A protein mimetic peptide for promoting the proliferation and stemness of tongue cancer cells and its application

Abstract

The present invention is applicable to the field of biomedical technology, and provides a protein mimic peptide that promotes the proliferation and stemness of tongue cancer cells and its application. The amino acid sequence of the protein mimic peptide is shown in SEQ ID NO.1. The protein mimic peptide provided by the present invention can promote the proliferation and stemness of tongue cancer cells under three-dimensional culture conditions, and can be used to construct a three-dimensional culture model of tongue cancer cells. It opens up a new path for basic research on tongue cancer, drug development, and the formulation of individualized treatment plans, and has broad application prospects and important scientific significance.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a protein mimetic peptide promoting the proliferation and stemness of tongue cancer cells and an application thereof. Background Art

[0002] Tongue squamous cell carcinoma is a common oral and maxillofacial malignancy, accounting for about 52% of all oral cancers. Although the overall incidence of oral cancer has shown a slight downward trend in recent years, the incidence of tongue cancer has been increasing. Tongue cancer is characterized by a high recurrence rate and lymph node metastasis rate, which seriously threatens the life and health of patients. At present, the treatment of tongue cancer is still mainly surgical treatment, supplemented by radiotherapy / chemotherapy, but these treatments have serious complications and do not significantly improve the patient's 5-year survival rate. Therefore, it is still necessary to find and develop more effective tumor therapeutic drugs and treatments through basic research. Basic tumor research and anti-tumor drug development require accurate, reliable and reproducible pharmacodynamic models to support them.

[0003] In vitro cell culture is one of the important means in pharmacodynamic evaluation. The traditional two-dimensional cell culture model refers to immersing cells in a culture medium, and the cells extend and grow along a two-dimensional plane on the surface of the culture bottle / dish. However, this culture method cannot simulate the spatial structure and growth environment of tumors in vivo, and cannot truly reflect the pathogenesis and drug response of tumors in clinical practice. The three-dimensional culture tumor model is to construct tumor cells into a three-dimensional multicellular aggregate in vitro, simulate the spatial structure and microenvironment of solid tumors, and is an ideal tumor research model. However, there is still a lack of three-dimensional cell culture models that can simulate the specific microenvironment of tongue cancer. In addition, under the three-dimensional culture state, cells survive longer but proliferate more slowly, which limits the clinical and basic research based on the three-dimensional tumor culture model. Therefore, it is necessary to construct a growth environment suitable for tongue cancer cells, which plays a role in maintaining cell stemness and promoting cell proliferation in the three-dimensional culture of tongue cancer cells. For this reason, the present invention proposes a protein mimetic peptide that promotes the proliferation and stemness of tongue cancer cells and its application. Summary of the invention

[0004] The purpose of the present invention is to provide a protein mimetic peptide for promoting the proliferation and stemness of tongue cancer cells and its application, aiming to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A protein mimicking peptide for promoting the proliferation and stemness of tongue cancer cells, wherein the amino acid sequence of the protein mimicking peptide is shown in SEQ ID NO.1.

[0007] An application of the protein mimetic peptide described above in constructing a three-dimensional culture model of tongue cancer cells.

[0008] Furthermore, the protein mimetic peptide promotes the proliferation, stemness and spheroidization ability of tongue cancer cells in a three-dimensional culture state.

[0009] Furthermore, the concentration of the protein mimetic peptide is 15 uM.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The protein mimetic peptide (hereinafter referred to as polypeptide) provided by the present invention can promote the proliferation and stemness of tongue cancer cells under three-dimensional culture conditions, and can be used to construct a three-dimensional culture model of tongue cancer cells. It has opened up a new path for basic research, drug development and formulation of individualized treatment plans for tongue cancer, and has broad application prospects and important scientific significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 HPLC diagram of the peptide.

[0013] Figure 2 This is the ESI-MS (electrospray ionization mass spectrometry) diagram of the peptide; the horizontal axis is m / z (mass-to-charge ratio) and the vertical axis is Intensity (peak intensity).

[0014] Figure 3 The formation of tongue cancer cell spheres in the control group and the experimental group; (A) is a picture of tongue cancer cell spheres cultured in medium containing and without polypeptide under an inverted microscope, and (B) is the statistics of the sphere formation efficiency of tongue cancer cell spheres in (A).

