Fig. 6.

miR-140 targets Dnpep in mouse chondrocytes. (A) Experimental strategy. RNA was prepared from the Ago2-IP fraction and total RNA from wild-type and Mir140-null primary chondrocytes and subjected to expression profiling. RNA enriched in the Ago2-IP fraction was first identified in wild-type (a) and Mir140-null (c) chondrocytes; genes that were enriched in the Ago2-IP fraction in wild-type but not in Mir140-null chondrocytes were selected. Among them, those upregulated in Mir140-null chondrocytes (b) were subjected to further investigation. (B) RNA immunoprecipitation followed by RT-PCR (RIP-RT-PCR) shows the specific association between Hmga2 and Dnpep transcripts and Ago2 in wild-type cells. (C) Dnpep is associated with Argonaute 2 (Ago2) in wild-type but not in Mir140-null chondrocytes. Ago2 association was determined by comparing transcript levels of Ago2 immunoprecipitants with those of total RNA (Input in panel B). Transcript levels were expressed relative to that of GAPDH of each fraction. Ago2-IP enrichment ratios were calculated by dividing normalized transcript levels in the Ago2-IP RNA by those in total RNA. The relative enrichment of Dnpep in the Ago2-IP fraction is significantly less in Mir140-null chondrocytes, whereas Hmga2, a quintessential target of let-7 miRNAs, is enriched in the Ago2-IP fractions of both Mir140-null and wild-type chondrocytes (*, P < 0.05, n = 4, t test). (D, E) Dnpep is upregulated in Mir140-null primary rib chondrocytes both at the RNA level (D) and at the protein level (E).