A molecular marker for osteosarcoma and its application

Abstract

The present invention discloses an osteosarcoma molecular marker and its application. The osteosarcoma molecular marker is spindle and centriole associated protein 1, the amino acid sequence of which is shown in SEQ ID No: 1. Tissue sequencing revealed that the expression level of spindle and centriole associated protein 1 having the amino acid sequence of SEQ ID No: 1 was significantly higher in osteosarcoma tissue than in normal bone tissue. After knocking down the protein, osteosarcoma proliferation, invasion, and migration were significantly inhibited, and apoptosis was increased. Therefore, the molecular marker can be used as an osteosarcoma marker, playing a role in assisting the diagnosis of osteosarcoma in its early stages, enabling patients to receive timely treatment and preventing the problem of osteosarcoma metastasis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to an osteosarcoma molecular marker and application thereof. Background Art

[0002] Osteosarcoma is the most common malignant bone tumor. It develops from mesenchymal cells and rapidly grows due to the direct or indirect formation of osteoid and bone tissue during the cartilage stage. Bone tumors primarily occur in longer bones and smaller portions of soft tissue, primarily affecting adolescents aged 10-20 years. Osteosarcoma is characterized by high morbidity and early metastasis rates, and low cure survival rates. The high early metastasis rate is primarily manifested in early metastasis to the lungs, which are insensitive to existing chemotherapy drugs and contribute significantly to the high mortality rate associated with bone tumors. Currently, the primary diagnostic methods for osteosarcoma include X-rays, magnetic resonance imaging (MRI), angiography, and tomography. However, these methods often exhibit significant lags, leading to delayed diagnosis and missed treatment opportunities.

[0003] Therefore, finding a means to diagnose osteosarcoma early has become a technical problem that needs to be solved urgently. With the continuous development of biomedicine, molecular diagnosis and targeted therapy have provided new ideas for the diagnosis and treatment of osteosarcoma. The existence or quantitative change of molecular markers helps to judge the nature of the tumor and provide guidance for the diagnosis and treatment of the tumor. At present, there are not many markers that can be used to assist in the diagnosis of osteosarcoma. It is necessary to find more markers to provide a reference for the clinical diagnosis of osteosarcoma. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is that the means for diagnosing osteosarcoma in the prior art have a certain lag, and thus a molecular marker for diagnosing and treating osteosarcoma and its application are proposed.

[0005] In order to solve the above technical problems, the technical solution of the present invention is:

[0006] The present invention provides an osteosarcoma molecular marker. The osteosarcoma molecular marker is spindle and centriole associated protein 1. The amino acid sequence of the spindle and centriole associated protein 1 is shown in SEQ ID No: 1.

[0007] Preferably, the spindle and centriole associated protein 1 is highly expressed in osteosarcoma tissue.

[0008] The present invention also provides an application of the molecular marker in preparing an auxiliary diagnosis product for osteosarcoma.

[0009] Preferably, the osteosarcoma cell lines are 143B and HOS.

[0010] Preferably, the auxiliary diagnostic product is a kit or a test paper.

[0011] The present invention also provides an application of the molecular marker as a target in screening anti-osteosarcoma drugs.

[0012] Another aspect of the present invention provides a shRNA for inhibiting the expression of the molecular marker. The gene sequence of the shRNA is shown in SEQ ID No: 2.

[0013] The present invention also provides a use of the shRNA in preparing a medicine for treating osteosarcoma.

[0014] Preferably, the drug is a compound, and the drug is used to inhibit the proliferation or metastasis of osteosarcoma cells.

[0015] The above technical solution of the present invention has the following advantages over the prior art:

[0016] (1) The present invention provides a molecular marker for osteosarcoma, which is spindle and centriole-associated protein 1, the amino acid sequence of which is shown in SEQ ID No. 1. Tissue sequencing revealed that the expression level of spindle and centriole-associated protein 1 having the amino acid sequence of SEQ ID No. 1 in osteosarcoma tissue was significantly higher than that in normal bone tissue. After knocking down the protein, osteosarcoma proliferation, invasion, and migration were significantly inhibited, and apoptosis was increased. This allows the molecular marker to be used as a marker for osteosarcoma, playing an auxiliary role in the diagnosis of osteosarcoma in the early stages of osteosarcoma, allowing patients to receive timely treatment and preventing the problem of osteosarcoma metastasis.

