Research On The Role Of Let-7b Targeting EZH2 In The Invasion And Metastasis Of Ovarian Cancer

ObjectiveTo explore more regulatory effect of hsa-Let-7b at post-transcriptional level through bioinformatics approaches for hsa-Let-7b target prediction and analysis.MethodInformation related to the human of let-7b was performed by using mi RBase online database.Access entry the hsa-let-7b-5p by using TARGETSCAN-VERT?PICTAR-VERT and RNA22-HSA three mi RNA target database forecast analysis of targets of human let-7b possible binding sites found in the potential targets of human let-7b.Ontology Gene(GO)enrichment and Encyclopedia of genes and genomes Kyoto(KEGG)pathway analysis were performed on the corresponding targets by DAVID database.The targets which were closely related to the invasion ability of ovarian cancer cells were selected as the research objects.ResultsThrough TARGETSCAN-VERT?PICTAR-VERT and RNA22-HSA three kinds of mi RNA target prediction software to predict the target of Let-7b,three software intersection Let-7b targets was 269.Found that the enrichment of the targets of human Let-7b-5p in collagen,extracellular matrix components,receptor complexes,extracellular matrix,fibrous collagen,part of plasma membrane,plasma membrane integral eight cell components(P < 0.01),involved in protein binding,growth factor binding,components of extracellular matrix,transformation growth factor beta receptor activity of 13 molecular function(P < 0.01)involving cell development process,cell metabolism regulation,regulation of the biosynthesis process,regulation of signal transduction,protein translation after modification,organ development 100 biological processes(P < 0.01).Analyzing the the KEGG pathway found the target gene of human Let-7b-5p significantly enriched in the MAPK signal transduction pathway,pancreatic cancer,chronic myeloid cells of leukemia,focal adhesions,JAK-STAT pathway,type 2 diabetes,cancer pathway,extracellular matrix interaction melanoma,calcium signaling pathway(P < 0.05);attention and ovarian cancer invasion is closely related to the gene transfer function.Finally,select of zeste Enhancer 2 repressive complex Polycomb 2subunit(EZH2)as Let-7b possible targets for further study.ConclusionLet-7b involves a wide range of regulation,EZH2 was likely to be a target of Let-7b.ObjectiveTo investigate the expression of Let-7b and EZH2 in ovarian tumor tissue,and analyze the relationship between them and clinical pathological characteristics.MethodFrom January 2014 to 2015 December,93 cases of ovarian tissue were collected from patients who had undergone surgery at the Department of Gynecology in the First Affiliated Hospital of Guangxi Medical University.The expression levels of Let-7b and EZH2 in ovarian tissue were examined by quantitative real-time PCR and immunohistochemistry.The relationship between Let-7b and EZH2 in normal ovarian tissues,benign ovarian tumor,borderline ovarian tumors and ovarian cancer tissue were also assessed.RESULTSThe expression level of let-7b in ovarian cancer was lower than any group,and the expression level of EZH2 in ovarian cancer was higher than any group.The expression level of let-7b significantly correlated with histological type,histological grade,lymph node metastasis and FIGO stage((P<0.05),but it showed no correlation with age.There was a correlation between the high expression of EZH2 and histological type,histological grade,lymph node metastasis and FIGO stage((P<0.05),but it showed no correlation with age.CONCLUSIONThe expression levels of Let-7b and EZH2 were closely related to the occurrence and the development of ovarian cancer,and the Let-7b expression was inversely correlated with the EZH2 in ovarian tissues.ObjectiveTo investigate the influence of Let-7b expression on the biological behavior of ovarian cancer A2780 and SKOV3 cell lines.MethodOvarian cancer A2780 and SKOV3 cell were transfected with artificial synthesized Let-7b mimics and inhibitor by cationic liposome.And then set the negative control group and the control group.Transfection efficiency was detected by Quantitative Real-time PCR.The difference of cell proliferation between transfected cells and not were assessed by Cell Counting Kit-8(CCK8),cell migration and invasion ability were tested by Transwell chamber and Wound Healing Assay,cells apoptosis were detected by flow cytometry.Results1.Efficiency of cell transfectionRT-PCR detection of other results,we can see that the effective transfection of each oligo and other mimics transfected group effective over expression of the other,and other inhibitor transfection group,the other for low expression.2.The impact of Let-7b mimics and Let-7b inhibitor on the biological behavior of ovarian cancer A2780 cells and SKOV3 cell lines.