Fig. 7
Alox5 is a novel target of the let-7 family of miRNAs. a Sequence comparison of the let-7 family members and the sequence partially complementary to the seed sequence of let-7 (shaded) found in the 3′-UTR of Alox5 mRNAs. b Schematic diagram of the Alox5-reporter construct (pISO-AL5) in which let-7 binding sequence in the Alox5 3′UTR (hatched box) was cloned at the 3′ end of the firefly luciferase gene upstream of SV40 poly(A). c pISO-AL5 or empty pISO vector was cotransfected into HEK293 cells with 10 nM let-7e or miR-142 (control) mimic or vehicle alone (bottom). Relative activities of the reporter were shown as a ratio of pISO-AL5/pISO (Mean ± SEM); p values were generated by unpaired Student’s t test . n = 6. d Macrophage RAW 264.7 cells were transfected with 18 nM miR-142 (control), let-7e, or miR-125b mimic, followed by qRT-PCR analysis of Alox5 mRNAs (bottom). Results were plotted as Mean ± SEM; p values were generated by unpaired Student’s t test. e AGMs from E10.5 Ctr or cKO embryos were transfected with miRNA, followed by CFU assay. Colony number was plotted as Mean ± SD (right); p values were generated by unpaired Student’s t test . For miR-142, n = Ctr: 9 embryos, cKO: 5 embryos. 2 litters. For let-7e, n = Ctr: 23 embryos, cKO: 6 embryos. 4 litters. For miR-9-5p, n = Ctr: 7 embryos, cKO: 4 embryos. 2 litters. NS, not significant. ND, not detectable
