Application of MOTS-c polypeptide and analogue thereof in reversing TRAIL drug resistance of tumor cells by activating MEF2A pathway

Abstract

The invention discloses an application of MOTS-c polypeptide and analogues thereof in reversing TRAIL drug resistance of tumor cells by activating an MEF2A pathway, and belongs to the technical field of biomedicine.By activating the MEF2A pathway, the MOTS-c polypeptide remarkably improves the sensitivity of the TRAIL drug-resistant tumor cells to drugs, and has a good application prospect. In vitro experiments and in vivo experiments, the compound shows good anti-tumor TRAIL drug resistance pharmacological characteristics, so that tumor diseases can be effectively prevented and / or treated. The compound can be used as a new generation of tumor chemotherapy sensitizer, and has a wide application prospect in the field of biological medicines.

Description

Technical Field

[0001] The invention discloses the use of MOTS-c polypeptide and analogs thereof for reversing TRAIL resistance of tumor cells by activating MEF2A pathway, and belongs to the technical field of biomedicine. Background Art

[0002] Hepatocellular carcinoma (HCC) is a common malignant tumor, mainly caused by persistent liver damage caused by factors such as hepatitis B and C virus infection, aflatoxin exposure, and long-term drinking. These factors lead to chronic inflammation of liver cells, which gradually develops into cirrhosis and may eventually evolve into hepatocellular carcinoma. According to statistics, there are about 800,000 new cases worldwide each year, most of which occur in Asia and Africa. For HCC patients, the current main treatments include surgical resection, liver transplantation, local ablation therapy, etc. However, due to the highly invasive and metastatic characteristics of HCC, the 5-year survival rate of patients is usually less than 20%. In addition, many patients are already in the advanced stage when diagnosed, missing the best time for treatment, further reducing the possibility of cure.

[0003] In recent years, mitochondrial 12S rRNA type c (MOTS-c), a peptide hormone encoded by mitochondria and widely present in various tissues, has attracted much attention due to its multiple functions in tumor biology. MOTS-c is not only involved in the regulation of cell metabolism, but also plays an important role in cell stress response and immune regulation. Studies have shown that the expression of MOTS-c is affected by multiple factors, such as oxidative stress, DNA damage and metabolic changes in mitochondria. Specifically, when cells face oxidative stress or DNA damage, mitochondria release MOTS-c to cope with these adverse conditions and maintain cell homeostasis. Studies have found that MOTS-c can promote mitochondrial biogenesis and enhance mitochondrial function, thereby improving the energy supply efficiency of cells. At the same time, it can also inhibit microglial activation and reduce inflammatory responses during cancer progression. Inflammatory response is an important pro-carcinogenic factor in the tumor microenvironment. By inhibiting inflammatory response, MOTS-c helps slow down the development of tumors. In addition, exogenous MOTS-c has been shown to effectively inhibit the proliferation, migration and invasion of ovarian cancer cells, while inducing cell cycle arrest and apoptosis. This suggests that MOTS-c may become a potential anti-cancer drug target.

[0004] The rapid expansion of tumors leads to a significant increase in the demand for oxygen by tumor cells, while the blood supply cannot meet this increased demand, thus triggering a state of hypoxia inside the tumor. Notably, hypoxia plays a crucial role in accelerating cancer progression. Studies have shown that under hypoxic conditions, cancer cells are resistant to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL is a transmembrane protein that can induce apoptosis by interacting with ligand receptors including death receptor 4 (DR4) and DR5. After TRAIL binds to DR4 or DR5, these receptors recruit and bind to a protein called fas-associating through the death domain (FADD). The FADD protein then recruits and activates caspase-8, which triggers a series of enzymatic reactions that ultimately lead to the degradation of cellular components and programmed cell death. Based on the potential of TRAIL receptor activation mechanism to induce apoptosis, a variety of cancer treatments targeting TRAIL receptor agonists are currently under development.

[0005] Actin-related protein 2A (MEF2A) is a transcription factor that regulates cell fate and adaptive responses. Further studies have confirmed its gene mutation in cancer progression, thereby inhibiting the process of tumorigenesis. Recent studies have shown that Houttuynia cordata Thunb induces HCC cell apoptosis by activating the hypoxia-inducible factor-1A (HIF-1A) / forkhead box O3 (FOXO3) / MEF2A pathway, suggesting that MEF2A plays a key role in the apoptotic mechanism of HCC cells. Through predictive analysis using the JASPAR online database (https: / / jaspar.genereg.net / ), we found possible binding sites of MEF2A to DR4 and DR5. Based on this, we speculated that MEF2A can inhibit the malignant progression of HCC by regulating TRAIL-induced tumor cell apoptosis through transcription.

