Application of CIR1 as a molecular marker in the diagnosis, treatment and prognosis of lung cancer bone metastasis

Abstract

The present invention discloses the application of CIR1 as a molecular marker in the diagnosis, treatment and prognosis evaluation of lung cancer bone metastasis, and belongs to the field of medical tumor diagnosis and treatment. The present invention found that CIR1 is highly expressed in tissues and cells of lung cancer bone metastasis, and is accompanied by a poor prognosis. In vivo and in vitro experiments confirmed that CIR1 downregulation can significantly inhibit the proliferation and migration ability of lung cancer cells, thereby inhibiting bone metastasis; at the same time, CIR1 downregulation can significantly inhibit the differentiation and maturation of lung cancer cells on osteoclasts, greatly reducing the occurrence of osteolytic destruction. Therefore, the present invention proposes to use a reagent for detecting the expression level of CIR1 in the preparation of a kit for detecting whether lung cancer has bone metastasis and / or a prognostic kit for predicting the tendency of lung cancer bone metastasis; and to use a CIR1 inhibitor in the preparation of a drug for preventing, alleviating and / or treating lung cancer bone metastasis, providing a new idea for the diagnosis, prognosis and treatment of lung cancer bone metastasis.

Description

Technical Field

[0001] The present invention belongs to the field of medical tumor diagnosis and treatment, and specifically relates to the application of CIR1 as a molecular marker in the diagnosis, treatment and prognosis evaluation of lung cancer bone metastasis. Background Art

[0002] Tumor bone metastasis is a common tumor complication, an extremely complex and sophisticated multi-step pathological process that includes a series of events that occur in a strictly sequential order: tumor cells grow and proliferate in the primary lesion and acquire metastatic characteristics, entering the circulatory system; tumor cells tend to move toward bone tissue in the circulatory system; circulating tumor cells colonize in bone tissue; tumor cells colonized in bone tissue adapt to the bone microenvironment and survive, and under specific conditions wake up, proliferate, and ultimately form visible metastatic lesions.

[0003] The mechanisms of bone metastasis are currently well-studied in breast cancer and prostate cancer. Under normal physiological conditions, osteoclast-mediated bone resorption and osteoblast-mediated bone formation in the bone microenvironment are in a dynamic equilibrium. When tumor cells metastasize to this stage, this dynamic equilibrium is disrupted by their involvement. Tumor cells can directly activate osteoclast precursors by secreting osteolytic factors such as PTHrP, IL-11, IL-6, IL-8, TGF-β, TNF, and EGF, and indirectly promote osteoclast differentiation and maturation by manipulating the ratio of OPG and RANKL. This significantly increases bone resorption, causing osteolytic destruction and providing space for further tumor growth. Simultaneously, osteoclasts, under the influence of osteoclasts, release large amounts of growth factors such as TGF-β, IGFs, and calcium from the bone matrix. These factors are then utilized by lung cancer cells in the bone microenvironment to further promote their growth. Tumor cells exploit and modify the bone microenvironment, inducing bone destruction while promoting their own malignant proliferation, thus forming a vicious cycle of bone metastasis.

[0004] Lung cancer is a common malignancy in my country. In its advanced stages, lung cancer can metastasize to various organs, causing corresponding symptoms and often causing significant pain and even life-threatening consequences for patients. The most common metastases are brain metastases and bone metastases. However, our understanding of the regulatory mechanisms underlying bone metastasis remains limited, and we lack rapid and effective non-invasive diagnostic methods and therapeutic intervention targets for evaluating bone metastases. Currently, the diagnosis of lung cancer bone metastasis is primarily based on imaging modalities such as bone scans and CT scans. However, due to radiation damage and economic factors, real-time monitoring is not feasible. Treatments that inhibit bone destruction, such as bisphosphonates and RANKL inhibitors, are often combined with standard cancer treatments to mitigate bone-related events. However, these treatments are not specific for lung cancer bone metastasis, and 30-50% of patients receiving these treatments will still develop new bone metastases. Furthermore, their effectiveness in prolonging survival in patients with lung cancer bone metastases is limited.