[0015] Figure 4 The results of cell activity detection using CCK8 method.

[0016] Figure 5 The results of cell proliferation detection by EdU staining; (A) is the picture of EdU-positive cells of tongue cancer cells cultured in medium without polypeptide and medium containing polypeptide under fluorescence microscope, and (B) is the statistics of the number of EdU-positive cells of tongue cancer cells in (A).

[0017] Figure 6 Results of clone formation experiments.

[0018] Figure 7 Results of three-dimensional culture of tumor cell spheres; (A) is the cell ATP content when the peptide concentration in the matrix gel is between 0.15-15 uM, and (B) is the cell picture under a microscope when there is no peptide in the matrix gel and the peptide concentration is 15 uM. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0020] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0021] Periostin (POSTN) is an extracellular matrix protein that has the function of maintaining the stemness of tumor cells. Studies have shown that it is highly expressed in oral squamous cell carcinoma tissues and has the function of promoting tumor proliferation and infiltration in oral cancer. ITGB1 is an integrin that is highly expressed in oral cancer. The activation of ITGB1 can promote tumor stemness. At the same time, ITGB1 is also a receptor for POSTN. Therefore, the present invention uses molecular docking and molecular dynamics simulation technology to design a POSTN mimetic peptide, i.e., a protein mimetic peptide, based on the interaction site between POSTN and ITGB1. Its amino acid sequence is as follows:

[0022] KRMLTKDLKNGMIIPSMYNNLGLFINHYPN.

[0023] Example 1. Synthesis and detection of polypeptides;

[0024] The peptide was commissioned to be synthesized by Shanghai Jier Biochemical Co., Ltd. The peptide was synthesized using the Fmoc solid phase synthesis method, connecting the first amino acid at the end of the peptide chain to an insoluble resin, connecting from the C-terminus to the N-terminus of the peptide chain, and performing the following steps:

[0025] Step 1, synthesis: First, mix the resin corresponding to the first amino acid at the C-terminus and an appropriate amount of dichloromethane (DCM) in a glass sand core reaction column to swell the resin. About 10 mL of DCM is required for each gram of resin. After swelling for 30 minutes, the DCM is removed and then washed twice with N,N-dimethylformamide (DMF). Then add the decapping liquid, stir under nitrogen protection for 30 minutes, then drain and wash with DMF. According to the peptide sequence, starting from the second amino acid at the C-terminus, condensation reactions are carried out in sequence. During each condensation, protected amino acids, dicyclohexylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt) and DMF are added to the resin, and the molar ratio of the feed amount to the resin is 3:1. React at 34±2℃ for 1.5 hours, drain the reaction solution, wash the resin with DMF 3 times, and drain. Repeat the above steps until the last amino acid is condensed, remove the protective group Fmoc, wash the resin with methanol 3 times, drain the resin, and finally obtain a dry peptide resin. Use cutting fluid to cut off the peptide chain connected to the resin, seal the reaction for 2 h, and separate the resin and the polypeptide with a sand core funnel. Then perform post-treatment, add anhydrous ether to the cutting fluid to precipitate the polypeptide, centrifuge, discard the supernatant, wash the polypeptide with anhydrous ether, and drain to obtain the crude polypeptide.