[0006] Based on the above research results, we explored whether MOTS-c can alleviate the resistance of HCC cells to TRAIL-induced apoptosis under hypoxic conditions by upregulating MEF2A expression. TRAIL (TNF-related apoptosis-inducing ligand) is a protein naturally present in the human body that can selectively induce apoptosis in cancer cells without harming normal cells. However, under hypoxic conditions, HCC cells often become resistant to TRAIL, resulting in poor treatment effects. If MOTS-c can enhance the sensitivity of HCC cells to TRAIL by upregulating MEF2A expression, this will provide a new approach for HCC treatment. Summary of the invention

[0007] In order to overcome the shortcomings of the prior art, the present invention provides the use of MOTS-c polypeptide and its analogs for reversing TRAIL resistance of tumor cells by activating MEF2A pathway.

[0008] The technical solution of the present invention is as follows:

[0009] The present invention provides a use of a MOTS-c polypeptide and its analogs for reversing TRAIL resistance of tumor cells by activating the MEF2A pathway, characterized in that the use is for preparing a drug for preventing and treating tumor diseases, and the amino acid sequence of the MOTS-c polypeptide is: MRWQEMGYIFYPRKLR (SEQ ID NO.1).

[0010] Preferably, the derivative is a polypeptide derivative or analogue having the same activity as the amino acid sequence of SEQ ID NO.1 after substitution, deletion or addition of one or several amino acid residues;

[0011] Preferably, the tumor disease includes cancer;

[0012] Preferably, the cancer includes at least one of the following: liver cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, cervical cancer, leukemia, lymphoma, melanoma, glioblastoma, neuroblastoma, sarcoma or gastric cancer;

[0013] Preferably, the dosage form of the drug is an oral dosage form;

[0014] Preferably, the dosage form of the drug is a non-oral dosage form.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The MOTS-c polypeptide of the present invention significantly increases the sensitivity of TRAIL-resistant tumor cells to drugs by activating the MEF2A pathway, and exhibits good anti-tumor TRAIL resistance pharmacological properties in both in vitro and in vivo experiments, thereby effectively preventing and / or treating tumor diseases. The present invention can be used as a new generation of tumor chemotherapy sensitizers and has broad application prospects in the field of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the selection of experimental cells and induction time according to an embodiment of the present invention.

[0018] Figure 2 Schematic diagram of the selection of experimental cells and induction time in the embodiment of the present invention

[0019] Figure 3 This is a schematic diagram of MOTS-c promoting TRAIL-induced HCC cell apoptosis under hypoxic conditions in an example of the present invention.

[0020] Figure 4 This is a schematic diagram of an embodiment of the present invention in which MOTS-c promotes TRAIL-induced HCC cell apoptosis by upregulating MEF2A under hypoxic conditions.

[0021] Figure 5 This is a schematic diagram of an embodiment of the present invention in which MOTS-c promotes TRAIL-induced HCC cell apoptosis by upregulating MEF2A under hypoxic conditions.

[0022] Figure 6 This is a schematic diagram of an embodiment of the present invention in which MOTS-c promotes TRAIL-induced HCC cell apoptosis by upregulating MEF2A under hypoxic conditions.

[0023] Figure 7 This is a schematic diagram of an embodiment of the present invention wherein MEF2A targets DR4 and DR5 in HCC cells.

[0024] Figure 8 This is a schematic diagram of an embodiment of the present invention in which MOTS-c induces apoptosis of HCC cells under hypoxia by activating MEF2A and upregulating DR4 and DR5.

[0025] Fig. 9 This is a schematic diagram of an embodiment of the present invention in which MOTS-c induces apoptosis of HCC cells under hypoxia by activating MEF2A and upregulating DR4 and DR5.

[0026] Fig.10 This is a schematic diagram of an embodiment of the present invention in which MOTS-c induces apoptosis of HCC cells under hypoxia by activating MEF2A and upregulating DR4 and DR5.

[0027] Fig.11 This is a schematic diagram of an embodiment of the present invention in which MOTS-c effectively promotes TRAIL-induced HCC cell apoptosis by upregulating MEF2A in vivo.

[0028] Fig.12 This is a schematic diagram of an embodiment of the present invention in which MOTS-c effectively promotes TRAIL-induced HCC cell apoptosis by upregulating MEF2A in vivo. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1: Cell culture and treatment.

