Long Non-Coding RNA CD27-AS1-208 Facilitates Melanoma Progression by Activating STAT3 Pathway

Abstract

Melanoma is the most lethal skin cancer that originates from epidermal melanocytes. Recently, long non-coding RNAs (lncRNAs) are emerging as critical regulators of cancer pathogenesis and potential therapeutic targets. However, the expression profile of lncRNAs and their role in melanoma progression have not been thoroughly investigated. Herein, we firstly obtained the expression profile of lncRNAs in primary melanomas using microarray analysis and unveiled the differentially-expressed lncRNAs compared with nevus. Subsequently, a series of bioinformatics analysis showed the great involvement of dysregulated lncRNAs in melanoma biology and immune response. Further, we identified lncRNA CD27-AS1-208 as a novel nuclear-localized factor with prominent facilitative role in melanoma cell proliferation, invasion and migration. Mechanistically, CD27-AS1-208 could directly interact with STAT3 and contribute to melanoma progression in a STAT3-dependent manner. Ultimately, the role of CD27-AS1-208 in melanoma progression in vivo was also investigated. Collectively, the present study offers us a new horizon to better understand the role of lncRNAs in melanoma pathogenesis and demonstrates that CD27-AS1-208 up-regulation contributes to melanoma progression by activating STAT3 pathway. Targeting CD27-AS1-208 in melanoma cells can be exploited as a potential therapeutic approach that needs forward validation in clinical trials in the future.

Keywords: CD27-AS1-208; LncRNAs; STAT3; melanoma; therapeutic targets.

Figure 1

Characterization of lncRNAs expression profile in primary melanoma compared with nevus. (A) Hierarchical clustering results of differentially-expressed lncRNAs in primary MM and Nevus. “Green” indicates relative low expression while “red” indicates relative high expression. (B) Percentage of differentially-expressed lncRNAs in the subgroups, sorted by their genome location and context. (C) GO analysis of differentially-expressed coding genes co-expressed with differentially-expressed lncRNAs was performed in biological processes. (D) KEGG pathway analysis of differentially-expressed coding genes co-expressed with differentially-expressed lncRNAs was performed. The vertical and horizontal axis represents the biological processes/pathways and −log10 (P value) of the corresponding biological process/pathways, respectively. GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; Sig, significant; UE, up-regulated expression; DE, down-regulated expression.

Figure 2

The expression of lncRNA CD27-AS1-208, which locates in the nucleus, is increased during melanoma progression and significantly correlation with Ki-67 level. (A) Validation of differentially-expressed lncRNAs with a new cohort of samples. (B) The expression level of CD27-AS1-208 in melanocytes and melanoma cell lines. (C, D) The expression level of CD27-AS1-208 in the cytoplasm and nucleus of melanoma cells. (E) The location of CD27-AS1-208 in primary melanoma and nevus. (F) Immunofluorescence staining of Ki-67 in indicated melanoma tissues. (G) Correlation between CD27-AS1-208 level and Ki67 staining in melanoma tissue. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, NHEM or cytoplasm as control; ^#HMS as control, ^# P < 0.05, ^## P < 0.01; ^&PIG1 as control, ^& P < 0.05, ^&& P < 0.01. Scale bar = 10μm. ns., not significant.

Figure 3

Knockdown of lncRNA CD27-AS1-208 inhibits melanoma cell proliferation, migration and invasion. (A) The knockdown efficiency of CD27-AS1-208 after 48h of transfection. (B) Clone formation assays of A2058 and A375 cells transfected with the indicated siRNA. (C) The migratory ability of A2058 and A375 cells transfected with the indicated siRNA was assessed by the wound healing assay. (D) A2058 and A375 cells transfected with siCD27-AS1-208 or NC siRNA were subjected to the matrigel invasion assay. Scale bar = 100μm. Data represent the mean ± SD of triplicates. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001. NC, negative control.

Figure 4

The facilitative role of CD27-AS1-208 depends on the activation of STAT3 pathway. (A, B) The expression levels of pSTAT3(Y705) in A2058 and A375 cell lines after 48h of transfection. (C) The mRNA levels of the target genes of the STAT3 pathway were detected by qRT-PCR after 48h of transfection. (D) Colony formation assays of A2058 and A375 cells transfected with the indicated siRNA alone or in combination with IL-6 (20ng/ml). (E) The invasive capacity of A2058 and A375cells transfected with the indicated siRNA alone or in combination with IL-6 (20ng/ml) was assessed by matrigel invasion assay. Scale bar = 100μm. Data represent the mean ± SD of triplicates. ^** P < 0.01, ^** P < 0.001, ***P < 0.001. NC, negative control.

Figure 5

Knockdown of CD27-AS1-208 suppresses the growth of melanoma xenografts. (A) Photographs of mice and xenograft tumors of A2058 cells with indicated treatment after 50 days of implantation. (B) Tumor weight of mice was calculated 50 days after implantation. (C) Growth curves of xenograft tumors with indicated treatment. (D) Immunofluorescence staining analysis of Ki-67 in isolated tumors with indicated treatment. ^* P < 0.05; Scale bar = 10μm.