Fig. 4

Association of miR-142-3p expression with mTOR activation and PRAS40 expression. A. Prediction of the miRNAs binding to PRAS40 3'UTR in those decreased in patients with TSC. B. Alignment of miR-142-3p to PRAS40’s 3'UTR. C, D. Luciferase assays. pCIneo-Luc (−), pCIneo-Luc-PRAS40 (wild type), or pCIneo-Luc-PRAS40 mutant (mutant type) reporter vector and nonspecific control miRNA (nc), miR-142-3p mimic (miR-142-3p) or miR-142-3p inhibitor (miR-142-3p I) together with Renilla luciferase expression vector phRL-SV40 were cotransfected into cells. The experiments were performed in triplicate. E, F. Tsc2^−/− MEFs were transfected with nonspecific control miRNA (−), miR-142-3p mimic (miR-142-3p) or miR-142-3p inhibitor (miR-142-3p I). Immunoblotting (E) and real-time PCR analysis (F) were performed. G–K. Real-time PCR analysis. Tsc2^+/+ or Tsc2^−/− MEFs were cultured in serum free media for 24 h (G). Kidney tissues from Tsc2^f/f; ksp-cre or Tsc2^f/+; ksp-cre mice (H). Tsc2^−/− MEFs were treated with or without rapamycin (5 nM) for 24 h (I). Tsc2^−/− MEFs were transfected with control shRNA, mTOR shRNA1, or mTOR shRNA2 (J). Kidney tissues from Tsc2^f/f; ksp-cre treated with or without rapamycin (K). L. Cell viability assay of Tsc2^−/− MEFs transfected without (−, null) or with nonspecific control miRNA (nc), miR-142-3p mimic (miR-142-3p) or miR-142-3p inhibitor (miR-142-3p I). M–N. Tsc2^−/− MEFs were cotransfected with negative control (−) or miR-142-3pmimic (miR-142-3p) together with empty vector or PRAS40 expression vector. PRAS40 protein level was analyzed by Immunoblotting analysis (M), and cell viability assays were performed (N). The quantification values of the band density were labeled above the bands. Bars, SD. *P < 0.05, **P < 0.01, ***P < 0.001 from Student’s t-test or one-way ANOVA