[0031] Experimental materials: HCC cell lines (Huh7, HepG2, Li-7, HCCLM3, and SNu387) were provided by EK-Bioscience, Shanghai, China. Huh7, HepG2, and HCCLM3 cells were cultured in Dulbecco's modified Eagle's medium provided by Biosun (Shanghai), and other cell lines were cultured in RPMI-1640 provided by Biosun. All cells were cultured in a medium containing 10% fetal bovine serum (Biosun) and 1% penicillin / streptomycin (Cytiva, Shanghai) at 37°C and 5% CO2. The culture medium was changed every 1 to 2 days. In order to simulate the hypoxic environment, the cells were exposed to a gas environment containing 94% nitrogen, 5% carbon dioxide, and 1% oxygen, and certain time points (4, 8, 12, 16, and 20 hours) were set. The control group was cells cultured under normal conditions containing 95% air and 5% carbon dioxide. In addition, Huh7 and HCCLM3 cells were treated with different concentrations of MOTS-c (China Peptide Co., Ltd., Shanghai) for 24 h (control group was treated with saline) and then subjected to hypoxia. HCC cells were incubated at 37°C for 18 h using the ATP-competitive AMPK inhibitor Compound C (MedChemExpress, Princeton, NJ, USA). Songon Biotech (Shanghai, China) provided small hairpin RNA of MEF2A (sh-MEF2A, targeting sequence 5'-CCTCCACTATCGGAGGAAGAGGAAT-3'), DR4 overexpression plasmid (oe-DR4, constructed using 42-1448 bp of DR4 gene), DR5 overexpression plasmid (oe-DR5, constructed using 138-1460 bp of DR5 gene), MEF2A overexpression vector (MEF2A, constructed using 346-1845 bp of MEF2A gene) and corresponding controls (sh-NC and oe-NC). Huh7 and HCCLM3 cells were seeded in 12-well plates. ThermoFisher's Lipofectamine 2000 reagent (Waltham, MA, USA), overexpression plasmids and shRNAs were mixed with Opti-MEM medium (Thermo Fisher) and then added to the cells according to the instructions and subjected to MOTS-c and hypoxia treatment.

[0032] Example 2: Hypoxia treatment reduced the protein expression of MEF2A, DR4 and DR5 in HCC cell lines.

[0033] Experimental methods: HCC cell lines (Huh7, HepG2, Li-7, HCCLM3, and SNu387) were provided by EK-Bioscience, Shanghai, China. Huh7, HepG2, and HCCLM3 cells were cultured in Dulbecco's modified Eagle's medium provided by Biosun (Shanghai), and other cell lines were cultured in RPMI-1640 provided by Biosun. All cells were cultured in a medium containing 10% fetal bovine serum (Biosun) and 1% penicillin / streptomycin (Cytiva, Shanghai) at 37°C and 5% CO2. The culture medium was changed every 1 to 2 days. To simulate a hypoxic environment, cells were exposed to a gas environment containing 94% nitrogen, 5% carbon dioxide, and 1% oxygen, and certain time points (4, 8, 12, 16, and 20 hours) were set. The control group was cells cultured under normal conditions containing 95% air and 5% carbon dioxide. In addition, Huh7 and HCCLM3 cells were treated with different concentrations of MOTS-c (China Peptide Co., Ltd., Shanghai) for 24 h (the control group was treated with saline) and then subjected to hypoxia.

[0034] Cell viability was analyzed by MTT assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Solerbo, Beijing, China) was added to each well of Huh7 and HCCLM3 cells seeded in a 96-well plate and incubated for 4 h. Then, the cells were incubated with 110 μL of color development solution for 10 min. Finally, the optical density at 490 nm was measured using an enzyme-linked immunosorbent assay (Thermo Fisher Scientific).

[0035] Western immunoblotting was performed. Total protein lysates were obtained from Huh7 and HCCLM3 cell samples and tumor tissues using RIPA buffer (Thermo Fisher Scientific). Twenty micrograms of lysates were electrophoresed on polyacrylamide gels (Thermo Fisher Scientific) and then transferred to PVDF membranes. Subsequently, the membranes were incubated with specific antibodies against MEF2A (Thermo Fisher Scientific, #PA5-27380), DR4 (Thermo Fisher Scientific, #PA1-30795), DR5 (Thermo Fisher Scientific, #PA5-19895), FADD (Thermo Fisher Scientific, #PA5-17808), caspase-8 (Thermo Fisher Scientific, #MA1-41280), and β-actin (Thermo Fisher Scientific, #PA1-16889). Protein bands were visualized using the EasyBlotECL kit from Songon Biotech. Protein grayscale was analyzed using Image J software from the National Center for Biotechnology Information (NIH, Bethesda, MD, USA).

