Application of an osteoclastin biomarker in the diagnosis, treatment and prognosis of breast cancer bone metastasis

Abstract

The present invention belongs to the technical field of biomedical detection, and specifically relates to an application of an osteoclastin biomarker in the diagnosis, treatment and prognosis of breast cancer bone metastasis. The gene sequence of the osteoclastin is shown in NR_003148.3 of the NCBI gene library. The osteoclastin promotes breast cancer bone metastasis by inducing osteoclastogenesis, and is significantly increased in breast cancer-bone metastasis patient tissues and serum, and can predict the risk of bone metastasis in patients, thereby achieving the effect of preventing bone metastasis. The reagent or kit for detecting the expression amount of the biomarker osteoclastin can only be detected after bone metastasis occurs, compared to the existing detection kit, which can only be detected after bone metastasis occurs. The osteoclastin of the present invention can be more characteristic and sensitive to predict the occurrence of breast cancer bone metastasis, diagnose breast cancer bone metastasis, predict disease progression, evaluate the therapeutic effect, guide the use of drugs and the prognosis evaluation kit, and as a target for treating bone metastasis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical detection, and specifically relates to an application of an osteoclastin biomarker in the diagnosis, treatment and prognosis of breast cancer bone metastasis. Background Art

[0002] Female breast cancer (BC) has become the leading cause of cancer morbidity worldwide, and most breast cancer deaths are due to complications caused by distant organ recurrence or tumor metastasis. Among them, the most common metastatic site of breast cancer is bone, occurring in 70% of breast cancer bone metastasis patients.

[0003] Clinically, breast cancer bone metastasis usually manifests as osteolytic bone metastasis, which is abnormal bone destruction caused by increased bone resorption mediated by osteoclasts. In the clinical treatment of bone metastasis, drugs that inhibit osteoclast differentiation / resorption are often used: bisphosphonates (BPs) and denosumab. However, the use of these two drugs can only be palliative treatment and has no significant effect on patient prognosis. At the same time, BPs can significantly delay bone metastasis in patients while also promoting the metastasis of tumor cells to visceral organs. Therefore, screening and identifying the key nodes of breast cancer bone metastasis and exploring its molecular mechanism will provide an important scientific basis for the diagnosis and treatment of breast cancer bone metastasis.

[0004] Due to the development of deep sequencing technology and the development of proteomics and computational methods, a large number of microproteins / short peptides have been found to be encoded by many previously unannotated non-protein coding transcripts, such as long non-coding RNAs (LncRNAs) and pseudogene RNAs. Increasing evidence shows that microproteins / short peptides encoded by non-coding RNAs are bioactive molecules that inhibit or promote tumorigenesis and / or tumor progression, including the development of breast cancer. Although several biological peptides encoded by lncRNAs have been found to be involved in the occurrence and development of breast cancer, their functional characteristics in breast cancer-bone metastasis have not yet been found. Therefore, as an emerging class of cell regulators, the hidden role of microproteins / short peptides encoded by lncRNAs in breast cancer-bone metastasis may become a new therapeutic strategy.

[0005] Therefore, it is crucial to find microproteins / short peptides encoded by lncRNA to predict breast cancer bone metastasis and evaluate its prognosis. Summary of the invention

[0006] In view of the above problems, the object of the present invention is to provide an application of an osteoclastin biomarker in the diagnosis, treatment and prognosis of breast cancer bone metastasis.

[0007] The technical contents of the present invention are as follows:

[0008] The present invention provides an application of osteoclastin as a biomarker or target for diagnosis, treatment and prognosis of breast cancer bone metastasis;

[0009] The osteoclastin is a self-named name, and its gene sequence is shown in NR_003148.3 of the NCBI gene library. It is a short peptide encoded by TPM3P9. The osteoclastin promotes the formation of osteoclasts, thereby promoting breast cancer bone metastasis, and can predict the risk of bone metastasis in patients, thereby achieving the effect of preventing bone metastasis.

[0010] The present invention also provides a reagent for detecting the expression level of osteoclastin for use in preparing a product for diagnosis and / or prognosis analysis of breast cancer bone metastasis, wherein the osteoclastin is as described above;

[0011] The method of detecting the expression level of osteoclastin includes detecting in blood and tissues;

[0012] The method of detecting the expression level of osteoclastin includes detecting the serum protein content in the blood, including the expression level of TPM3P9 in the serum;

[0013] The breast cancer bone metastasis diagnosis and / or prognosis analysis product includes one or more of a detection reagent, a test kit, a test strip, and a gene chip;

[0014] The kit includes a western blot or ELISA kit, preferably an ELISA kit with self-coated antibodies;

[0015] The breast cancer bone metastasis diagnosis and / or prognosis analysis product has at least one of the following functions:

[0016] (1) Prediction of the occurrence of breast cancer bone metastasis;

[0017] (2) Diagnosis of breast cancer bone metastasis;

[0018] (3) Prediction of progression of breast cancer bone metastasis;

[0019] (4) Screening and division of risk groups for breast cancer bone metastasis.

