The Inhibitory Effect Of Ebastine On The Growth Of Melanoma Cells And The Role Of YTHDF2 In Melanoma

BackgroundMelanoma is a malignant tumor derived from skin melanocytes.Although its incidence is relatively low,it is characterized by early metastasis,high mortality,low lethal age,and poor prognosis,which seriously threatens human life.In the early stage of the disease,optimistic results can be obtained through surgical treatment,but once move to the metastatic stage,it is difficult to obtain satisfactory treatment effect even after radiotherapy and chemotherapy.Therefore,it is particularly important for clinical treatment of melanoma to explore molecular mechanism of the occurrence and development of melanoma,and to find new therapeutic methods and molecular targets.Ebastine is a classic potent H1-Histamine Receptor(H1-Histamine Receptor)antagonist with long-acting antihistamine effect and excellent performance in anti-allergy,antiinflammatory and other aspects.It is a commonly used drug for chronic urticaria,allergic rhinitis and other allergic diseases.More and more studies have shown that Ebastine also plays an important role in tumor treatment,but there is no relevant report on its role in melanoma.Autophagy is a phenomenon of "self-digestion" in eukaryotic organisms and a type Ⅱprogrammed cell death.Self-renewal of cells and tissues is achieved by removing damaged or excess organelles,pathogens and protein aggregates,maintaining homeostasis and cell survival.Studies have shown that autophagy is closely related to the occurrence and development of tumors,affecting tumor-host interactions by promoting stress adaptation and inhibiting the activation of innate and adaptive immune responses.Several studies have demonstrated that tumor cells can degrade oncoproteins through autophagy to inhibit the occurrence and development of tumors.Searching for drugs that can cause "autophagic death" has become a hot spot in the study of autophagy.N6-adenylate(m6A)methylation is the most common internal RNA modification in eukaryotic organisms,and it is also a dynamic and reversible post-transcriptional modification.It is installed by m6A methyltransferases,demethylases,and m6A binding proteins.YT521-B homology domain family 2(YTHDF2)is an important member of m6A "readers".It mediates the degradation of mRNA by recognizing specific m6A modification sites.YTHDF2 has been found to be involved in the development of a variety of cancers,including bladder cancer,hepatocellular carcinoma,gastric cancer,breast cancer,osteosarcoma,cervical cancer,prostate cancer,pancreatic cancer,leukemia,etc.ObjectiveBased on the current research that Ebastine plays a role in the treatment of various tumors,we further study its effect and mechanism on melanoma,so as to provide more theoretical basis for the treatment of melanoma.To clarify the expression of YTHDF2 in melanoma tissue and normal skin tissue;to study the effect of YTHDF2 on the biological behavior of melanoma cells,and to further explore its regulatory mechanism;to further clarify the effect of m6A methylation on melanoma in order to find more effective therapeutic targets.Method1.To culture human melanoma cells A375 and M14 in vitro and cell viability was detected by cell counting kit(CCK-8)and IC50 was calculated.2.The effect of Ebastine on autophagic flux in melanoma cells was detected by transfecting mCherry-GFP-LC3B fluorescent plasmid,and the number of fluorescent spots in different experimental groups was counted to judge the development of autophagic flux.3.To clarify the regulation of Ebastine on autophagy and signaling pathways in melanoma cells,western blot was used to detect the changes of autophagy-related proteins(LC3 and Beclin1)and AKT/mTOR pathway proteins.4.The expression of YTHDF2 in normal skin tissue and melanoma tissue was detected by immunohistochemistry,and the relationship between its expression level and clinical parameters was analyzed.5.YTHDF2 overexpression and interference cell lines was constructed by lentivirus and plasmid.The effect of YTHDF2 on the proliferation ability of melanoma cells was detected by CCK-8 and plate cloning assay;Transwell assay was used to detect the migration ability of YTHDF2 on melanoma cells.6.The downstream target genes of YTHDF2 were predicted by using the ENCORI database,confirmed by western blot and RNA binding protein immunoprecipitation assay,and MCL1 was selected as the downstream target gene.Results1.The median inhibition concentration(IC50)of Ebastine in melanoma cells A375 and M14 determined by CCK-8 was 3.353μmol/L and 3.785μmol/L,choosing 2.0μmol/L and 4.0μmol/L Ebastine for follow-up experiments;The Ebastine concentration of 2μmol/L was selected to treat melanoma cells for CCK-8 cell proliferation experiment.Compared with the control group,the cell viability of the Ebastine-treated group was significantly reduced.2.After transfection with the mCherry-EGFP-LC3B dual-fluorescent plasmid,Laser confocal microscopy results show:compared with the control group,the autophagy level of melanoma cells in the Ebastine-treated group was significantly increased,The number of autophagosome and autolysosome increased significantly.3.The results of western blot showed that compared with the control group,the expression of autophagy-related proteins LC3-Ⅱ/Ⅰ and Beclinl was significantly increased after Ebastine treatment.Significant reductions in p-AKT/AKT and p-mTOR/mTOR were found after treatment of melanoma cells with different concentrations of Ebastine.4.The results of immunohistochemistry showed that the expression of YTHDF2 in melanoma tissue was significantly higher than that in normal skin tissue;statistical analysis showed that the expression level of YTHDF2 was not correlated with the age,gender and lymph node metastasis of melanoma patients.5.Compared with the control group,the proliferation and migration abilities of the cells were significantly decreased after stable overexpression YTHDF2 in melanoma cells;after silencing YTHDF2,the proliferation and migration abilities of melanoma cells were significantly improved.6.We choose MCL1 as candidate target genes by querying ENCORI database and reading related literatures;according to the results of western blot and RNA-binding protein immunoprecipitation,MCL1 was finally selected as the downstream target gene for research.Conclusion1.By inhibiting the AKT/mTOR pathway,Ebastine activates the autophagy pathway in melanoma cells and reduces the viability of melanoma cells.2.YTHDF2 was highly expressed in melanoma tissues.YTHDF2 plays a biological role in inhibiting tumor proliferation and migration in melanoma and regulates the expression of MCL1.