Fig. 5: SMAD5-AS1 served as a molecular sponge for miR-135b-5p.
a Schematic diagram showed the putative miR-135b-5p binding sites with the SMAD5-AS1. The sequences of wild-type SMAD5-AS1 and mutant SMAD5-AS1 were listed as well. Luciferase reporter gene assays were performed to measure the luciferase activity in TMD8 cells. **P < 0.01. b Anti-Argonaute 2 (AGO2) RNA immunoprecipitation (RIP) assays were used in TMD8 cells to determine SMAD5-AS1 and miR-135b-5p RNA enrichment in immunoprecipitated (IP) complex. Anti-immunoglobulin G (IgG) was used as the control. SMAD5-AS1 and miR-135b-5p were enriched preferentially in miRNA ribonucleoprotein complexes (miRNPs)1,2,3 containing AGO2 compared with anti-IgG immunoprecipitates. **P < 0.01 c The biotinylated miR-135b or its mutant (miR-135b-mut) was transfected into TMD8 cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was applied to quantify the RNA levels of SMAD5-AS1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Scatter plot showed the relative ratios of the input of IP. SMAD5-AS1 was more enriched in the wild-type miR-135b-5p compared with that in the mutant-type miR-135b-5p with broken SMAD5-AS1 binding site **P < 0.01. d The relative expression of miR-135-5p in diffuse large B cell lymphoma (DLBCL) cells transfected with SMAD5-AS1-overexpressed plasmid/control plasmid and shSMAD5-AS1 plasmid/control plasmid. e RT-qPCR and Cell Counting Kit-8 (CCK8) assay were applied to measure the relative expression of SMAD5-AS1 and cell proliferation in TMD8 cells at 36 h after transfection with control, miR-135b mimic or miR-135b inhibitor, SMAD5-AS1 overexpression plasmid or shSMAD5-AS1 plasmid, and miR-135b mimic + Lv-SMAD5-AS1 or miR-135b inhibitor + shSMAD5-AS1. *P < 0.05. N = 3 independent experiments