[0005] Therefore, this field urgently needs further research and understanding of the regulatory mechanism of lung cancer bone metastasis. Discovering new and efficient lung cancer bone metastasis-specific expression targets is of great significance for the early diagnosis, treatment and prognosis of lung cancer bone metastasis. Summary of the Invention

[0006] In order to address the problems in the field of limited understanding of the mechanism of lung cancer bone metastasis and the lack of non-invasive diagnostic methods and therapeutic intervention targets for rapid and effective evaluation of lung cancer bone metastasis, the purpose of the present invention is to provide a CIR1 as a molecular marker for the diagnosis, treatment and prognosis evaluation of lung cancer bone metastasis. The CIR1 is used as a target for early prediction of the occurrence of lung cancer bone metastasis, diagnosis of lung cancer bone metastasis, prediction of disease progression, evaluation of treatment effects, guidance of drug use and prognosis evaluation.

[0007] Based on the above, the present invention first provides the use of CIR1 as a molecular marker in the diagnosis, treatment and prognosis evaluation of lung cancer bone metastasis.

[0008] On the other hand, the present invention also provides the use of a CIR1 inhibitor in the preparation of a drug for preventing, alleviating and / or treating bone metastasis of lung cancer.

[0009] Preferably, the CIR1 inhibitor is selected from: substances that can fully or partially inhibit the expression of CIR1 gene; or substances that can fully or partially inhibit the function of CIR1 protein.

[0010] Preferably, the substance includes: protein, oligonucleotide, oligonucleotide expression vector, small molecule compound or gene intervention tool capable of knocking out or knocking down CIR1 gene expression.

[0011] Preferably, the CIR1 inhibitor is used to reduce the proliferation and migration ability of lung cancer cells.

[0012] Preferably, the CIR1 inhibitor is used to inhibit the maturation-promoting effect of lung cancer cells on osteoclasts.

[0013] On the other hand, the present invention also provides the use of a reagent for detecting the expression level of CIR1 in the preparation of a reagent or kit for diagnosing and evaluating the prognosis of lung cancer bone metastasis.

[0014] On the other hand, the present invention also provides a kit for diagnosing and / or evaluating the prognosis of lung cancer bone metastasis, which comprises a reagent capable of detecting the expression level of CIR1 protein or mRNA in a sample.

[0015] Preferably, the kit comprises a qRT-PCR, Northern blot, FISH, ISH, Western blot or ELISA kit.

[0016] Preferably, the sample comprises a human blood sample or a tissue sample.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present study found that CIR1 is highly expressed in tissues and cells with lung cancer bone metastasis, which is associated with a poor prognosis. In vitro and in vivo experiments confirmed that downregulating CIR1 significantly inhibited the proliferation and migration of lung cancer cells, thereby suppressing bone metastasis. Furthermore, downregulating CIR1 significantly inhibited the effect of lung cancer cells on the differentiation and maturation of osteoclasts, significantly reducing the occurrence of osteolytic destruction.

[0019] Based on the above findings, the present invention proposes to use a reagent for detecting CIR1 expression levels in the preparation of a kit for detecting whether lung cancer has bone metastasis and / or a prognostic kit for predicting the tendency of lung cancer to bone metastasis; and to use a CIR1 inhibitor in the preparation of a drug for preventing, alleviating and / or treating lung cancer bone metastasis, providing new ideas for the diagnosis, prognosis and treatment of lung cancer bone metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The invention relates to the screening of directional bone metastasis cell lines, wherein

[0021] A: Screening of stable cell lines containing Luciferase;

[0022] B: Establishment of a lung cancer bone metastasis mouse model and screening of bone metastatic lung cancer cells;

[0023] C: Detection of the bone metastasis ability of A549-BM cells.

[0024] Figure 2Indicates the expression of CIR1 in tissues and cells of lung cancer bone metastasis, among which,

[0025] A: qRT-PCR detection of CIR1 mRNA expression level in non-small cell lung cancer cells;

[0026] B: Western blot detection of CIR1 protein expression level in non-small cell lung cancer cells;

[0027] C: Immunohistochemistry was used to detect the expression level of CIR1 in clinical samples and quantitative analysis was performed;

[0028] D: Prognostic analysis of CIR1 expression levels in lung cancer patients.

