Application of CoL10A1 and kit

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of CoL10A1 and a kit. The CoL10A1 has high expression in BC tissue, low expression in normal tissue and high expression in mRNA and protein level in BCBM tissue, so that the application of a reagent for detecting CoL10A1 expression in preparation of a breast cancer brain metastasis diagnosis product is provided, and the diagnosis product can also diagnose breast cancer.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of CoL10A1 and a kit. Background Art

[0002] Breast cancer (BC) is one of the most common cancers in women worldwide, and the incidence and mortality of breast cancer are among the highest among malignant tumors. Early breast cancer patients will develop distant metastases, with the brain being one of the most common sites of metastasis. Brain metastases also occur in HER2-positive and triple-negative breast cancer (TNBC) patients. With the development of drugs such as chemoradiotherapy, targeted therapy, and immune checkpoint inhibitors, the overall survival time of patients with advanced breast cancer has been significantly prolonged, but the survival time of patients with breast cancer brain metastasis (BCBM) has not been significantly improved, and the prognosis remains poor. Therefore, there is an urgent need for accurate prognostic prediction models to address BCBM patients' concerns about survival and help optimize their treatment strategies.

[0003] The molecular mechanisms involved in BCBM include cancer cell survival, migration, and adhesion, the role of the blood-brain barrier in extravasation, and the interaction between cancer cells and resident cells in the brain. Current treatment options for BCBM include local treatments such as surgery, whole-brain radiotherapy, stereotactic radiotherapy, and chemotherapy, and the specific choice depends on the clinical manifestations 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 of BCBM patients remains poor. The complexity of BCBM suggests that it is necessary to develop new ways to judge breast cancer brain metastasis. Summary of the invention

[0004] In order to solve the above problems, the present invention provides the application and kit of CoL10A1.

[0005]

[0006] Preferably, the diagnostic product comprises reagents used in a qPCR method or reagents used in a Western Blot method.

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

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

[0009] The invention relates to the use of the reagent for inhibiting the expression of CoL10A1 in the preparation of a product for treating brain metastasis of breast cancer.

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

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

[0012] Preferably, the reagent for inhibiting the expression of CoL10A1 is a 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 present invention is beneficial in that: The present invention shows that CoL10A1 is highly expressed in BC tissues and lowly expressed in normal tissues. CoL10A1 is highly expressed in BCBM tissues at the mRNA and protein levels. Therefore, the use of a reagent for detecting CoL10A1 expression in the preparation of a diagnostic product for breast cancer brain metastasis is proposed, and the diagnostic product can also diagnose breast cancer.

[0016] The present invention finds that CoL10A1 can promote the proliferation, migration and invasion of BC cells. The constructed cancer brain metastasis model shows the location of brain metastases in the brain. The growth of brain metastases in the CoL10A1 knockout group is significantly slow. Therefore, the use of an agent that inhibits the expression of CoL10A1 in the preparation of a product for the treatment of breast cancer brain metastasis is proposed. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The results showed that CoL10A1 was highly expressed in BCBM and BC tissues. A was the qPCR detection of CoL10A1 mRNA expression in BMBC tissues (n=19) and control tissues (n=5). B was the Western Blot detection of CoL10A1 protein expression in BMBC samples (n=19) and control tissues (n=5). C was the qPCR detection of CoL10A1 mRNA expression in BC (n=4) and paracancerous tissues (n=4). D was the Western Blot detection of CoL10A1 protein expression in BC samples (n=4) and paracancerous tissues (n=4). *p<0.05, ***p<0.001.

[0018] Figure 2 Shows the expression of CoL10A1 in BC cells, A is qPCR detection of CoL10A1 mRNA expression in BC cells, B is Western Blot detection of CoL10A1 protein expression in BC cell lines, C is qPCR detection of CoL10A1 knockdown efficiency in MDA-MB-468 cells CoL10A1 group (siNC, siRNA-1, siRNA-2, siRNA-3), D is Western Blot detection of CoL10A1 knockdown efficiency in MDA-MB-468 cells (siNC, siRNA-1, siRNA-2, siRNA-3), compared with the control group, *p<0.05, ***p<0.001.

