Cell permeation peptide Y119E and application thereof in inhibiting growth of cis-platinum drug-resistant tumors

Abstract

The invention relates to a cell permeable peptide Y119E and application thereof in inhibiting the growth of cis-platinum drug-resistant tumors, and is characterized in that a cell penetrating peptide TAT is used as a carrier, a cell permeable peptide containing PGAM1 Y119 phosphorylated cell permeable peptide (Y119E) is transported into tumor cells, and the cell permeable peptide (Y119E) contains the Y119 phosphorylated cell permeable peptide (Y119E) with the sequence of YGRKRRQRRR-KIWRRSEDVP, and the cell permeable peptide (Y119E) has the sequence of Y119 phosphorylated cell permeable peptide (Y119E) with the sequence of YGRKRRQRRR-KIWRRSEDVP. The invention relates to an application of a cell permeation oligopeptide Y119E in a drug for inhibiting the growth of cisplatin chemotherapy drug-resistant tumors. The traditional Chinese medicine composition can effectively inhibit tumor subcutaneous growth in nude mice.

Description

Technical Field

[0001] The invention relates to a cell-penetrating peptide Y119E and application thereof in inhibiting the growth of cisplatin chemotherapy-resistant tumors, and belongs to the field of medicine. Background Art

[0002] 1. The anticancer drug cisplatin inhibits tumor cell proliferation by damaging DNA

[0003] Cisplatin is the first generation of platinum anti-tumor drugs. Its history can be traced back to the 18th century, when scientists used it as an inert electrode due to its inactive chemical properties. 1,2 It was not until 1965 that Barnett Rosenberg discovered the inhibitory effect of cisplatin on cell proliferation, and platinum gradually came into people's attention as an anti-cancer drug. After clinical trials, it was approved by the US Food and Drug Administration in 1978 and put on the market. 1,2 , and was then widely used in the treatment of malignant tumors such as testicular cancer, ovarian cancer, lung cancer, head and neck tumors, and it occupies an important position in chemotherapy drugs, especially in the treatment of testicular cancer and ovarian cancer, with initial cure rates of 100% and about 85% respectively. 2 After being taken up by cells, cisplatin is hydrolyzed and activated, and then directly targets DNA molecules. Cisplatin coordinates with the 7th single atom on guanine and adenine in DNA molecules to form intrachain crosslink products, thereby maintaining DNA unwinding or bending, reducing DNA stability and preventing DNA replication. 1 . Not only that, cisplatin can also induce cells to produce ROS (reactive oxygen species), further leading to DNA damage and cell apoptosis. Cisplatin has the characteristics of simple structure, clear mechanism, low toxicity, broad anti-cancer spectrum, strong effect, and synergistic effect with various anti-tumor drugs. It has successfully set off a wave of research on platinum drugs and is still the first-line drug for the clinical treatment of solid tumors.

[0004] 2. DNA damage repair system increases tumor cell resistance to cisplatin

[0005] Although cisplatin is very successful in treating lung cancer and other cancers, cellular resistance to cisplatin is a major challenge currently faced by the study of platinum drugs. Once the human body develops resistance, it means that the efficacy of the drug is reduced, and it is necessary to increase the dosage or change the drug, which will increase the cytotoxicity of cisplatin and a series of negative effects. Existing studies have explored some mechanisms of resistance in vitro, which can be mainly attributed to the following: on the one hand, cisplatin may be inactivated by proteins or small biological molecules in the cell; more importantly, on the other hand, the cisplatin-DNA cross-linking products are repaired by the DNA damage repair system in the cell. 3. Most cells contain a DNA damage repair mechanism, that is, there will be repair proteins to repair damaged DNA, and the anti-cancer effect of cisplatin is reflected in the destruction of the normal structure of DNA. Therefore, if you want to successfully kill cancer cells, you must overcome the need to avoid the repair of cisplatin-DNA cross-linking products by repair proteins. Therefore, the mechanism of cisplatin resistance in clinical tumors is still a scientific issue of great research value in clinical practice. Studying the mechanism of cell resistance to cisplatin and finding ways to inhibit cell resistance is the best chemosensitization strategy. Combination therapy of cisplatin and its resistance-reversing drugs will more effectively promote the application of cisplatin.

[0006] 3. "Aerobic glycolysis" provides material guarantee and reducing power for tumor cell DNA repair

[0007] The continuous division of tumor cells poses a challenge to their metabolism 4 : First, it requires doubling of important cellular components, that is, enhancing the synthesis of biomacromolecules; second, it requires sufficient energy supply; third, it requires sufficient reducing power to maintain the redox balance in the cell. In order to "meet" the "challenges" brought by proliferation, tumor cells undergo metabolic reprogramming to meet the material and other requirements of rapid proliferation. Compared with differentiated cells, rapidly proliferating tumor cells consume a large amount of glucose for glycolysis metabolism rather than aerobic oxidation even under conditions of sufficient oxygen, and produce a large amount of lactic acid. This metabolic mode is called "aerobic glycolysis" 5-9 , also known as the "Warburg effect" 5-9Aerobic glycolysis provides the following advantages for the rapid proliferation of tumor cells: First, there are two very important branch pathways in the glycolysis metabolic pathway: ① The pentose phosphate pathway entered by the intermediate product glucose-6-phosphate, whose main metabolites are ribose-5-phosphate (R-5-P) and reduced coenzyme II (NADPH), etc. R-5-P is the main precursor molecule for nucleotide biosynthesis; and NADPH can provide reducing power for the synthesis of biological macromolecules and can also be used to balance the reactive oxygen free radicals produced during rapid cell proliferation; ② The serine synthesis pathway entered by the intermediate product 3-phosphoglycerate, whose main metabolites are serine and glycine, can be used for protein biosynthesis, and can also produce one-carbon units in the process of serine to glycine conversion for nucleotide biosynthesis. It can be seen that the pentose phosphate pathway and the serine synthesis pathway, as important branch pathways of glycolysis, are both involved in the synthesis of nucleotides, providing indispensable material guarantees for DNA replication and damage repair. In addition, the NADPH produced in the pentose phosphate pathway can effectively counteract ROS and reduce DNA oxidative damage caused by ROS. The above evidences all indicate that tumor metabolic reprogramming is involved in regulating cellular DNA damage repair, and is likely to further participate in regulating the chemoradiotherapy resistance process related to DNA damage repair.

