Figure 7
Inhibition of in vitro kallikrein 1b26 (klk1b26) translation by synthetic miRNAs partially complementary with the 5'-region of klk1b26 mRNA. (A) Sequences of miRNAs and their complementary sites in the 5'-region of klk1b26 mRNA. The numbers above the klk1b26 mRNA sequence indicate the nucleotide positions from the transcription start site (GenBank: NM 010644). (B) Inhibition of in vitro translation of klk1b26 by the miRNAs and antisense RNA of F21 forward primer (asR-F21). Messenger RNA (500 ng) purified from male submandibular glands (SMGs) was preincubated with or without miR-325, miR-1497a or asR-F21 (10 μM each) in a translation mixture. The mixture was then incubated with a reticulocyte lysate for in vitro protein synthesis, and [35S]methionine-labeled klk1b26 protein was analyzed by SDS-PAGE as described in Methods. N, 325, 1497a, and asR stand for the absence of synthetic RNA, miR-325, miR-1497a, and asR-F21, respectively. Representative results are shown. Similar results were obtained in three independent experiments. (C) Effects of synthetic miRNAs and antisense RNA of F21 forward primer (asR-F21) on reverse transcription (RT)-PCR for klk1b26 mRNA using various forward primers targeting the 5'-terminal region. Sequences of the primers are described in Table 1. PCR was carried out with each forward primer in combination with the R552 reverse primer in the absence or presence of the synthetic RNAs. N, 325, 1497a, and asR indicated the absence of synthetic RNA (3 μM), miR-325, miR-1497a, and asR-F21, respectively. Representative results are shown. Similar results were obtained in three independent experiments.