[0020] The present invention also provides an application of an osteoclastin inhibitor or target in the preparation of a drug for inhibiting breast cancer bone metastasis

[0021] The beneficial effects of the present invention are as follows:

[0022] The application of osteoclastin as a biomarker or target for diagnosis, treatment and prognosis of breast cancer bone metastasis of the present invention, the osteoclastin promotes breast cancer bone metastasis by inducing osteoclastogenesis, and significantly increases in the tissue and serum of patients with breast cancer-bone metastasis, and can predict the risk of patients with bone metastasis, thereby achieving the effect of preventing bone metastasis. Compared with the existing detection kits that can only detect after bone metastasis occurs, the osteoclastin of the present invention can be used as a kit for early prediction of the occurrence of breast cancer bone metastasis, diagnosis of breast cancer bone metastasis, prediction of disease progression, evaluation of treatment effects, guidance of drug use and prognosis evaluation, and as a target for the treatment of bone metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the high expression of osteoclastin in the supernatant of breast cancer cell lines with specific bone metastasis;

[0024] Figure 2 Phase contrast micrographs of osteoclast precursor cells, TRAP staining images, and quantitative results;

[0025] Figure 3 This is a field emission scanning electron micrograph of the cytoskeleton of osteoclast precursor cells;

[0026] Figure 4 This is a graph showing the results of bone resorption assay of osteoclast precursor cells cultured on bone slices;

[0027] Figure 5 The standardized bioluminescence imaging signal (BLI) of bone metastasis and Kaplan-Meier survival curve of mice in the designated experimental groups without bone metastasis;

[0028] Figure 6 Representative mouse bone lesions and histological images;

[0029] Figure 7 This is the result of ELISA analysis of osteoclastin expression in the serum of healthy subjects and patients;

[0030] Figure 8 Representative images of osteoclastin expression in breast tissue and different breast cancer tissues;

[0031] Fig. 9 This is the Kaplan-Meier analysis of the high / low expression of osteoclastin and the bone metastasis-free survival time of breast cancer patients. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0033] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0034] Example 1

[0035] Osteoclastin, a biomarker for diagnosis, treatment and prognosis of breast cancer bone metastasis

[0036] Primary breast cancer cells from patients were isolated using a mouse bone metastasis model for bone metastasis research. Cells were obtained by amplifying tissues from patients with breast cancer bone metastasis in mice, and the cell culture conditions were DMEM containing 10% fetal bovine serum. Osteoclast precursor cells were obtained from normal mouse bone marrow, and the cell culture conditions were 1640 culture medium containing 10% fetal bovine serum.

[0037] The culture medium containing primary breast cancer cells PBCs#2 / #3 / #5 and specific bone metastasis breast cancer cells BMBCs#2 / #3 / #5 was obtained for quantitative mass spectrometry detection and analysis. The results are as follows Figure 1 As shown, Figure a is a volcano analysis diagram of abnormally expressed proteins in the serum of primary breast cancer cells PBCs#2 / #3 / #5 compared with the serum of specific bone metastasis breast cancer cells BMBCs#2 / #3 / #5. It can be seen that compared with the serum of primary breast cancer cells PBCs#2 / #3 / #5, the osteoclastin level in the serum of specific bone metastasis breast cancer cells BMBCs#2 / #3 / #5 is significantly increased, which shows that osteoclastin can be used in the diagnosis of breast cancer bone metastasis.

[0038] The correlation between serum osteoclastin levels and breast cancer bone metastasis in mice was analyzed by ELISA experiments. Blood samples from breast cancer patients were collected into serum separation tubes. After clot formation, the tubes were centrifuged at 2000g for 10 minutes, and serum was collected. The samples were diluted to 25% and measured. To dilute the serum, it was stored below -20°C to avoid repeated freeze-thaw cycles. The serum samples were stored at room temperature for about 1.0h to allow them to be completely thawed.

[0039] The ELISA experiment uses an ELIS kit coated with antibodies for detection and analysis. The preparation of the antibody-coated kit is as follows:

[0040] 1) Coating: Dilute the biotin-coupled anti-human IL-2 antibody to 5 μg / mL with CBS coating solution at pH 9.6, add 50 μL to each well of the ELISA plate, and coat overnight at room temperature, 4°C or 37°C;

[0041] 2) Washing: discard the coating solution (pat dry vigorously, then dry with dust-free paper), wash the plate three times with 1× PBST washing solution at pH 7.2-7.4 (fill each well), each time for 3 min to 5 min;

[0042] 3) Blocking: add 250 μL of blocking solution (1% BSA) to each well and block at 37°C for 2 h.

[0043] Blood samples were tested according to the instructions of the ELISA kit to determine the osteoclastin levels in human serum and breast cancer cell culture medium.

[0044] The results are as follows Figure 1 As shown in b, compared with the serum of mice inoculated with PBCs#2 / #3 / #5 cells in the breast, the level of osteoclastin in the serum of mice inoculated with BMBCs#2 / #3 / #5 cells in situ in the breast was significantly increased, indicating that the level of osteoclastin in serum increases before bone metastasis of breast cancer. It can be seen that osteoclastin in serum can be used for the diagnosis of bone metastasis of breast cancer.

