Amplification of PVT-1 is involved in poor prognosis via apoptosis inhibition in colorectal cancers

Abstract

Background: We previously conducted gene expression microarray analyses to identify novel indicators for colorectal cancer (CRC) metastasis and prognosis from which we identified PVT-1 as a candidate gene. PVT-1, which encodes a long noncoding RNA, mapped to chromosome 8q24 whose copy-number amplification is one of the most frequent events in a wide variety of malignant diseases. However, PVT-1 molecular mechanism of action remains unclear.

Methods: We conducted cell proliferation and invasion assays using colorectal cancer cell lines transfected with PVT-1siRNA or negative control siRNA. Gene expression microarray analyses on these cell lines were also carried out to investigate the molecular function of PVT-1. Further, we investigated the impact of PVT-1 expression on the prognosis of 164 colorectal cancer patients by qRT-PCR.

Results: CRC cells transfected with PVT-1 siRNA exhibited significant loss of their proliferation and invasion capabilities. In these cells, the TGF-β signalling pathway and apoptotic signals were significantly activated. In addition, univariate and multivariate analysis revealed that PVT-1 expression level was an independent risk factor for overall survival of colorectal cancer patients.

Conclusion: PVT-1, which maps to 8q24, generates antiapoptotic activity in CRC, and abnormal expression of PVT-1 was a prognostic indicator for CRC patients.

Figure 1

Positive correlation between chromosome 8q24 copy-number and PVT-1 or MYC expression. (A) Chromosome 8q24 copy-number (horizontal axis) and PVT-1 expression (vertical axis) analysed by array-CGH and gene expression array in 130 colorectal cancer cases. Dot plots indicate each case. (B) Chromosome 8q24 copy-number (horizontal axis) and PVT-1 expression (vertical axis) in 50 colorectal cancer cell lines (the Cancer Cell Line Encyclopedia database). Dot plots indicate each cell line. (C) Chromosome 8q24 copy-number (horizontal axis) and MYC expression (vertical axis) analysed by array-CGH and gene expression array in 130 colorectal cancer cases. Dot plots indicate each case. (D) Chromosome 8q24 copy-number (horizontal axis) and MYC expression (vertical axis) in 50 colorectal cancer cell lines (the CCLE database). Dot plots indicate each cell line.

Figure 2

Knockdown of PVT-1 promoted apoptosis in colorectal cancer cells. (A) qRT–PCR analysis showed that PVT-1 expression levels of both cell lines transfected with PVT-1 siRNA were significantly lower than negative control (nc) cells. (B) Cell proliferation ratio of RKO cells (left panel) and HCT116 cells (right panel) with or without PVT-1 knockdown. (C) Invasive properties of RKO cells (left panel) and HCT116 cells (right panel) with or without PVT-1 knockdown. (D) A total of 1187 genes with significant alteration of expression levels (649 upregulated and 538 downregulated) by knockdown of PVT-1(P<0.05). Representative genes are shown in the right side of the heat map. (E) Western blot (left, RKO; right, HCT116) analyses of cells transfected with PVT-1 siRNA, and negative control cells show the activation of SMAD4 and cleavage of caspase3. Histograms represent the means±s.d. of three independent experiments. nc, cells transfected with negative control siRNA. si-1, cells transfected with PVT-1 siRNA-1. si-2, cells transfected with PVT-1 siRNA-2. *, statistically significant (P<0.05).

Figure 3

Abundant PVT-1 expression is involved in poor prognosis in colorectal cancer. (A) qRT–PCR analyses of PVT-1expression levels on tumour tissues and paired normal tissues from 164 colorectal cancer samples. (B) Kaplan–Meier overall survival curves for 164 patients with CRC classified according to PVT-1 expression level. (C) qRT–PCR analyses of MYC expression levels on tumour tissues and paired normal tissues from 164 colorectal cancer samples. (D) Kaplan–Meier overall survival curves for 164 patients with CRC classified according to MYC expression level.