An androgen reduced transcript of LncRNA GAS5 promoted prostate cancer proliferation

<div><p>Prostate cancer (PCa) becomes a leading cause of death in males nowadays. Recent reports showed that androgen-responsive long non-coding RNAs played important roles in tumorigenesis and progression of PCa. In this study, we focused on a special transcript of GAS5 (ENST00000456293.5, GAS5-007), which was reported as a tumor suppressor. Here, we demonstrated GAS5-007 was reduced by androgen treatment and inhibited by AR. Next, we explored the expression level of GAS, finding the expression of it in PCa tissue was higher than normal tissue in both public databases and human tissue samples. Functional analysis of GAS5 showed it was related to regulating translational elongation, protein biosynthesis, and transcription. Moreover, we observed GAS5-007 knockdown inhibited the proliferation, cell cycle and promoted cell apoptosis of PCa. We also constructed a GAS5-miRNA network to explain the different roles of different GAS5 transcripts in PCa. This study provides novel insights to identify potential diagnostic biomarker and therapy target for prostate cancer in clinical treatment.</p></div

LncRNA GAS5 promoted cell proliferation, cell cycle and inhibited cell apoptosis in PCa cells.

<p>(A) The efficiency of siGAS5 was confirmed by qRT-PCR. (B) Knockdown of GAS5 inhibited the proliferation of LNCaP cells. (C) Cell cycle assay was performed in LNCaP cells. Cells were transfected with NC or siGAS5, stained with PI and evaluated with FACScalibur flow cytometer. Knockdown of GAS5 inhibited cell cycle. (D) Cell apoptosis assay was performed in PC-3 cells. Cells were transfected with si-NC or si-GAS5, stained with PI and FITC. GAS5 knockdown increased the percentage of cells in both early apoptosis and late apoptosis. The cell cycle and apoptosis analysis results presented as mean ± SD (n = 3). Significance was defined as p < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001).</p

The expression of GAS5 in prostate cancer.

<p>(A) The relative expression level of GAS5 in tumor tissue compared with normal tissue in public gene expression data GSE8511, GSE55945 (B) and GSE38241 (C). (D) The relative mRNA level of GAS5 in 52 normal tissue samples compared with 419 tumor tissue samples from TCGA database. (E) Relative expression level of GAS5 was measured in normal prostate epithelial cell line WPMY-1 and four prostate cancer cell lines, LNCaP, 22RV1, DU145 and PC-3 by qRT-PCR. (F) The relative expression of GAS5 in 11 normal prostate tissues and 14 prostate tumor tissues were measured by qRT-PCR. Statistical analyses between groups were performed using an ANOVA analysis. Significance was defined as p < 0.05 (*, p < 0.05; **, p < 0.01; ***, p < 0.001).</p

GO biological process and KEGG pathway enrichment analysis of LncRNA GAS5.

<p>(A) Gene co-expression networks of GAS5 were constructed according to Pearson correlation coefficients. (B) GO biological process analysis of GAS5-positive genes and GAS5-negative genes (E). (C) KEGG pathway enrichment analysis of GAS5-positive genes and GAS5-negative genes (F) using MAS 3.0. (D) The PPI network.</p

Differences of targeted miRNA of GAS5-001, GAS5-002 and GAS-007.

<p>(A) Intersections of GAS5-001, GAS5-002 and GAS5-007 targeted miRNA. (B) Targeted miRNA networks of GAS5-001, GAS5-002 and GAS5-007.</p

The regulation of AR on the expression of LncRNA GAS5.

<p>(A) qRT-PCR analysis of GAS5’s expression in LNCaP cells treated with DHT in time series of 0h, 2h, 8h, 24h, 48h. (B) qRT-PCR analysis of GAS5’s expression in LNCaP cells treated with DHT in dose series of 0nM, 0.1nM, 1nM, 10nM, 100nM, 1000nM. (C) The efficiency of siAR on the expression of AR was confirmed by qRT-PCR and the expression of GAS5 was tested after transfection of siAR compared with NC in LNCaP cells. (D) The correlation of mRNA level between GAS5 and AR. (E) The correlation of regulation between GAS5 and AR. Significance was defined as p < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001).</p

Global regulation of AR signaling by miRNAs.

<p>.A. miRNA-mediated AR signalling network. B. miRNA dominant regulation. The Pie chart for miRNAs shows their dominant regulation on identified targets. 1, 2, 3, 4, 5, 6 and 8 represent the numbers of miRNAs, which co-regulate on the same target mRNA.</p

Flowchart of strategy.

<p>This is the outline of the whole procedure for analysing microarray data to construct AR network in this study. Detailed steps are provided in the methodology and result sections.</p

miR-19a's regulation on identified targets and prostate cancer cell viability.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056592#pone-0056592-g004" target="_blank">Figure 4A</a>. RT-PCR analysis of miR-19a’s identified significant targets: SUZ12, RAB13, SC4MOL, ABCA1 and PSAP. Fold change between miR-19a transfected samples and miR-NC (control) was presented with significance assessment. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056592#pone-0056592-g004" target="_blank">Figure 4B</a>. Luciferase assays for miR-19a’s regulation on targets: SUZ12, RAB13, SC4MOL, ABCA1 and PSAP. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056592#pone-0056592-g004" target="_blank">Figure 4C–D</a>. miR-19a’s contribution to prostate cancer cell viability. C. LNCaP cells cultured in androgen-depleted medium were treated with DHT, miR-19a and miR-NC separately, or in a combinational way. D. LNCaP cells cultured in androgen-depleted medium were treated with DHT, anti-miR-19a (si19a) and anti-miR-NC (siNC) separately, or in a combinational way. In both figures, the cell viability was measured by MTT assay during 4 days. MTT absorbance at each time point was presented.</p

Time discriminator for distinguishing early and late response stages.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056592#pone-0056592-g002" target="_blank">Figure 2A</a>. Time course profile of differentially expressed miRNA number. The dashed line refers to time discriminator for distinguishing early and late response stages. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056592#pone-0056592-g002" target="_blank">Figure 2B</a>. Expression profile of miRNAs’ early- and late-response to DHT stimuli. Differential expression relative to 0 h is represented by . separates miRNA response into early and late stages. miRNAs are clustered into 4 groups: early upregulated (1), late upregulated (2), early downregulated (3) and late downregulated (4). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056592#pone-0056592-g002" target="_blank">Figure 2C</a>. Venn diagram for number distribution of early responsive miRNAs and late responsive miRNAs. The red part denotes the androgen-resonsive miRNAs with response happened solely at the early stage, the blue part denotes miRNAs with response solely at the late stage, and the purple part denotes miRNAs with response both at the early and late stages. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056592#pone-0056592-g002" target="_blank">Figure 2D</a>. Predicted ARE enrichment in early and late responsive miRNA genes. AREs are in the ±10 kb sequences flanking 5′-start site of pre-miRNAs.</p