YY1-induced lncRNA00511 promotes melanoma progression via the miR-150-5p/ADAM19 axis

Abstract

Increasing evidence indicates that long noncoding RNAs (lncRNAs) are therapeutic targets and key regulators of tumors development and progression, including melanoma. Long intergenic non-protein-coding RNA 511 (LINC00511) has been demonstrated as an oncogenic molecule in breast, stomach, colorectal, and lung cancers. However, the precise role and functional mechanisms of LINC00511 in melanoma remain unknown. This study confirmed that LINC00511 was highly expressed in melanoma cells (A375 and SK-Mel-28 cells) and tissues, knockdown of LINC00511 could inhibit melanoma cell migration and invasion, as well as the growth of subcutaneous tumor xenografts in vivo. By using Chromatin immunoprecipitation (ChIP) assay, it was demonstrated that the transcription factor Yin Yang 1 (YY1) is capable of binding to the LINC00511 promoter and enhancing its expression in cis. Further mechanistic investigation showed that LINC00511 was mainly enriched in the cytoplasm of melanoma cells and interacted directly with microRNA-150-5p (miR-150-5p). Consistently, the knockdown of miR-150-5p could recover the effects of LINC00511 knockdown on melanoma cells. Furthermore, ADAM metallopeptidase domain expression 19 (ADAM19) was identified as a downstream target of miR-150-5p, and overexpression of ADAM19 could promote melanoma cell proliferation. Rescue assays indicated that LINC00511 acted as a competing endogenous RNA (ceRNA) to sponge miR-150-5p and increase the expression of ADAM19, thereby activating the PI3K/AKT pathway. In summary, we identified LINC00511 as an oncogenic lncRNA in melanoma and defined the LINC00511/miR-150-5p/ADAM19 axis, which might be considered a potential therapeutic target and novel molecular mechanism the treatment of patients with melanoma.

Keywords: ADAM19; LINC00511; PI3K/AKT; ceRNA; melanoma.

Figure 1

LINC00511 was over-expressed in melanoma cells. A. The results from the GEO database showed that LINC00511 expression was significantly upregulated in melanoma tissues compared with normal tissues. The fold change (FC) of genes was assessed by log transformation. |log FC| > 2 and adjusted P < 0.05 were defined as the screened threshold. B. Based on the TCGA dataset, LINC00511 expression was found to be increased in melanoma tissues. C. The expression of LINC00511 in melanoma tissues and nor-tumor tissues by ISH (scale bars, 500 µm). D. Melanoma cells exhibited elevated levels of LINC00511 expression. E. Relative LINC00511 levels were in melanoma cells transfected with si-NC and si-LINC00511-1, -2. F, G. The proliferation of transfected melanoma cells was assessed using CCK8 and EdU experiments. H. The effect of transfection with si-NC, si-LINC00511-1, and -2 on the rate of apoptosis was evaluated using TUNEL analysis. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; ns, not significant.

Figure 2

LINC00511 was over-expressed in melanoma cells. A. Metastasis ability was measured by wound-healing assay. B, C. The migration and invasion abilities of A375 and SK-Mel-28 cells were detected (scale bars, 100 µm). D. Relative expression levels of protein markers were observed in A375 and SK-Mel-28 cells transfected with si-NC, si-LINC00511-1, and -2 by western blot. E. The tumor’s morphology, growth curve, volume, and weight were observed. F. The expression of Ki-67 was assessed by IHC (scale bars, 200 µm). G. The levels of LINC00511 were measured in xenograft tissues by qRT-PCR. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; ns, not significant.

Figure 3

Transcription factor YY1 mediates the upregulation of LINC00511. A. The intersection of PROMO, hTFtarget, and JASPAR databases was utilized to identify transcription factors upstream of LINC00511. B. The DNA motif of YY1 on the promoter LINC00511. C, D. The expression of YY1 in A375 and SK-Mel-28 cells after LINC00511 knockdown was detected by qRT-PCR and western blot. E, F. The efficiency of YY1 knockdown in A375 and SK-Mel-28 cells was determined by qRT-PCR and western blot. G. Adopted qRT-PCR assay to detect the relative expression of LINC00511 by YY1 depletion. H. The ChIP assay confirmed the binding affinity between YY1 and the LINC00511 promoter. I. The luciferase activity of the wild/mutant LINC00511 promoter was detected upon YY1 depletion. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; ns, not significant.

Figure 4

LINC00511 acted as a sponge for miR-150-5p. A. The localization of LINC00511 was predicted using the lncRNA subcellular localization predictor, lncLocator. B. The LINC00511 molecule was mostly located in the cytoplasm. C. FISH assay was conducted to determine the subcellular localization of LINC00511 in A375 and SK-Mel-28 cells. D. The expression levels of miR-150-5p were identified using qRT-PCR in A375 and SK-Mel-28 cells with LINC00511 knockdown. E. Luciferase activity was measured in A375 and SK-Mel-28 cells co-transfected with miR-NC and miR-150-mimics containing LINC00511-wt or LINC00511-mut. F. The RIP assay revealed that both LINC00511 and miR-150-5p expressions were enhanced in the mixture immunoprecipitated by anti-Ago2. G. The miRNA pull-down assays confirmed the binding ability between LINC00511 and miR-150-5p. H. Scatter-plots show a negative correlation between LINC00511 and miR-150-5p at the mRNA level in 499 SKCM tissues. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; ns, not significant.