[0045] The osteoclasts of the present invention promote the generation of osteoclasts, thereby promoting the bone metastasis of breast cancer, and can predict the risk of bone metastasis in patients, thereby achieving the effect of preventing bone metastasis. It can be seen from the results and analysis of the following figures:

[0046] Figure 2 Osteoclast precursor cells were treated with recombinant osteoclastin, and then the osteoclasts were stained for TRAP (left) and quantified (right). It can be seen that compared with osteoclast precursor cells treated with BSA, osteoclast precursor cells treated with recombinant osteoclastin showed TRAP + The number of multinucleated cells increased significantly and the TRAP activity increased significantly; these results suggest that osteoclastin plays an important role in osteoclastogenesis.

[0047] Figure 3 The field emission scanning electron micrographs of actin filaments in osteoclast precursor cells show that the density of actin filaments between osteoclasts was increased in the group treated with recombinant osteoclastin; the density of actin filaments between osteoclasts was increased in the group with high osteoclast expression in PBCs#3 cells; while the density of actin filaments between osteoclasts was reduced in the control group. After knocking out osteoclastin in BMCCs#3 cells, the density of actin filaments between osteoclasts was reduced. This result once again proves the important role of osteoclastin in osteoclast differentiation.

[0048] Figure 4The middle is the result of detecting osteoclasts absorbing bone. The bone slices were subjected to scanning electron microscopy / SEM (left) and the number of absorption pits on each bone slice was quantified (right). The results showed that compared with osteoclasts treated with BSA, osteoclasts treated with recombinant osteoclastin promoted osteoclasts to absorb bone. This result proves the ability of osteoclasts to promote osteoclasts to absorb bone.

[0049] Figure 5 The BLI signal of bone metastasis in the designated experimental group and the Kaplan-Meier survival curve of bone metastasis-free mice were normalized. Breast cancer cells were injected into mice by left ventricular injection to observe the metastasis of tumor cells in the mice. The results showed:

[0050] Overexpression of osteoclastin significantly enhanced the bone metastasis ability of PBCs#3 and PBCs#5 cells, which was manifested by earlier bone metastasis, greater bone metastasis tumor burden, more severe osteolytic bone lesions, and significant regulation of bone parameters, including increased trabecular spacing and trabecular pattern factors, and reduced trabecular volume, trabecular number, and trabecular thickness. These results demonstrate that osteoclastin promotes bone metastasis of breast cancer cells.

[0051] Figure 6 Middle, left: μCT (trabecular bone) images and histological (TRAP and ALP staining) images of representative mouse bone sites, BLI, μCT and histological (H&E staining) images of mouse bone lesions after injection of breast cancer cells at the tumor metastasis stage; right: Bone CT parameters, TRAP in bone metastasis + Quantification of osteoclasts and ALP osteoblasts;

[0052] The above results prove that breast cancer cells secrete osteoclasts to promote osteolytic bone metastasis of breast cancer. However, knocking down osteoclasts inhibits the bone metastasis and osteolysis of breast cancer cells, showing no bone metastasis burden and no osteolytic lesions, indicating that targeting osteoclasts is an important node for inhibiting breast cancer bone metastasis.

[0053] Figure 7 The osteoclastin levels in serum of 11 healthy subjects, 19 breast cancer patients without bone metastasis, and 32 breast cancer patients with bone metastasis were analyzed by ELISA. Compared with healthy subjects and breast cancer patients without bone metastasis, the osteoclastin levels in serum of breast cancer patients with bone metastasis were significantly increased.

[0054] Figure 8Representative images (left) and quantification (right) of immunohistochemistry of osteoclastin in normal human breast tissue, breast cancer in situ tissue without bone metastasis, breast cancer in situ tissue with bone metastasis, and breast cancer tissue at the site of bone metastasis. Scale bar 100 μm. The breast cancer tissue of the patient was fixed with formalin and then prepared into paraffin sections for immunohistochemical staining. The results showed that the expression level of osteoclastin in breast cancer tissue with bone metastasis was significantly increased compared with normal breast tissue and breast cancer tissue without bone metastasis.

[0055] Fig. 9 Kaplan-Meier analysis of high / low expression of osteoclastin and bone metastasis-free survival time in breast cancer patients (n=68; P=0.002, log-rank test). The results showed that high levels of osteoclastin significantly promoted bone metastasis of breast cancer.

[0056] Based on the above experimental results and the figures, the osteoclastin of the present invention functions as a biomarker or target for diagnosis, treatment and prognosis of breast cancer bone metastasis.

[0057] Compared with the currently used detection kits which can only detect after bone metastasis occurs, the osteoclastin described in the present invention can more specifically and sensitively predict the occurrence of breast cancer bone metastasis at an early stage, diagnose breast cancer bone metastasis, predict disease progression, evaluate treatment effects, guide drug use and prognosis evaluation, and serve as a target for the treatment of bone metastasis.

Claims

1. Application of osteoclast knockout reagents in the preparation of drugs for the treatment of breast cancer bone metastasis.

2. The use according to claim 1, characterized in that: The gene sequence of osteoclastin is shown in NR_003148.3 of the NCBI gene library.

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