Use of col10a1 and kits

Abstract

The application belongs to the technical field of biology and particularly relates to application and a kit of CoL10A1. CoL10A1 is highly expressed in BC tissues and lowly expressed in normal tissues, CoL10A1 is highly expressed at mRNA and protein levels in BCBM tissues, therefore, application of a reagent for detecting CoL10A1 expression in preparation of a breast cancer brain metastasis diagnosis product is proposed, and the diagnosis product can also be used for diagnosing breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of CoL10A1 and its reagent kit. Background Technology

[0002] Breast cancer (BC) is one of the most common cancers among women worldwide, ranking among the top malignant tumors in both incidence and mortality. Early-stage breast cancer patients often experience distant metastases, with the brain being one of the most common sites. Brain metastases also occur in HER2-positive and triple-negative breast cancer (TNBC) patients. While advancements in radiotherapy, chemotherapy, targeted therapy, and immune checkpoint inhibitors have significantly improved the overall survival of patients with advanced breast cancer, survival for patients with breast cancer brain metastases (BCBM) has not improved significantly, and their prognosis remains poor. Therefore, accurate prognostic prediction models are urgently needed to address the survival concerns of BCBM patients and help optimize their treatment strategies.

[0003] The molecular mechanisms involved in brain metastases (BCBM) include cancer cell survival, migration, and adhesion; the role of the blood-brain barrier in extravasation; and the interaction between cancer cells and intrinsic brain cells. Current treatment options for BCBM include surgery, whole-brain radiotherapy, stereotactic radiotherapy, and local therapies such as chemotherapy, with the specific choice depending on clinical presentation and the number of brain metastases. However, due to the limited permeability of the blood-brain barrier, chemotherapy resistance, and the heterogeneity between primary breast cancer and BCBM, the prognosis for patients with BCBM remains poor. The complexity of BCBM underscores the need to develop new methods for identifying breast cancer brain metastases. Summary of the Invention

[0004] To address the above problems, this invention provides applications of CoL10A1 and a kit.

[0005]

[0006] Preferably, the diagnostic product includes reagents used in qPCR or Western blotting methods.

[0007] Preferably, the reagents used in the qPCR method include sequences as shown in SEQ ID NO.7-8.

[0008] A kit comprising the reagent for detecting CoL10A1 expression as described in claim 1.

[0009] The application of the reagent that inhibits the expression of CoL10A1 in the preparation of a product for the treatment of brain metastases in breast cancer.

[0010] Preferably, the reagent for inhibiting CoL10A1 expression is the sequence shown in SEQ ID NO.1-2, SEQ ID NO.3-4 or SEQ ID NO.5-6.

[0011] Preferably, the reagent for inhibiting CoL10A1 expression is the sequence shown in SEQ ID NO.3-4.

[0012] Preferably, the reagent for inhibiting CoL10A1 expression is the sequence shown in SEQ ID NO.5-6.

[0013] Preferably, the therapeutic product inhibits breast cancer.

[0014] Preferably, the therapeutic product inhibits the growth of brain metastases.

[0015] Compared with the prior art, the advantages of the present invention are:

[0016] This invention relates to the high expression of CoL10A1 in breast cancer (BC) tissues and low expression in normal tissues. CoL10A1 is highly expressed at both the mRNA and protein levels in BCBM tissues. Therefore, this invention proposes the application of reagents for detecting CoL10A1 expression in the preparation of diagnostic products for breast cancer brain metastases, and these diagnostic products can also diagnose breast cancer.

[0017] This invention discovered that CoL10A1 can promote the proliferation, migration, and invasion of BC cells. Using a constructed cancer brain metastasis model, the location of brain metastases in the brain was shown. The growth of brain metastases in the CoL10A1 knockout group was significantly slower. Therefore, the application of the reagent that inhibits the expression of CoL10A1 in the preparation of breast cancer brain metastasis treatment products is proposed. Attached Figure Description

[0018] Figure 1The results show that CoL10A1 is highly expressed in BCBM and BC tissues. A shows the mRNA expression of CoL10A1 in BMBC tissues (n=19) and control tissues (n=5) detected by qPCR. B shows the protein expression of CoL10A1 in BMBC samples (n=19) and control tissues (n=5) detected by Western Blot. C shows the mRNA expression of CoL10A1 in BC (n=4) and adjacent normal tissues (n=4) detected by qPCR. D shows the protein expression of CoL10A1 in BC samples (n=4) and adjacent normal tissues (n=4) detected by Western Blot. *p<0.05, ***p<0.001.

