Journal: Molecular Cancer
Article Title: MicroRNA-141 enhances anoikis resistance in metastatic progression of ovarian cancer through targeting KLF12/Sp1/survivin axis
Figure Lengend Snippet: KLF12 is a direct target of miR-141. a The downstream targets of miR-141 were predicted by an in silico study using three bioinformatics algorithms (PicTar, Miranda, and TargetScanHuman 6.0). b Ontology analysis using miRNA_Targets database 22 identified those predicted targets are involved in metabolic process, biological regulation, localization and cellular process etc. c The putative miR-141 targets which may be associated with anoikis resistance of ovarian cancer cells are categorized into six functional aspects. The red highlighted genes are the novel targets, while the black color genes have been reported as the miR-141 direct targets. d Western blot analysis showed that enforced expression of miR-141 (+) could reduce, while depletion of endogenous miR-141 by anti-miR-141 (+) could enhance the expressions of KLF12 and SIK1 only as compared with their controls (−) in ovarian cancer cells. But the expressions of other putative downstream targets; FUS, E2F3 and MAP2K4 had little or no change when the level of miR-141 was altered. e Luciferase reporter assay was performed in HEK293 cells and showed that the 3’ UTR of KLF12 luciferase activity was significantly inhibited by miR-141 expression dependently. f A schematic diagram shows the putative binding sites for miR-141 on the whole 3'UTR region of KLF12. Transient transfection of miR-141 mitigated the expression of KLF12 3’UTR-luciferase reporter activity. The KLF12 3’UTR with C1 binding site showed the highest suppression by miR-141. g ( Left panel ) The schematic diagram shows the most conserved and common pairing of wild-type and its mutant target regions of KLF12 and miR-141. ( Right panel ) The bar chart shows that miR-141 remarkably reduced the wild-type, whereas the KLF12_3’UTR mutant showed no change in luciferase reporter activity. h QPCR showed that miR-141 could reduce the expression of KLF12 mRNA in ovarian cancer cell lines. i Stable expression of miR-141 significantly reduced the expression of KLF12 in A2780cp and OVCA433 cells, while depletion of miR-141 using anti-miR-141 restored the expression of KLF12 in SKOV3 cells
Article Snippet: To confirm the oncogenic functions of miR-141, a miR-141 precursor-expressing plasmid (pmR-ZsGreen1 vector, Clontech) was stably transfected into three advanced ovarian cancer cell lines: A2780CP (p53 mutated) (141-C2 and 141-C8), OVCA433 (p53 wildtype) (141-C5 and 141-C10) and SKOV3 (p53 deleted) (141-C1 and 141-C4) (Fig. , left panel ).
Techniques: In Silico, Functional Assay, Western Blot, Expressing, Luciferase, Reporter Assay, Activity Assay, Binding Assay, Transfection, Mutagenesis, Real-time Polymerase Chain Reaction