Article Title: Non-canonical signaling pathway of SNAI2 induces EMT in ovarian cancer cells by suppressing miR-222-3p transcription and upregulating PDCD10
Figure Lengend Snippet: miR-222-3p targets PDCD10 to repress cell migration by downregulating the Wnt/ β -catenin signaling pathway. (A) qPCR analysis of β -catenin and TCF4 expression in HO 8910 PM cells (PDCD10-overexpressing groups) and SKOV3 cells (PDCD10-silenced groups). ( B ) Activity of the Wnt/ β -catenin signaling pathway in HEK-293T cells detected by the TOP flash/FOP flash dual-luciferase reporting system. HEK-293T cells were transfected with TOP flash/FOP flash plasmid and pRL-SV40 vector followed by transfection with or without PDCD10, and siRNA-NC/PDCD10, siRNA-03/PDCD10, or siRNA-04. Firefly luciferase activities were tested by a luminometer, and the activity of the Wnt/ β -catenin signaling pathway was recorded as TOP/FOP. ( C ) Western blot analysis of PDCD10 and β -catenin protein expression following overexpression of PDCD10 in HO 8910 PM cell fractions. β -actin was utilized as a marker for the cytosolic fractions. ( D ) HO 8910 PM cells transduced with or without PDCD10 were subjected to immunofluorescence with human-specific β -catenin antibodies. ( β -catenin in red). Bar, 10 µm. ( E ) β -catenin nuclei-intensity (detected by Operetta High-Content Imaging System (Perkin Elmer) in HO 8910 PM cell). ( F ) Western blot analysis of PDCD10, β -catenin translation levels in HO 8910 PM cells, or after coculturing with miR-ctrl mimic or miR-222-3p mimic in the presence or absence of PDCD10 (Left). The relative expression rate was calculated by Image J (Right). Data (A-B and E-F) represent the mean ± SD in different assays (n=3), *, P< 0.05; **, P< 0.01, determined by unpaired two-tailed t-test.
Article Snippet: PDCD10, SNAI2, E-cad, VIM and β- catenin used in this study were from following sources: antibodies against PDCD10 and GAPDH were purchased from Abcam and Utibody, respectively (ab110531 and UM4002, respectively); antibodies against SNAI2 and GAPDH were acquired from Affinity (AF4002) and Utibody (UM4001), respectively; antibodies against E-cad, VIM, and β- catenin (20874-1-AP, 22018-1-AP and 10366-1-AP, respectively) were obtained from Proteintech.
Techniques: Migration, Expressing, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Western Blot, Over Expression, Marker, Transduction, Immunofluorescence, Imaging, Two Tailed Test