p53 Represses the Oncogenic Sno-MiR-28 Derived from a SnoRNA
Fig 4
sno-miR-28 alters p53 protein stability through TAF9B and forms a feed-forward loop between p53, sno-miR-28, and TAF9B.
(A) Western blots are shown to determine the protein levels of p53 and p21 after overexpression of sno-miR-28 and/or induction of p53 by Ponasterone A (PonA) in inducible H1299 cell line, compared with cells transfected with negative control RNA (ncRNA) and/or uninduced H1299 cells. (B) p53 mRNA levels in MCF10A and PonA-treated H1299 cells after overexpression of sno-miR-28 are shown by RT-PCR, compared with scrambled negative control (ncRNA). (C) The mRNA expression levels of p53 downstream targets are shown after overexpression of sno-miR-28 (or negative control) in H1299 cells upon p53 activation. (D) sno-miR-28 (or negative control) was either expressed (mimic) or inhibited (LNA) in MCF10A cells and the protein expression of p53 was determined by Western blot. (E) The mRNA expression levels of p53 downstream targets are shown after overexpression of sno-miR-28 in MCF10A cell line, compared with ncRNA. (F,G) TAF9B was knocked using an siRNA in H1299 cells compared with a negative control. Successful knockdown is shown at both the protein (F) and mRNA (G) levels. (H) CDKN1A expression was determined by RT-PCR after TAF9B was knockdown. ** p<0.01 versus controls for all experiments. β-actin is included as a loading control for all Western blots. “-”mark in sno-miR-28 mimic and anti-sno-miR-28 LNA experiments represent negative control transfections using a scrambled ncRNA.