Fig 2: LncRNA-TTN-AS1 regulated TTN expression by activating TTN promoter activity and stabilizing TTN mRNA.a Methylation status (left panel) and relative expression (right panel) of lncRNA-TTN-AS1 in different cell lines. b Upper panel: a schematic map of TTN promoter region and luciferase reporter. Bottom panel: dual luciferase report assay was used to detect TTN promoter activity in 293T cells. c The half-life of TTN mRNA was measured by qRT-PCR after blocking new RNA synthesis with a-amanitin and normalized to 18s rRNA. d, e Subcellular distribution of TTN protein and mRNA in B16F10 cells treated with overexpressed TTN-AS1 plasmid or vector were analyzed by immunofluorescence (d) and nuclear/cytoplasmic fractionation (e) assays. Scale bar, 10 µm. Experiments were performed three times and data were presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Student t test. f Schematic representation of the mechanism by which lncRNA-TTN-AS1 interacts with TTN.

Fig 3: LncRNA-TTN-AS1 regulated TTN expression in SKCM cells.a, b Kaplan–Meier analysis of overall survival in SKCM case with high versus low TTN expression (n = 198) (a), and lncRNA-TTN-AS1 expression (n = 165) (b). c Significant correlation between lncRNA-TTN-AS1 and TTN expression in seven SKCM cell lines was calculated with Spearman test. d, e LncRNA-TTN-AS1 and TTN expression in B16F10 cells with downregulation (d) or overexpression (e) of lncRNA-TTN-AS1 were detected by qRT-PCR. f, g TTN protein level in B16F10 cells with downregulation (f) or overexpression (g) of lncRNA-TTN-AS1 was analyzed by western blotting, and ß-actin was used as a loading control. h Subcellular localization of TTN and lncRNA-TTN-AS1 in B16F10 cell lines were measured by nuclear/cytoplasmic fractionation assays. Experiments were performed three times and data were presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Student t test.

Fig 4: TTN and lncRNA-TTN-AS1 promoted SKCM cell migration and invasion in vitro.a Wound healing assay showed the migrating ability of B16F10 cells after transfection with shTTN, shTTN-AS1, or vector. Scale bar, 200 µm. b Transwell migration and invasion assays showed the migrating and invasive ability of B16F10 cells after transfection with shTTN, shTTN-AS1, or vector. Scale bar, 200 µm. Experiments were performed three times and data were presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Student t test.

Fig 5: TTN and lncRNA-TTN-AS1 induced SKCM cell proliferation and suppressed SKCM cell apoptosis in vitro.a, b The proliferation and viability of B16F10 cells after transfection with shTTN, shTTN-AS1, or vector were tested by colony formation (a) and CCK-8 (b) assays. Scale bar, 400 µm. c The cell-cycle progression was measured by PI staining in B16F10 cells after transfection with shTTN, shTTN-AS1, or vector. d The cell apoptosis percentage was examined using Annexin V-APC/PI in B16F10 cells after transfection with shTTN, shTTN-AS1, or vector. e, f Cell cycle-associated proteins (e) and proapoptotic proteins (f) were analyzed in B16F10 cells after transfection with shTTN, shTTN-AS1, or vector, and ß-actin was used as a loading control. Results were analyzed with ImageJ software. Experiments were performed three times and data were presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Student t test.