[0017] (2) The shRNA provided by the present invention can inhibit the expression of spindle and centriole-associated protein 1, and can be used as a drug for treating osteosarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0019] Figure 1 (a) shows the gene expression in osteosarcoma and normal bone tissue;

[0020] Figure 1 (b) shows the KM curve of overall survival of patients with osteosarcoma with high SPICE1 expression and patients with osteosarcoma with low SPICE1 expression;

[0021] Figure 1 (c) shows the KM curve of progression-free survival of osteosarcoma patients with high SPICE1 expression and low SPICE1 expression;

[0022] Figure 2 (a)-(b) shows the results of CCK-8 assay to detect the proliferation of HOS cells and 143B cells in the knockdown group and control group;

[0023] Figure 3 This is the result of the plate clone formation experiment to detect the clone formation of HOS cells and 143B cells in the knockdown group and the control group;

[0024] Figure 4 This is the result of flow cytometry apoptosis assay for HOS cells and 143B cells in the knockdown group and control group.

[0025] Figure 5 These are the results of migration assays to detect the migration of HOS cells and 143B cells in the knockdown and control groups;

[0026] Figure 6 These are the results of invasion assays to detect the invasion of HOS cells and 143B cells in the knockdown and control groups;

[0027] Figure 7 is the q-PCR test result of virus knockdown of shRNA recombinant expression vector and control group;

[0028] Figure 8 These are the results of western blotting detection of virus knockdown in the shRNA recombinant expression vector and the control group. DETAILED DESCRIPTION

[0029] Example 1

[0030] This embodiment provides an osteosarcoma molecular marker. Specifically, the osteosarcoma molecular marker is spindle and centriole-associated protein 1 (SPICE1). SPICE1 is a protein localized to spindle microtubules in mitosis and centrioles throughout the cell cycle. SPICE1 is a regulatory factor required for centriole duplication, bipolar spindle formation, and chromosome assembly in mitosis. Its amino acid sequence is shown in SEQ ID No: 1:

[0031] MSFVRVNRCGPRVGVRKTPKVKKKKTSVKQEWDNTVTDLTVHRATPEDLVRRHEIHKSKNRALVHWELQEKALKRKWRKQKPETLNLEKRRLSIMKEILSDQYQMQDVLEKSDHLIAAAKELFPRRRTGFPNVTVAPDSSQGPIVVNQDPITQSIFNESVIEPQALNDVDGEEEGTVNSQSGESENENELDNSLNSQSNTNTDRFLQQLTEENFELISKLWTDIQQKIATQSQITPPGTPSSALSSGEQRAALNATNAVKRLQTRLQPEESTETLDSSYVVGHVLNSRKQKQLLNKVKRKPNLHALSKPKKNISSGSTTSADLPNRTNSNLDVLKHMIHEVEHEMEEYERWTGREVKGLQSSQGLTGFTLSLVSSLCRLVRYLKESEIQLRKEVETRQQLEQVLGDHRELIDALTAEILRLREENAATQARLQQYMVTTDEQLISLTHAIKNCPVINNRQEIQASESGATGRRVMDSPERPVVNANVSVPLMFREEVAEFPQEELPVKLSQVPDPPDNMNLAKNFPAHIFEPAVLLTPPRQKSNLKFSPLQDVLRRTVQTRPAPRLPPTVEIIEKEQNWEEKTLPIDTDIQNSSEENRLFTQRWRVSHMGEDLENKTQAPFVNLSQPLCNSHSNTQQSRSPTFSEELPVLGDGQQLRTNESLIQRKDIMTRIADLTLQNSAIKAHMNNIIEPRGEQGDGLRELNKQESASDMTSTFPVAQSLTPGSMEERIAELNRQSMEARGKLLQLIEQQKLVGLNLSPPMSPVQLPLRAWTEGAKRTIEVSIPGAEAPESSKCSTVSPVSGINTRRSSGATGNSCSPLNATSGSGRFTPLNPRAKIEKQNEEGWFALSTHVS。

