lncRNA-ATB functions as a competing endogenous RNA to promote YAP1 by sponging miR-590-5p in malignant melanoma

Abstract

The critical long non‑coding RNAs (lncRNAs) involved in the carcinogenesis and progression of malignant melanoma (MM) have not been fully investigated. In the present study, it was identified that lncRNA activated by transforming growth factor‑β (lncRNA‑ATB) was upregulated in MM tissues and cells compared with benign nevus cells and human melanocytes, via comparative lncRNA screening from Gene Expression Omnibus datasets and reverse transcription‑quantitative polymerase chain reaction analysis. Furthermore, lncRNA‑ATB promoted the cell proliferation, cell migration, and cell invasion of MM cells in vitro, and tumor growth in vivo. It was additionally identified that lncRNA‑ATB attenuated cell cycle arrest and inhibited cellular apoptosis in MM cells. Finally, it was demonstrated that lncRNA‑ATB functions as a competing endogenous RNA (ceRNA) to enhance Yes associated protein 1 expression by competitively sponging microRNA miR‑590‑5p in MM cells. In conclusion, the present study revealed the expression and roles of lncRNA‑ATB in MM, and indicated that lncRNA‑ATB functions as a ceRNA to promote MM proliferation and invasion by sponging miR‑590‑5p.

Figure 1

lncRNA-ATB is upregulated in MM cell lines of malignant melanoma. (A) Vectorgram analysis of lncRNAs in MM and BN specimens. The x-axis indicates the Log₂ fold-change in lncRNA expression in MM tissues, and the y-axis indicates the Log₂ fold-change of lncRNA expression in BN tissues. Red, lncRNAs upregulated by ≥2-fold in MM compared with BN. Green, lncRNAs upregulated by ≥2-fold in BN compared with MM. Blue, lncRNAs upregulated by <2-fold in MM compared with BN or lncRNAs upregulated by <2-fold in BN compared with MM. Data from the GEO datasets (GSE3189): Differentially expressed lncRNAs from the GEO dataset GSE3189 with FDR <0.01 and |logFC| >2 were identified using the R package. The raw P-value was corrected using the Benjamini and Hochberg method to circumvent the multi-test bias. A fold-change value >2 or <0.25 and FDR <0.01 were selected as cutoff criteria for differentially expressed lncRNAs. (B) Volcano plot of lncRNAs in MM and BN. The x-axis indicates the Log₂ fold-change in lncRNA expression between MM and BN tissues, while the y-axis indicates the Log₁₀ of the adjusted P-value for each lncRNA. Values above the red line were identified to be statistically significant. (P<0.01) following application of the Benjamini and Hochberg method. lncRNA-ATB expression level was >4-fold increased between cases and controls (P<0.001 vs. BN). (C) Relative expression of lncRNA-ATB in human epidermal melanocytes and MM cells. ^*P<0.05 vs. HEMa-LP cells. (D) Knockdown efficiency of lncRNA-shRNA in MM cells. Data are from three experiments and are presented as the mean ± standard deviation. ^*P<0.05 vs. respective lncRNA-ATB NC group (Student's t-test). MM, malignant melanoma; BN, benign nevi; lncRNA, long noncoding RNA; ATB, activated by transforming growth factor-β; GEO, Gene Expression Omnibus; FDR, false discovery rate; shRNA, short hairpin RNA; NC, negative control.

Figure 2

Effects of lncRNA-ATB on the cell proliferation and apoptosis of MM cells. (A) The effects of lncRNA-ATB shRNA on the proliferation of A2058 cells were detected by CCK-8 assays. (B) Effects of lncRNA-ATB on the proliferation of A375 cells were detected by CCK-8 assays. (C) Effects of lncRNA-ATB on the cellular apoptosis of MM cells were detected by flow cytometry using an Annexin V/PI kit, and (D) the results were quantified. Data are from three experiments and are presented as the mean ± standard deviation. ^*P<0.05 vs. respective NC group (Student's t-test). MM, malignant melanoma; lncRNA, long noncoding RNA; shRNA, short hairpin RNA; NC, negative control; PI, propidium iodide; FITC, fluorescein isothiocyanate; OD, optical density; CCK-8, cell counting kit-8; ATB, activated by transforming growth factor-β.