(1)Cell proliferation ability was analyzed by CCK8 assayTo detect the cell proliferation of A2780 and SKOV3 cells for each group at five points of(0h,24 h,48h,72 h,96h)by CCK8 assay,respectively.The results showed that cell proliferation capacity of both of A2780 and SKOV3 cell transfected with let-7b mimics were decreased while was compared to the control group(P < 0.05),by contrast,cell proliferation capacity of both of A2780 and SKOV3 transfected with let-7b inhibitor in each group were enhanced while was compared to the control group(P < 0.05).(2)Wound Healing AssayResults showed that in A2780 and SKOV3 cells cell lines,who was transfected Let-7 b mimics migration slower than the blank control group in 48 h,the difference was statistically significant(P < 0.05).Transfection Let-7 b inhibitor of cell migration faster than the blank control group,the difference was statistically significant(P < 0.05).(3)Invasion ability of cellTo detect invasion ability to A2780 and SKOV3 cells after transfection 48 hours in each group by counting and photographing the penetrating cells.The results showed that the number of A2780 and SKOV3 cells transfected with let-7b mimics were significantly decreased compared with blank group(P <0.05),by contrast,the number of A2780 and SKOV3 cells transfected with let-7b inhibitor were significantly increased compared with blank group(P <0.05).(3)Apoptosis were detected by flow cytometryThe results showed that in the ovarian cancer A2780 cell line,the let-7b mimics group early apoptosis was obviously higher than that of blank control group B and NC-mimics difference was statistically significant,there was no difference between let-7b inhibitor group,group B and NC-inhibitor group(P >0.05).And in ovarian cancer cell line SKOV3,let-7b mimics group early apoptosis was obviously higher than that of blank control group B and NC-mi difference was statistically significant(P < 0.05)that let-7b inhibitor group early apoptosis was significantly lower than that in group B and group NC-inhibitor,the difference has statistical significance(P < 0.05).ConclusionOverexpress Let-7b can inhibit the proliferation,invasion and metastasis of ovarian cancer cells,and down regulated Let-7b can enhance the proliferation,invasion and metastasis of ovarian cancer cells.Let-7b might be involved in the occurrence and development of ovarian cancer as a tumor suppressor gene.ObjectiveTo verify whether NIRF was targeted by let-7a through experimental approaches.MethodIn the two objective ovarian experimental cancer cell line A2780 and SKOV3 were evaluated the transfection efficiency,in cells with high transfection efficiency construct containing EZH2 and EZH2 target gene 3 'UTR pmir Glo carrier(EZH2-WT carrier)3' UTR predicted locus mutation pmir Glo carrier(EZH2-MUT carrier).Let-7b Mimics and co transfection of experimental target cells.At the same time,the inhibitor Let-7b positive control group(Let-7b inhibitor NC)and mimics Let-7 negative control group(Let-7mimics NC)were constructed.Luciferase activity in each group was detected by dual luciferase reporter system.Transient transfection technique was used to detect the expression of Let-7b in ovarian cancer cell line A2780 and ovarian cancer cell line SKOV3.After Western blot,the expression of EZH2 protein was detected after transfection.ResultsThe 2 experiment cells were observed by inverted fluorescence microscope,the A2780 by NC-FAM after transfection can reach about 80%,can meet the requirements of following experiments,the luciferase assays in A2780 cells.By sequencing the luciferase reporter plasmid pmir Glo-EZH2-3 UTR wild type(WT)was successfully constructed and transfected the mimic-let-7b+pmir Glo-EZH2-3 UTR wt fluorescence signal intensity of the weakest transfected with the strongest fluorescence signal intensity mimic-NC+pmir Glo-EZH2-3 '-UTR WT(P < 0.05)difference has statistical significance.And transfected with the fluorescence signal intensity mimic-let-7b+ pmir GloEZH2-3 UTR mut and mimic-NC +pmir Glo-EZH2-3 UTR mut differ little(P >0.05),that let-7b specific binding to EZH2-3 UTR play a role,the inhibition of gene expression and translation.Western blot analysis showed that compared with untransfected cells,transfection of let-7b mimics the cells of EZH2 expression was significantly decreased(P < 0.05),the expression of EZH2 in cell transfection of let-7b inhibitor significantly increased(P < 0.05).ConclusionEZH2 3 'UTR contains Let-7b control information,Let-7b through the target to bind to the 3' UTR region of EZH2,EZH2 is a target gene of Let-7b,Let-7b negative regulation of EZH2 gene expression.Overexpression Let-7b in ovarian cancer cells may inhibit the proliferation and invasion and metastasis of ovarian cancer cells by inhibiting the expression of EZH2 protein.Let-7b may play an important role in the proliferation and metastasis of ovarian cancer invasion process.