[0008] 4.PGAM1 is a key regulatory enzyme that coordinates aerobic glycolysis and branch metabolism

[0009] "Aerobic glycolysis" metabolism is regulated by metabolic enzymes. Studies have shown that changes in the expression or activity of glycolytic metabolic enzymes are closely related to rapid tumor proliferation, metastasis, immune escape or clinical drug resistance, and may serve as potential targets for cancer treatment. Phosphoglycerate mutase (PGAM1) is one of the metabolic enzymes in the glycolysis pathway, catalyzing the mutual conversion of 3-phosphoglycerate (3-PG) and 2-phosphoglycerate (2-PG). Because it catalyzes a reversible reaction, PGAM1 has not received much attention for a long time. However, studies in recent years have shown that the expression of PGAM1 is negatively regulated by the tumor suppressor factor TP53, and is generally highly expressed and significantly activated in a variety of tumors, such as lung cancer, breast cancer, bile duct cancer, renal clear cell carcinoma, prostate cancer and glioma. 10,11 . The results of clinical data show that the expression level of PGAM1 is negatively correlated with the prognosis of tumor patients, and positively correlated with the tumor tissue grade and severity, playing an important role in the development of tumors. In terms of its mechanism, PGAM1 not only ensures the smooth progress of glycolysis, but more importantly, its substrate 3-PG is a competitive inhibitor of the key enzyme G6PD in the pentose phosphate pathway, and its product 2-PG is an activator of the key enzyme PHGDH in the serine synthesis pathway. Activating PGAM1 can lead to a decrease in 3-PG content, relieving the inhibition of the pentose phosphate pathway, while the 2-PG content is increased, activating the serine synthesis pathway. 10,11Therefore, although PGAM1 is not a rate-limiting enzyme, it can simultaneously regulate the branch pathway of glycolysis, promote DNA damage repair, resist oxidative stress damage, and is closely related to processes such as tumor proliferation and metastasis, making it an attractive target for cancer treatment.

[0010] 5. PGAM1 activity is positively correlated with cisplatin resistance in non-small cell lung cancer

[0011] Recent studies have shown that in patients with non-small cell lung cancer, high expression of PGAM1 is significantly associated with poor prognosis. The PGAM1 inhibitor HKB99 can effectively inhibit the proliferation and migration of non-small cell lung cancer and induce its apoptosis. As mentioned above, PGAM1 can enhance the two branch pathways of the pentose phosphate pathway and the serine synthesis pathway in the "aerobic glycolysis" metabolism, which in turn provides material guarantee and reducing power for tumor cell DNA repair, and may reverse the resistance of chemotherapeutic drugs such as cisplatin. This establishes a connection between the high expression of PGAM1 in non-small cell lung cancer and the resistance to cisplatin. Our previous results showed that PGAM1 is highly expressed and upregulated in non-small cell lung cancer A549 cells and tissues, promoting the production of nucleotides and NADPH, inhibiting PGAM1 expression or reducing PGAM1 activity to increase cisplatin-induced DNA damage to A549 / DDP cells, and enhancing the anti-tumor effect of cisplatin on A549 / DDP cells.

[0012] Cell-penetrating peptides (CPPs) are a large class of sequences consisting of 10 to 30 amino acids, also known as protein transduction domains or transduction peptides. So far, the cell-penetrating peptides discovered include: Pep-1, pVEC, MAP, pAntp, Transportan, Polyarginines, TAT, etc. Among them, TAT is a very classic cell-penetrating peptide with a high membrane penetration efficiency. 12,13 TAT contains 11 amino acids with an amino acid sequence of YGRKKRRQRRR. TAT can be localized in both the nucleus and cytoplasm and has been widely used as a delivery tag for biomacromolecules. 14 . Summary of the invention

[0013] The purpose of the present invention is to provide a cell penetrating peptide Y119E and its application in inhibiting the growth of cisplatin chemotherapy-resistant tumors. According to the molecular mechanism of PGAM1-PKM2 binding, a cell penetrating peptide (Y119E) that simulates Y119 phosphorylation is developed based on the sequence of PGAM1. It is known that TAT itself has no effect on cell proliferation and survival, which provides a guarantee for cell penetrating peptide drugs, and the cell penetrating peptide can effectively inhibit the interaction of PGAM1-PKM2, thereby inhibiting the growth of cisplatin chemotherapy-resistant tumors.

[0014] The technical solution of the present invention is implemented as follows: a cell penetrating peptide Y119E, characterized in that: based on the sequence of PGAM1, the cell penetrating peptide TAT is used as a carrier to transport the cell penetrating peptide containing the Y119 phosphorylation site into the cell, comprising a Y119 phosphorylated cell penetrating peptide (Y119E), whose sequence is YGRKKRRQRRR-KIWRRSEDVP.

[0015] The cell-penetrating peptide Y119E inhibits the growth of cisplatin chemotherapy-resistant tumors.