[0019] Figure 2 The expression of CoL10A1 in BC cells is shown. A is the expression of CoL10A1 mRNA in BC cells detected by qPCR; B is the protein expression of CoL10A1 in BC cell lines detected by Western Blot; C is the knockdown efficiency of CoL10A1 in MDA-MB-468 cells (siNC, siRNA-1, siRNA-2, siRNA-3) detected by qPCR; D is the knockdown efficiency of CoL10A1 in MDA-MB-468 cells (siNC, siRNA-1, siRNA-2, siRNA-3) detected by Western Blot. Compared with the control group, *p<0.05, ***p<0.001.

[0020] Figure 3 The results show that knockdown of CoL10A1 inhibits the proliferation of MDA-MB-468 cells. A is cell proliferation assay using CCK-8, ***p<0.001. B is the expression of proliferation-related proteins (PCNA, c-MYC, and Ki67) detected by Western blotting. C is the quantitative analysis of proliferation-related proteins, ***p<0.001. D is the cell proliferation assay using a plate colony assay.

[0021] Figure 4 The results show that knocking down CoL10A1 inhibits the migration and invasion of MDA-MB-468 cells. A represents cell migration detected by scratch assay (100×), B represents cell migration detected by Transwell assay, C represents quantitative analysis of cell migration rate, **p<0.01, ***p<0.001, D represents quantitative analysis of cell migration rate, *p<0.05, ***p<0.001.

[0022] Figure 5This shows that knocking down CoL10A1 inhibits the ability of MDA-MB-468 cells to cross endothelial cells. A represents the ability of MDA-MB-468 cells to cross the hCMEC / D3 cell layer as detected by Transwell (100×). MDA-MB-468 cells are stained red with CM-DIL, and cell nuclei are stained blue with DAPI. B represents the quantitative analysis of the number of cells that crossed the hCMEC / D3 cell layer. *p<0.05, ***p<0.001.

[0023] Figure 6 The results show that the EMT signaling pathway is involved in CoL10A1-induced migration and invasion of MDA-MB-468 cells. A shows the detection of EMT signaling pathway-related proteins by Western Blot, and B shows the quantitative analysis of EMT signaling pathway-related proteins. **p<0.01, ***p<0.001.

[0024] Figure 7 The image shows the inhibition of tumor formation in mice by CoL10A1 knockdown. Image A represents brain metastases from the shNC group. In the image, 1 represents 1.553e+05, 2 represents 1.485e+06, 3 represents 2.817e+06, 4 represents 4.149e+06, 5 represents 5.479e+06, 6 represents 6.811e+06, 7 represents 8.143e+06, 8 represents 9.473e+06, 9 represents 1.080e+07, 10 represents 1.214e+07, and 11 represents 1. Image 347e+07, B represents a representative image of brain metastases in the shCoL10A1 group, where 1 represents 9.993e+05, 2 represents 5.899e+06, 3 represents 1.080e+07, 4 represents 1.570e+07, 5 represents 2.060e+07, 6 represents 2.550e+07, 7 represents 3.040e+07, 8 represents 3.530e+07, 9 represents 4.020e+07, 10 represents 4.510e+07, and 11 represents 5.000e+07. Detailed Implementation

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0026] CoL10A1, or Collagen type X alpha 1 chain, is a gene encoding the X-type collagen alpha 1 chain. It plays a crucial role in the human body, serving as a specific marker for mast chondrocytes and being essential for endochondrial bone formation. During skeletal development, CoL10A1 expression is closely related to chondrocyte proliferation and hypertrophic differentiation. CoL10A1 expression is upregulated in various malignant tumors, including lung cancer, gastric cancer, and pancreatic cancer, and is associated with poor prognosis. High CoL10A1 expression is associated with tumor progression, invasion, and metastasis. CoL10A1 promotes tumor cell proliferation, invasion, and migration by activating multiple signaling pathways, such as the TGF-β1 / Smad, MEK / ERK, and focal adhesion kinase signaling pathways. CoL10A1 can activate the epithelial-mesenchymal transition (EMT) signaling pathway, which plays a key role in tumor invasion and metastasis. As mentioned above, CoL10A1 plays a key role in tumor development, but its role in brain metastasis of breast cancer remains unclear.

