Long Non-coding RNA PVT1 Promotes Cell Proliferation and Migration by Silencing ANGPTL4 Expression in Cholangiocarcinoma

Abstract

Cholangiocarcinoma (CCA) is the most common biliary tract malignancy, with a low survival rate and limited treatment options. Long non-coding RNAs (lncRNAs) have recently been verified to have significant regulatory functions in many kinds of human cancers. It was discovered in this study that the lncRNA PVT1, whose expression is significantly elevated in CCA, could be a molecular marker of CCA. Experiments indicated that PVT1 knockdown greatly inhibited cell migration and proliferation in vitro and in vivo. According to RNA sequencing (RNA-seq) analysis, PVT1 knockdown dramatically influenced target genes associated with cell angiogenesis, cell proliferation, and the apoptotic process. RNA immunoprecipitation (RIP) analysis demonstrated that, by binding to epigenetic modification complexes (PRC2), PVT1 could adjust the histone methylation of the promoter of ANGPTL4 (angiopoietin-like 4) and, thus, promote cell growth, migration, and apoptosis progression. The data verified the significant functions of PVT1 in CCA oncogenesis, and they suggested that PVT1 could be a target for CCA intervention.

Keywords: ANGPTL4; cholangiocarcinoma; lncRNA PVT1.

Figure 1

The lncRNA PVT1 Is Overexpressed in Cholangiocarcinoma Tissues (A) Hierarchical clustering analysis of lncRNAs that were differentially expressed (fold change > 2; p < 0.05) in cholangiocarcinoma tissues and normal tissues. (B) Overlap of dysregulated lncRNAs in GEO datasets. (C) PVT1 was detected in 17 pairs of CCA tissues by qRT-PCR. The levels of PVT1 in CCA tissues were significantly higher than those in non-tumorous tissues.

Figure 2

PVT1 Promotes Cell Proliferation and Migration in Cholangiocarcinoma Cells (A) qRT-PCR was used to determine the expression of PVT1 after siRNA transfection in the HuCCT1 and RBE cell lines. (B) Colony formation assays were used to determine the colony-forming ability of si-PVT1-transfected cells. (C) CCK-8 assays were used to determine the viability of si-PVT1-transfected cholangiocarcinoma cells. (D) Transwell assays showed that PVT1 knockdown inhibited cholangiocarcinoma cell migration. The error bars indicate the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Knockdown of PVT1 Causes Apoptosis by Promoting Cell-Cycle Arrest In Vitro (A) Fluorescence-activated cell sorting (FACS) analysis of the effect of PVT1 on apoptosis. (B) FACS analysis of the effect of PVT1 on cell cycle progression. The error bars indicate the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Figure 4

PVT1 Regulates CCA Cell Proliferation In Vivo (A) Tumors established with the mice in the scrambled control or sh-PVT1 group. (B) The size of tumors in the scrambled control or sh-PVT1 group. (C) After cell injection, the tumor volumes were calculated every 4 days. (D) The tumor weights are presented as the means ± SD. The error bars indicate the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

RNA-Seq after PVT1 Knockdown in HuCCT1 Cells (A) Mean-centered hierarchical clustering analysis of 1,295 transcripts that were altered (≥1.5-fold change) in si-NC-treated cells and siRNA-PVT1-treated cells, assessed in triplicate. (B) Gene ontology analysis for all genes with altered expression. (C and D) The mRNA levels of the altered genes were selectively confirmed by qRT-PCR in PVT1-knockdown HuCCT1 (C) and RBE (D) cells. (E) The mRNA levels of the altered genes were selectively confirmed by qRT-PCR in PVT1-overexpressing cells. The error bars indicate the means ± SD. *p < 0.05, **p < 0.01; ns, not significant.

Figure 6

PVT1 Binds with EZH2 to Coregulate Target Genes, Especially ANGPTL4 (A) After nuclear and cytosolic separation, RNA expression levels were measured by qRT-PCR. GAPDH was used as a cytosolic marker and U1 was used as a nuclear marker. (B) Fluorescent images of RBE cells treated with anti-PVT1 (red), anti-18S (red), and anti-U6 (red) RNA probes. DAPI staining is shown in blue. (C) The probability of interaction of EZH2 and PVT1 was determined with an online tool (http://pridb.gdcb.iastate.edu/RPISeq/index.html). Predictions with probabilities >0.5 were considered positive. RPI-seq predictions are based on random forest (RF) or support vector machine (SVM). (D) An RIP experiment for EZH2 was performed, and the coprecipitated RNA was subjected to qRT-PCR for PVT1. (E) Expression and co-localization of EZH2 and PVT1 in HuCCT1 cells. Representative fluorescent images show HuCCT1 cells treated with fluorescently labeled anti-EZH2 antibody (green) and anti-PVT1 RNA (red). DAPI staining indicates the cell nuclei (blue). (F) Methylation-related genes were detected by qRT-PCR in the HuCCT1 and RBE cell lines after knockdown of EZH2. (G) Methylation-related genes were detected by qRT-PCR in the HuCCT1 and RBE cell lines after overexpression of EZH2. (H) The correlation between EZH2 and ANGPTL4 expression was detected by analyzing GEO: GSE26566 data. (I) The altered protein levels of ANGPTL4 were selectively confirmed by western blot analysis in cells with knockdown of PVT1 or EZH2. (J) The altered protein levels of ANGPTL4 were selectively confirmed by western blotting in cells overexpressing PVT1 or EZH2. The error bars indicate the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

Figure 7

PVT1 Binds with EZH2 in the Nucleus and Epigenetically Silences ANGPTL4, Inhibiting Cell Proliferation and Migration in CCA Cell Lines (A) Expression level of ANGPTL4 in cholangiocarcinoma based on the analysis of GEO: GSE26566 data. (B) ANGPTL4 expression was determined in 17 pairs of CCA tissues by qRT-PCR. (C–F) HuCCT1 and RBE cells transfected with vector/ANGPTL4/pcDNA-PVT1 and cells transfected with PVT1 followed by transfection with ANGPTL4. After transfection, the cells were analyzed by CCK-8 assays (C and D) and transwell assays (E and F). (G and H) ChIP of EZH2 and H3K27me3 of the promoter region of the ANGPTL4 locus after siRNA treatment with si-NC and si-PVT1 2# (G) or overexpression of PVT1 (H) in HuCCT1 cells. qPCR was performed to quantify the ChIP assay products. Enrichment was quantified relative to the input controls. Antibodies directed against IgG were used as a negative control. (I) Proposed model by which PVT1 regulates ANGPTL4 expression to promote CCA tumor growth. The error bars indicate the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.