Figure 3

Effects of lncRNA-ATB on the cell cycle distribution of malignant melanoma cells. (A) Effects of lncRNA-ATB shRNA on the cell cycle distribution of A2058 cells were detected by flow cytometry using PI. (B) Effects of lncRNA-ATB on the cell cycle distribution of A375 cells were detected by flow cytometry using PI. (C) Quantification of the results in A2058 cells. (D) Quantification of the results in A375 cells. Data are from three experiments and presented as mean ± SD. ^*P<0.05 vs. respective NC group (Student's t-test). PI, propidium iodide; lncRNA, long noncoding RNA; shRNA, short hairpin RNA; ATB, activated by transforming growth factor-β; NC, negative control.

Figure 4

Effects of lncRNA-ATB on the cell migration and invasion of MM cells. (A) Effects of lncRNA-ATB shRNA on the cell migration of A2058 and A375 cells were detected by Transwell assays (magnification, ×200). (B) Effects of lncRNA-ATB shRNA on the cell invasion of A2058 and A375 cells were detected by Transwell assays (magnification, x200). (C) Analysis of the migration and invasion data from A2058 cells. (D) Analysis of the migration and invasion data from A375 cells. Data are from three experiments and are presented as the mean ± standard deviation. ^*P<0.05 and ^**P<0.01 vs. respective NC group (Student's t-test). NC, negative control; shRNA, short hairpin RNA; lncRNA, long noncoding RNA; ATB, activated by transforming growth factor-β.

Figure 5

Effects of lncRNA-ATB on the tumorigenic ability and lung colonization of MM cells. (A) Tumors collected from the lncRNA-ATB shRNA-luciferase groups and A2058 lncRNA-ATB NC-luciferase groups. (B) Tumor growth curve of A2058 lncRNA-ATB shRNA-luciferase cells and A2058 lncRNA-ATB NC-luciferase cells in nude mice. (C) Tumor weights of lncRNA-ATB shRNA-luciferase and A2058 lncRNA-ATB NC-luciferase groups at day 35. (D) Effects of lncRNA-ATB shRNA on lung metastasis were measured using a Xenogene IVIS Kinetic imaging system every 7 days subsequent to injecting B16/F10 lncRNA-ATB shRNA-luciferase cells and B16/F10 lncRNA-ATB NC-luciferase cells intravenously into C57/B6 mice. Data are from three experiments and are presented as the mean ± standard deviation. ^*P<0.05 vs. respective NC group (Student's t-test). NC, negative control; shRNA, short hairpin RNA; lncRNA, long noncoding RNA; ATB, activated by transforming growth factor-β.

Figure 6

lncRNA-ATB sponges miR-590-5p to promote YAP1 expression in MM cells. (A) lncRNA-ATB and its putative binding sequence in miR-50-5p. (B) Efficacy of miR-590-5p mimics or inhibitors in MM cells. (C) Relative expression of miR-590-5p in the same sample pulled down by biotinylated lncRNA-ATB and NC probe. (D) Relative expression of miR-590-5p in MM cells transfected with lncRNA-ATB shRNA and lncRNA-ATB overexpressing plasmid. (E) Relative luciferase activity in 293 cells transfected with WT or Mut lncRNA-ATB plasmid. (F) Relative expression of lncRNA-ATB in MM cells transfected with miR-590-5p mimics or inhibitors. (G) Immunoblotting analysis and (H) relative quantification of YAP1 expression levels in MM cells transfected with lncRNA-ATB shRNA or overexpressing plasmid. Data are from three experiments and are presented as the mean ± standard deviation. ^*P<0.05 and ^**P<0.01 vs. respective NC group (Student's t-test). MM, malignant melanoma; NC, negative control; shRNA, short hairpin RNA; lncRNA, long noncoding RNA; ATB, activated by transforming growth factor-β; WT, wild-type; Mut, mutant; YAP1, Yes associated protein 1; miR, microRNA; Bio, biotinylated.