[0027] The experiments involved in this invention are performed according to the following procedures:

[0028] (1) Cell proliferation experiment

[0029] Cells from the control group and the CoL10A1 knockdown group (5 × 10⁻⁶ cells) were respectively divided into two groups. 3 Cells were seeded in 96-well plates and incubated for 2 hours at 0, 24, 48, and 72 hours with 10 µL of CCK-8 (HY-K0301, MCE). Absorbance was measured at 450 nm. The effect of CoL10A1 knockdown on cell colony formation ability was examined using a plate colony assay.

[0030] (2) Cell migration experiment

[0031] Cells from the control group and the CoL10A1 knockdown group (5 × 10⁻⁶ cells) were respectively divided into two groups. 5 Cells were seeded into 6-well plates. The next day, when the cell density reached approximately 90%, cells were streaked using a 10µL pipette tip, keeping the tip as vertical as possible. The cells were washed three times with PBS to remove the streaked cells, and serum-free medium was added for further culture. Photos were taken at 0h and 24h.

[0032] (3) Transwell experiment

[0033] Cells from the control group and the CoL10A1 knockdown group (5 × 10⁶ cells) were used to divide the cells into two groups. 4Cells were seeded in the upper chamber of a Transwell (3422, Costa) cell culture medium, and 600 µL of complete culture medium was added to the lower chamber for further culture. After 24 hours, the culture medium was discarded, the upper chamber was removed, and the cells on the back of the upper chamber were gently wiped away with a cotton swab. The cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS, and stained with crystal violet for 30 minutes. The cells were then observed and photographed under a microscope.

[0034] For cell invasion assays, hCMEC / D3 (1×10⁻⁶) was used. 5 Cells were seeded in the upper chamber of a transwell, and 600 µL of complete culture medium was added to the lower chamber. The cells were then incubated in an incubator. On day 2, control and CoL10A1 knockdown groups (5 × 10⁶ cells / year) were seeded separately. 4 The cells were stained with CM-DIL (1973357, Invitrogen) according to the manufacturer's instructions. The treated cells were then seeded onto an hCMEC / D3 cell layer. After 48 hours, the culture medium was discarded, the upper chamber was removed, and the cells on the reverse side of the upper chamber were gently wiped away with a cotton swab. The cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS, and stained with DAPI for 30 minutes. The cells were then observed and photographed under a fluorescence microscope.

[0035] Example 1

[0036] CoL10A1 expression in BCBM and BC tissues

[0037] RNA and protein were extracted from BMBC tissues and BC cells using RNAiso Plus (9108, Takara) and protein lysis buffer (01771197, Yaxin), respectively. The levels of CoL10A1 RNA and protein were detected using qPCR and Western Blot. The results are as follows: Figure 1 As shown.

[0038] The primer sequences for qPCR are as follows:

[0039] CoL10A1-F: 5'-TTTGGGGTTTCGCCCTATCC-3', designated as SEQ ID NO.7

[0040] CoL10A1-R: 5'-CTGGGGGTGAGGGGACTAAA-3', denoted as SEQ ID NO.8;

[0041] Actin-F: 5'-CCTTCCTTCCTGGGCATGG-3', denoted as SEQ ID NO.9;

[0042] Actin-R: 5'-GATCTTCATTGTGCTGGGTGC-3', denoted as SEQ ID NO.10.

[0043] The reaction system (20µL) is shown in Table 1:

[0044] Table 1 Reaction System

[0045]

[0046] The PCR cycle is shown in Table 2:

[0047] Table 2 PCR Cycles

[0048]

[0049] To verify the expression of CoL10A1, this invention further validated this result using clinical samples. The results showed that CoL10A1 expression in breast cancer tissues was higher than in adjacent normal tissues, such as... Figure 1 CD.

[0050] Furthermore, this invention analyzed CoL10A1 expression in BCBM samples and control samples. The results showed that CoL10A1 was highly expressed at both the mRNA and protein levels in BCBM tissues, such as... Figure 1 AB.

[0051] This invention analyzed the expression of CoL10A1 in breast cancer cell lines MCF7, MDA-MB-231, MDA-MB-453, MDA-MB-468, BT474, HS578T, and normal human breast epithelial cells MCF10A. Figure 2 As shown in AB. The results showed that, except for BT474 cells and MDA-MB-231 cells, the mRNA expression of CoL10A1 in other breast cancer cells was higher than that of MCF10A. In addition, the protein expression of CoL10A1 in breast cancer cells was also higher than that of MCF10A.

[0052] These results indicate that CoL10A1 is highly expressed in BCBM tissues.