[0026] Step 2, purification: Use a capillary to take a trace amount of crude peptide in a 0.5 mL fine tube, and dissolve the crude peptide with water in an ultrasonic until it is clear. Then use a 5-100% gradient condition to perform preliminary analysis on HPLC (high pressure liquid chromatography), the column model is 4.6*250 mm, kromasil C18-5, and the flow rate is 1 mL / min. After the instrument is balanced, use a washed micro-injection needle to take 10 uL of crude peptide liquid, and add the sample to the chromatograph through the injection valve for crude product analysis. Set the preparation gradient according to the retention time of the crude product analysis sample in the chromatographic column, (peak time-retention time)*[(final concentration-initial concentration) / time]=Cacetonitrile%. Subsequently, use the set gradient to balance the high pressure liquid preparative chromatograph, the column is a 150DAC dynamic axial column, the filler model is C18 10 uM, and the preparation flow rate is 350 mL / min. After fully dissolving the crude peptide sample with pure water by ultrasonication, filter it and inject it into the chromatographic column of the preparative chromatograph. Elute the crude peptide sample according to the set gradient, collect the liquid sample corresponding to the target product peak according to the peak situation, and verify whether it is the target peptide by LC-MS. Use a rotary evaporator to remove the organic solvent and most of the water in the liquid sample, and control the temperature at around 40°C. Put the spun liquid sample into a freezer and freeze it into ice cubes, then put it into a pre-cooled dryer for drying. After the sample is frozen into powder, weigh it and use HPLC again to detect the purity of the purified peptide sample. High performance liquid chromatography (HPLC) Figure 1) The results showed that a single peak with a strong signal appeared at the retention time of 11.615min, and the peptide peak area accounted for 96.61% of the total peak area, indicating that the purity of the synthetic peptide was 96.61%. The theoretical molecular weight of the peptide was 3537.196, and the electrospray ionization mass spectrometry analysis results ( Figure 2 ) showed that the peak values ​​of the hexavalent peak, pentavalent peak, tetravalent peak, trivalent peak and divalent peak were 590.55, 708.45, 885.25, 1179.90 and 1769.40, respectively, which basically matched the peak values ​​of the polypeptide.

[0027] Example 2: Synthetic polypeptides promote tongue cancer cell stemness;

[0028] CAL27 cells digested with trypsin were inoculated in low-adhesion six-well plates at a density of 500 cells per well. The culture medium of the control group (Control) consisted of DMEM containing 10 ng / mL bFGF, 20 ng / mL EGF and 1% double antibody. The culture medium of the experimental group was supplemented with a peptide with a final concentration of 1.5 uM on the basis of the culture medium of the control group. After 7 days of culture, the cell spheroidization efficiency was observed and calculated under an inverted microscope. The cell spheroidization efficiency = the number of cell spheres with a diameter greater than 75 uM in each well / the total number of cells originally inoculated in each well. The results are shown in Figure 2. Figure 3 As shown in the figure, the spheroid formation efficiency of tongue cancer cell spheres formed in the experimental group was higher than that in the control group. This result indicated that the peptide could significantly promote the stemness and spheroid formation ability of tongue cancer cells.

[0029] Example 3, the activity of synthetic polypeptide in promoting the proliferation of tongue cancer cells (using CCK8 method to detect cell activity);

[0030] The trypsin-digested CAL27 cells were inoculated at a density of 3000 cells / well in a 96-well plate, and the culture medium was replaced after 24 hours of culture. The culture medium of the control group was a culture medium without polypeptide (high glucose + 10% serum and 1% double antibody), and the final concentration of polypeptide in the experimental group culture medium was between 0.015-15 uM. After 24 hours of culture, 10 uL CCK-8 solution was added to each well, and the culture plate was placed in an incubator and incubated for 1 hour. The absorbance (OD) at 450nm was measured with a microplate reader. Cell viability % = (absorption value of the experimental group - absorption value of the blank group) / (absorption value of the control group - absorption value of the blank group) × 100%. The results are as follows Figure 4 As shown, when the polypeptide concentration is between 0.15-15uM, as the polypeptide concentration increases, the cell activity gradually increases, indicating that within this concentration range, the cell proliferation activity increases with the increase of polypeptide concentration.

[0031] Example 4: Synthetic polypeptide promotes tongue cancer cell proliferation (using EdU staining to detect cell proliferation);