[0032] Tissue sequencing revealed that spindle and centriole-associated protein 1 (SPICE1) is highly expressed in osteosarcoma tissue, with expression levels significantly higher in osteosarcoma than in normal bone tissue. Combined with database analysis, these results indicate that patients with high SPICE1 expression have a lower 5-year survival rate. In vitro knockdown of SPICE1 inhibited osteosarcoma proliferation, invasion, and migration, while increasing apoptosis.

[0033] In this embodiment, the database analysis required for bioinformatics analysis specifically refers to the TCGA database (The Cancer Genome Atlas) and the GEO (GENE EXPRESSION OMNIBUS) database, and the survival data are visualized using the Kaplan-Meier Plotter (https: / / kmplot.com / ) website. Enter the sarcoma cancer section, enter the required pathology type in the Histology field, and output the data visualization based on the database inclusion. Expression level, overall survival (OS), and disease-free survival (DFS) are selected as the export format. The results are shown in Figures 1(a)-(c).

[0034] Figure 1(a) shows the gene expression in osteosarcoma and normal bone tissue. The test results are as follows: the selected threshold is |log2(FC)|>1 & p.adj<0.05. There are 1,126 genes that meet this threshold, of which 337 are highly expressed (logFC is positive) and 789 are underexpressed (logFC is negative).

[0035] Figure 1 (b)-(c) shows the KM curve. Statistical analysis was performed using the statistical analysis and visualization software R (version 3.6.3), including the R packages: survminer package [version 0.4.9] (for visualization) and survival package [version 3.2-10] (for statistical analysis of survival data); molecule: SPICE1 [ENSG00000163611]; group: 0-50 vs 50-100; prognosis type: Overall Survival; disease: osteosarcoma; data: TPM format data from the mRNA-seq data file of the TARGET (https: / / ocg.cancer.gov / programs / target) Osteosarcoma project; data transformation method: log2 transformation; data filtering: remove controls / normals and retain clinical information.

[0036] In Figure 1(b), curve 1 is the overall survival curve of patients with osteosarcoma with low SPICE1 expression, and curve 2 is the overall survival curve of patients with osteosarcoma with high SPICE1 expression; in Figure 1(c), curve 1 is the non-progression survival curve of patients with osteosarcoma with low SPICE1 expression, and curve 2 is the non-progression survival curve of patients with osteosarcoma with high SPICE1 expression.

[0037] As can be seen, patients with osteosarcoma with high SPICE1 expression have shorter overall survival and progression-free survival than those with low SPICE1 expression. This suggests that SPICE1 can be used as a marker to aid in the diagnosis of osteosarcoma, potentially enabling its application in osteosarcoma diagnostic products.

[0038] Example 2

[0039] Cell functional experiment: culture and transfection of osteosarcoma cells.

[0040] 1. Cell culture

[0041] The 143B cells and HOS cells to be cultured and tested were obtained from the Cell Bank of the Chinese Academy of Sciences.

[0042] 1.1 Cell recovery

[0043] (1) Preheat the water bath to 37°C, quickly remove the cell cryopreservation tube from the liquid nitrogen tank, and use a water bath to warm the cell cryopreservation solution to quickly return to temperature and melt.

[0044] (2) Place the thawed cell cryopreservation solution in a biosafety cabinet, aspirate it with a Pasteur pipette, and place it in a 15ml centrifuge tube pre-filled with 2ml of complete culture medium. Turn on the centrifuge and centrifuge at 800rpm for three minutes, then remove the supernatant.

[0045] (3) Resuspend the cells in 3 ml of complete culture medium and transfer them into a clean culture flask. Make up the volume of culture medium to 5 ml, shake the cells, and place them in a 37°C 5% CO2 incubator for observation the next day.