[0029] Figure 3 It indicates that the loss of CIR1 expression reduces the occurrence of bone metastasis, among which,

[0030] A: Western blot assay to detect the efficiency of CIR1 knockout in H460-BM using CRISPR-Cas9 technology;

[0031] B: Bone metastasis mouse models were established by left ventricular injection (IC injection) or external iliac artery injection (IIA injection) of H460-BM and H460-BM CIR1 KO cells. The effect of CIR1 knockout on the bone metastasis ability of H460-BM cells was analyzed (n = 6 per experimental group). The left image shows representative images of bioluminescence imaging and X-ray (BLI / X-ray) bone metastasis detection 4 weeks after cell injection; the right image shows the quantitative image of bioluminescence imaging intensity.

[0032] C: Immunohistochemical analysis of Ki67 expression in bone metastatic lesions 4 weeks after extrailiac injection of H460-BM and H460-BM CIR1 KO cells;

[0033] D: The effect of CIR1 knockout on the dormancy of early bone-colonizing cancer cells was analyzed using an extrailiac injection bone metastasis model. One week after transplantation of H460-BM cells and H460-BM CIR1 KO cells, immunofluorescence (IF) staining was used to detect CK8+ (red), EdU+ (green, left), or P27+ (green, right) in sections of early bone micrometastases.

[0034] E: High-precision micro-computed tomography (micro-CT) (left) and three-dimensional reconstruction (right) comparison of femoral metastasis lesions formed by H460-BM cells and H460-BM CIR1 KO cells, and calculation of bone volume / tissue volume (BV / TV);

[0035] F: Tartaric acid-resistant acid phosphatase (TRAP) staining marks mature osteoclasts in osteolytic lesions (left), and quantification of TRAP-positive cells at the tumor boundary (right). Arrows indicate TRAP-positive cells; T indicates tumor; B indicates bone tissue.

[0036] Figure 4 It indicates that low expression of CIR1 reduces the occurrence of bone metastasis, among which,

[0037] A: Western blot and qRT-PCR were used to detect the knockdown efficiency of CIR1 in H460-BM;

[0038] B: The number of living cells in H460-BM and H460-BM CIR1 KD cells was detected by MTT assay at 0, 24, 48, 72, and 96 hours;

[0039] C: Using H460-BM cells as a control, the proliferation ability of H460-BM CIR1 KD cells in soft agar was tested, and soft agar colony growth analysis was performed after 2-3 weeks.

[0040] D: Transwell chamber was used to detect the invasion ability of 8000 H460-BM and H460-BM CIR1 KD cells for 12 hours;

[0041] E: 2×10 5 The cells were injected into nude mice through the left ventricle. Four weeks later, the location and intensity of cell fluorescence in the nude mice were detected using small animal in vivo imaging technology.

[0042] F: Immunohistochemistry was performed on metastatic bone tissue sections to detect the expression level of Ki67;

[0043] G: Micro-CT was used to scan the femurs of mice with bone metastasis, perform three-dimensional bone reconstruction, analyze various bone density indicators, and calculate the bone volume / tissue volume (BV / TV);

[0044] H: TRAP staining was performed on metastatic bone tissue sections to detect the degree and number of osteoclast differentiation.

[0045] Figure 5 It indicates that high expression of CIR1 promotes bone metastasis, among which,

[0046] A: Western blot analysis of CIR1 expression levels in H460-BM, H460, and H460 CIR1 OE cells;

[0047] B: Comparison of the bone metastasis capacity of H460 cells and H460 CIR1 OE cells by left ventricular injection and external iliac artery injection (n = 6 per experimental group). Left: Representative images of bioluminescence imaging and X-ray (BLI / X-ray) bone metastasis detection 4 weeks after left ventricular injection and external iliac artery injection of H460 cells or H460 CIR1 OE cells; right: quantification of bioluminescence imaging intensity.