[0053] Example 2

[0054] Biological functions of CoL10A1 in BC cells

[0055] The expression of CoL10A1 in in vitro cultured BC cell line MDA-MB-468 was inhibited by siRNA silencing technology. Cell RNA and protein were extracted, and the expression level of CoL10A1 was analyzed and verified by qPCR and Western Blot.

[0056] 1. siRNA silencing technology

[0057] Logarithmic growth phase MDA-MB-468 cells were seeded into 6-well plates and cultured. On the second day, when the cell density reached 60-70%, transfection was performed. EP tubes containing siRNA powder were centrifuged at 5000 rpm for 1 minute at 4°C. DEPC water was added to dissolve the siRNA powder according to the instructions, and the tubes were then placed on ice. The transfection complex was prepared as follows: tube ① 125µL Opti-MEM + 5µL Lip3000, tube ② 125µL Opti-MEM + 5µL siRNA. Tubes ① and ② were gently inverted to mix. The diluted reagent from tube ② was added to tube ① and incubated at room temperature for 15 minutes. The complex from tubes ① and ② was then added to the 6-well plates and cultured for 8-12 hours. The culture medium was replaced with complete cell culture medium and cultured for another 8-12 hours. RNA was extracted after 24 hours for q-PCR to assess interference efficiency, and protein was extracted after 48 hours for Western blotting or cell function assays.

[0058] The siRNA sequence is as follows:

[0059] siRNA-1 (1828): SS Seq: 5'-GCAACAGCAUUAUGACCCA-3', denoted as SEQ ID NO.1, ASSeq: 5'-UGGGUCAUAAUGCUGUUGC-3', denoted as SEQ ID NO.2;

[0060] siRNA-2 (228): SS Seq: 5'-CCUACACCAUAAAGAGUAA-3', denoted as SEQ ID NO.3, ASSeq: 5'-UUACUCUUUAUGGUGUAGG-3', denoted as SEQ ID NO.4;

[0061] siRNA-3 (2013): SS Seq: 5'-CCAUCAUCGAUCUCACAGA-3', denoted as SEQ ID NO.5, ASSeq: 5'-UCUGUGAGAUCCAUGAUGG-3', denoted as SEQ ID NO.6.

[0062] When used, SEQ ID NO.1 to SEQ ID NO.6 of the present invention have a TT base attached to the 3' end to increase stability.

[0063] This invention transfected MDA-MB-468 cells with three different siRNAs (siRNA-1, siRNA-2, and siRNA-3). The results showed that these siRNAs significantly reduced the expression of CoL10A1 at both the mRNA and protein levels in MDA-MB-468 cells. Figure 2 The CD results showed that siRNA silencing technology could inhibit the expression of CoL10A1 in MDA-MB-468 cells, indicating that siRNA silencing was successful.

[0064] 2. Knockdown of CoL10A1 inhibits breast cancer cell proliferation.

[0065] CCK8 assay was used to study the effect of CoL10A1 knockout on cell proliferation. The results are as follows: Figure 3 As shown, the results indicated that CoL10A1 knockout inhibited the proliferation of MDA-MB-468 cells compared to the siNC group (p<0.001). This invention also analyzed the expression of proliferation-related proteins such as PCNA, c-MYC, and Ki67. The results showed that the expression of proliferation proteins was significantly reduced in the CoL10A1 knockout group. Colony formation assays were used to detect the clonogenic ability of MDA-MB-468 cells, and the results showed that clonogenic ability was inhibited in the CoL10A1 knockout group. This invention found that CoL10A1 knockout inhibits the clonogenic ability of MDA-MB-468 cells.

[0066] The above research results indicate that CoL10A1 plays an important role in the proliferation of breast cancer cells.

[0067] 3. Knocking down CoL10A1 inhibits breast cancer cell migration and invasion.

[0068] Further verification of the role of CoL10A1 in the migration and invasion of breast cancer cells yielded the following results: Figure 4 As shown in the figure. Scratch assays were used to examine the effect of CoL10A1 on the migration of MDA-MB-468 cells. The results showed that, compared with the siNC group, transfection with siRNA significantly reduced the migration of MDA-MB-468 cells (**p<0.01; **p<0.001). Transwell assays indicated that, compared with the control group, the cell migration rate in the siRNA transfection group was significantly reduced (*p<0.05; ***p<0.001). Downregulation of CoL10A1 can inhibit the migration and invasion of breast cancer cells.

[0069] 4. CoL10A1 affects blood-brain barrier permeability and tumor cell invasion.