[0046] 1.2 Cell culture medium replacement

[0047] (1) Take out the culture flask, place it under a microscope to observe the cell status and record and take pictures.

[0048] (2) Disinfect the culture flask with alcohol and place it in a biosafety cabinet. Use a Pasteurized tube to remove the original culture medium. Replace the tube with a new one and draw 2 ml of PBS buffer solution into the culture flask. Gently shake the culture flask and wash the cell culture surface with PBS buffer solution. This step can be repeated twice.

[0049] (3) Add 5 ml of α-MEM medium containing 10% fetal bovine serum (FBS) to the culture flask (T25 culture flask).

[0050] (4) Place the culture flask back into the incubator and culture the cells as usual. Change the cell culture medium every two days.

[0051] 1.3 Cell passaging

[0052] (1) Take out the cell culture flask from the incubator and observe the cells under an inverted microscope. Pay attention to the degree of cell growth and confluence. When the cells grow to more than 85%, they can be passaged.

[0053] (2) Use a Pasteur pipette to remove the original culture medium, wash the cells with 1 ml of PBS, and then remove the washing solution.

[0054] (3) Add 1 ml of 0.25% trypsin-EDTA digestion solution to the culture flask and gently shake the culture flask to allow the trypsin to infiltrate the entire cell culture surface.

[0055] (4) During digestion, the cell morphology can be observed with an inverted microscope. When the cells shrink into a round shape and a bright ring appears around them, 2 ml of complete culture medium can be used to terminate the digestion.

[0056] (5) Gently blow the cell wall surface in the culture flask with cell suspension to disperse the cells from the culture flask as much as possible, transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.

[0057] (6) Resuspend the cell pellet in 1 ml of complete culture medium and transfer 250 μl to a new culture flask. This is considered a 1:4 subculture. Cells subcultured at this density can grow for three days. Continue to incubate the cells as usual and observe the cell status the next day.

[0058] 1.4 Cell cryopreservation

[0059] (1) Take cells in the logarithmic growth phase, discard the original culture medium, and wash twice with 2 ml PBS.

[0060] (2) Add 1 ml of trypsin and digest for 2 minutes, then add complete culture medium to terminate the digestion.

[0061] (3) Gently blow the adherent cells to make them fall off, transfer the cell suspension to a centrifuge tube, centrifuge at 800 rpm for 5 minutes, and gently aspirate the supernatant.

[0062] (4) Add 1 ml of pre-prepared pre-cooled cell freezing solution, gently blow to resuspend the cells, transfer them into cryopreservation tubes, mark the relevant information, place them at 4°C for 30 minutes, at -20°C for 2 hours, and in a refrigerator at -80°C overnight. The next day, place them in liquid nitrogen for long-term storage.

[0063] 1.5 Cell Count

[0064] (1) Rinse the blood cell counting chamber and cover glass, wipe with alcohol and dry.

[0065] (2) Digest the cells, centrifuge, and resuspend in 1 ml of complete culture medium.

[0066] (3) Dilute the cell suspension at a certain multiple. It is recommended to dilute it 3 times when A549 cells have grown all over the culture flask.

[0067] (4) Pipette about 10ul of cell suspension and add it to the sample loading area of ​​the blood cell counting plate.

[0068] (5) Count the number of cells in the four 4x4 squares of the hemocytometer under a 4x objective lens.

[0069] (6) Calculate the concentration of the cell suspension according to the following formula, and then estimate the total cell number based on the volume of the original cell suspension and the dilution factor.

[0070] Cell concentration = (number of cells in the four large squares of the counting plate / 4) × dilution factor × 10 4 pcs / ml.

