructions), and incubate it on a shaker at room temperature for 1 hour. (7) Exposure: discard the secondary antibody, wash the membrane with TBST for 5 min × 3 times; mix ECL colorimetric solution A and B in a volume ratio of 1:1, take out the membrane and press dry the water with a paper towel, add the colorimetric solution and cover it evenly, and incubate for a few seconds before exposure.

[0138] Western blot results of cell lines overexpressing FIBCD1 are shown in Figure 5As shown in Figure B, compared with the control group (OE-NC, blank plasmid), the expression of FIBCD1 protein in MDA-MB-231 cells transfected with pcDNA3.1-FIBCD1 plasmid was significantly increased. Fig. 9 As shown in B, compared with the control group (si-NC), the expression level of FIBCD1 in MDA-MB-231 cells transfected with siRNA was significantly decreased, indicating that the two siRNAs had a significant inhibitory effect.

[0139] Example 4 CCK8 cell proliferation assay

[0140] After 48 hours of transfection, the cells after transient overexpression or knockdown were routinely digested, resuspended, counted, and plated in 96-well plates for cell proliferation detection using the CCK8 kit. The number of cells per well was adjusted to 1×10 4 After 5 cells were added, 200 μL of complete medium was added to each well (note: divided into high-sugar and low-sugar culture conditions), and 96-well plates were inoculated with 5 parallel wells in each group at 4 time points. The edge wells were supplemented with PBS to maintain the humidity balance in the plate and placed in the incubator for culture. A time gradient was set, and the proliferation experiment took 0, 24, 48, 72, and 96h as observation points. 10 μL of CCK8 was added to each well, and the plate was returned to the incubator for culture for 2h. The absorbance value OD450 was measured at a wavelength of 450nm with an enzyme marker, and statistical analysis was performed with 0h as the benchmark. In this case, overexpression means using cloned plasmids to enhance expression; knockdown means using siRNA to interfere with expression; and the control means cells transfected with blank plasmids.

[0141] The results of the cell proliferation experiment showed that FIBCD1 overexpression could significantly enhance the growth of breast cancer cells and was affected by high and low sugar culture conditions. Figure 6 As shown; wherein, the orange solid line is the test result of MDA-MB-231 cells overexpressing FIBCD1 under high glucose culture conditions (25mM Glucose), marked as "OE-FIBCD1(25mM)", the green solid line is the test result of MDA-MB-231 cells in the empty group under high glucose culture conditions (25mM Glucose), marked as "OE-NC(25mM)", the orange dotted line is the test result of MDA-MB-231 cells overexpressing FIBCD1 under low glucose culture conditions (5mM Glucose), marked as "OE-FIBCD1(5mM)", the green dotted line is the test result of MDA-MB-231 cells in the empty group under low glucose culture conditions (5mM Glucose), marked as "OE-NC(5mM)".

[0142] Conversely, inhibiting the expression of FIBCD1, i.e. knocking down FIBCD1, can significantly inhibit the growth of breast cancer cells, and the results are as follows: Fig.10 As shown; the purple solid line is the test result of MDA-MB-231 cells expressing FIBCD1 normally under high glucose culture conditions, marked as "si-NC (25mM)", the green solid line is the test result of MDA-MB-231 cells after the first siRNA interference under high glucose culture conditions, marked as "si-FIBCD1#1 (25mM)", and the blue solid line is the test result of MDA-MB-231 cells after the second siRNA interference under high glucose culture conditions, marked as "si-FIBCD1#2 (25 mM)", the purple dotted line is the test result of MDA-MB-231 cells that normally express FIBCD1 under low-glucose culture conditions, marked as "si-NC(5mM)", the green dotted line is the test result of MDA-MB-231 cells after the first siRNA interference under low-glucose culture conditions, marked as "si-FIBCD1#1(5mM)", and the blue dotted line is the test result of MDA-MB-231 cells after the second siRNA interference under low-glucose culture conditions, marked as "si-FIBCD1#2(5mM)".