[0048] C: Immunohistochemical analysis of Ki67 expression in bone metastatic lesions 4 weeks after extrailiac injection of H460 and H460 CIR1 OE cells;

[0049] D: The effect of overexpression of CIR1 on the dormancy of early bone-colonizing cells was analyzed using an extrailiac injection bone metastasis model. One week after H460-BM and H460 CIR1 OE cell transplantation, immunofluorescence (IF) staining was used to detect CK8+ (red) and EdU+ (green) in sections of early bone micrometastases.

[0050] E: High-precision micro-computed tomography (micro-CT) (left) and three-dimensional reconstruction (right) comparing femoral metastatic lesions formed by H460 cells and H460 CIR1 OE cells;

[0051] F: TRAP staining marks mature osteoclasts in osteolytic lesions (left), quantification of TRAP-positive cells at the tumor border (right, n=8, 3 mice per group), arrows indicate TRAP-positive cells; T indicates tumor; B indicates bone tissue. DETAILED DESCRIPTION

[0052] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0053] Corepressor interacting with RBPJ (CIR1) was first discovered as an RBPJ-binding protein. It recruits the transcriptional repressor RBPJ to the histone deacetylase complex, shutting down nuclear transcriptional activation of the Notch receptor and negatively regulating the Notch signaling pathway. Other studies have found that in autosomal retinitis pigmentosa, CIR1 acts as an RNA-binding protein, exerting its pathogenic effects by binding to the pathogenic gene PAP-1 and affecting its splicing. Currently, the functional role of CIR1 in biology and pathology remains unknown.

[0054] As previously mentioned, to address the limited understanding of the mechanisms of lung cancer bone metastasis in the field and the lack of rapid and effective non-invasive diagnostic methods and therapeutic intervention targets for evaluating lung cancer bone metastasis, the applicants of the present invention, through extensive screening and research, discovered that CIR1 is highly expressed in tissues and cells with lung cancer bone metastasis, and is associated with a poor prognosis.

[0055] Subsequent in vitro and in vivo experiments confirmed that CIR1 downregulation significantly inhibited the proliferation and migration of lung cancer cells, thereby suppressing bone metastasis. Furthermore, CIR1 downregulation significantly inhibited the effect of lung cancer cells on the differentiation and maturation of osteoclasts, significantly reducing the occurrence of osteolytic destruction. Ultimately, this study provides the first application of CIR1 as a molecular marker in the diagnosis, treatment, and prognosis of lung cancer bone metastasis.

[0056] On the other hand, the present invention also provides the use of a CIR1 inhibitor in the preparation of a drug for preventing, alleviating and / or treating bone metastasis of lung cancer.

[0057] Preferably, the CIR1 inhibitor is selected from: substances that can fully or partially inhibit the expression of CIR1 gene; or substances that can fully or partially inhibit the function of CIR1 protein.

[0058] It is further explained that the substance that can fully or partially inhibit the expression of the CIR1 gene can be achieved by interrupting the transcription of the CIR1 gene and / or blocking the translation of the mRNA of the CIR1 gene; the substance that can fully or partially inhibit the efficacy of the CIR1 protein should at least be understood as a substance that can inhibit the activity, effective duration and stability of the CIR1 protein.

[0059] Preferably, the substance includes: protein, oligonucleotide, oligonucleotide expression vector, small molecule compound or gene intervention tool capable of knocking out or knocking down CIR1 gene expression.

[0060] It is further explained that the genetic intervention tool for knocking out or knocking down the expression of the CIR1 gene can be any intervention tool that can knock out or knock down the CIR1 gene at the genetic level, such as RNAi fragments (such as siRNA, shRNA, miRNA) or gene editing tools (such as CRISPR-Cas9, ZFN, TALENs, etc.).

[0061] Preferably, the CIR1 inhibitor is used to reduce the proliferation and migration ability of lung cancer cells.

[0062] Preferably, the CIR1 inhibitor is used to inhibit the effect of lung cancer cells on promoting differentiation and maturation of osteoclasts.

[0063] On the other hand, the present invention also provides the use of a reagent for detecting the expression level of CIR1 in the preparation of a reagent or kit for diagnosing and / or evaluating prognosis of lung cancer bone metastasis.