[0016] The cell-penetrating peptide Y119E inhibits tumors including non-small cell lung cancer.

[0017] The positive effect of the present invention is that it can effectively inhibit the interaction between PGAM1 and PKM2, thereby inhibiting the growth of cisplatin chemotherapy-resistant tumors. After intraperitoneal injection of cell-penetrating peptide Y119E, the growth rate of cisplatin chemotherapy-resistant tumor cells in nude mice subcutaneously is significantly inhibited.

[0018] PGAM1 pY119 phosphorylation, as a hallmark modification of tumors, specifically exists in lung cancer, breast cancer, gastric cancer, acute T-lymphocytic leukemia and other tumors, and can be regarded as a potential target for tumor treatment. To this end, we selected non-small cell lung cancer resistant to cisplatin chemotherapy for testing. The specific experimental example is as follows:

[0019] (1) Take cisplatin chemotherapy-resistant non-small cell lung cancer A549 / DDP cells (5×10 6 ) and resuspended in 100 μL PBS;

[0020] (2) Eighteen 6-8 week old female nude mice (BALB / c-nude) were selected and the cell suspension was injected into the axilla (subcutaneously) of the nude mice;

[0021] (3) Tumor-bearing mice were randomly divided into an experimental group and a control group (nine nude mice in each group). The experimental group mice were intraperitoneally injected with cell-permeable peptide Y119E every other day; the control group mice were intraperitoneally injected with a peptide containing only TAT as a negative control;

[0022] (4) Regularly measure the long diameter (L) and short diameter (W) of the nude mouse tumor and calculate the long diameter (L) and short diameter (W) of the nude mouse tumor according to the formula (LW) 2 / 2Calculate the volume of the tumor;

[0023] (5) After 20 days, the tumor was removed from the mouse subcutaneously and a tumor volume growth curve was drawn. The specific experimental data and statistical results are as follows:

[0024]

[0025] (7) Tumor weight and statistical results are as follows:

[0026]

[0027] The above results indicate that, compared with the cell-penetrating peptide TAT, Y119E effectively inhibits the subcutaneous growth of cisplatin chemotherapy-resistant non-small cell lung cancer cells in nude mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Cisplatin-resistant A549 / DDP cells did not respond to cisplatin-induced ROS production.

[0029] Figure 2 Cisplatin-resistant A549 / DDP cells do not respond to cisplatin-induced DNA damage.

[0030] Figure 3 Cisplatin-resistant A549 / DDP cells did not respond to cisplatin-induced apoptosis.

[0031] Figure 4 The phosphorylation level of PGAM1 Y119 in cisplatin-resistant A549 / DDP cells.

[0032] Figure 5 To build A549 / DDP WT and A549 / DDP Y119F Stable cell lines.

[0033] Figure 6 For A549 / DDP Y119F Cells re-responsive to cisplatin-induced ROS production.

[0034] Figure 7 For A549 / DDP Y119F Cells reprogrammed to respond to cisplatin-induced DNA damage.

[0035] Figure 8 For A549 / DDP Y119F Cells re-respond to cisplatin-induced apoptosis.

[0036] Fig. 9 is the amino acid sequence of the cell-penetrating peptide Y119E.

[0037] Fig.10 The cell-permeable peptide Y119E inhibits the interaction between PGAM1 and PKM2.

[0038] Fig.11 This is a diagram of subcutaneous tumor-bearing mice and the drug delivery pattern of cell-penetrating peptide Y119E.

[0039] Fig.12 The cell-penetrating peptide Y119E inhibited tumor growth in nude mice.

[0040] Fig.13 The cell-penetrating peptide Y119E inhibited tumor growth (volume) in nude mice.

[0041] Fig.14 The cell-penetrating peptide Y119E inhibited tumor growth (mass) in nude mice. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with the accompanying drawings and embodiments: Figure 1-14 As shown; a cell penetrating peptide Y119E, characterized in that: based on the sequence of PGAM1, the cell penetrating peptide TAT is used as a carrier to transport the cell penetrating peptide containing the Y119 phosphorylation site into the cell, comprising a Y119 phosphorylated cell penetrating peptide (Y119E), whose sequence is YGRKKRRQRRR-KIWRRSEDVP.

[0043] The cell-penetrating peptide Y119E inhibits the growth of cisplatin chemotherapy-resistant tumors.

[0044] The cell-penetrating peptide Y119E inhibits tumors including non-small cell lung cancer.

[0045] Example 1

[0046] Materials and Methods

[0047] 1. Cell Culture

[0048] Before doing cell experiments, you need to wipe the clean bench with 75% alcohol cotton, put the reagents and materials needed for culture in the clean bench, turn on the UV mode, and sterilize for more than 30 minutes before use. A549 and A549 / shPGAM1 cells use F12 culture medium, A549 / DDP, A549 / DDP / PGAM1 WT and A549 / DDP / PGAM1 Y119F The cells were cultured in 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin double antibody solution in a constant temperature incubator at 37°C and 5% CO2.

[0049] According to Tables 1, 2, 3 and 4, prepare F12 and 1640 culture media, PBS buffer and trypsin required for cell culture.

[0050] Table 1 F12 medium formula (pH 7.2-7.4)

[0051]

[0052] Table 2 1640 medium formula (pH 7.2-7.4)

[0053]

[0054] Table 3 1× PBS formula (pH 7.2-7.4)

[0055]

[0056]

[0057] Table 4 10× Trypsin Formula

[0058]

[0059] 2. Cell Protein Extraction

[0060] (1) Digest the cells to be lysed with 1× trypsin, place them in the corresponding 1.5 mL EP tube, and centrifuge at 300 g for 5 min at 4°C.