[0071] 2. Cell transfection (lentiviral transfection)

[0072] 2.1 Construction and sequencing of shRNA recombinant expression vector

[0073] (1) Construction of SPICE1 (NM_144718) interference plasmid:

[0074] First, a negative control shRNA fragment, shCtrl (scrambled shRNA), was constructed. This shRNA does not target any gene and serves only as a negative control. The gene sequence of the scrambled shRNA (sh-con) is shown in SEQ ID No. 3: 5-TTCTCCGAACGTGTCACGT-3. A recombinant shRNA expression vector (sh-SPICE1) was constructed, with the gene sequence shown in SEQ ID No. 2: 5-GACTGATCTAACCGTTCAT-3. This shRNA inhibits the expression of the molecular marker SPICE1. The specific construction process involves introducing the designed sequence into a lentiviral vector. After cloning and transfection of target cells, viral particles are harvested, purified, concentrated, and titered for future use. (The lentiviral vector construction process was completed by Shanghai Genetech.)

[0075] 2.2 Lentiviral transfection of cells

[0076] (1) 24 hours before lentiviral transfection, adherent cells were cultured at 2×10 4 Place the cells / well into a six-well plate. Shake the six-well plate to prepare for transfection the next day.

[0077] (2) Replace the original culture medium with 1 ml of fresh culture medium containing 1X stimulating medium P, add an appropriate amount of virus suspension (MOI value = number of virus particles / number of cells = 40), and continue to culture at 37°C.

[0078] (3) 16 hours after transfection, replace the virus-containing culture medium with fresh conventional culture medium and continue culturing.

[0079] (4) Continue culturing for 2-3 days and determine the cell transfection efficiency by observing the GFP positivity rate under a fluorescence microscope.

[0080] 3. CCK-8 assay to detect cell proliferation

[0081] 3.1 Experimental Principle of CCK-8 Assay

[0082] The CCK-8 reagent (Cell Countin GKit 8 Cell Technology Reagents) contains WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonatophenyl)-2H-tetrazolium monosodium salt), a water-soluble tetrazolium salt that is easy to store. In the presence of the electron carrier 1-methoxy-PMS, WST-8 is reduced by deoxygenases within cells to a water-soluble yellow product with an absorbance at 450 nm. The absorbance of the product is proportional to the number of viable cells. This kit has high sensitivity and low cytotoxicity, making it widely used.

[0083] 3.2 Sample addition and detection sequence

[0084] Logarithmically growing osteosarcoma cell lines HOS and 143B were taken, digested, resuspended and counted, and then plated into 96-well plates. Each different cell type was plated into four groups on day 0, day 1, day 2, and day 3, with five replicate wells in each group, 1500 cells in each well, and 100 μl of 10% FBS culture medium. Six hours after plating, 10 μl of CCK-8 reagent was added to each of the five replicate wells on day 0. After incubation at 37°C for 3 hours, the absorbance at 450 nm was detected using a microplate reader. Thereafter, 10 μl of CCK-8 reagent was added to the corresponding wells at the same time point from the first to the third day after plating. The absorbance at 450 nm was also detected using a microplate reader after incubation at 37°C for 3 hours. The data were statistically plotted as a line graph, and the experiment was repeated more than three times. (The results are shown in Figure 2 (a)-(b)). The horizontal axis in the figure is time (days), and the vertical axis is the absorbance value of the solution at a wavelength of 450nm (OD450). By adding CCK8 solution every day and detecting the OD value at a wavelength of 450nm, the numerical value of the cell number is obtained. It can be seen that the growth of cell number in the knockdown group is significantly lower than that in the control group.

[0085] 4. Plate colony formation assay to detect cell proliferation ability

[0086] Osteosarcoma cell lines HOS and 143B in the logarithmic growth phase were taken, digested with trypsin, resuspended and counted, and 1000 cells per well of the differently treated cells were plated into six-well plates and cultured with 2 ml of complete medium. The growth of cell colonies was observed every day. When clone colonies with more than 50 cells were formed, samples were collected. The culture medium in the six-well plate was aspirated, washed three times with PBS, fixed with 4% paraformaldehyde for about 30 minutes, washed three times with PBS, and then stained with 1% crystal violet for 20 minutes. The excess dye was washed with PBS, and pictures were taken and information was collected using an inverted microscope. The number of clone colonies was counted using Image J analysis software, and the cell clone formation rate was calculated as the number of clone colonies / the number of cells plated into the plate. The experiment was repeated three times (the results are shown in the figure). Figure 3 As shown in Figure 3 ), the cell clone formation ability was detected by knocking down the SPICE1 gene in HOS and 143B cells. The results showed that the clone colonies of sh-SPICE1 were significantly reduced compared with the control group.

