The Screening,Identification And Functional Study Of Cisplatin Resistance Related MiRNAs Regulated By Exosomes In Ovarian Cancer Cell Line A2780 Based On High-throughput Sequencing

Objective:To investigate the influence of exosomes derived from supernatant of ovarian cancer cisplatin resistant cell line A2780/DDP on its parent cell line A2780,detect the difference of exosomes mi RNA in supernatant of A2780/DDP and A2780 cells,and search for exosomes mi RNA targets related to cisplatin resistance of ovarian cancer.The related role of mi RNA in ovarian cancer was also discussed.Methods:1.Exosomes derived from supernatant of A2780/DDP cells were obtained by overspeed centrifugation or kit method;The morphology and size of cell supernatant extract were observed by Transmission Electron Microscope.The particle size of cell supernatant extract was detected by Nano Particle Size Analyzer.Western Blot was used to detect the expression of CD63,CD9,Ep CAM and Annexin V in cell supernatant extracts.2.The extracted exosomes from supernatant of A2780/DDP cells were co-cultured with A2780 cell line and the expression levels of autophagy related genes such as P62,LC3-I and LC3-II in the treated A2780 cell line were detected by Western Blot.The survival rate of A2780 cells after treatment was detected by CCK-8 method.Immunofluorescence microscopy was used to observe the uptake of exosomes from supernatant of A2780/DDP cells after Dil staining in A2780 cell line after Dio staining.3.Exosomes from supernatant of A2780 and A2780/DDP cells were extracted,and the mi RNA expression profile of differential exosomes was detected by high-throughput sequencing(HTS)technology.Target genes of related exosome mi RNAs were predicted using mi RDB and Targetscan online database.The related functions and pathways of exosome mi RNA were predicted by GO and KEGG databases,respectively.4.Predicted the correlation between Hsa-Mi R-675-3p and cisplatin IC50 in ovarian cancer.Ovarian cancer samples were extracted from UCSC-Xena platform and mi RNA-m RNA network was constructed with GENCODE and TCGA database.R language combined with five algorithms including ESTIMATE,GSVA,CIBERSORT,MCPcounter and x Cell predicted the association between hsa-mi R-675-3p and the immune microenvironment of ovarian cancer.Results:1.Transmission electron microscopy showed that the supernatant extracts of ovarian cancer cell line A2780 and A2780/DDP cells were vesicles with a diameter of 40-120 nm.The diameter of supernatant extract from cisplatin-resistant ovarian cancer cell line A2780/DDP was 40-120 nm.Western blot showed that CD63,CD9,Ep CAM and Annexin V were detected on the supernatant extract of cisplatin-resistant ovarian cancer cell line A2780/DDP.2.The ovarian cancer cisplatin-resistant cell line A2780/DDP derived exosome was co-cultured with human ovarian cancer cisplatin-sensitive cell line A2780.Western Blot showed that the autophagy related proteins P62,LC3-1 and LC3-II of cisplatin-sensitive ovarian cancer cell line A2780 were increased in cisplatin-sensitive ovarian cancer cell line A2780.CCK8 results of cell proliferation and toxicity test showed that the drug resistance level IC50 increased in the experimental group.Immunofluorescence results showed that the exosome derived from cisplatin-resistant ovarian cancer cell line AA2780/DDP could be taken up by human cisplatin-sensitive ovarian cancer cell line A2780.3.High-throughput sequencing(HTS)results showed that 103 mi RNAs were up-regulated and 23 mi RNAs down-regulated in the exosome derived from A2780 and A2780/DDP cells.Ten exosomal mi RNAs upregulated in cisplatin-sensitive ovarian cancer cell line A2780/DDP were screened,and the downstream target genes were predicted by online mi RDB and Targetscan,and the downstream target genes of exosomal mi RNAs were classified by Venn diagram.The results of gene ontology GO analysis showed that biological process(BP)included cellular process,developmental process and anatomical structure development.Cell composition(CC)includes cellular processes,developmental processes and the regulation of anatomical structure development.Molecular function(MF)includes protein binding,transcriptional activity and DNA binding.KEGG analysis of Kyoto Encyclopedia of Genes and Genomes showed that PI3K-Akt signaling pathway,Wnt signaling pathway and Erbb signaling pathway may be involved in the process of ovarian cancer drug resistance.4.Real-time quantitative PCR showed that the expression level of Hsa-mi R-675-3p in the exosome derived from cisplatin-resistant ovarian cancer cell line A2780/DDP was the highest,followed by Hsa-mi R-429 and Hsa-mi R-371a-5p.PPI analysis of protein interaction network showed that FMR1 and CD86 were central genes.The results of GDSC database analysis showed that the expression levels of Hsa-mi R-675-3p and Hsa-mi R-429 were positively correlated with the expression levels of IC50 at half inhibitory concentration.Analysis results of mi RNA-m RNA regulatory network showed that drug-resistant related proteins ANXA1,AREG,DUSP1,DUSP,EGR1,HRAS,IL6,ILK,MAFB,MFAP5,MSLN,NR4A1,PINK1,PRKCDBP,PTGER3,SNCA,STIM1 and TGFBI were positively correlated with Hsa-mi R-675-3p(P < 0.05).The results of immunorelated microenvironment analysis of ovarian cancer showed that upregulation of Hsa-mi R-675-3p showed a certain correlation with epithelial cells of ovarian cancer(P<0.05).Conclusions:1.Exosomes can be successfully isolated from supernatant extracts of human ovarian cancer cell line A2780/DDP and A2780 cells.2.Exosomes derived from supernatant of cisplatin-resistant ovarian cancer cell line A2780/DDP can be taken up by downstream cisplatin-sensitive ovarian cancer cell line A2780,and influence the levels of cisplatin-resistant and autophagy.3.Hsa-mi R-675-3p derived from the exosome of ovarian cancer cell line A2780/DDP is a potential target of cisplatin resistance in ovarian cancer.