[0064] On the other hand, the present invention also provides a kit for diagnosing and / or evaluating the prognosis of lung cancer bone metastasis, which comprises a reagent capable of detecting the expression level of CIR1 protein or mRNA in a sample.

[0065] Preferably, the kit comprises a qRT-PCR, Northern blot, FISH, ISH, Western blot or ELISA kit.

[0066] Preferably, the sample comprises a human blood sample or a tissue sample.

[0067] The following is a further description of the concept of the present invention in conjunction with specific experiments, but the scope of protection of the present invention is not limited thereto.

[0068] The experimental methods and conditions used in the following examples were all performed according to conventional methods or manufacturer's instructions unless otherwise specified. The materials and reagents used in the following experimental examples were all commercially available unless otherwise specified.

[0069] Materials and Methods

[0070] 1. Experimental Materials

[0071] (1) Cell lines: Non-metastatic human lung cancer cell lines A549, H1975, LLC, H1299, and H460 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0072] In addition, bone metastasis-prone cells were screened based on a purchased non-metastatic human lung cancer cell line: A549 cells were injected into nude mice via the left ventricle. After some cells metastasized to the bones, the cancer cells were isolated from the bone tissue and reinjected into the nude mice. After several cycles, the bone metastasis-prone lung cancer cell line BM549 (also designated A549-BM) was obtained. Similarly, bone metastasis-prone lung cancer cell lines BM1975, BM-LLC, BM1299, and H460-BM were screened and obtained.

[0073] It should be noted that, in this application, "H460-BM cells" are also expressed as "BM460 cells" or "SPC cells" and can be used interchangeably.

[0074] (2) Clinical tissue samples: Lung cancer tissue samples and adjacent tissue samples obtained through puncture biopsy and surgery, as well as clinical pathological data, were collected from lung cancer patients at Shanghai Chest Hospital between 2010 and 2011; lung cancer bone metastasis samples and clinical pathological data were collected from spinal bone metastasis lesion resection for pain relief from tumor compression at Zhongshan Hospital Affiliated to Fudan University between 2016 and 2021. The clinical samples and clinical pathological data of lung cancer patients in this study were obtained with the consent of the patients and approval of the ethics committee.

[0075] (3) Experimental animals: BALB / c athymic nude mice, 6–8 weeks old and 18–20 g, were purchased from Shanghai Slack Laboratory Animal Co., Ltd. All mouse experiments in this study were approved by the Animal Care Committee of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences.

[0076] 2. Experimental methods

[0077] (1) Tartrate-fast acid phosphatase (TRAP) staining

[0078] Preparation of TRAP experiment related reagents:

[0079] Solution A (0.1 M acetate buffer, pH 5.0): Dissolve 1.3608 g of sodium acetate (CH3COONa) in 100 mL of distilled water and adjust the pH to 5.0 with glacial acetic acid (CH3COOH);

[0080] Solution B (hexazo parafuchsin): Dissolve 2g of sodium nitrite (NaNO2) in 50mL of deionized water to obtain a 4% sodium nitrite solution. Dissolve 2.5g of parafuchsin (C19H17N3) in 50mL of deionized water. Add 7.5mL of concentrated hydrochloric acid. Heat to 90°C to dissolve. Filter through a 0.22μm filter to obtain the parafuchsin solution. Store in a brown bottle at 4°C. Before use, mix the 4% sodium nitrite and parafuchsin solution in equal proportions to obtain Solution B.

[0081] Solution C: Dissolve and mix 20 mg of naphthol AS-BI phosphate in 1 mL of N,N-dimethylformamide.

[0082] Staining method: After dewaxing and rehydrating bone sections, place sections in TRAP incubation solution (18 mL of Solution A + 1 mL of Solution B + 1 mL of Solution C, mix thoroughly, adjust the pH to 5.0 with 1 M NaOH or 1 M hydrochloric acid, add 0.282 g of sodium potassium tartrate (NaKC4H4O6), dissolve thoroughly, and filter through a 0.22 μm filter to obtain TRAP incubation solution). Incubate at 37°C for 50 minutes. Terminate the reaction when osteoclasts appear wine-red under a microscope. Rinse with distilled water. Stain sections with Harris hematoxylin for 5 minutes, rinse with running water, differentiate with 1% hydrochloric acid-alcohol for several seconds, rinse with running water, then blue with 0.6% ammonia solution, rinse with running water. Dehydrate normally. Clear with xylene three times for 5 minutes each, air dry, mount, and examine under a microscope.

