LncRNA GAS5 is a critical regulator of metastasis phenotype of melanoma cells and inhibits tumor growth in vivo

Abstract

The present study intended to demonstrate the effects of long noncoding RNA growth arrest-specific transcript 5 (GAS5) on the migration and invasion of melanoma cells. We first detected the expression of GAS5 among four kinds of melanoma cell lines, followed by constructing GAS5-knocked down and overexpressed stable cells. Next, we evaluated the effects of GAS5 on cell migration and invasion using wound healing and gelatin zymography assays. Finally, melanoma cells with different GAS5 expression were injected into nude mice, and the tumor volumes were recorded and tumor tissues were analyzed after sacrificing the mice. This study systematically examined the function of GAS5 in mediating melanoma metastasis and revealed that GAS5 plays an anticancer role in melanoma via regulating gelatinase A and B, both in vitro and in vivo.

Keywords: GAS5; gelatinase; lncRNA; melanoma; metastasis.

Figure 1

The gene structure of GAS5 and its expression level among different melanoma cells. Notes: (A) Twelve exons and numerous snoRNAs are present in GAS5 gene, and two types of mature GAS5 transcript are present, GAS5a and GAS5b, in which the latter is predominant in many mammal cells. (B) qRT-PCR analysis of GAS5 expression among various melanoma cells. Compared with Hacat, A375 harbors the highest GAS5 expression while SK-Mel-110 expresses the least. The data are represented as mean ± SD, ^**P<0.01 and ^***P<0.001, three independent experiments. Abbreviations: bp, base pair; GAS5, growth arrest-specific transcript 5; qRT-PCR, quantitative real-time polymerase chain reaction; snoRNA, small nucleolar RNA.

Figure 2

The establishment of stable GAS5 knockdown and overexpression in melanoma cells. Notes: (A) ZsGreen1 expression was examined using an Olympus IX51 fluorescent microscope under UV light (right) and white light conditions (left) in A375-GAS5si and SK-Mel-110-GAS5over. All the comparative pictures in A375-GAS5si and SK-Mel-110-GAS5over were taken at the same magnifications using fluorescent microscopy. Magnification 40×10. Using qRT-PCR analysis, GAS5 expression was measured following the treatment of A375 with siRNA (B) and SK-Mel-110 (C) with full length of GAS5. The data are represented as mean ± SD; ^**P<0.01 and ^***P<0.001, three independent experiments. Abbreviations: GAS5, growth arrest-specific transcript 5; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 3

GAS5 inhibits migration of melanoma cells. Notes: Migration of melanoma cells after knocking down and overexpressing GAS5. (A) Wound healing assay was performed and compared between A375 and A375-GAS5si, SK-Mel-110 and SK-Mel-110-GASover cell lines. The photos of wounds were taken at different times. The red lines indicate the wound widths which were measured. Original magnification ×40; bars =500 μm. (B) The quantitative analysis of the percentage of wound width was shown in the line chart. Three independent experiments were conducted and similar results were obtained. ^**P<0.01; ^***P<0.001. Abbreviation: GAS5, growth arrest-specific transcript 5.

Figure 4

Gelatin zymography of gelatinase A and B among melanoma cells. Notes: (A) Detecting MMP2 and MMP9 protein expression among A375, A375-GAS5si, SK-Mel-110, and SK-Mel-110-GAS5over cells by Western blot. (B) Gelatine zymography of total protein lysates from A375, A375-GAS5si, SK-Mel-110, and SK-Mel-110-GAS5over. Both proactive and active forms of MMP2 and MMP9 were detected and are marked by arrows (left). Densitometric quantification of the activity of MMP2 and MMP9 in the A375, A375-GAS5si, SK-Mel-110, and SK-Mel-110-GAS5over melanoma cells. Gel zymography analysis showed that both MMP2 and MMP9 activities were significantly higher in A375-GAS5si versus A375 cells (P<0.01). Conversely, overexpressing GAS5 attenuated the MMP2 and MMP9 activity (right). The figures are representative examples of three independent experiments. The values are significantly different from the control (^*P<0.05 and ^**P<0.01). Abbreviations: GAS5, growth arrest-specific transcript 5; MMP, matrix metalloproteinase.

Figure 5

GAS5 inhibited melanoma growth in vivo. Notes: A375, A375-GAS5si, SK-Mel-110, and SK-Mel-110-GAS5over cells (5×10⁶ per mouse) were implanted into nude mice. (A) The tumor nodules were detached, photographed, and weighed after sacrificing mice at the end of the experiment on day 26. (B) The growth curves show the calculated tumor sizes in mice of each group (n=3 per group), and data are presented as mean ± SD at each time point. The tumor size was calculated and plotted every 6 days and there was no visible tumor formation in the first week. Compared with A375, the A375-GAS5si group formed larger tumors (P=0.001) while the SK-Mel-110-GAS5over group inhibited the tumor growth relative to the SK-Mel-110 group (P<0.001). (C) The tumor mass was weighed after sacrificing on day 26 and representative images were presented. Knocking down GAS5 dramatically elevated the tumor mass (P<0.05), whereas overexpressing GAS5 significantly limited the tumor mass (P<0.05). (D) Soluble protein extracts from xenograft mice were subjected to immunoblotting for the indicated gelatinase A (MMP2). (E) Soluble protein extracts from xenograft mice were subjected to immunoblotting for the indicated gelatinase B (MMP9). Data are expressed as mean ± SD for each group. ^*P<0.05, ^**P<0.01, and ^***P<0.001. Abbreviations: GAS5, growth arrest-specific transcript 5; MMP, matrix metalloproteinase.