Research Paper Volume 12, Issue 17 pp 17459—17479

Figure 5. Regulatory networks between LINC00160 and SAA1 in RCC cells. (A) RT-qPCR analysis of LINC00160 in the subcellular fractions of resistant cells. U6 and GAPDH acted as nuclear and cytoplasmic markers respectively. (B) Transcriptional factors were selected from PROMO and GeneCards databases. (C) RNA immunoprecipitation (RIP) experiments were performed using anti-TFAP2A antibody in resistant cells. (D) 5 predicted bound sites of SAA1 promoter region. (E) Western blotting analysis of TFAP2A after knockdown. β-actin served as the loading control. (F) Luciferase activity assays of SAA1 promoter regions in resistant cells after LINC00160 and TFAP2A knockdown respectively. (G) Chromatin immunoprecipitation (CHIP) assay of TFAP2A enrichment at SAA1 promoter region relative to control IgG in resistant cells. Each experiment was performed at least three times and data was represented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. P>0.05 is denoted by ns. LINC00160, long non-coding RNA 160; SAA1, serum amyloid A1; RCC, renal cell carcinoma; RT-qPCR, quantitative real time polymerase chain reaction; U6, RNA U6 Small Nuclear 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RIP, RNA immunoprecipitation; TFAP2A, transcription factor AP-2 alpha; CHIP, chromatin immunoprecipitation.