Skip to main content
. 2015 Jul 21;43(15):7462–7479. doi: 10.1093/nar/gkv735

Figure 1.

Figure 1.

The primate-specific microRNA miR-944 is highly expressed in keratinocytes. (A) Schematic representation of human TP63 and MIR944. TP63 exon numbers are noted. Alternative promoters (P1 and P2) give rise to the TAp63 and ΔNp63 transcript isoforms, respectively. MIR944 (red hairpin) resides within intron 4 of human TP63. Analysis using the University of California, Santa Cruz (UCSC) Genome Brower demonstrates the evolutionary conservation of the TP63 genomic fragments harboring exon 4 and MIR944 in primates and vertebrates. (B) The positions of the qPCR primers used in this study are indicated on human TP63. ‘TAp63’, ‘ΔNp63’, and ‘p63’ probes can detect exons 2 and 3, 3′ and 4, and 4 and 5, respectively. The ‘Int3-Ex4’ and ‘Ex4-Int4’ probes can detect the intron 3′–exon 4 junction region and the exon 4–intron 4 junction region, respectively. (C) Expression levels of TAp63 mRNA, ΔNp63 mRNA, and miR-944 in various human cell lines were analyzed using RT-qPCR. TAp63 mRNA and ΔNp63 mRNA expression levels were normalized to peptidylprolyl isomerase A (PPIA) mRNA expression, and miR-944 expression was normalized to the expression of the RNU48 small RNA. Data represents the mean ± SD of triplicate biological samples. The values of ΔNp63, the relative expression of miR-944 obtained from keratinocytes, and the relative expression of TAp63 obtained from WM115 cells were all set as 1. (D) Human primary keratinocytes were transfected with 50 nM siRNA against DROSHA (si-DROSHA), DGCR8 (si-DGCR8), or a negative control siRNA (si-NC). After 2 days, RNA was extracted, and expression levels were analyzed using RT-qPCR. DROSHA and DGCR8 mRNA levels were normalized to the expression levels of ribosomal protein large P0 (RPLP0) mRNA, and miR-944 expression was normalized to RNU48 small RNA levels. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus si-NC, unpaired Student's t-test. (E) Human primary keratinocytes and WM266-4 melanoma cells were immunoprecipitated with the AGO2 or IgG antibody. RNA was extracted from immunoprecipitates, and miR-944 expression was analyzed. As a positive control, miR-125b expression was also examined. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus IgG, unpaired Student's t-test.