[0061] (2) Prepare LSB cell lysis buffer according to Table 5.

[0062] (3) Remove the supernatant, add 200 μL LSB cell lysis buffer and 1 μL nuclease, and blow evenly with a pipette until the solution becomes clear and no longer sticky. Then let it stand at room temperature for 1 h to allow it to fully lyse.

[0063] (4) Boil the sample in a metal bath at 100°C for 10 min and then separate it instantly. After packaging, store it at -80°C for later use.

[0064] Table 5 1×LSB buffer recipe

[0065]

[0066] 3. Protein Immunoblotting

[0067] (1) Prepare 10% separation gel according to Table 6. After mixing, add it to the gel plate immediately to a distance of 2.5 cm from the top. Then add 1 mL of anhydrous ethanol to press the gel surface. Let it stand for more than 30 minutes. After solidification, pour off the anhydrous ethanol.

[0068] Table 6 10% separation gel formula

[0069]

[0070] (2) Prepare 5% concentrated gel according to Table 7. After mixing, add it to the gel plate immediately and insert the comb. Be careful not to form bubbles. Let it stand for 30 minutes. Remove the comb, rinse the gel clean to prevent the gel fragments in the wells from interfering with the sample loading, and place the cleaned gel in the electrophoresis tank.

[0071] Table 7 5% concentrated gel formula

[0072]

[0073] (3) Prepare 10× electrophoresis buffer and 10× transfer buffer according to Tables 8 and 9, respectively.

[0074] Table 8 10× electrophoresis fluid formula

[0075]

[0076] Table 9 10× transfer solution formula

[0077]

[0078] (4) Prepare 1× electrophoresis buffer: 1800 mL DDW and 200 mL 10× electrophoresis buffer, mix well and pour into the electrophoresis tank until the gel is covered; prepare 1× transfer buffer: 700 mL DDW, 200 mL methanol and 100 mL 10× transfer buffer, mix well and place at -20°C for later use.

[0079] (5) Add samples and protein molecular weight standards (Marker) to the loading wells, and fill the remaining wells with 1× Loading Buffer.

[0080] (6) Close the lid of the electrophoresis tank, turn on the switch, perform constant voltage electrophoresis, set the voltage to 80V, wait until the marker produces a red band and the band is horizontal and neat, then change the voltage to 120V.

[0081] (7) When the color indicator of the Loading Buffer reaches the bottom of the electrophoresis tank, pause the electrophoresis and remove the gel plate and wash it.

[0082] (8) Transfer: According to the molecular weight of the marker, cut the corresponding target protein band and place it in the old transfer solution. Cut out the PVDF membrane of the corresponding size to the protein band, place it in methanol solution to activate for 1 min, take it out and place it in the transfer solution. Place the gel and PVDF membrane in the order of "black gel and white membrane" in the transfer clamp, clamp the sponge-filter paper-membrane-gel-filter paper-sponge, and place it in the transfer tank. Pour in the pre-cooled 1× transfer solution, add ice cubes, and transfer the membrane at a constant current of 275mA for 75min.

[0083] (9) Blocking: Place the PVDF membrane in the blocking solution and block at room temperature for about 1 hour. Prepare the blocking solution and 10× TBS buffer solution according to Tables 10 and 11, respectively.

[0084] Table 10 Blocking solution formula

[0085]

[0086] Table 11 10×TBS formula

[0087]

[0088] (10) Incubation with primary antibody: Mix 900 mL DDW, 100 mL TBS, and 1 mL Tween to make a 1× TBST solution. Dilute the primary antibody with 1× TBST at a ratio of 1:1000, place the membrane in the primary antibody dilution solution, and incubate on a horizontal shaker for 1 h or overnight at 4°C.

[0089] (11) Incubation with secondary antibody: Wash the membrane three times with 1× TBST, 5 min each time. Dilute the secondary antibody with 1× TBST at a ratio of 1:5000 or 1:10000, place the membrane in the secondary antibody dilution solution, incubate on a horizontal shaker for 50 min, and then wash three times with 1× TBST, 5 min each time.

[0090] (12) Exposure and development: Place the PVDF membrane in an appropriate position on the developing plate, absorb the remaining liquid with filter paper, and evenly add ECL developer for exposure.

[0091] 4. Total RNA Extraction

[0092] (1) Cells were cultured in a 6-well plate. When the cell density reached 90%, total RNA could be extracted from the cells.

[0093] (2) Discard the old culture medium, gently rinse the cells twice with PBS, add 1 mL of Trizol to cover the cell surface at once, and gently blow the cells off with a regular pipette tip. Repeat the process until there is no stickiness. Transfer the cells to a 1.5 mL EP tube and let stand at room temperature for 10 min to allow the cells to completely lyse.

[0094] (3) Use an RNA-grade pipette tip to add 200 μL of chloroform, shake vigorously for about 30 seconds, and let stand at room temperature for 5 minutes.

[0095] (4) Place in a pre-cooled centrifuge, centrifuge at 4°C, 12,000 rpm for 15 min. After centrifugation, pipette 400 μL of supernatant into an RNA-grade EP tube, add 400 μL of isopropanol, mix thoroughly, gently invert up and down 8 to 10 times, and let stand at room temperature for 20 min.

[0096] (5) Centrifuge at 4°C, 12,000 rpm, for 10 min. Aspirate the supernatant, being careful not to aspirate the precipitate at the bottom of the EP tube.

[0097] (6) Add 1 mL of 75% alcohol prepared with DEPC water along the tube wall to wash the RNA precipitate.

[0098] (7) Centrifuge at 4°C, 12,000 rpm, for 5 min and aspirate the supernatant.

