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Circ_C20orf11 Regulates The Mechanism Of YWHAZ Involvement In Cisplatin Resistance In Ovarian Cancer

Posted on:2023-11-26Degree:DoctorType:Dissertation
Country:ChinaCandidate:J YinFull Text:PDF
GTID:1524306827453104Subject:Oncology
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Objective: Chemotherapy resistance in ovarian cancer cells is an important cause of treatment outcome and prognosis.We propose to investigate the tumour drug resistance-associated gene YWHAZ to verify its regulatory mechanism with upstream non-coding RNAs in ovarian cancer and to validate the role of this regulatory mechanism in the process of drug resistance development in ovarian cancer cells.Methods:Prediction of upstream regulatory genes of YWHAZ using TCGA,GEO,GEPIA databases and online tools star Base and circ Bank;q RT-PCR assay for predicted gene expression to screen suitable experimental cell lines;DDP gradient concentration culture method(0.625,1.25,2.5,5,10,20 μM)to culture drug-resistant cell lines and use MTT assay was used to calculate the IC50 of the cells;double luciferase assay was performed to verify the target binding ability between the target genes;RNA interference technology was used to silence circ_C20orf11 and mi R-527 in the cells and stable culture;q RT-PCR was used to detect the expression levels of YWHAZ,mi R-527,mi R-423-5p and circ_C20orf11 under different conditions.The expression of extracellular vesicle markers CD9,CD63 and TSG101,tumor cell membrane proteins YWHAZ and PD-L1 were detected by Western Blot;cell cloning assay was performed to detect cell proliferation and flow cytometry to detect apoptosis;transmission electron microscopy and nanoparticle tracer were used to identify the morphology and particle size of EVs in cell culture supernatant and human serum The expression of macrophage-associated cytokines TNF-α,i NOS,CD206,IL-6,IL-10 and Arg-1 was measured by q RT-PCR.A nude mouse transplantation tumor model to detect the effect of circ_C20orf11 on ovarian tumor growth and tumor microenvironment in vivo.Results: 1)The expression levels of mi R-527,mi R-423-5p and circ_C20orf11 were significantly different in ovarian normal cells and ovarian epithelial malignant tumor cells.The expression levels of circ_C20orf11were the highest in ovarian cancer cell line SKOV3,A2780 and control ovarian normal epithelial cells(P<0.001);there were significant differences in the expression levels of drug-resistant cells SKOV3/DDP and A2780/DDP(P<0.01),and the expression of circ_C20orf11 in drug-resistant cells was significantly was significantly higher than that of ovarian cancer cells;2)IC50 values of SKOV3 cells treated with the same concentration of DDP were significantly different from those of SKOV3/DDP cells and A2780 cells from A2780/DDP cells(P<0.01),and IC50 values of drug-resistant cells were more than twice as high as those of sensitive cells;3)IC50 values of SKOV3/DDP cells and A2780/DDP cells after silencing circ_C20orf11 cells and A2780/DDP cells were treated with DDP,IC50 values decreased significantly compared with those before silencing,the number of cell clones decreased significantly and apoptosis increased significantly.circ_C20orf11 had a target binding site with mi R-527/mi R-423-5p and mi R-527/mi R-423-5p had a target binding site with YWHAZ.transfection of mi R-527-inhibitor was able to reverse the functional changes of cells after silencing circ_C20orf11,and a significant increase in the number of cell clones and a significant decrease in apoptosis could be observed;4)circ_C20orf11 is a pro-oncogene,and silencing circ_C20orf11 in drug-resistant cells,the expression of both YWHAZ and PD-L1 The expression of both YWHAZ and PD-L1 was significantly decreased(P<0.01)after transfection with mi R-527-inhibitor(P<0.05).The tumorigenic assay in nude mice showed a reduction in the size and weight of silenced circ_C20orf11 transplanted tumors,a significant increase in the expression of the oncogene mi R-527(P<0.01),and a significant decrease in the expression of both YWHAZ and PD-L1(P<0.01);5)The mean diameter of EVs isolated from cell supernatants was 100 nm,and that of EVs isolated from human-derived serum Both specific markers CD9,CD63 and TSG101 were expressed,and the expression level of circ_C20orf11 in serum EVs from drug-resistant patients was significantly different(P<0.01)and significantly increased compared to that of non-drug-resistant patients.EVs were co-cultured with macrophages and EVs were able to be taken up by macrophages and EVs with low expression of circ_C20orf11 induced more IL-6 secretion from macrophages,while IL-10 secretion was reduced.EVs that silenced circ_C20orf11 cells were able to induce a significant increase in TNF-α,IL-6 and i NOS expression in macrophages(P<0.01),increased M1-type polarization CD206,IL-10 and Arg-1 expression was reduced(P<0.05)and M2-type polarization was reduced.Conclusion: In ovarian cancer circ_C20orf11 is a pro-oncogene that 1)targets and regulates YWHAZ through mi R-527/mi R-423-5p to participate in cell proliferation and apoptosis,thereby enhancing the ability of ovarian cancer cells to tolerate DDP;2)induces macrophage M2-type polarization in the tumor microenvironment through EVs to participate in immune regulation and promote tumor progression and drug resistance.The above two may be possible mechanisms by which circ_C20orf11 regulates DDP resistance in ovarian cancer.
Keywords/Search Tags:Ovarian cancer, chemo-resistance, circular RNA, Extracellular vesicles, Tumor-associated macrophages
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