[0143] Example 5 Cell migration experiment

[0144] After 48 hours of transfection, the cells after transient overexpression or knockdown were digested as usual, resuspended in PBS, and counted. First, 700 μL of complete medium was added to the lower chamber of the 24-well plate (matching the chamber), and the transwell chamber was gently placed into the lower chamber with the medium added to avoid bubbles. The cell suspension was blown and 500 μL per well was added to the chamber. Incubate at 5% CO 2 Culture in an incubator for 24 hours. Take out the chamber, wash it with PBS, and then place it in a 24-well plate (matching the chamber) with 700 μL of methanol to fix it. After 30 minutes, take out the chamber and air-dry it appropriately. Stain the cells that migrated to the basal surface with 0.5% crystal violet solution for 30 minutes, then wipe the cells inside the chamber with a cotton swab, wash it twice with PBS, and air-dry it appropriately. Observe under a microscope and select 4× and 10× magnifications of 5 fields of view (upper, lower, left, right, and middle) for photography, and count with Image-J software.

[0145] The cell migration ability of MDA-MB-231 cells overexpressing FIBCD1 and expressing FIBCD1 normally under high glucose culture conditions (25mM Glucose) and low glucose culture conditions (5mM Glucose) was tested respectively. The experimental results showed that FIBCD1 overexpression can significantly enhance the migration ability of breast cancer cells (MDA-MB-231), and high glucose culture conditions can further promote cell migration. Figure 7 As shown in A in the figure, under the microscope, the number of cell migration in the "OE-FIBCD1 (25mM)" group was the largest, significantly higher than the number of cell migration in the "OE-FIBCD1 (5mM)" group, and also significantly higher than the number of cell migration in the "OE-NC (25mM)" group, while the number of cell migration in the "OE-NC (5mM)" group was significantly lower than that in the other three groups; the corresponding statistical results are shown in Figure 7 As shown in B.

[0146] Correspondingly, inhibiting the expression of FIBCD1, i.e. knocking down FIBCD1, can significantly inhibit the migration ability of breast cancer cells, and is affected by high and low sugar culture conditions. Fig.11 As shown; among them, Fig.11 As shown in A in the figure, under the microscope, the number of cell migration in the "si-FIBCD1#1(25mM)" group and the "si-FIBCD1#2(25mM)" group was significantly lower than that in the "si-NC(25mM)" group. The number of cell migration in the "si-FIBCD1#1(5mM)" group and the "si-FIBCD1#2(5mM)" group was also significantly lower than that in the "si-NC(5mM)" group. The corresponding statistical results are shown in Fig.11 As shown in B.

[0147] Example 6 Cell invasion assay

[0148] After 48 hours of transfection, the cells after transient overexpression or knockdown were digested as usual, resuspended in PBS, and counted. 2 hours before the start of the experiment, 60 μL of diluted matrigel glue (dilution concentration of 200-300 μg / mL) was added to the membrane of the chamber and placed in 5% CO 2 The cells were polymerized into gel in an incubator, and 700 μL of culture medium containing 15% FBS serum was added to the lower chamber of a transwell chamber covered with extracellular matrix, and the chamber was gently placed on it; 200 μL of the prepared cell suspension was added to the upper chamber, and the chamber was cultured in an incubator for 24 h; the upper chamber was taken out and placed in another clean 24-well plate, washed with PBS for 3 times, 200 μL of pure methanol was added, the cells were fixed for 15 min, then they were aspirated, and 200 μL of crystal violet was added for staining for 15 min; the crystal violet was aspirated, and PBS was added for washing 2 times; 5 fields of view were randomly selected using phase contrast microscopy to calculate the number of cells.

[0149] The cell invasion ability of MDA-MB-231 cells overexpressing FIBCD1 and expressing FIBCD1 normally under high glucose culture conditions (25mM Glucose) and low glucose culture conditions (5mM Glucose) was tested respectively. The experimental results showed that FIBCD1 overexpression can significantly enhance the invasion ability of breast cancer MDA-MB-231 cells, and high glucose culture conditions will further promote cell invasion. Figure 8 As shown in A in the figure, under the microscope, the number of cells invading in the "OE-FIBCD1 (25mM)" group was the largest, significantly higher than the number of cells invading in the "OE-FIBCD1 (5mM)" group, and also significantly higher than the number of cells invading in the "OE-NC (25mM)" group, while the number of cells invading in the "OE-NC (5mM)" group was significantly lower than the other three groups; the corresponding statistical results are shown in Figure 8 As shown in B.