[0083] (2) Construction of a mouse model of lung cancer bone metastasis

[0084] Before the experiment, mice were anesthetized with isoflurane or tribromoethanol, and different mouse tumor models were established according to different methods. For intracardiac injection (IC injection), cancer cells were resuspended in 100 μl of PBS and injected into the left ventricle of the mouse. For extrailiac injection (IIA injection), cancer cells were resuspended in 50 μl of PBS and injected into the external iliac artery of the mouse. After one week, early bone microlesions and tumor dormancy were detected in the mice. After four weeks, bioluminescence imaging and X-ray (BLI / X-ray) were used to monitor the progression of bone metastasis and radiolucent damage of osteolytic lesions.

[0085] After one or four weeks, the mice were killed, and the leg bones were removed and fixed with PFA. Non-bone tissues could be directly embedded and sliced. After micro-CT scanning of bone tissue, it was decalcified for 2 weeks and then embedded and sliced.

[0086] Example 1: Screening and obtaining lung cancer cell lines with directional bone metastasis

[0087] To study the molecular mechanism of lung cancer bone metastasis, this application established a lung cancer bone metastasis mouse model and screened bone metastasis lung cancer cells. First, a lentivirus containing the luciferase gene was used to infect non-small cell lung cancer A549 cells. After several rounds of resistance screening, a stable cell line containing luciferase was obtained ( Figure 1 A). Subsequently, a lung cancer bone metastasis mouse model (left ventricle injection model) was constructed, and the stable cell line containing Luciferase was injected into the nude mouse through the left ventricle using a small animal ultrasound diagnostic instrument. The localization of lung cancer cells in the bone tissue of nude mice was determined by small animal in vivo imaging technology. Afterwards, bone metastasis lung cancer cells A549-BM were successfully screened using the left ventricle injection model. Specifically, A549 (Luciferase) cells were injected into the left ventricle of mice, and the localization of lung cancer cells was determined by small animal in vivo imaging technology. Lung cancer cells located in bone tissue were isolated and then injected into the left ventricle of mice again after in vitro screening and culture. After 10 cycles, all A549 cells injected into nude mice were located in bone tissue, and they were defined as bone metastasis lung cancer cells A549-BM ( Figure 1 B).

[0088] Finally, this example verified the bone metastasis ability of A549-BM. Using the left ventricular injection model, A549 cells and A549-BM cells were injected into the left ventricle of mice, respectively. After 30 days, in vivo imaging of small animals showed that metastatic lesions were detected in the limb joints of mice in the A549-BM cell group. Micro-CT examination found that the nude mice injected with A549-BM cells had more severe bone destruction ( Figure 1C), suggesting that A549-BM has specific bone metastasis potential and can form lesions similar to human lung cancer bone metastasis; the left ventricular injection lung cancer bone metastasis mouse model can well simulate the occurrence process of lung cancer bone metastasis, providing a cell and animal model for the subsequent work of this project.

[0089] Referring to the method of this example, bone metastatic lung cancer cells BM1975, BM-LLC, BM1299 and H460-BM were screened and obtained respectively.

[0090] Example 2: Analysis of CIR1 as a marker in the diagnosis, treatment and prognosis of lung cancer bone metastasis

[0091] (I) CIR1 is highly expressed in lung cancer bone metastasis tissues and cells

[0092] In the non-small cell lung cancer bone metastasis type lung cancer cells BM549, BM1975, BM-LLC and their corresponding parent cells A549, H1975, LLC screened in Example 1, the mRNA and protein expression levels of CIR1 were detected by qRT-PCR and Western blotting techniques, respectively. Figure 2 As shown in Figures AB, the mRNA and protein expressions of CIR1 in bone metastatic lung cancer cells were significantly increased compared with those in the parent cells without bone metastasis.