Figure 5

miR-150-5p directly targeted ADAM19 in melanoma. A. The intersection of miRDB, Target Scan Human, ENCORI, and miRWalk databases was used to identify ADAM19 as the downstream target of miR-150-5p. B. The top ten genes were selected for qRT-PCR analysis of their expression in A375 and SK-Mel-28 cells. C. Based on the TCGA dataset, ADAM19 expression is found to be increased in melanoma tissues. D. Overall survival analysis based on the TCGA dataset. E, F. The expression of ADAM19 in melanoma tissues and nor-tumor tissues by IHC (scale bars, 500 µm; scale bars, 100 µm). G. Luciferase activity assay was used to confirm luciferase reporter. H. RIP assays were carried out to demonstrate the coexistence of miR-150-5p and ADAM19 in RISCs. I. ADAM19 was pulled down by the biotin-miR-150-5p probe, but not by the biotin-miR-150-NC probe. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; ns, not significant.

Figure 6

Silencing ADAM19 suppressed melanoma progression in vitro and in vivo. A. The efficiency of si-ADAM19 knockdown was determined by qRT-PCR. B. The protein level of ADAM19 was illustrated by Western blot assay after knocking down ADAM19 in A375 and SK-Mel-28 cells. C, D. CCK8 and EdU experiments were used to evaluate the proliferation rate of transfected melanoma cells. E. The influence of ADAM19 knockdown on the rate of apoptosis was assessed by TUNEL analysis. F. Metastatic ability was assessed using the wound-healing assay. G. Relative expression levels of Bax, Bcl-2, MMP9, and PCNA were observed in A375 and SK-Mel-28 cells with ADAM19 knockdown. H. The tumor’s morphology, growth curve, volume, and weight were observed. I. The levels of ADAM19 in xenograft tissues were measured using qRT-PCR. J. The expression of Ki-67 was examined through IHC (scale bars, 200 µm). ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; ns, not significant.

Figure 7

ADAM19 reversed the impact of miR-150-5p on melanoma cells. A. Expression levels of miR-150-5p were identified in melanoma cells after ADAM19 overexpression. B, C. Relative expression levels of ADAM19 are observed in A375 and SK-Mel-28 cells transfected with miR-150-5p. D, E. CCK8 and EdU assays to proliferation rate of transfected cells. F. The influence of ADAM19 and miR-150-5p mimics on the rate of apoptosis was evaluated through TUNEL analysis. G. Scratch wound healing assay is used to evaluate migration capacity. H. Relative expression levels of ADAM19, Bax, Bcl-2, MMP9, PCNA were observed in A375 and SK-Mel-28 cells transfected with ADAM19 and miR-150-5p mimics. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; ns, not significant.

Figure 8

LINC00511 acted as a sponge for miR-150-5p to up-regulate ADAM19 expression. A. The expression levels of ADAM19 in A375 and SK-Mel-28 cells transfected with si-LINC00511 and miR-150-5p inhibitor were identified using qRT-PCR. B. Relative expression levels of ADAM19, Bax, Bcl-2, MMP9, PCNA were observed in A375 and SK-Mel-28 cells transfected with si-LINC00511 and miR-150-5p inhibitor. C, D. CCK8 and EdU assays to proliferation rate of transfected cells. E. The influence of si-LINC00511 and miR-150-5p inhibitor on the rate of apoptosis was evaluated through TUNEL analysis. F. Metastasis ability was measured using wound-healing assay in melanoma cells transfected with si-LINC00511and miR-150-5p inhibitor. G. Gross appearance, tumor volume, and tumor weight of each group. H. IHC staining results (Ki-67). ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; ns, not significant.

Figure 9

LINC00511 activated the PI3K/AKT pathway in melanoma cell through the miR-150-5p/ADAM19 axis. A. KEGG enrichment analysis. B. The effects of the LINC00511 gene on PI3K, p-PI3K, p-Akt and Akt expression in A375 and SK-Mel-28 cells were detected by western blot analysis. C-E. The relative protein expression of PI3K, p-PI3K, p-Akt and Akt was analyzed in A375 and SK-Mel-28 cells transfected with si-NC/si-LINC00511+inhibitor NC/miR-150-5p inhibitor, si-NC/si-ADAM19/+inhibitor NC/miR-150-5p inhibitor, si-NC/si-LINC00511+NC/ADAM19 respectively. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; ns, not significant.

Figure 10

A proposed model in which YY1-induced LncRNA00511 promotes melanoma progression by regulating ADAM19 expression and PI3K/AKT signaling through competitive binding with miR-150-5p.