[0019] Figure 3 The results show that knockdown of CoL10A1 inhibited the proliferation of MDA-MB-468 cells. A is the detection of cell proliferation by CCK-8, ***p<0.001, B is the detection of the expression of proliferation-related proteins (PCNA, c-MYC and Ki67) by Western Blot, C is the quantitative analysis of proliferation-related proteins, ***p<0.001, and D is the detection of cell proliferation by plate cloning experiment.

[0020] Figure 4 The results show that knockdown of CoL10A1 inhibited the migration and invasion of MDA-MB-468 cells. A is the scratch assay for cell migration (100×), B is the Transwell assay for cell migration, C is the quantitative analysis of cell migration rate, **p<0.01, ***p<0.001, D is the quantitative analysis of cell migration rate, *p<0.05, ***p<0.001.

[0021] Figure 5Knockdown of CoL10A1 inhibits the ability of MDA-MB-468 cells to pass through endothelial cells. A is the Transwell assay for the ability of MDA-MB-468 cells to pass through the hCMEC / D3 cell layer (100×). MDA-MB-468 cells were stained red with CM-DIL and cell nuclei were stained blue with DAPI. B is the quantitative analysis of the number of cells passing through the hCMEC / D3 cell layer. *p<0.05, ***p<0.001.

[0022] Figure 6 The results show that the EMT signaling pathway is involved in the migration and invasion of MDA-MB-468 cells induced by CoL10A1. A is Western Blot detection of EMT signaling pathway-related proteins. B is quantitative analysis of EMT signaling pathway-related proteins. **p<0.01, ***p<0.001.

[0023] Figure 7 It shows that knockdown of CoL10A1 inhibits tumor formation in mice. A is a representative image of brain metastases in the shNC group, where 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. A is 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 DESCRIPTION

[0024] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0025] CoL10A1, or collagen type X alpha 1 chain, is a gene encoding type X collagen alpha 1 chain. It plays an important role in the human body. CoL10A1 is a specific marker for hypertrophic chondrocytes and is essential for endochondral bone formation. During skeletal development, the expression of CoL10A1 is closely related to the proliferation and hypertrophic differentiation of chondrocytes. CoL10A1 is upregulated in a variety of malignant tumors, including lung cancer, gastric cancer, and pancreatic cancer, and is associated with poor prognosis. High expression of CoL10A1 is associated with tumor progression, invasion, and metastasis. CoL10A1 promotes tumor cell proliferation, invasion, and migration by activating multiple signaling pathways, such as 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 breast cancer brain metastasis remains unclear.

[0026] The experiment involved in the present invention was carried out according to the following operation: (1) Cell proliferation assay The cells of the control group and the CoL10A1 knockdown group (5×10 3 ) were inoculated in a 96-well plate, and 10 μL of CCK-8 (HY-K0301, MCE) was added at 0h, 24h, 48h, and 72h, respectively, for incubation for 2h, and the absorbance was measured at a wavelength of 450nm. The effect of knocking down CoL10A1 on the cell clone formation ability was detected by plate cloning experiment.

[0027] (2) Cell migration assay The cells of the control group and the CoL10A1 knockdown group (5×10 5 ) were inoculated in a 6-well plate. The next day, when the cell density reached about 90%, a line was drawn with a 10µL pipette tip. The tip was kept vertical as much as possible and could not be tilted. The cells were washed with PBS three times, the drawn cells were removed, and serum-free medium was added to continue the culture. Photos were taken at 0h and 24h.