[0087] 5. Detection of cell apoptosis by flow cytometry

[0088] Osteosarcoma cell lines 143B and HOS in the logarithmic growth phase were washed with PBS, digested with EDTA-free trypsin, counted, and 4x10 4 Cells were resuspended in 100 μl of pre-cooled binding buffer, and 5 μl of Annexin V was added. After incubation in the dark for 5 minutes, 10 μl of PI staining solution (Propidium Iodide (PI)) was added. Flow cytometry was used to detect cell clustering under different fluorescence channels (APC-PERCP) immediately (the results are shown in Figure 2). Figure 4 As shown in Figure 3), the test results showed that compared with the control group, the number of apoptotic cells in sh-SPICE1 was significantly increased.

[0089] 6. Invasion and migration assay to detect cell invasion and migration ability

[0090] Transwell chambers were used for cell invasion and migration experiments. Basement membrane was coated and hydrated to prepare transwell chambers. Cells in the logarithmic growth phase were resuspended in serum-free medium and the cell concentration was adjusted to 1×10 5Cells were inoculated into chambers with 200 μl of the cell suspension at a concentration of 150 μg / ml. The chambers were then placed in a 24-well plate containing 600 μl / well of α-MEM medium containing 15% fetal bovine serum and cultured for 24 hours. The chambers were removed, the upper chamber fluid was aspirated, and the cells were rinsed twice with PBS. The cells were fixed with 95% alcohol for 10 minutes. Unmigrated cells on the upper side of the chamber membrane were wiped clean with a cotton swab. The cells were stained with crystal violet for 20 minutes and washed twice with PBS. Cells that migrated to the dorsal side of the chamber membrane were counted under an inverted microscope. Ten random fields of view were counted and the average was calculated. The experiment was repeated three times (the results are shown in Figure 2). Figure 5-6 As shown in Figure 3), the test results showed that after knocking down the SPICE1 gene in 143B and HOS cells, the cell invasion and migration abilities were tested. The results showed that compared with the control group, the number of invasive and migrating cells in sh-SPICE1 was significantly reduced.

[0091] 7. Viral knockdown effect

[0092] The negative control group shRNA fragment (sh-con) and the shRNA recombinant expression vector (sh-SPICE1) were tested for their knockdown effects on the virus. The test results are as follows: Figure 7 As shown in the figure, the efficiency of 143B and HOS cells infected with SPICE1 virus was detected by q-PCR experiments. The results showed that the mRNA level of sh-SPICE1 was significantly reduced compared with the control group. The test results showed that sh-SPICE1 had a significant knockdown effect on osteosarcoma virus and can be used to prepare drugs for the treatment of osteosarcoma. Specifically, the drug for the treatment of osteosarcoma is a compound that can play a therapeutic effect by inhibiting the proliferation and metastasis of osteosarcoma cells. In addition, Figure 8 As shown, the experiment detected the efficiency of 143B and HOS cells after infection with SPICE1 virus. The results showed that the protein level of sh-SPICE1 was significantly decreased compared with the control group.

[0093] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of a shRNA for inhibiting the expression of osteosarcoma molecular markers in the preparation of a drug for treating osteosarcoma, characterized in that: The osteosarcoma molecular marker is spindle and centriole associated protein 1, the amino acid sequence of the spindle and centriole associated protein 1 is shown in SEQ ID No: 1, and the gene sequence of the shRNA is shown in SEQ ID No:

2.

2. The use according to claim 1, characterized in that The spindle and centriole-associated protein 1 is highly expressed in osteosarcoma tissue.

3. The use according to claim 2, characterized in that The medicine is used for inhibiting the proliferation or metastasis of osteosarcoma cells.

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