[0150] Correspondingly, inhibiting the expression of FIBCD1, i.e. knocking down FIBCD1, can significantly inhibit the invasion ability of breast cancer cells, and is affected by high and low sugar culture conditions. Fig.12 As shown; among them, Fig.12 As shown in A in the figure, under the microscope, the number of cell invasion in the "si-FIBCD1#1(25mM)" group and the "si-FIBCD1#2(25mM)" group was significantly lower than that in the "si-NC(25mM)" group. The number of cell invasion in the "si-FIBCD1#1(5mM)" group and the "si-FIBCD1#2(5mM)" group was also significantly lower than that in the "si-NC(5mM)" group. The corresponding statistical results are shown in Fig.12 As shown in B.

[0151] Example 7 Effect of metformin on FIBCD1 expression

[0152] Breast cancer cells MDA-MB-231 were cultured under high glucose conditions (25 mM Glucose), and different concentrations of metformin (0, 2.5, 5, 10 mM) were added to treat the cells for 3 days. Cell proteins were then collected and detected by western blot. The specific operation steps were performed according to 3.4 of Example 3.

[0153] The results are as follows Fig.13 As shown: After metformin treatment, the expression of FIBCD1 protein in MDA-MB-231 cells was significantly decreased in a dose-dependent manner.

[0154] Example 8 Effect of overexpression / knockdown of FIBCD1 on the expression of cell proliferation-related factor MCM5

[0155] After 48 hours of transfection, the cells that were transiently overexpressed / knocked down were used to extract cellular RNA using the RNA extraction kit TaKaRaMiniBESTUniversal RNA Extraction Kit (Cat.#:9767). The specific operation was referred to the instruction manual. The cDNA was prepared according to 1.2.7 (3) of Example 1. The expression level of the MCM5 gene after overexpression / knockdown of FIBCD1 was measured by qRT-PCR method. The specific operation was carried out according to 1.2.7 (4) of Example 1.

[0156] The experimental results are as follows Fig.14 As shown in the results, the gene expression of cell proliferation-related factor MCM5 was significantly upregulated after overexpression of FIBCD1 ( Fig.14 In contrast, after knocking down FIBCD1, the gene expression of MCM5 was significantly downregulated ( Fig.14 (as shown in B in the figure).

[0157] Example 9 Interference with FIBCD1 inhibits tumor growth in breast cancer and diabetic model mice

[0158] 9.1 Virus Packaging

[0159] Virus packaging was performed using 293T cells using the virus packaging helper plasmids psPAX2 and pMD2G. 24 h before transfection, 2.5 × 10 6 Cells. Mix 4μg of target plasmid, 3μg of psPAX2, and 1μg of pMD2.G in 600μL Opti-MEM, mix gently, and leave at room temperature for 5 minutes. Dissolve 20μL lipo2000 in 600μL serum-free culture medium, mix gently, and leave at room temperature for 5 minutes. Mix the above two tubes, mix gently, and leave at room temperature for 20 minutes, then add to the culture dish and mix gently. Incubate at 37°C, 5% CO 2 Culture in an incubator. After 6 hours, replace with complete medium, continue to culture for 48 hours, collect the supernatant into a 15mL centrifuge tube, and store at 4°C. Add 10mL of complete medium, continue to culture in an incubator for 24 hours, collect the supernatant and mix it with the virus liquid collected the day before. Filter the lentiviral supernatant with a 0.45μm filter, centrifuge at 80000g and 4°C for 3 hours, collect the precipitate, add an appropriate amount of culture medium to dissolve the lentiviral precipitate, aliquot the virus liquid, and store at -80°C.

[0160] 9.2 Construction of stable cell lines with FIBCD1 interference

[0161] 24 hours before virus infection, cells were plated in culture dishes. The virus was diluted 200 times with fresh culture medium, 8 μg / mL polybrene was added, cells were infected for 8 hours, fresh culture medium was replaced, and culture was continued for 72 hours; then fresh complete culture medium was replaced, which was mixed with a certain concentration of puromycin, which was the lowest concentration that could almost kill the target cells in 3 days. Cultured in a constant temperature incubator, successfully transfected target cells were screened, and interference effects were detected by qRT-PCR and Western blot.