[0028] (3) Transwell experiment The cells of the control group and the CoL10A1 knockdown group (5×10 4 ) were inoculated in the upper chamber of Transwell (3422, Costar), and 600 µL of complete medium was added to the lower chamber for continued culture. After 24 hours, the medium was discarded, the upper chamber was removed, and the cells on the back of the upper chamber were gently wiped with a cotton swab and washed three times with PBS. They were fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS, and stained with crystal violet for 30 minutes. Observed and photographed under a microscope.

[0029] For cell invasion assay, hCMEC / D3 (1×10 5 ) cells were seeded in the upper chamber of the transwell, and 600 µL of complete culture medium was added to the lower chamber, and the cells were cultured in an incubator. On the second day, cells (5×10 4 ) was stained with CM-DIL (1973357, Invitrogen) according to the instructions. The treated cells were inoculated on the hCMEC / D3 cell layer. After 48 hours, the culture medium was discarded, the upper chamber was removed, and the cells on the back of the upper chamber were gently wiped with a cotton swab and washed three times with PBS. Fix with 4% paraformaldehyde for 15 minutes, wash three times with PBS, and stain with DAPI for 30 minutes. Observe and take pictures under a fluorescence microscope.

[0030] Example 1 Expression of CoL10A1 in BCBM and BC tissues RNAiso Plus (9108, Takara) and protein lysis buffer (01771197, Yazyme) were used to extract RNA and protein from BMBC tissues and BC cells, respectively. qPCR and Western Blot methods were used to detect CoL10A1 RNA and protein levels. The results are shown in Figure 1 shown.

[0031] The primer sequences for qPCR are as follows: CoL10A1-F: 5'-TTTGGGGTTTCGCCCTATCC-3', recorded as SEQ ID NO. 7 CoL10A1-R: 5'-CTGGGGGTGAGGGGACTAAA-3', recorded as SEQ ID NO. 8; Actin-F: 5'-CCTTCCTTCCTGGGCATGG-3', recorded as SEQ ID NO.9; Actin-R: 5'-GATCTTCATTGTGCTGGGTGC-3', recorded as SEQ ID NO.10.

[0032] The reaction system (20 µL) is shown in Table 1: Table 1 Reaction system PCR cycles are shown in Table 2: Table 2 PCR cycles In order to verify the expression of CoL10A1, the present invention further verified this result using clinical samples. The results showed that the expression of CoL10A1 in breast cancer tissue was higher than that in adjacent paracancerous tissues. Figure 1 CD.

[0033] In addition, the present invention analyzed the expression of CoL10A1 in BCBM samples and control samples. The results showed that CoL10A1 was highly expressed in BCBM tissues at the mRNA and protein levels, such as Figure 1 AB.

[0034] The present invention analyzes the expression of CoL10A1 in breast cancer cell lines MCF7, MDA-MB-231, MDA-MB-453, MDA-MB-468, BT474, HS578T and human normal breast epithelial cells MCF10A. Figure 2 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 in MCF10A. In addition, the protein expression of CoL10A1 in breast cancer cells was also higher than that in MCF10A.

[0035] These results indicate that CoL10A1 is highly expressed in BCBM tissues.

[0036] Example 2 Biological functions of CoL10A1 in BC cells The expression of CoL10A1 in the BC cell line MDA-MB-468 cells cultured in vitro 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 methods.

[0037] 1. siRNA silencing technology MDA-MB-468 cells in the logarithmic growth phase were inoculated in 6-well plates. On the second day, the cell density reached 60-70% and transfection was performed. The EP tube containing siRNA powder was centrifuged at 4°C, 5000rpm for 1 minute, and DEPC water was added to fully dissolve it according to the instructions and placed on ice for later use. Transfection complex system was configured: tube ① Opti-MEM 125µL + Lip3000 5µL, tube ② Opti-MEM 125µL + siRNA 5µL, tube ① and tube ② were inverted upside down to mix, and the diluted reagent in tube ② was added to tube ①, and incubated at room temperature for 15 minutes; then the complex of tube ① and tube ② was added to the 6-well plate and continued to be cultured. After 8-12 hours, the complete cell culture medium was replaced and continued to be cultured. After 24 hours, RNA can be extracted for q-PCR experiment to detect interference efficiency, and protein can be extracted for Western Blot experiment or cell function experiment after 48 hours.

