Fig 2: LINC01134 directly binds the AKT1S1 promoter and activates AKT1S1 expression. (A) AKT1S1 mRNA levels in LINC01134-stably-overexpressed and control HCCLM3 cells were detected by qRT-PCR. (B) AKT1S1 mRNA levels in LINC01134-stably-silenced and control HCCLM3 cells were detected by qRT-PCR. (C) AKT1S1 protein levels in LINC01134-stably-overexpressed and control HCCLM3 cells were detected by western blot. (D) AKT1S1 protein levels in LINC01134-stably-silenced and control HCCLM3 cells were detected by western blot. (E) Subcellular localization of LINC01134 and control transcripts was analyzed by qRT-PCR in biochemically fractionated HCCLM3 cells. NEAT1 was used as a chromatin-associated lncRNA control. MALAT1 was used as a nucleoplasm-localized lncRNA control. GAPDH mRNA was used as a cytoplasmic control. (F) The highly adversely complementary region between LINC01134 (464–753 nt) and AKT1S1 promoter (−1,802 to −1,498 bp). (G) ChIRP assays with LINC01134 capture probes were conducted in HCCLM3 cells. The enrichment of the AKT1S1 promoter and GAPDH promoter was detected by qRT-PCR. (H) After transient co-transfection of wild-type or complementary region mutated LINC01134 overexpression plasmids with firefly luciferase reporter containing the AKT1S1 promoter and pRL-TK (encoding renilla luciferase) into HCCLM3 cells, dual luciferase reporter assays were conducted to assess AKT1S1 promoter activity. Results are shown as the relative ratio of firefly luciferase activity to renilla luciferase activity. (I) After transient co-transfection of LINC01134-specific shRNAs with firefly luciferase reporter containing the AKT1S1 promoter and pRL-TK into HCCLM3 cells, dual luciferase reporter assays were conducted to assess AKT1S1 promoter activity. Results are shown as the relative ratio of firefly luciferase activity to renilla luciferase activity. Results are shown as the mean ± SD of three independent experiments. **p < 0.01, ***p < 0.001, ns, not significant, by unpaired two-sided Student’s t-test (A) or one-way ANOVA followed by Dunnett’s multiple comparisons test (B,G–I).

Fig 3: The autophagy levels and related molecular pathways assessed in TSC2-/- MEFs that received VO-OHpic treatment. (A) Western blot assays of LC3, p-PTEN and p-PRAS40 in TSC2-/- MEFs, with or without VO-OHpic treatment. Quantitative analysis of (B) LC3-I and (C) LC3-II expression levels in TSC2-/- MEFs, with or without VO-OHpic treatment. Quantitative analysis of (D) p-PTEN/PTEN and (E) p-PRAS40/PRAS40 relative expression levels in TSC2-/- MEFs, with or without VO-OHpic treatment. *P<0.05, **P<0.01 and ***P<0.001. VO-OHpic, hydroxyl(oxo)vanadium 3-hydroxypiridine-2-carboxylic acid; +V, VO-OHpic treatment; MEF, murine embryonic fibroblasts; p, phosphorylated; TSC, tuberous sclerosis complex; LC3, human microtubule-associated protein 1 light chain 3; PRAS40, proline-rich Akt substrate 40 kDa.

Fig 4: Inhibition of AKT1S1 reverses the roles of LINC01134 in HCC. (A) AKT1S1 mRNA and protein levels in LINC01134-stably-overexpressed and concurrently AKT1S1-stably-silenced SK-HEP-1 cells were detected by qRT-PCR and western blot. (B) AKT1S1 mRNA and protein levels in LINC01134-stably-overexpressed and concurrently AKT1S1-stably-silenced HCCLM3 cells were detected by qRT-PCR and western blot. (C) Transwell migration assays were conducted to assess the migration ability of LINC01134-stably-overexpressed and concurrently AKT1S1-stably-silenced SK-HEP-1 and HCCLM3 cells. Representative images of migratory cells were shown. Scale bar = 100 μm. (D) Transwell invasion assays were conducted to assess invasion ability of LINC01134-stably-overexpressed and concurrently AKT1S1-stably-silenced SK-HEP-1 and HCCLM3 cells. Representative images of invasive cells were shown. Scale bar = 100 μm. (E–G) LINC01134-stably-overexpressed and concurrently AKT1S1-stably-silenced HCCLM3 cells were intrasplenically injected into nude mice. At the 28th day after injection, the number (E) and size (F) of liver metastatic nodules were detected by HE staining (G). (H) Luciferase-labeled LINC01134-stably-overexpressed and concurrently AKT1S1-stably-silenced HCCLM3 cells were injected into the tail veins of nude mice. At the 28th day after injection, lung metastasis was assessed via detecting luciferase signal intensities. Results are shown as the mean ± SD of three independent experiments (A–D) or n = 6 mice in each group (E–H). *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, by one-way ANOVA followed by Tukey’s multiple comparisons test (A–D) or Kruskal–Wallis test followed by Dunn’s multiple comparisons test (E,F,H).

Fig 5: PTEN may regulate autophagy by allowing for a bypass of PTEN/PRAS40 signaling in TSC2-/- cells and influence cell proliferation. PTEN could suppress the activation of mTOR and promote autophagy not only by inhibiting Akt/Rheb/mTOR pathway, but also by activating the PTEN/PRAS40 pathway.