[0036] Experimental results: Figure 1-2 As shown in the results, hypoxia treatment reduced the protein expression of MEF2A, DR4, and DR5 in HCC cell lines. Among them, the expression of Huh7 and HCCLM3 decreased more significantly, and Huh7 and HCCLM3 were selected for further study in this study. Subsequently, Huh7 and HCCLM3 cells were exposed to hypoxia for 4, 8, 12, 16, and 20 hours, respectively, to determine the appropriate induction time. The results showed that the survival ability of HCC cells was the highest after 12 hours of hypoxia treatment.

[0037] Example 3: MOTS-c promotes TRAIL-induced HCC cell apoptosis under hypoxic conditions by activating MEF2A.

[0038] Experimental method: Based on the above Example 2, Huh7 and HCCLM3 cells were treated with 20 μM MOTS-c under hypoxic conditions for 12 h for further analysis. Using the above-mentioned protein immunoblotting method, the PVDF membrane was incubated with specific antibodies against MEF2A (Thermo Fisher Scientific, #PA5-27380), DR4 (Thermo Fisher Scientific, #PA1-30795), DR5 (Thermo Fisher Scientific, #PA5-19895), FADD (Thermo Fisher Scientific, #PA5-17808), caspase-8 (Thermo Fisher Scientific, #MA1-41280) and β-actin (Thermo Fisher Scientific, #PA1-16889). The protein bands were developed using the EasyBlot ECL kit from SongonBiotech. Protein grayscale was analyzed using Image J software from the National Center for Biotechnology Information (NIH, Bethesda, MD, USA).

[0039] Experimental results: Figure 3 As shown in the results, MOTS-c significantly promoted the apoptosis of hepatocellular carcinoma (HCC) cells under hypoxia. In addition, hypoxia reduced the expression levels of MEF2A, DR4, DR5, FADD, and caspase-8, while MOTS-c effectively attenuated these effects.

[0040] Example 4: MOTS-c promotes TRAIL-induced HCC cell apoptosis by upregulating MEF2A under hypoxic conditions.

[0041] Experimental methods: Next, we investigated whether MOTS-c activated MEF2A to regulate TRAIL-induced HCC cell apoptosis under hypoxia. Cell transfection technology was used. Shanghai Songon Biotech Biotechnology Co., Ltd. provided small hairpin RNA of MEF2A (sh-MEF2A, targeting sequence 5'-CCTCCACTATCGGAGGAAGAGGAAT-3'). Huh7 and HCCLM3 cells were seeded in 12-well plates. Using Lipofectamine 2000 reagent (Waltham, MA, USA) from Thermo Fisher, overexpression plasmids and shRNAs were mixed with Opti-MEM medium (Thermo Fisher), then added to the cells according to the instructions, and MOTS-c and hypoxia treatment were performed.

[0042] Tunel staining was used to detect cell apoptosis. The culture medium of Huh7 and HCCLM3 cells was removed, and Tunel working solution was added to the culture wells, incubated for 0.5 hours, and then fixed according to the specific protocol of TUNEL apoptosis detection kit (#T2130, Solebio) for Tunel staining. Finally, the cells were analyzed for fluorescence intensity using a fluorescence microscope (Thermo Fisher) under the conditions of excitation wavelength / emission wavelength of 490 / 525nm. For flow cytometric analysis of cell apoptosis, Huh7 and HCCLM3 cells were washed with cold phosphate buffer and then suspended in binding buffer. According to the instructions of Solarbio commercial kit (#CA1020), cells were incubated with Annexin V-FITC and propidium iodide in binding buffer for 15 minutes. Cell apoptosis was detected using a flow cytometer (Thermo Fisher Scientific). The expression levels of related proteins were detected by the aforementioned protein immunoblotting method.