[0038] The siRNA sequences are: siRNA-1 (1828): SS Seq: 5'-GCAACAGCAUUAUGACCCA-3', recorded as SEQ ID NO.1, ASSeq: 5'-UGGGUCAUAAUGCUGUUGC-3', recorded as SEQ ID NO.2; siRNA-2 (228): SS Seq: 5'-CCUACACCAUAAAGAGUAA-3', recorded as SEQ ID NO. 3, ASSeq: 5'-UUACUCUUUAUGGUGUAGG-3', recorded as SEQ ID NO. 4; siRNA-3 (2013): SS Seq: 5'-CCAUCAUCGAUCUCACAGA-3', recorded as SEQ ID NO.5, ASSeq: 5'-UCUGUGAGAUCCAUGAUGG-3', recorded as SEQ ID NO.6.

[0039] When used, SEQ ID NO. 1 to SEQ ID NO. 6 of the present invention are connected to the 3' end with base TT to increase stability.

[0040] The present invention used three different siRNAs (siRNA-1, siRNA-2, siRNA-3) to transfect MDA-MB-468 cells, and the results showed that these siRNAs could significantly reduce the expression of CoL10A1 in MDA-MB-468 cells at the mRNA and protein levels. Figure 2 CD results showed that siRNA silencing technology could inhibit the expression of CoL10A1 in MDA-MB-468 cells, indicating that siRNA silencing was successful.

[0041] 2. Knockdown of CoL10A1 inhibits breast cancer cell proliferation CCK8 assay was used to study the effect of CoL10A1 knockout on cell proliferation. Figure 3 As shown, the results showed that compared with the siNC group, CoL10A1 knockout inhibited the proliferation of MDA-MB-468 cells (p<0.001). The present 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 in the CoL10A1 knockout group was significantly reduced. The cloning ability of MDA-MB-468 cells was detected using a colony formation assay, and the results showed that the cloning ability of the CoL10A1 knockout group was inhibited. The present invention found that knocking out CoL10A1 inhibited the cloning ability of MDA-MB-468.

[0042] The above research results indicate that CoL10A1 plays an important role in breast cancer cell proliferation.

[0043] 3. Knockdown of CoL10A1 inhibits breast cancer cell migration and invasion The effect of CoL10A1 on breast cancer cell migration and invasion was further verified. Figure 4 As shown. The scratch assay detected the effect of CoL10A1 on the migration of MDA-MB-468 cells. The results showed that compared with the siNC group, the migration of MDA-MB-468 cells was significantly reduced after siRNA transfection (**p<0.01; **p<0.001). The Transwell results showed 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.

[0044] 4. CoL10A1 affects blood-brain barrier permeability and tumor cell invasion The ability of CoL10A1 to penetrate the blood-brain barrier of breast cancer cells was further verified. Figure 5 As shown. First, a layer of hCMEC / D3 cells was laid in the upper chamber of the Transwell chamber. After 24 hours, MDA-MB-468 cells stained with CM-DIL were added to the hCEMC / D3 cell layer and cultured in an incubator. After 48 hours, the MDA-MB-468 cells that passed through were counted under a fluorescence microscope. The results showed that the ability of MDA-MB-468 cells to pass through hCEMC / D3 cells could be significantly reduced after transfection of siRNA. Knockdown of CoL10A1 inhibited the migration and invasion of breast cancer cells.