[0162] 9.3 Establishment of mouse model of breast cancer combined with diabetes

[0163] (1) Preparation of citric acid buffer: Add 2.1 g of citric acid (FW: 210.14) to 100 mL of double distilled water to prepare solution A; add 2.94 g of sodium citrate to 100 mL of double distilled water to prepare solution B. Mix solutions A and B in a 1:1 ratio and adjust the pH value to 4.2-4.5. (2) Preparation of streptozotocin: Dissolve STZ in citric acid buffer at a concentration of 0.1 g / L for injection, and inject the corresponding STZ according to the fasting body weight. The injection should be completed within 30 minutes. (3) Modeling of BALB / C mice: Each modeling was fasted for more than 16 hours, and streptozotocin solution was intraperitoneally injected at a dose of 50 mg / kg for 5 consecutive days. Blood sugar was measured on the 10th day. The mice were fed with high-fat and high-sugar feed throughout the whole process. (4) After the successful establishment of the diabetes model, the animals were randomly divided into two groups (7 in each group): sh-NC group and sh-FIBCD1 group. (5) Collect cells to determine the interference effect, and inject 100 μL of cell suspension (containing 3×10 6 cells). (6) Tumor observation: Tumor size was measured seven days after injection, and mouse weight and tumor volume were recorded twice a week. Tumor volume was calculated according to the formula V = (length × width2) / 2, and tumor growth curve was drawn. (7) When the tumor size was close to 1 cm 3 At 4 pm, the mice were killed, the tumors were removed, their sizes and weights were measured, and the differences in tumor growth between the two groups of mice were compared.

[0164] The experimental results are as follows Fig.15 The results showed that compared with the control group (sh-NC group), interference with FIBCD1 could significantly inhibit the tumor growth of breast cancer and diabetic mice, and the tumor size was significantly inhibited (such as Fig.15 There was a significant difference in tumor volume (as shown in A Fig.15 The tumor weight decreased significantly (as shown in B). Fig.15 This indicates that shRNA (SEQ ID No. 4) interfering with FIBCD1 has a good tumor-suppressing effect on BC-DM model mice.

[0165] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. Use of a substance that inhibits the expression of FIBCD1 marker in the preparation of a drug for preventing and / or treating breast cancer complicated with diabetes.

2. The use according to claim 1, characterized in that The amino acid sequence of the protein encoded by the FIBCD1 marker is shown in SEQ ID No.

1.

3. The use according to claim 1, characterized in that The substance that inhibits the expression of the FIBCD1 marker includes at least one of a protein, a polypeptide and a small molecule compound that inhibits the activity of the protein encoding the FIBCD1 marker.

4. The use according to claim 1, characterized in that The substance that inhibits the expression of the FIBCD1 marker includes a substance that knocks down the gene of the FIBCD1 marker or a substance that knocks out the gene of the FIBCD1 marker.

5. The use according to claim 4, characterized in that: The substance for knocking down the gene of the FIBCD1 marker includes at least one of miRNA, siRNA, dsRNA, and shRNA that silences the gene of the FIBCD1 marker.

6. The use according to claim 5, characterized in that: The siRNA for silencing the gene of the FIBCD1 marker comprises at least one of the sequences shown in SEQ ID No. 2-3; And / or, the shRNA for silencing the gene of the FIBCD1 marker includes the sequence shown in SEQ ID No.

4.

7. The use according to claim 4, characterized in that: The material for knocking out the gene of the FIBCD1 marker includes a CRISPR / Cas9 gene editing system for knocking out the gene of the FIBCD1 marker.

8. Use of a reagent for detecting the expression of the FIBCD1 marker in the preparation of a kit for diagnosing and / or evaluating the prognosis of breast cancer complicated with diabetes.

9. The use according to claim 8, characterized in that The amino acid sequence of the protein encoded by the FIBCD1 marker is shown in SEQ ID No.

1.

10. The use according to claim 8 or 9, characterized in that: The reagent comprises a primer pair for amplifying the nucleotide sequence of the FIBCD1 marker, and the nucleotide sequence of the primer pair is shown as SEQ ID No.5 and SEQ ID No.6.

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