miR-34 and p53: New Insights into a Complex Functional Relationship
Fig 6
miR-34a-KO HCT116 cells have a normal p53 response to genotoxic stress.
(A) Sequence of the miR-34a region in chromosome 1 in miR-34a-KO HCT116 cells compared to wild-type (WT) cells. The mature miR-34a sequence is in red, with the seed sequence underlined. The bottom panel shows the Northern blot for miR-34a in WT and 34a-KO HCT116 cells. U6 is a loading control. (B) miR-34a levels by qRT-PCR in WT or 34a-KO cells, untreated or DOX-treated for 16 hr. N/D, non-detectable. (C) Induction of miR-34b and miR-34c in WT and 34a-KO HCT116 cells after DOX treatment. Cells were treated with DOX as in (B). miR-34b/c levels were analyzed by qRT-PCR. (D) Proliferation of WT, p53-KO and 34a-KO cells by MTT cell proliferation assay. (E) qRT-PCR analysis of mRNA levels of p53 transcriptional targets in WT or 34a-KO HCT116 cells treated or not with DOX for 16 hr, relative to the untreated WT sample. The inset shows a representative immunoblot for p53 and some p53 transcriptional targets. α-tubulin is a loading control. (F) Apoptosis, assessed by annexin V/PI staining, in untreated or DOX-treated (48 hr) WT or 34a-KO HCT116 cells (DOX fluorescence in the PI channel increases “PI staining” in non-apoptotic cells). Representative dot plots are at left and the mean +/- STDEV of 3 independent experiments is at right. (G) Immunoblot of miR-34a target proteins in WT and 34a-KO HCT116 cells. The numbers on the left indicate the average signal intensity, normalized to the loading control, in 34a-KO/WT cells from 3 independent experiments. All experiments were performed at least 3 times and the graphs show the mean +/- STDEV from replicate experiments (*, p<0.05; **, p<0.01, relative to WT cells, 2-tailed Student’s t-test).