[0093] In addition, the protein expression of CIR1 was detected in the clinical tissue samples collected in this application by immunohistochemical staining. Figure 2 As shown in Figure C: Compared with adjacent adjacent tissues and in situ lung cancer tissues, the protein expression of CIR1 in lung cancer bone metastasis tissue samples was significantly increased.

[0094] These results indicate that CIR1 is highly expressed in lung cancer bone metastasis tissues and cells. Therefore, CIR1 expression levels can be used to diagnose lung cancer bone metastasis. For example, the mRNA or protein expression level of CIR1 in a lung cancer tissue sample can be used as an indicator, or the mRNA or protein expression level of CIR1 in a lung cancer tissue sample can be compared with that of a control tissue sample. If the indicator exceeds a set threshold, bone metastasis is determined.

[0095] (II) High expression of CIR1 indicates short survival time

[0096] Based on the TCGA database, lung cancer patients in the database were divided into CIR1 high expression group and CIR1 low expression group, and further combined with the patients' prognosis and survival time analysis, the results showed that compared with the CIR1 low expression group, lung cancer patients in the CIR1 high expression group showed a worse prognosis ( Figure 2 D).

[0097] Because there is no medical information in the database on whether lung cancer patients have bone metastasis, but based on some previous and subsequent studies of this application, this application can reasonably speculate that high expression of CIR1 indicates a greater risk of bone metastasis and a shorter survival time.

[0098] Example 3: Loss of CIR1 expression reduces the occurrence of bone metastasis

[0099] To further verify whether CIR1 plays a role in bone metastasis, this application used CRISPR-Cas9 technology to construct H460-BM CIR1KO cells with stable CIR1 gene knockout in H460-BM cells, and Western blot experiments verified the knockout efficiency ( Figure 3 A). Using the method for constructing an animal model of lung cancer bone metastasis described above, H460-BM cells and H460-BM CIR1 KO cells were injected into the left ventricle and external iliac artery of mice, respectively. A series of subsequent analyses, including monitoring of bone metastasis progression and bone damage detection, were performed.

[0100] The results are as follows Figure 3 As shown: One week after H460-BM CIR1 KO cells (CIR1 KO in the figure) and H460-BM cells (CIR1 WT in the figure) were transplanted, immunofluorescence assays revealed that compared with the CIR1 WT group, the nucleoside analogue Edu intake in the CIR1 KO group was significantly reduced, and the expression of the cell dormancy marker P27 was significantly increased ( Figure 3 D); 4 weeks later, BLI / X-ray imaging showed that in both bone metastasis mouse models, H460-BM cells could metastasize to the bone, while H460-BM CIR1KO cells lost the ability to metastasize to the bone ( Figure 3 B); further, immunohistochemical experiments were used to detect the expression of cell proliferation marker Ki67. The results showed that the proliferation ability of H460-BM cells with CIR1 knockout (i.e., H460-BM CIR1 KO cells) in bone tissue was also inhibited to a certain extent ( Figure 3 C).

[0101] In addition, we performed micro-CT analysis on the femur of mice and quantitative analysis of bone volume fraction. The results showed that compared with H460-BM cells, the bone destruction ability of H460-BM CIR1 KO cells was significantly weakened ( Figure 3 E); TRAP staining showed that H460-BM could promote the differentiation and maturation of osteoclasts, but after knocking out the CIR1 gene, the promoting effect disappeared ( Figure 3 F).

[0102] The above experimental results show that knocking out the CIR1 gene can promote lung cancer cells to enter a dormant state in the early stage, and then inhibit the directional bone metastasis ability and bone destruction ability of lung cancer cells, ultimately reducing the probability of bone metastasis of lung cancer cells.