[0099] (8) Repeat steps (6) and (7).

[0100] (9) Open the lid and dry at room temperature for about 5 minutes. Add 10 μL of DEPC water to the sediment, soak for 3 minutes, and then gently blow to mix.

[0101] (10) Detect the RNA concentration, pay attention to the values ​​and absorption peak positions at A260 and A280, and then place the RNA on ice for subsequent reverse transcription.

[0102] 5. Reverse Transcription

[0103] (1) Prepare the genomic DNA removal reaction solution according to Table 12, mix the reaction solution with a pipette, and then place it in a preheated 42°C metal bath for incubation for 2 minutes to remove the genomic DNA.

[0104] (2) Prepare the reverse transcription reaction solution according to Table 13, mix well and incubate in a preheated 37°C water bath for 15 min, transfer to an 85°C metal bath and incubate for 5 s to obtain cDNA.

[0105] (3) Detect cDNA concentration. The obtained cDNA sample can be stored in a -80℃ refrigerator for long-term storage.

[0106] Table 12 Reaction solution for removing genomic DNA

[0107]

[0108] Table 13 Reverse transcription reaction solution

[0109]

[0110] 6. Wild-type plasmid construction

[0111] (1) Primer design: Primers were designed using Primer 5.0.

[0112] (2) Recover the target fragment by agarose gel electrophoresis: First, prepare the PCR reaction solution according to the PCR reaction system shown in Table 14, and perform the PCR reaction according to the procedure. Secondly, mix the above PCR product and 10×LoadingBuffer in a ratio of 9:1, and terminate the reaction. Prepare 1% agarose gel for electrophoresis, and set the electrophoresis conditions to 120V 30min. Then, place the agarose gel in a scanning gel imager, look for the target band under ultraviolet light, cut it off and put it in a 5mL EP tube. Finally, recover the DNA according to the agarose gel DNA recovery kit, and use a spectrophotometer to determine the fragment concentration.

[0113] Table 14 PCR reaction solution

[0114]

[0115] (3) Double enzyme digestion and product recovery: Prepare double enzyme digestion reaction solution (using EcoR I and BamH I as an example) according to Table 15. The digestion product is recovered using a common DNA product purification kit, and finally the concentration is measured using a spectrophotometer.

[0116] Table 15 Double enzyme digestion reaction system

[0117]

[0118] (4) Ligation: Prepare the ligation reaction solution as shown in Table 16. The reaction condition is 16°C overnight.

[0119] Table 16 Ligation Reaction Solution

[0120]

[0121]

[0122] (5) Transformation and plating: First, add the ligation product to 100 μL of DH5α competent medium and place on ice for 30 minutes. Heat shock in a 42°C water bath for 90 seconds and place on ice for another 5 minutes. Next, add 900 μL of resistance-free LB medium and culture at 37°C with shaking at 160 rpm for 45 minutes to 1 hour. Then, centrifuge at 12,000 rpm for 1 minute, remove part of the supernatant, and use the remaining 100 μL of liquid to resuspend the precipitate. Finally, add the suspension to the LB plate, spread it evenly with a spreader rod, and then place it in a 37°C incubator and culture it upside down for 12 to 16 hours.

[0123] (6) Picking and shaking bacteria: Select a single colony of appropriate size and shape, and use a small pipette to pick it out and put it into 5 mL of LB liquid culture medium containing resistance. Incubate overnight at 37°C and 160 rpm with shaking for 14 to 16 hours.

[0124] (7) Plasmid extraction: Before plasmid extraction, preserve the strain. In the clean bench, aspirate 500 μL of 40% glycerol and 500 μL of bacterial solution and mix them evenly, then store them in a -80°C refrigerator. The remaining bacterial solution is extracted according to the instructions of the Kangwei Endotoxin-free Plasmid Extraction Kit, the concentration is determined, and then stored in a -20°C refrigerator.

[0125] (8) Plasmid identification: The plasmid was sequenced by Coomassie Inc., and the sequencing results were compared using SeqMan software. The strains of the plasmids with successful sequence alignment were expanded and cultured for later use.

[0126] 7. Construction of mutant plasmids

[0127] (1) Primer design: Primers were designed using Primer 5.0.

[0128] (2) Using the wild-type plasmid constructed in the above steps as a template, one-step PCR is performed to obtain a large number of target fragments. The one-step PCR system is shown in Table 2.19.

[0129] (3) Add DNP1 and digest at 37°C for 3 h.

[0130] (4) The subsequent steps are the same as 2.2.7.1(5) to (8).

[0131] 8. Lentiviral packaging

[0132] The cells used for lentiviral packaging are mainly HEK293T cells.

[0133] (1) 12 hours before transfection, cells should be counted using a cell counter and plated in a 10 cm cell culture plate to a cell count of 1×10 7 When the cell density reaches 80%, cell transfection is performed.

[0134] (2) Before transfection, the cells should be replaced with a new medium. The old medium should be discarded, and the cells should be gently rinsed twice with PBS. The medium should be replaced with 4 mL of serum-free and antibody-free medium and placed in a cell culture incubator for continued culture.

[0135] (3) During transfection, the corresponding plasmid and liposome PEI should be diluted first, and the corresponding number of sterilized 1.5 mL EP tubes should be prepared. Transfection reagents A and B should be prepared respectively. 250 μL of DMEM medium without dual antibodies and plasmids should be added to tube A. The mass ratio of packaging plasmid PAX: core plasmid: packaging plasmid PMD should be 3:4:1, that is, 7.5 μg of packaging plasmid PAX, 10 μg of core plasmid and 2.5 μg of packaging plasmid PMD should be added to a 10 cm cell culture dish. 250 μL of culture medium without dual antibodies and 10 μg of PEI should be added to tube B. Gently mix the liquid in the tube with a pipette and let it stand at room temperature for 5 minutes.

