Fig 1: Circ_C16orf62 was expressed at low level in GC cells. (A) Expression of circ_C16orf62 in human normal gastric epithelial cell line (GES-1) and GC cell lines (MKN-74, HGC-27 and AGS) detected by RT-qPCR assay. (B, C) TUBB2A levels in human normal gastric epithelial cell line (GES-1) and GC cell lines (HGC-27 and AGS) were measured by RT-qPCR and western blot assays. (D, E) Relative levels of circ_C16orf62 and C16orf62 mRNA were tested in HGC-27 and AGS cells treated with or without RNase R. (F, G) The cellular localization of circ_C16orf62 in AGS and HGC-27 cells was analyzed by subcellular fractionation assay. * P<0.05.
Fig 2: Circ_C16orf62 and TUBB2A were decreased in GC tissues. (A, B) RT-qPCR assay was applied to measure expression of circ_C16orf62 and TUBB2A in 32 pairs of GC tumor tissues and adjacent normal tissues. (C) The expression association between circ_C16orf62 and TUBB2A in GC tumor tissues was analyzed by Pearson correlation analysis. * P<0.05.
Fig 3: Circ_C16orf62 elevated TUBB2A expression by sponging miR-421 in GC cells. (A, B) TUBB2A level was detected in AGS and HGC-27 cells transfected with NC, circ_C16orf62, circ_C16orf62+ miR-NC, circ_C16orf62+ miR-421, circ_C16orf62+ si-NC and circ_C16orf62+si-TUBB2A. * P<0.05.
Fig 4: TUBB2A inhibited proliferation, migration and invasion of GC cells in vitro. (A, B) TUBB2A level was detected by RT-qPCR and western blot assays in AGS and HGC-27cells transfected with pcDNA or TUBB2A. (C–E) MTT and colony formation assays were applied to assess the proliferative ability in pcDNA or TUBB2A-transfected AGS and HGC-27cells. (F, G) Transwell assay was applied to assess the capacities of migration and invasion in pcDNA or TUBB2A-transfected AGS and HGC-27cells. * P<0.05.
Fig 5: Circ_C16orf62 and TUBB2A bound to miR-421in GC cells. (A) The binding sites between circ_C16orf62 and miR-421 were predicted by bioinformatics software StarBase3.0. (B, C) The binding relationship between circ_C16orf62 and miR-421were testified by dual-luciferase reporter assay. (D, E) MiR-421 level was measured in AGS and HGC-27 transfected with NC, circ_C16orf62, si-NC and si-circ_C16orf62#1. (F) The binding sequences between TUBB2A and miR-421 were predicted by bioinformatics software Targetscan. (G, H) Relative luciferase activity was determined by dual-luciferase reporter assays in AGS and HGC-27cells co-transfected with reporter plasmid (TUBB2A-wt/TUBB2A-mut) and miR-421 or miR-NC. (I) Transfection efficiency of miR-421 mimic or anti-miR-421 was detected by RT-qPCR assay. (J, K) The effects of miR-421 overexpression or miR-421 knockdown on TUBB2A mRNA level were tested by RT-qPCR assay. (L, M) TUBB2A protein level was examined in miR-421 or anti- miR-421-transfected AGS and HGC-27 cells. * P<0.05.
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