[0043] Experimental results: Figure 4-6 As shown in Figure 2, MEF2A knockdown promoted the survival of HCC cells. Conversely, under hypoxic conditions, MOTS-c inhibited the survival of Huh7 and HCCLM3 cells, but this effect was attenuated after MEF2A knockdown or administration of the AMPK inhibitor Compound C ( Figure 4 ). Similarly, MEF2A downregulation alleviated apoptosis of HCC cells under hypoxic conditions, while MOTS-c induced apoptosis in HCC cells. However, MEF2A knockdown or administration of Compound C reversed MOTS-c-induced apoptosis in HCC cells ( Figure 5 , Figure 7). In addition, MOTS-c increased the levels of p-AMPK, MEF2A, DR4, DR5, FADD, and caspase-8 in hypoxia-induced HCC cells. Conversely, MEF2A knockdown reduced the levels of MEF2A, DR4, DR5, FADD, and caspase-8 in MOTS-c-stimulated HCC cells under hypoxia, but had no effect on AMPK phosphorylation. However, the AMPK inhibitor Compound C reversed the promoting effect of MOTS-c on AMPK phosphorylation in hypoxia-induced HCC cells ( Figure 6 ). Therefore, the above data indicate that MOTS-c enhances TRAIL-induced apoptosis of hepatoma cells under hypoxia by activating MEF2A.

[0044] Example 5: MEF2A targets DR4 and DR5 in HCC cells.

[0045] Experimental methods: Shanghai Songon Biotech Biotechnology Co., Ltd. provided DR4 overexpression plasmid (oe-DR4, constructed using 42-1448bp of DR4 gene), DR5 overexpression plasmid (oe-DR5, constructed using 138-1460bp of DR5 gene) and MEF2A overexpression vector (MEF2A, constructed using 346-1845bp of MEF2A gene).

[0046] Dual luciferase reporter gene assay. The possible binding sites of MEF2A to DR4 and DR5 were predicted using the JASPAR online database, and wild-type (WT) and mutant (MUT) DR4 and DR5 reporter plasmids were constructed based on the predicted binding sites. In brief, the DR4 or DR5 sequence containing the binding site was amplified by polymerase chain reaction and then inserted into the pGL3 vector. The resulting vectors were named DR4-WT and DR5-WT, respectively. The binding sites in the DR4 and DR5 sequences were mutated by Shanghai Songon Biotech Biotechnology Co., Ltd., and DR4-MUT and DR5-MUT were then generated using the mutated DR4 and DR5 sequences. HCC cells dispersed in culture medium were introduced with reporter plasmids, MEF2A overexpression plasmids, or corresponding negative controls (NC). Subsequently, the cells were cultured for 48 hours, and the samples were analyzed using a commercial kit (Solarbio, product number #D0010).

[0047] Chromatin immunoprecipitation (ChIP) assay. Formaldehyde was added to each dish to crosslink HCC cells according to the specific method of the ChIP kit provided by Wanlei Bio (Shenyang, China). The cells were mixed with glycine and protease inhibitors and disrupted using ultrasound. The cells were mixed with ProteinA+G magnetic beads and then immunoprecipitated with MEF2A antibody (ThermoFisher, #PA5-27380), RNA polymerase II antibody (Wanlei Bio), and normal IgG antibody (Wanlei Bio). The melting reaction was performed by adding NaCl to the sample, and the DNA fragments were recovered by the Wanleibio PCR purification kit. Finally, the precipitated DNA was analyzed by agarose gel electrophoresis.

[0048] Experimental results: The JASPAR online database was used to predict the possible binding sites of MEF2A to DR4 and DR5. The results are as follows Figure 8 As shown. ChIP experiments showed that MEF2A has a strong affinity for the promoter regions of DR4 and DR5 ( Fig. 9 Subsequently, in Huh7 and HCCLM3 cells, the luciferase activity of the DR4-WT or DR5-WT and MEF2A co-transfection groups was significantly increased compared with the DR4-WT or DR5-WT and NC co-transfection groups, while the luciferase activity of the DR4-MUT or DR5-MUT and MEF2A co-transfection groups was not significantly different from that of the DR4-MUT or DR5-MUT and NC co-transfection groups ( Fig.10 ). Therefore, MEF2A is able to specifically target death receptors DR4 and DR5 in HCC cells.

[0049] Example 6: MOTS-c induces apoptosis of HCC cells under hypoxia by upregulating DR4 and DR5 via activating MEF2A.

[0050] Experimental methods: The regulatory role of DR4 and DR5 in MOTS-c-mediated TRAIL-induced apoptosis of hepatocellular carcinoma (HCC) cells was further explored. The cell activity was detected by the MTT method, cell apoptosis was analyzed by flow cytometry, the cells were further detected by Tunel staining, and the expression level of related proteins was detected by apoptosis protein immunoblotting.