[0136] (4) Mix the plasmid and liposomes thoroughly and let stand at room temperature for 15 min.

[0137] (5) Tilt the culture dish slightly so that all the culture medium is at the bottom. Use a pipette to gently add the plasmid and PEI mixture into the culture dish and return it to the cell culture incubator for further culture.

[0138] (6) 6 hours after transfection, discard the old culture medium in the cell culture dish and replace it with complete culture medium.

[0139] (7) Virus supernatant can be collected 48 hours after transfection, and then collected every 24 hours for a total of three times. The collected virus supernatant should be temporarily stored in a 4°C refrigerator.

[0140] (8) After all the virus supernatants are collected, they are first placed in a centrifuge at 800 rpm for 5 minutes. The purpose of this step is to remove the cell debris in the virus supernatant. Then, it is filtered into a new 50 mL centrifuge tube using a 0.22 μm filter membrane. The virus is then added to the ultrafiltration tube several times. The ultrafiltration tube needs to be rinsed with PBS in advance. Centrifuge at 3000 g, 4°C, until no liquid is filtered out. The liquid in the ultrafiltration tube is the virus concentrate. The virus is packaged in 1.5 mL EP tubes, 100 μL per tube, marked, sealed with sealing film, and stored in a -80°C refrigerator for later use.

[0141] 9. Lentivirus infection of cells

[0142] (1) Count cells before infection.

[0143] (2) Take a certain amount of cell suspension so that the number of cells is 1×10 3 , added to a 1.5mL EP tube containing 100μL of virus concentrate, and 5μg / mL of polybrene was added at the same time. Finally, an appropriate amount of double-antibody-free F12 medium was added to make the total volume 500μL, and it was allowed to stand at room temperature for 15min.

[0144] (3) Add the incubated virus-cell mixture into a 24-well plate and mark it.

[0145] (4) After 24 hours of infection, discard the liquid in the well plate, rinse the cells twice with PBS, and replace with complete medium. Then, change the medium every 24 hours. After 72 hours, a second infection can be performed, following the same steps as above.

[0146] 10. Detection of intracellular ROS levels

[0147] (1) Cell plating: The cell density in a 12-well plate is 1.25×10 5 / well, use 1 mL of complete medium to culture for 1 day.

[0148] (2) Treatment with or without drug for 48 hours.

[0149] (3) Half an hour before harvesting cells, add Cell Rox dye at a ratio of 1:500. No need to change the solution and continue culturing.

[0150] (4) Discard the old culture medium, digest with 1× trypsin for 2 min, terminate the digestion with a small amount of serum, transfer the cell suspension to a centrifuge tube, and centrifuge at 4°C, 300g, for 5 min to collect the cells.

[0151] (5) Wash cells twice with PBS, 4°C, 300g, 5 min.

[0152] (6) Resuspend the cells in 200 μL PBS and analyze by flow cytometry.

[0153] 11. Detection of intracellular DNA damage level (immunofluorescence technique)

[0154] (1) Cell plating: Place the cell slides in a 6-well plate in advance and plate the cells at a density of 3×10 5 / well and culture overnight.

[0155] (2) The next day, cisplatin was added and stimulated for 24 hours. After 24 hours, the cell slides were transferred to a new six-well plate, placed on ice, and washed once with pre-cooled PBS for 2 minutes.

[0156] (3) Wash once with 1 mL of 1×CSK permeabilization solution for 2 min.

[0157] (4) Wash once with 1 mL 1×CSK-0.5% Triton for 5 min.

[0158] (5) Wash again with 1 mL of 1×CSK permeabilization solution for 2 min, and then wash once with ice-cold PBS for 2 min.

[0159] (6) Fix with 4% paraformaldehyde at room temperature for 7 min, and wash three times with PBS at room temperature, each time for 2 min.

[0160] (7) Use 5% BSA-PBST blocking solution and block for 30 min at room temperature or overnight at 4°C.

[0161] (8) Incubation with primary antibody: Wash three times with PBST, 2 min each time, dilute the primary antibody with blocking solution at a ratio of 1:200, stick a sealing film on the lid of the culture dish, add 50 μL of primary antibody on the sealing film, remove the slide, control the water, place the slide with the cell surface facing the sealing film, and incubate overnight at 4°C in the dark.

[0162] (9) Incubation with secondary antibody: Wash three times with PBST, 2 min each time, dilute the secondary antibody at a ratio of 1:200 with blocking solution, add 50 μL to each slide and incubate at room temperature in the dark for 0.5 h.

[0163] (10) Wash three times with PBST, each time for 2 min. To stain the cell nuclei, dilute DAPI with 1× PBS at a ratio of 1:2000, add 2 mL of DAPI staining solution to each well, and stain for 2 min in the dark.

[0164] (11) Discard the DAPI diluent and add 2 mL of 1× PBS to wash three times, 2 min each time. Take out the slide and wash it with DDW for 5 s, then control the water.

[0165] (12) Mounting: Add 7 μL of Mowiol 488 mounting solution to each slide, and slowly cover the cell surface with the Mowiol 488 mounting solution to avoid bubbles. Keep the slides away from light for more than 4 hours and use a fluorescence microscope to take pictures and analyze.

[0166] 12. Detection of intracellular DNA damage level (flow cytometry)

[0167] (1) Cell plating: The cell density in a 12-well plate is 1.25×10 5 / well, use 1mL normal complete medium to culture for 1 day.

[0168] (2) Treatment with or without drug for 48 hours.

