Fig. 7: Pcsk9 is a direct target of Lin28b/let-7.
Before the experiment started, CAFs were cultured with 15376T-CM for 6 days to induce Lin28b expression. CM for CAFs culture was replaced daily (d–j, m–p). a–d 14837CAFs (a, b), Lin28b-WT-expressing 14837CAFs (a, b), 15376CAFs (c, d), and Lin28b-WT-expressing 15376CAFs (c, d) were cultured under high glucose (25 mM) or low glucose (2 mM). The levels of Lin28b and Pcsk9 were measured by western blotting (a, c). The levels of Pcsk9 in the supernatants were examined by ELISA (b, d). Representative of n = 3 independent experiments (a, c). e, f 15376CAFs and Fzd4-KO 15376CAFs were cultured under high glucose (25 mM) or low glucose (2 mM). The levels of Fzd4, Lin28b, and Pcsk9 were measured by western blotting (e). The levels of Pcsk9 in the supernatants were examined by ELISA (f). Representative of n = 3 independent experiments (e). g, h Fzd4+ CAFs and Fzd4- CAFs were cultured under high glucose (25 mM) or low glucose (2 mM). the levels of Fzd4, Lin28b, and Pcsk9 were measured by western blotting (g). The levels of Pcsk9 in the supernatants were examined by ELISA (h). Representative of n = 3 independent experiments (g). i The levels of Pcsk9 in 15376CAFs, Lin28b-KO 15376CAFs, flag-Lin28b-WT(r)-expressing 15376CAFs and flag-Lin28b-MU(r)-expressing 15376CAFs were measured by western blotting. Representative of n = 3 independent experiments. j 15376CAFs were transfected with let-7a agomir or let-7 sponge vector. The protein levels of Lin28b and Pcsk9 were measured by western blotting. Representative of n = 3 independent experiments. k, l Sequence alignment of the putative let-7a binding sites, and sketch of the construction of wild-type or mutant Pcsk9 3’UTR (k). The relative luciferase activity was analyzed after the pmir-GLO-Pcsk9 3’UTR (wild-type or mutant) vectors were co-transfected into 293T cells with let-7a agomir or agomir nc (l). P-value by one-way ANOVA with Tukey’s multiple comparison test. m, n The mRNA expression level (m) and mRNA stability (n) of Pcsk9 in 15376CAFs, Lin28b-KO 15376CAFs, flag-Lin28b-WT(r)-expressing 15376CAFs and flag-Lin28b-MU(r)-expressing 15376CAFs were measured by real-time qPCR. o, p 15376CAFs (o), Lin28b-KO 15376CAFs (o), flag-Lin28b-WT(r)-expressing 15376CAFs (p), and flag-Lin28b-MU(r)-expressing 15376CAFs (p) were collected and polysomes were fractionated on sucrose density gradients. Amount of Pcsk9 mRNA in various polysome fractions was analyzed by RT-PCR and normalized to 5S rRNA level. q, r 15376T cells were orthotopically injected into WT and FSP-Cre;Lin28bfl/fl mice. After 2 weeks, the tumors were analyzed by Pcsk9 and α-SMA IHC staining. Representative images are shown. Scale bar: 30 μM (q). Pcsk9 IHC scores in CAFs were plotted (n = 10 views per group). Data are shown as mean±s.d. r Three biologically independent experiments were performed (b, d, f, h, l–p). Data are shown as mean ± s.d. P-value were determined by one-way ANOVA with Tukey’s multiple comparison test (b, d, f, h, l–p) or two-tailed unpaired Student’s t-tests (r).