[0032] Trypsin-digested CAL27 cells were inoculated at a density of 30,000 cells / dish in a laser confocal dish, and the culture medium was replaced after 24 hours of culture. The culture medium of the control group was a culture medium without peptides, and the final concentration of peptides in the culture medium of the experimental group was 15 uM, and the culture was cultured for 72 hours. After the culture was completed, the culture dish was removed from the incubator, the culture medium was discarded, and the culture medium containing EdU was added, and the cells were incubated in the incubator for another 2 hours. After the incubation was completed, the culture medium was discarded, the cells were fixed with 4% paraformaldehyde for 30 minutes, the cells were washed twice with BSA, the cells were incubated with 0.5% Triton X-100 for 20 minutes, and then washed twice with BSA. 500 uL Click reaction solution was added to each well, the culture plate was gently shaken to ensure that the reaction mixture could evenly cover the sample, and incubated at room temperature in the dark for 40 minutes. After the incubation was completed, the Click reaction solution was aspirated and the cells were washed 3 times with blocking solution, each time for 3-5 minutes. Then, diluted Hoechst 33342 dye solution was added and incubated for 30 min in the dark, and then the cells were washed twice with PBS. Finally, a fluorescence microscope was used to take pictures, and the EdU and Hoechst 33342 fluorescence pictures were overlapped and marked as Merge pictures. The number of EdU positive cells was analyzed using Image J software. The EdU staining results are shown in Figure 2. Figure 5 As shown, the number of EdU-positive cells in tongue cancer cells cultured with the addition of polypeptide was significantly higher than that in the control group, indicating that the polypeptide significantly promoted the proliferation of tongue cancer cells.

[0033] Example 5: Clone formation experiment;

[0034] The trypsin-digested CAL27 cells were seeded at a density of 300 cells / well in a six-well plate, and the culture plate was placed in an incubator for 24 hours before the culture medium was replaced. The culture medium of the control group was a culture medium without polypeptides, and the final concentration of polypeptides in the experimental group culture medium was 1.5 uM. The culture was continued for 10 days until the number of cells in most single clones exceeded 50. During the culture process, the culture medium was replaced every 3 days, and the cell status was observed. After 10 days of culture, 1 mL of 4% paraformaldehyde was added to fix the cells for 15 minutes, and then washed with PBS. 1 mL of crystal violet stain was added to each well, and the cells were washed with PBS after staining for 15 minutes, and then photographed. The experimental results are as follows Figure 6 As shown, the polypeptide significantly promoted the formation of tongue cancer cell clones, indicating that the polypeptide significantly promoted the proliferation and stemness of tongue cancer cells.

[0035] Example 6: Three-dimensional culture of tumor cell spheres;

[0036] CAL27 cells were digested with trypsin and plated at 3 × 10 4The cells were inoculated at a density of 100 μL / mL in a matrix gel containing different concentrations of peptides (0-150 μM). The wells were inverted and placed in a 37°C incubator for 30 min, then placed upright and cultured with culture medium for 14 days. The cell morphology was observed under a microscope and photographed. The cell proliferation activity was detected by detecting the ATP level. After the culture was completed, the culture medium was removed, 100 μL of detection reagent was added to each well, and the cells were shaken at room temperature for 5 min to fully lyse the cells. Chemiluminescence detection was performed using a multifunctional microplate reader. The results are shown in Figure 7 As shown, Figure 7 Figure A shows that when the peptide concentration is between 0.15-15 uM, the cell ATP content gradually increases with the increase of peptide concentration, indicating that within this concentration range, the cell proliferation activity increases with the increase of peptide concentration. When the peptide concentration is 15 uM, the cell ATP content is the highest, indicating that the peptide at this concentration has the strongest ability to promote the proliferation of tongue cancer cells. Figure 7 B in the figure shows the cell pictures under microscope without peptide and at a peptide concentration of 15 uM. The results show that peptide can promote the size of tumor cell spheroids in three-dimensional culture state.

[0037] In summary, the polypeptide provided by the present invention can promote the proliferation and stemness of tongue cancer cells under three-dimensional culture conditions, and can be used to construct a three-dimensional culture model of tongue cancer cells. It has opened up a new path for basic research, drug development and formulation of individualized treatment plans for tongue cancer, and has broad application prospects and important scientific significance.

[0038] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A protein mimetic peptide that promotes the proliferation and stemness of tongue cancer cells, characterized in that: The amino acid sequence of the protein mimetic peptide is as follows: KRMLTKDLKNGMIIPSMYNNLGLFINHYPN.

2. Use of the protein mimetic peptide according to claim 1 in constructing a three-dimensional culture model of tongue cancer cells.

3. The use according to claim 2, characterized in that: The protein mimic peptide promotes the proliferation, stemness and spheroidization ability of tongue cancer cells in a three-dimensional culture state.

4. The use according to claim 3, characterized in that: The concentration of the protein mimetic peptide is 15 uM.

Images