[0169] (3) Discard the old culture medium, digest with 1× trypsin for 2 min, terminate the digestion with an appropriate amount of serum, gently blow up the cells, transfer them to a centrifuge tube, and collect the cells by centrifugation at 800 rpm for 5 min.

[0170] (4) Wash the cells twice with 1 mL 1× PBS, 300 g for 5 min each time.

[0171] (5) Wash once with 1 mL 1× CSK permeabilization solution, 300 g for 5 min.

[0172] (6) Permeabilize with 100 μL 1×CSK-0.2% Triton on ice for 5 min.

[0173] (7) Wash once with 1 mL of 1× CSK permeabilization solution at 300 g for 5 min.

[0174] (8) Wash the cells once with 1 mL of ice-cold 1× PBS at 300 g for 5 min.

[0175] (9) Discard the supernatant and add 100 μL of 4% paraformaldehyde for fixation for 7 min.

[0176] (10) Wash the cells once with 1 mL of room temperature 1× PBS at 300 g for 5 min.

[0177] (11) Discard the supernatant and add 200 μL of 5% BSA-PBST blocking solution. Block for 1 h at room temperature.

[0178] (12) Incubation with primary and secondary antibodies: dilute the primary antibody at a ratio of 1:200 with blocking buffer, dilute the secondary antibody at a ratio of 1:500 with blocking buffer, and incubate with 100 μL of the primary and secondary antibody mixture for 1 h.

[0179] (13) Wash the cells once with 1 mL of room temperature 1× PBS at 300 g for 5 min.

[0180] (14) Add 200 μL 1× PBS, resuspend the cells, and detect on flow cytometry.

[0181] 13. Cell Apoptosis Detection

[0182] (1) Cell plating: The cell density in a 12-well plate is 1.25×10 5 / well, use 1mL normal complete medium to culture for 1 day.

[0183] (2) Treatment with or without drug for 48 hours.

[0184] (3) Collect the cells, retain the old culture medium, and digest the cells with EDTA-free trypsin for about 5 minutes. Add a small amount of serum to terminate the digestion, transfer the cell suspension to a centrifuge tube, and centrifuge at 4°C, 300g for 5 minutes to collect the cells.

[0185] (4) Resuspend the cells in complete culture medium and incubate at 37°C for 30 min.

[0186] (5) Wash the cells twice with pre-cooled PBS, 300g for 5 min each time.

[0187] (6) Resuspend the cells in 100 μL of 1× Annexin V binding buffer and stain with 3–5 μL each of YF647-Annexin V and PI for 10–15 min in the dark.

[0188] (7) Resuspend the cells in 400 μL PBS and analyze by flow cytometry.

[0189] 14. Nude mouse tumor experiment

[0190] (1) Select female nude mice aged 6 to 8 weeks.

[0191] (2) The desired cells were digested, collected, and counted. 1×10 6 The cells were resuspended in 100 μL PBS and inoculated into the axilla of the left forelimb of nude mice.

[0192] (3) Subsequently, cisplatin was intraperitoneally injected every other day at a dose of 2 mg / kg. The volume of the tumor inoculated in each nude mouse was detected, calculated, and recorded.

[0193] (4) Two weeks after inoculation, the tumors were removed from the nude mice, and their volumes were detected and recorded.

[0194] result

[0195] It is known that inhibiting the expression of PGAM1 will reduce the level of intracellular DNA synthesis and NADPH production, and increase the level of reactive oxygen species ROS and DNA damage. That is, high expression of PGAM1 can promote intracellular DNA synthesis, increase the level of reducing power NADPH, and thus balance the level of reactive oxygen species ROS, reduce DNA damage, and ultimately promote cell proliferation. This phenotype is exactly the opposite of the phenotype of DNA damage and increased ROS levels mediated by cisplatin, a first-line chemotherapy drug commonly used in clinical practice. Therefore, we speculate that PGAM1 may be involved in the resistance of non-small cell lung cancer to cisplatin. To this end, we conducted a series of experiments and obtained the following results and conclusions:

[0196] (I) Verify that cisplatin-resistant cells are insensitive to cisplatin treatment, Figure 1-3 As shown. Among them:

[0197] Figure 1 .Cisplatin-resistant A549 / DDP cells do not respond to cisplatin-induced ROS production. (A) Non-small cell lung cancer A549 cells and cisplatin-resistant A549 / DDP cells were seeded into 12-well plates at appropriate densities, and cells were collected after cisplatin treatment (10μM) for 48h to detect intracellular ROS levels. (B) The results of three independent experiments in A were statistically analyzed. n=3, one way-ANOVA, ****, P<0.0001; ns, P>0.05, no statistical difference.

[0198] Figure 2 .Cisplatin-resistant A549 / DDP cells do not respond to cisplatin-induced DNA damage. (A) A549 and A549 / DDP cells were seeded into 12-well plates at appropriate densities, and cells were collected after cisplatin treatment (10 μM) for 48 h to detect the intracellular γH2Ax level, i.e., the level of DNA damage. (B) The results of three independent experiments in A were statistically analyzed. n=3, one way-ANOVA, ****, P<0.0001; ns, P>0.05, no statistical difference, scale bar: 20 μm.

[0199] Figure 3 .Cisplatin-resistant A549 / DDP cells do not respond to cisplatin-induced apoptosis. (A) A549 and A549 / DDP cells were seeded into 12-well plates at appropriate densities, and cells were collected after cisplatin treatment (10 μM) for 48 h to detect cell apoptosis levels. (B) The results of three independent experiments in A were statistically analyzed. n=3, one way-ANOVA, ****, P<0.0001; ns, P>0.05, no statistical difference.