[0051] Experimental results: Figure 11-12The results showed that the inhibitory effect of MOTS-c-induced Huh7 and HCCLM3 cell viability was weakened by the decrease of MEF2A expression, while this inhibitory effect was alleviated when DR4 or DR5 expression was increased. In addition, under hypoxia, MOTS-c treatment significantly promoted the apoptosis of Huh7 and HCCLM3 cells; however, after transfection of sh-MEF2A to knock down MEF2A, the apoptosis of Huh7 and HCCLM3 cells was inhibited, while overexpression of DR4 or DR5 partially reversed the above effects. It is worth noting that low expression of MEF2A inhibited the changes in MEF2A, DR4, DR5, FADD and caspase-8 levels in HCC cells induced by MOTS-c treatment under hypoxia, but after transfection of DR4 or DR5 overexpression plasmids, FADD, caspase-8, DR4 or DR5 were reversed. In summary, therefore, MOTS-c regulates the expression of DR4 and DR5 by upregulating MEF2A and promotes the apoptosis of HCC cells under hypoxia.

[0052] Example 7: MOTS-c effectively promotes TRAIL-induced HCC cell apoptosis in vivo by upregulating MEF2A.

[0053] Experimental methods: Tumor-bearing nude mouse model. Thirty 5-6-week-old male BALB / c nude mice were purchased from Hunan Shengyi Jingda Experimental Animal Co., Ltd. (located in Changsha, China). These nude mice were housed in a sterile environment with free access to food and water. Huh7 and HCCLM3 cells (5×10^6 each) were prepared into cell suspensions in phosphate-buffered saline (PBS) and injected subcutaneously into the axillary region of nude mice. The experimental mice were divided into three groups according to the treatment: 1. Normoxic group: maintained under normal environmental conditions before cell transplantation and treated with PBS (n=5); 2. MOTS-c group: placed under hypoxic conditions before cell transplantation, and then given 5 mg / kg of MOTS-c by intraperitoneal injection every day for 20 consecutive days (n=5); 3. Control group: also placed under hypoxic conditions before cell transplantation, but only received intraperitoneal injection of PBS (n=5). After 20 days, the mice were killed by cervical dislocation, and the formed nodules were collected for immunohistochemical analysis.

[0054] Immunohistochemistry (IHC) analysis. Primary tumors of HCC cells were embedded in paraffin and cut into 4-μm-thick sections. The slides were heated in a microwave oven and immunohistochemically stained using p-AMPK (#PA5-17831, Thermo Fisher), MEF2A (#PA5-27380, Thermo Fisher), DR4 (#PA1-30795, Thermo Fisher), and DR5 (#PA5-19895, Thermo Fisher) antibodies. The slides were counterstained with hematoxylin (Solarbio) and images were captured using an optical microscope (Thermo Fisher).

[0055] Experimental results: Figure 8-10 The results showed that hypoxia treatment inhibited the protein expression of MEF2A, DR4, DR5, FADD and caspase-8, while MOTS-c treatment reversed this trend. Immunohistochemistry experiments further confirmed that hypoxia treatment significantly reduced the positive expression rate of MEF2A, DR4 and DR5, while MOTS-c treatment significantly restored the expression levels of these proteins. In summary, MOTS-c treatment can effectively promote TRAIL-induced HCC cell apoptosis by upregulating MEF2A.

[0056] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily realized. Therefore, without departing from the general concept defined by the claims and equivalent scope, the present invention is not limited to the specific details and the illustrations shown and described here.

Claims

1. The use of MOTS-c polypeptide and its analogs to reverse TRAIL resistance of tumor cells by activating MEF2A pathway, characterized in that: The use is to prepare drugs for preventing and treating tumor diseases. The amino acid sequence of the MOTS-c polypeptide is: MRWQEMGYIFYPRKLR (SEQ ID NO.1).

2. The use according to claim 1, characterized in that The derivative is a polypeptide derivative or analogue having the same activity as the amino acid sequence of SEQ ID NO.1 after substitution, deletion or addition of one or several amino acid residues.

3. The use according to claim 1, characterized in that The tumor disease includes cancer.

4. The use according to claim 3, characterized in that The cancer includes at least one of the following: liver cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, cervical cancer, leukemia, lymphoma, melanoma, glioblastoma, neuroblastoma, sarcoma or gastric cancer.

5. The use according to claim 1, characterized in that: The dosage form of the drug is an oral dosage form.

6. The use according to claim 1, characterized in that: The dosage form of the drug is a non-oral dosage form.

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