| ObjectiveOvarian cancer is a gynecologic malignancy with third incidence rate but the highest fatality rate.Due to the unobvious clinical symptoms and extensive chemotherapy resistance,the therapeutic effect has not been ideal for many years.Among them,ovarian cancer chemotherapy resistance has been a key factor in the survival prognosis of ovarian cancer patients.Although there are many hypotheses about the occurrence of chemoresistance,the specific mechanism is not very clear.In this study,we analyzed the relationship between the level of leptin and survival in patients with epithelial ovarian cancer treated with different chemotherapy regimens in the database.The possible molecular mechanisms of leptin in the chemotherapeutic resistance of epithelial ovarian cancer were studied by the methods of cell chemotherapy in vitro,flow cytometry,chromatin immunoprecipitation and gene concentration analysis,and the possibility of leptin as a prognostic indicator of epithelial ovarian cancer treatment and prognosis was discussed.Methods1,1656 cases of epithelial ovarian cancer in TCGA database were grouped according to different chemotherapy regimens using Kaplan-Meier analysis technology and the effect of leptin expression on overall survival rate was analyzed.2.Multiple database analysis revealed that the expression of leptin had no direct effect on disease progression and survival prognosis of ovarian cancer patients.3.The appropriate curve concentration and IC50 value of five kinds of chemotherapeutic agents in two ovarian cancer cell lines were obtained by CCK8 assay.4,Vitro experiments showed that exogenous leptin recombinant protein could change the cytotoxicity of ovarian cancer cells by platinum combined with paclitaxel/docetaxel chemotherapy,further verification found that this effect only in the presence of paclitaxel/docetaxel have differences,suggesting that exogenous recombinant protein can decrease chemosensitivity to paclitaxel/docetaxel in ovarian cancer cells.5.Flow cytometry analysis showed that the use of paclitaxel chemotherapy drugs can increase the proportion of ovarian cancer cells in G2/M phase,while exogenous leptin recombinant protein can weaken this effect.6.The results of GSEA analysis showed that The patients with high expression of leptin treated with paclitaxel were enriched with HALLMARK-EPITHELIAL-MESENCHYMAL-TRANSITION(EMT)in the collection of characteristic genes of tumor markers and the KEGG gene set.7.The transcriptional factor site prediction and chromatin immunoprecipitation experiment verify that C/EBP a may be an important transcription factor of leptin,and can be combined with multiple binding sites at 0bp-3000bp in the starting area of leptin.(primer 1,301 bp to 655bp on ORF and primer 92742bp to 3000bp range).8.The results of luciferase reporter gene(lucifrase)activity detection showed that the luciferase activity of the upper leptin loci mutation group was significantly lower than that of the non mutant group after the same transfected C/EBP a overexpressed plasmid,and the difference was significant(p =0.004427191,p = 0.00896897,p = 8.8421 1E-05,p= 0.010116376).Conclusion1,Low leptin levels are more conducive to prognosis of ovarian cancer patients treated with platinum plus paclitaxel chemotherapy;2,Leptin recombinant protein reduced the killing effect of paclitaxel chemotherapy on ovarian cancer cells at the cell level;3,Leptin mediates the decrease in sensitivity of ovarian cancer cells to paclitaxel chemotherapy,which may be achieved by activating EMT;4,Transcription factor C/EBP a can bind to the upstream region of leptin and activate its transcriptional translation in ovarian cancer cells. |
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