[0103] Example 4: Low expression of CIR1 reduces the occurrence of bone metastasis

[0104] In this example, shRNA was used to stably reduce the expression of CIR1 in H460-BM cells to obtain H460-BM CIR1 KD cells. Western blot and qRT-PCR experiments verified the knockdown efficiency ( Figure 4 Subsequently, the malignancy of H460-BM (CIR1 WT in the figure) and H460-BM CIR1 KD (CIR1 KD in the figure) cells was identified by MTT assay. The results showed that the proliferation ability of CIR1KD lung cancer cells was significantly inhibited ( Figure 4 Low density experiments showed that the ability of CIR1 KD lung cancer cells to form clones was significantly inhibited ( Figure 4 C); Transwell experiments found that the invasion ability of CIR1 KD lung cancer cells was significantly inhibited ( Figure 4 D).

[0105] Furthermore, in this example, H460-BM cells and H460-BM CIR1 KD cells were injected into the left ventricle and external iliac artery of mice, respectively. Four weeks later, BLI / X-ray imaging showed that after CIR1 knockdown, the colonization and proliferation ability of tumor cells in bone tissue was significantly weakened ( Figure 4 E); Immunohistochemical experiments were used to detect the expression of cell proliferation marker Ki67. The results showed that after CIR1 knockdown, the proliferation ability of tumor cells in bone tissue was also inhibited to a certain extent ( Figure 4 G); Further, micro-CT analysis of the femur of mice showed that the bone destruction ability of CIR1 KD mice was significantly weakened compared with that of CIR1 WT group ( Figure 4 F); TRAP staining was performed on bone tissue sections to detect the number of osteoclasts. The results showed that after CIR1 knockdown, the number of mature osteoclasts at the interface between tumor cells and bone tissue was significantly reduced ( Figure 4 This suggests that CIR1 may be involved in regulating the effect of lung cancer cells on promoting the differentiation and maturation of osteoclasts.

[0106] The above experimental results collectively show that knocking down CIR can inhibit the proliferation and migration ability of lung cancer cells, while inhibiting the differentiation and maturation effects of lung cancer cells on osteoclasts and reducing bone damage ability.

[0107] Example 5: High expression of CIR1 promotes bone metastasis

[0108] In this example, a cell line H460-CIR1OE, which stably and highly expresses CIR1, was constructed from parental H460 cells. Western blot analysis was performed to compare the expression of CIR1 in H460-CIR1OE cells with that in H460-BM cells. The results showed that the expression of CIR1 in H460 CIR1 OE cells was slightly higher than that in H460-BM cells, but the difference was not significant ( Figure 5 A), which can be used for subsequent experiments.

[0109] H460 and H460-CIR1 OE cells were injected into nude mice via left ventricular injection and external iliac artery injection, respectively. One week later, immunofluorescence analysis revealed that compared with the control group (i.e., H460 cell-implanted group), the mice with high CIR1 expression (i.e., CIR1 OE group) had significantly increased Edu intake and significantly decreased P27 expression, a cell dormancy marker, in the CIR1 OE group. Figure 5 D). After 4 weeks, BLI / X-ray imaging showed that H460-CIR1 OE cells showed stronger bone tissue colonization and proliferation abilities compared with H460 cells ( Figure 5 B); Immunohistochemistry revealed that high expression of CIR1 could promote the expression of Ki67, a cell proliferation marker, in lung cancer cells ( Figure 5 C); Micro-CT analysis of mouse femurs and quantitative bone volume fraction showed that H460-CIR1 OE cells had significantly enhanced bone destruction ability compared with H460 cells ( Figure 5 TRAP staining also found that after high expression of CIR1, H460 cells can significantly promote the differentiation and maturation of surrounding osteoclasts ( Figure 5 F).

[0110] The above experimental results collectively indicate that CIR1 has the ability to promote bone metastasis of lung cancer.

[0111] In summary, after extensive research and experimental verification, the present invention ultimately proposes the use of reagents for detecting CIR1 expression levels in the preparation of kits for detecting whether lung cancer has bone metastasis and / or prognostic kits for predicting the tendency of lung cancer to bone metastasis; and the use of CIR1 inhibitors in the preparation of drugs for preventing, alleviating and / or treating lung cancer bone metastasis, providing new ideas for the diagnosis, prognosis and treatment of lung cancer bone metastasis.

[0112] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. Application of a reagent for detecting CIR1 expression level in the preparation of a diagnostic kit for lung cancer bone metastasis.

Images