[0070] Further verification of CoL10A1's ability to penetrate the blood-brain barrier in breast cancer cells yielded the following results: Figure 5As shown in the diagram, a layer of hCMEC / D3 cells was first seeded in the upper chamber of a Transwell chamber. After 24 hours, CM-DIL-stained MDA-MB-468 cells were added to the hCMEC / D3 cell layer and cultured in an incubator. After 48 hours, the number of MDA-MB-468 cells that passed through the cell layer was counted under a fluorescence microscope. The results showed that transfection with siRNA significantly reduced the ability of MDA-MB-468 cells to pass through hCMEC / D3 cells. Knockdown of CoL10A1 inhibited the migration and invasion of breast cancer cells.

[0071] 5. CoL10A1 regulates cell migration and invasion through the EMT signaling pathway.

[0072] EMT not only participates in normal developmental processes but also plays a crucial role in tumor metastasis and invasion. Previous studies have shown that the EMT signaling pathway is involved in regulating cell migration and invasion in various cancer types, such as glioblastoma, carcinoma, and leukemia. GSEA was used to evaluate pathways associated with CoL10A1 in the TCGA database. Results showed that CoL10A1 expression is related to the ECM receptor interaction signaling pathway. Ander-Levchenko's research found that changes in the ECM-like cell adhesion matrix can regulate EMT processes. Therefore, this invention hypothesizes that CoL10A1 may regulate BC cell migration and invasion through the EMT signaling pathway. This invention evaluates whether the EMT signaling pathway can regulate CoL10A1-regulated BC cell migration and invasion. The expression of EMT signaling pathway-related proteins, such as E-Cadherin, β-catenin, N-Cadherin, Vimentin, Slug, Snail, and ZEB1, was detected. The results showed increased expression of β-catenin and E-cadherin, and decreased expression of N-cadherin, vimentin, slug, snail, and ZEB1. These results indicate that CoL10A1 regulates cell migration and invasion through the EMT signaling pathway, such as... Figure 6 As shown.

[0073] Example 3

[0074] The effect of CoL10A1 on tumorigenesis in mice was that downregulation of CoL10A1 inhibited tumorigenesis in vivo.

[0075] This invention has demonstrated that CoL10A1 can promote the proliferation, migration, and invasion of BC cells. Furthermore, this invention has examined whether CoL10A1 has a pro-tumor effect in animal models. A cancer brain metastasis model was constructed by intracarotid artery injection of cells.

[0076] (1) Injection via the internal carotid artery

[0077] Female C57BL / 6N mice (6-8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were randomly divided into two groups: a 4T1 cell group (shNC) and a CoL10A1 knockdown 4T1 cell group (shCoL10A), with 3 mice in each group. Mice were anesthetized and fixed on rubber plates. 100 µL of cell suspension was slowly injected into the arterial lumen, and the mice's condition was monitored.

[0078] (2) IVIS imaging

[0079] Each mouse was intraperitoneally injected with 150 mg / kg D-luciferin (HY-12591, MCE). Five minutes later, the mice were anesthetized, and bioluminescence imaging was performed using an IVIS200. The exposure time was set to 20 seconds, and the light intensity was set to 80%. The light signals generated in vivo by luciferase and substrate were detected by the IVIS system to quantitatively analyze biological processes. Data were analyzed using IVIS software (IVIS in vivo imaging software).

[0080] Stable CoL10A1 knockout from 4T1 cells was injected into nude mice, revealing the location of tumors in the brain. The CoL10A1 knockout group showed significantly slower growth. Figure 7 As shown in the figure. The results indicate that CoL10A1 can promote tumor growth in a nude mouse model.

[0081] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0082] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0083] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a reagent that inhibits CoL10A1 expression in the preparation of a therapeutic product for brain metastases in breast cancer, characterized in that, The reagent used to inhibit CoL10A1 expression is siRNA-2 or siRNA-3; the sequence of siRNA-2 is shown in SEQ ID NO.3-4, and the sequence of siRNA-3 is shown in SEQ ID NO.5-6.

2. The application according to claim 1, characterized in that, The reagent used to inhibit CoL10A1 expression is siRNA-2.

3. The application according to claim 1, characterized in that, The reagent used to inhibit CoL10A1 expression is siRNA-3.

4. The application according to claim 1, characterized in that, The treatment product inhibits breast cancer.

5. The application according to claim 1, characterized in that, The therapeutic product inhibits the growth of brain metastases.

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