Figure 7.

miR-155 Inhibits the Expression of the Tumor Suppressor Gene P21WAF1/CIP1 in Liver Cancer Cells

(A) ChIP with anti-pCDK2 was performed in three stable cell lines: rLV-Hep3B, rLV-miR-155-Hep3B, and miR-155+rLV-H3F3A-Hep3B. (B) ChIP with anti-pCDK2 was performed. (C) ChIP using anti-RNA Pol II was performed. (D) ChIP using anti-RNA Pol II was performed. (E) Super-DNA-protein complex gel migration assay using biotin-labeled P21WAF1/CIP1 promoter cis-element probe (Biotin-P21WAF1/CIP1) and anti-pCDK2, anti-Biotin. (F) Super-DNA-protein complex gel migration assay using biotin-labeled P21WAF1/CIP1 promoter cis-element probe (Biotin-P21WAF1/CIP1) and anti-RNA Pol II, anti-Biotin. (G) The ability of pCDK2 to bind to the P21WAF1/CIP1 promoter-enhancer loop by chromosome configuration capture (3C)-ChIP. (H) The ability of RNA Pol II to bind to the P21WAF1/CIP1 promoter-enhancer loop was detected by the 3C-ChIP. (I) pEZX-MT-P21WAF1/CIP1-promoter-Luc luciferase reporter gene activity was detected. (J) Total RNA was detected by RT-PCR for the transcriptional capacity of P21WAF1/CIP1. (K) Western blotting was used to detect the expression of P21WAF1/CIP1. (L) qRT-PCR was used to detect miR-155 in three stable cell lines: rLV-Hep3B, rLV-miR-155-Hep3B, and miR-155+pGFP-V-RS-CAK. (a) Quantification and (b) western blot. (M) ChIP was performed in rLV-Hep3B, rLV-miR-155-Hep3B, and miR-155+pGFP-V-RS-CAK. (N) 3C-ChIP using anti-pCDK2, anti-RNA Pol II. (O) pEZX-MT-P21WAF1/CIP1-promoter luciferase reporter gene activity was examined. (P) The transcription of P21WAF1/CIP1 was detected by RT-PCR. (Q) Expression of P21WAF1/CIP1 was detected by western blotting. (R) Immunoprecipitation (IP) analysis with anti-CyclinA and anti-CDK2. (S) Western blotting with anti -E2F1.β-actin as an internal reference gene.