Fig. 4

MBNL1 is a direct target of miR-130b-3p. A Screening for miR-130b-3p target genes by prediction softwares TargetScan, RNA22, PicTar, miRDB and miRWalk. B Venn diagram of DEGs in three datasets. Dataset 1 represents downregulated DEGs in Tsc2 − / − vs. Tsc2 + / + MEFs. Dataset 2 represents decreased DEGs in AF of TSC patients vs. normal skin. Dataset 3 represents predicted target genes of miR-130b-3p. C Tsc2 + / + , Tsc2 − / − and rapamycin (20 nM, 24 h) treated Tsc2 − / − MEFs. D Tsc1 + / + , Tsc1 − / − and rapamycin (20 nM, 24 h) treated Tsc1 − / − MEFs. C and D Cell lysates and total RNA were subjected to western blot (left panels) and qRT-PCR (right panels) analyses. E Tsc2 − / − and Tsc1 − / − MEFs were transfected with the miR-130b-3p inhibitor or negative control (NC-inhibitor). F Tsc2 + / + and Tsc1 + / + MEFs were transfected with the miR-130b-3p mimics or negative control (NC-mimics). E and F MBNL1 and miR-130b-3p levels in the indicated cells were examined by western blot (up panels) or qRT-PCR (low panels). G Sequence alignment of the predicted miR-130b-3p binding site within the mouse MBNL1 3´-UTR and its mutated sequence for luciferase reporter assay. H Luciferase reporter assay was performed in HEK 293 T cells that were co-transfected with miR-130b-3p mimics or NC-mimics together with reporter vectors containing MBNL1 3´-UTR or mutated MBNL1 3´-UTR. Relative luciferase activities are presented. I RIP assay was performed using antibodies against AGO2 or IgG and the enrichment of miR-130b-3p and MBNL1 were detected by qRT-PCR in Tsc2 − / − or Tsc1 − / − MEFs. J RIP assay was performed using AGO2 or IgG antibodies to estimate the enrichment of MBNL1 in Tsc2 + / + MEFs infected with lentivirus expressing miR-130b-3p or a control lentivirus. Data indicate mean ± SD of triplicate samples. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s indicates no significant difference