Study On The Inhibitory Effect Of Exosomal MiRNA-214-5p On Ovarian Cancer

Objective Ovarian cancer is one of the three common gynecological malignant tumors,which has the characteristics of insidious early symptoms,high recurrence rate and lack of targeted treatment.Tumor microenvironment is the necessary condition for tumor occurrence and development.Mesenchymal stem cells,exosomes and related miRNA,as important components of microenvironment,participate an important role in biological processes of the tumor,such as tumor proliferation and migration,angiogenesis,immunosuppression and so on.The purpose of this study is to clarify the effect of exosomal miR-214-5p derived from human mesenchymal stem cells(MSC)on the biological behavior of ovarian cancer and its possible signal pathway,so as to provide new ideas for possible therapeutic targets of ovarian cancer.Methods 1.Cell culture Human ovarian cancer cells were cultured in DMEM medium containing 10%fetal bovine serum and 1%penicillin-streptomycin double antibody.Culture of MSC cells in complete medium without exocrine serum.2.Screening and verification of miRNA In previous studies,we have obtained the differential expression profile of exosomal miRNA secreted by ovarian cancer cells by High-Throughput Sequencing.And we selected four different miRNA to verify the expression of the miRNA in ovarian cancer cells(A2780 and SKOV3),including miR-486-5p,miR-214-5p,miR-200b-3p and miR-203a.CCK8 and Transwell migration experiments were used to identify the corresponding differences in biological behavior among the four miRNA,and the target genes for subsequent experiments were selected.3.Extraction and identification of the exosome The human mesenchymal stem cells were cultured in vitro.We obtained the cell supernatant and the exosome by ultracentrifugation.The exosome samples were observed by transmission electron microscope,and the size of the exosome was measured by nano-particle size analyzer.Then we use Western blot to analyze the expression of the tag proteins(CD9,CD81,TSG101)of the exosome.4.Observation of MSC-exo-miR-214-5p by fluorescence microscope Human mesenchymal stem cells were cultured with miR-214-5p which labeled by cy3 in vitro,and the exosomes were extracted and labeled with PKH67.The exosomes were co-incubated with SKOV3.The content of miRNA in ovarian cancer cells before and after co-incubation was determined by RT-PCR.5.Co-culture of exosomes derived from MSC with ovarian cancer cells After adding miRNA NC and miR-214-5p,MSC was cultured in vitro,and the exosome was extracted from the MSC.MSC-exo-miRNA NC and MSC-exo-miR-214-5p mimics were added to two groups of human ovarian cancer cells(A2780)without intervention and co-cultured to a certain cell density for follow-up experiments.The cells in SKOV3 group were treated with the same method.The above two types of four groups of cells were numbered and grouped to distinguish.6.The effect of MSC-exo-miRNA-214-5p on ovarian cancer We use CCK8,FACS experiment,Transwell migration assay and scratch assay to verify the effect of MSC-exo-miRNA-214-5p on ovarian cancer cells.Treat SKOV3 cells by the same method.7.Biological information analysis of miRNA-214-5p Search for human ovarian cancer related genes by using DisGeNET database and Phenopedia database in HuGE Navigator.Python defines the intersection of the two databases,and the Jvenn tool draws the Venn diagram.The possible target genes of miR-214-5p were predicted by the analysis software miRwalk.The gene aggregation part of the above two parts was selected and the GO and KEGG enrichment analysis were analyzed by DAVID database,and we used R-Studio to draw the bubble diagram of enrichment analysis.Results 1.Screening and verification of miRNA According to the results of CCK8 and Transwell migration assay,the proliferation and migration ability of ovarian cancer cells transfected with miR-214-5p were significantly lower than those of miR-486-5p,miR-200b-3p and miR-203a groups.So we chose miR-214-5p as the follow-up target gene.2.Extraction and identification of the exosome After the process and extraction of the exosome of human mesenchymal stem cells,tea saucer-like exocrine particles were obtained by ultracentrifugation.The size of the tea saucer-like exosome was detected by nano-particle size analyzer,which is about 141.0nm.Western Blot showed that the label proteins of the exosome were positive,including CD9,CD81 and TSG101.3.Transfer the marked miRNA into the exosome,co-incubation with cells Cy3-labeled miRNA can enter the MSC by electroporation,and enter the SKOV3 cells with the exosome.RT-PCR showed that the content of miRNA in exosomes and ovarian cancer cells increased4.The effect of exosomal miRNA-214-5p on ovarian cancer According to CCK8,FACS experiment,Transwell migration assay and scratch assay,the proliferation and migration ability of human ovarian cancer cells transfected with miR-214-5p mimics was weaker than that of the control groups,and the apoptosis rate was significantly higher than that of the control groups.5.Biological information analysis of miRNA-214-5p There were 1224 human ovarian cancer related genes in DisGeNET database and 1269 human ovarian cancer related genes in HuGE Navigator database.Python identified 382 overlapping genes in the two databases.The possible target genes of miR-214-5p were predicted by the analysis software miRwalk.Python again screened 382 repeats in miRwalk and finally identified 53 genes.The biological functions and signal pathways of the 53 genes were analyzed by DAVID database,and the bubble diagram of enrichment analysis was drawn by R-Studio.Conclusion 1.MSC-exo-miR-214-5p mediates cellular communication between ovarian cancer cells and MSC.2.MSC-exo-miR-214-5p can inhibit ovarian cancer cell’s proliferation and migration,and promote their apoptosis.3.Based on the analysis of the possible target genes of miR-214-5p,it was found that there were 53 target genes related to ovarian cancer.According to the corresponding bioinformatics analysis of the selected target genes,it is found that they have biological behaviors such as angiogenesis and cytokine activity,and may be involved in MAPK,RAP1 and other signal pathways,but the related mechanisms still need to be verified by follow-up experiments.