Fig 2: miR-185 Directly Binds to the 3′ UTRs of the RHEB and RICTOR mRNAspmirGLO vectors containing either the wild-type (WT) or mutant (mut) binding sites of miR-185-5p in RHEB (A) and RICTOR (B) mRNAs were constructed to conduct luciferase reporter assays as described previously. Dual-luciferase reporter assays were performed to verify binding between miR-185-5p and RHEB and RICTOR mRNA. The HSC line LX-2 was co-transfected with a pmirGLO vector containing either the WT or mut target sites plus either the miR-185-5p mimics or the mimic control oligonucleotide. Results of relative luciferase activity are shown as the mean ± SEM obtained from triplicate experiments. (C–E and G) RHEB and RICTOR mRNA and protein levels were analyzed by real-time qPCR (C and D) and western blotting (E and G), respectively. The mRNA and protein levels of RHEB and RICTOR were downregulated in LX-2 cells transfected with miR-185 mimics but upregulated when transfected with miR-185 inhibitors. (F and H) Western blotting data were quantified using the ImageJ software. Data shown represent the means ± SEM of independent experiments (unpaired two-sample Student’s t test, n = 3, **p < 0 0.01, *p < 0 0.05).

Fig 3: Interference of CBAP and TSC2 interaction impairs tumor cell growth.A–C, N26 efficiently inhibits mTORC1 signaling in Jurkat cells by interfering with the CBAP and TSC2 interaction. Lysates of GFP-N26-CBAP-expressing cells were subjected to immunoblotting (A) or were immunoprecipitated with anti-CBAP antibody (B). C, expression of N26-CBAP increases the TSC1 within Akt immune complexes. GFP (+) vector (Vec) cells and GFP-N26-CBAP-expressing cells were both sorted by flowcytometry and subjected to immunoprecipitation with anti-pan-Akt antibody. The immunoprecipitates were subjected to analysis described in Figure 1A. D and E, GFP-N26-CBAP increased the TSC2 GAP activity and inhibited Jurkat cell growth. Cell lysates of GFP(+) cells were used for In vitro GAP assays using non-isotope GTP-loaded GST-Rheb as substrates and anti-GTP-Rheb specific antibody to distinguish GTP-Rheb versus GDP-Rheb (D). Viable cell numbers in the GFP (+) population with or without rapamycin treatment were determined by trypan blue exclusion staining at indicated time points (E). One-way ANOVA was used to calculate statistical significance; ∗p < 0.05; ∗∗∗p < 0.001., n = 4. F, model of N26-CBAP interference of mTORC1 signaling in Jurkat cells via regulating stability of TSC1/2-Akt complexes. N26-CBAP perturbs CBAP-TSC2 interaction, leading to an increase of TSC1 binding to TSC2-Akt complex, increase of Rheb-GTP hydrolysis, and decrease of mTORC1 signaling.

Fig 4: Resveratrol activates autophagy through the miR-4654/Rheb axis. (A) Cell viability was analyzed in HSFBs in the different groups using an MTT assay. *P<0.05, **P<0.01, ***P<0.001. (B) RT-qPCR analysis of the expression levels of Rheb in HSFBs in the different groups. **P<0.01, ***P<0.001; ##P<0.01 vs. the resveratrol group. (C) Western blotting was used to analyze the protein expression levels of Rheb, LC3 and Beclin 1 in HSFBs in the different groups. *P<0.05, **P<0.01, ***P<0.001; #P<0.05, ##P<0.01 vs. the resveratrol group. Data are presented as the mean ± SD. N=3. HSFBs, hypertrophic scar-derived fibroblasts; LC3, microtubule-associated protein 1A/1B-light chain 3; Control, mimic-NC + inhibitor-NC + PBS + vector group; OE, overexpression; KD, knockdown; miR, microRNA.

Fig 5: CCl4-Induced Liver Fibrosis in Mice Downregulates miR-185 and Upregulates RHEB and RICTOR(A) The histopathological changes in livers 1 month after injection of CCl4 are shown by H&E staining, Masson’s trichrome staining, and Sirius red staining of sections from two representative livers. (B) Real-time qPCR analysis for α-SMA, COL1A1, COL1A2, COL3A1, RHEB, and RICTOR in two groups. mRNA levels increased in all genes. All results of relative expression values are shown as the mean ± SEM. (C) Western blotting analysis for α-SMA, fibronectin (FN), TGFR1, RHEB, RICTOR, and GAPDH in livers of representative mice from each group. The protein levels were upregulated in the CCl4 group. α-SMA and collagen are obviously increased, and the changes in RHEB and RICTOR levels are modest. (D) Data represent one of three experiments with similar results. Western blotting data were quantified using the ImageJ software. (E) Immunohistochemistry for α-SMA, RHEB, and RICTOR in liver sections from representative NC and CCl4 livers. In the model group, protein levels of α-SMA, RHEB, and RICTOR increased. (F) Real-time qPCR measured miR-185-5p expression in livers from NC and CCl4-treated mice. miR-185-5p expression was reduced in the CCl4 group.