[0045] 5. CoL10A1 regulates cell migration and invasion through the EMT signaling pathway EMT is not only involved in the normal development process of the body, but also plays an important role in the metastasis and invasion of tumors. Previous studies have shown that the EMT signaling pathway is involved in regulating cell migration and invasion of various types of cancers, such as glioblastoma, cancer, and cancer. GSEA was used to evaluate the pathways associated with CoL10A1 in the TCGA database. The results showed that CoL10A1 expression was related to the ECM receptor interaction signaling pathway. Ander-Levchenko's study found that changes in the ECM-like cell adhesion matrix can regulate the process of EMT. Therefore, the present invention speculates that CoL10A1 may regulate the migration and invasion of BC cells through the EMT signaling pathway. The present invention evaluates whether the EMT signaling pathway can regulate the migration and invasion of BC cells regulated by CoL10A1. The expression of proteins related to the EMT signaling pathway, such as E-Cadherin, β-catenin, N-Cadherin, Vimentin, Slug, Snail, and ZEB1, was detected. The results showed that the expression of β-catenin and E-Cadherin was increased, and the expression of N-Cadherin, Vimentin, Slug, Snail, and ZEB1 was decreased. These results suggest that CoL10A1 regulates cell migration and invasion through the EMT signaling pathway, such as Figure 6 shown.

[0046] Example 3 Effect of CoL10A1 on tumorigenesis in mice, showing that downregulation of CoL10A1 inhibits tumorigenicity in vivo The present invention has proved that CoL10A1 can promote the proliferation, migration and invasion of BC cells. In addition, the present invention has tested whether CoL10A1 has a tumor-promoting effect in an animal model. Cells were injected into the carotid artery to construct a cancer brain metastasis model.

[0047] (1) Internal carotid artery injection Female C57BL / 6N mice (6-8 weeks) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. The mice were randomly divided into 2 groups, 4T1 cell group (shNC) and CoL10A1 knockdown 4T1 cell group (shCoL10A), with 3 mice in each group. The mice were anesthetized and fixed on a rubber board. 100μL of cell suspension was slowly injected into the arterial cavity to monitor the status of the mice.

[0048] (2) IVIS imaging Each mouse was intraperitoneally injected with 150 mg / kg D-luciferin (HY-12591, MCE). After 5 minutes, the mice were anesthetized and bioluminescence imaging was performed using IVIS200. The exposure time was set to 20 seconds and the light intensity was set to 80%. Luciferase and substrate are used to generate light signals in vivo, and the IVIS system detects the intensity of these light signals to quantitatively analyze biological processes. The data were analyzed using IVIS software (IVIS In Vivo Imaging Software).

[0049] Stable knockout of CoL10A1 in 4T1 cells was injected into nude mice, showing the location of the tumor in the brain. The growth of the CoL10A1 knockout group was significantly slow, as shown in Figure 7 The results showed that CoL10A1 could promote tumor growth in nude mouse models.

[0050] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0051] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0052] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of reagents for detecting CoL10A1 expression in the preparation of diagnostic products for breast cancer brain metastasis.

2. The use according to claim 1, characterized in that: The diagnostic product includes reagents used in the qPCR method or reagents used in the Western Blot method.

3. The use according to claim 2, characterized in that: The reagents used in the qPCR method include the sequences shown in SEQ ID NO.7-8.

4. A kit, characterized in that: The invention comprises the reagent for detecting the expression of CoL10A1 as claimed in claim 1.

5. Use of the agent for inhibiting the expression of CoL10A1 as claimed in claim 1 in the preparation of a product for treating brain metastasis of breast cancer.

6. The use according to claim 5, characterized in that: The reagent for inhibiting the expression of CoL10A1 is a sequence shown by SEQ ID NO.1-2, SEQ ID NO.3-4 or SEQ ID NO.5-6.

7. The use according to claim 6, characterized in that: The reagent for inhibiting the expression of CoL10A1 is the sequence shown in SEQ ID NO.3-4.

8. The use according to claim 6, characterized in that: The reagent for inhibiting the expression of CoL10A1 is the sequence shown in SEQ ID NO.5-6.

9. The use according to claim 5, characterized in that: The therapeutic product inhibits breast cancer.

10. The use according to claim 5, characterized in that: The therapeutic product inhibits the growth of brain metastases.

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