[0200] (ii) The phosphorylation level of PGAM1 Y119 in cisplatin-resistant cells contributes to resistance to cisplatin treatment, e.g. Figure 4-8 As shown, where:

[0201] Figure 4 .PGAM1 Y119 phosphorylation level in cisplatin-resistant A549 / DDP cells. (A) A549 and A549 / DDP cells were seeded into 6-well plates at appropriate density. After stable growth, the cells were collected to obtain the total intracellular protein, and the intracellular PGAM1 protein was enriched using IP technology. The expression of PGAM1 in A549 and A549 / DDP cells and the corresponding phosphorylation modification were detected using WB technology, and quantitative statistical analysis was performed. (B) The results of three independent experiments in A were statistically analyzed. n = 3, student t-test, **, P < 0.01.

[0202] Figure 5 .Build A549 / DDP WT and A549 / DDP Y119F Stable cell lines. (A) Overexpression of PGAM1 in A549 / DDP cells and PGAM1 knockdown (KD) A549 / DDP cells WT PGAM1 Y119F , The protein level of PGAM1 was detected by WB technology and quantified. (B) Statistics of the results of three independent experiments in A. n = 3, student t-test, **, P < 0.01; ns, P > 0.05, no statistical difference.

[0203] Figure 6 .A549 / DDP Y119F Cells re-responded to cisplatin-induced ROS production. (A) A549 / DDP WT and A549 / DDP Y119F Cells were seeded into 12-well plates at appropriate density, and cells were collected after 48 h of cisplatin treatment (10 μM) to detect intracellular ROS levels. (B) Statistics of the results of three independent experiments in A. n = 3, one way-ANOVA, *, P < 0.05; ns, P > 0.05, no statistical difference.

[0204] Figure 7 .A549 / DDP Y119F Cells re-respond to cisplatin-induced DNA damage. (A) A549 / DDP WT and A549 / DDP Y119F Cells were seeded into 12-well plates at appropriate density, and cells were collected after 48 h of cisplatin treatment (10 μM) to detect intracellular γH2Ax levels, i.e., DNA damage levels. (B) Statistical results of three independent experiments in A. n = 3, one way-ANOVA, **, P < 0.01; ns, P > 0.05, no statistical difference.

[0205] Figure 8 .A549 / DDP Y119F Cells re-responded to cisplatin-induced apoptosis. (A) A549 / DDP WT and A549 / DDP Y119F Cells were seeded into 12-well plates at appropriate density, and cells were collected after 48 h of cisplatin treatment (10 μM) to detect cell apoptosis. (B) Statistical results of three independent experiments in A. n = 3, one way-ANOVA, ****, P < 0.0001; ns, P > 0.05, no statistical difference.

[0206] (III) The above results indicate that PGAM1 Y119 phosphorylation specifically exists in cisplatin-resistant lung cancer cells and helps to resist the therapeutic effect of cisplatin. We designed a cell-penetrating peptide Y119E to inhibit PGAM1-PKM2 binding at the cellular level and inhibit the growth of cisplatin-resistant lung cancer at the mouse level. Figure 10-14 As shown. Among them:

[0207] Fig. 9 .Amino acid sequence of cell-penetrating peptide Y119E.

[0208] Fig.10 .Cell-permeable peptide Y119E inhibits the interaction between PGAM1 and PKM2. (A) Cisplatin-resistant human non-small cell lung cancer cells A549 / DDP were selected and treated with TAT or Y119E, respectively. Then, antibodies were used to enrich PGAM1 protein, and the binding of PKM2 and the phosphorylation level of PGAM1 were detected by WB technology. (B) The results of three independent experiments in A were statistically analyzed. n = 3, student t-test, ****, P < 0.0001.

[0209] Fig.11 .Diagram of subcutaneous tumor-bearing and cell-penetrating peptide Y119E administration pattern in mice.

[0210] Fig.12 .Cell-penetrating peptide Y119E inhibits tumor growth in nude mice. Nine pairs of 6-8 week-old male nude mice were randomly selected, and A549 / DDP cells were transplanted into the armpits of nude mice by subcutaneous injection. Then TAT or Y119E were injected intraperitoneally every other day. On the 20th day after transplantation, the tumors were removed from the mice and photographed.

[0211] Fig.13 . Cell-penetrating peptide Y119E inhibits tumor growth (volume) in nude mice. Fig.12 The tumor volumes of mice treated with WT and WT were calculated regularly. n=9, student t-test, ****P<0.0001, ns, P>0.05, no statistical difference.

[0212] Fig.14 .The cell-penetrating peptide Y119E inhibits tumor growth (mass) in nude mice. Fig.12 The mice treated with α-glucose were weighed and the tumor formation was detected. n=9, student t-test, ****P<0.0001.

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Claims

1. A cell penetrating peptide Y119E, characterized in that: The cell-penetrating peptide TAT was used as a carrier to deliver a cell-penetrating peptide containing PGAM1 phosphorylated at position Y119 into cells, including a Y119 phosphorylated cell-penetrating peptide (Y119E) with a sequence of YGRKKRRQRRR-KIWRRSEDVP.

2. A cell-penetrating peptide Y119E is used as a drug to inhibit the growth of cisplatin chemotherapy-resistant tumors.

3. The use of a cell-penetrating peptide Y119E according to claim 2 in the treatment of cisplatin chemotherapy-resistant tumor growth, characterized in that The tumor includes lung cancer.

4. The use of a cell-penetrating peptide Y119E according to claim 3 in the pharmaceutical application of inhibiting the growth of cisplatin chemotherapy-resistant tumors, characterized in that The lung cancer is non-small cell lung cancer.

Images