Knockdown of long non‑coding RNA PVT1 reverses multidrug resistance in colorectal cancer cells

Abstract

Multidrug resistance (MDR) is one of the primary causes of chemotherapy failure in colorectal cancer (CRC), and extensive biological studies into MDR are required. The non‑coding RNA plasmacytoma variant translocation 1 (PVT1) has been demonstrated to be associated with low survival rates in patients with CRC. However, whether PVT1 serves a critical function in the MDR of CRC remains to be determined. To determine the association between PVT1 expression and 5‑fluorouracil (5‑FU) resistance in CRC, the expression levels of PVT1 mRNA in 5‑FU‑resistant CRC tissues and cell lines (HCT‑8/5‑FU and HCT‑116/5‑FU) were assessed by a reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Cytotoxicity was evaluated using a Cell Counting Kit‑8 assay and apoptosis rates were assessed via flow cytometry. In the present study, PVT1 mRNA was highly expressed in 5‑FU‑resistant CRC tissues and cell lines. HCT‑8/5‑FU and HCT‑116/5‑FU cells transfected with small interfering RNA PVT1 and treated with 5‑FU exhibited higher apoptotic rates and lower survival rates. By contrast, overexpression of PVT1 in HCT‑8 and HCT‑116 cells transfected with lentiviral vector‑PVT1‑green fluorescent protein and treated with 5‑FU exhibited lower apoptosis rates and higher survival rates. RT‑qPCR and western blotting demonstrated that the overexpression of PVT1 increased the mRNA and protein expression levels of multidrug resistance‑associated protein 1, P‑glycoprotein, serine/threonine‑protein kinase mTOR and apoptosis regulator Bcl2. The present study indicates that PVT1 overexpression may promote MDR in CRC cells, and suggested that inhibition of PVT1 expression may be an effective therapeutic strategy for reversing MDR in CRC.

Figure 1.

PVT1 is associated with 5-FU-resistance. (A) The expression levels of PVT1 mRNA in CRC tissues from 5-FU-sensitive and 5-FU-resistant patients. (B) The expression levels of PVT1 mRNA in 5-FU-resistant HCT-8 and 5-FU-resistant HCT-116 cells. All values are presented as the mean ± standard deviation. ***P<0.001. PVT1, plasmacytoma variant translocation 1; 5-FU, 5-fluorouracil; CRC, colorectal cancer.

Figure 2.

Knockdown of PVT1 reverses drug resistance in 5-FU-resistant CRC cells. (A) PVT1 mRNA levels in HCT-8/5-FU and HCT-116/5-FU following silencing of PVT1. (B) The effect of PVT1 knockdown on the viability of HCT-8/5-FU cells transfected with siPVT1 and treated with 5-FU. (C) The effect of PVT1 knockdown on the viability of HCT-116/5-FU cells transfected with siPVT1 and treated with 5-FU. The proportion of apoptotic cells in HCT-8/5-FU cells HCT-116/5-FU cells transfected with siPVT1 was determined by (D) flow cytometry and (E) quantified. All values are presented as the mean ± standard deviation. **P<0.01 and ***P<0.001 vs. respective NC. PVT1, plasmacytoma variant translocation 1; 5-FU, 5-fluorouracil; CRC, colorectal cancer; OD, optical density, PI, propidium iodide; NC, negative control; si, small interfering.

Figure 3.

Overexpression of PVT1 suppresses apoptosis in CRC cells. (A) LV-PVT1-GFP-transfected cells overexpressed PVT1. (B) The effect of PVT1 overexpression on the viability of HCT-8 cells transfected with LV-PVT1-GFP and treated with 5-FU. (C) The effect of PVT1 overexpression on the cell viability of HCT-116 cells transfected with LV-PVT1-GFP and treated with 5-FU. The percentage of apoptotic cells in HCT-8/5-FU cells and HCT-116/5-FU cells transfected with LV-PVT1-GFP was analyzed by (D) flow cytometry and (E) quantified. All values are presented as the mean ± standard deviation. **P<0.01 and ***P<0.001 vs. respective NC. PVT1, plasmacytoma variant translocation 1; CRC, colorectal cancer; 5-FU, 5-fluorouracil; LV-PVT1-GFP, lentiviral vector-PVT1-green fluorescent protein; PI, propidium iodide; OD, optical density; oePVT1, overexpression of PVT1; NC, negative control.

Figure 4.

PVT1 influences the expression of genes and proteins associated with MDR. mRNA expression levels of MRP1, P-gp, mTOR and Bcl-2 in (A) the HCT-8 cells transfected with LV-PVT1-GFP and (B) the HCT-116 cells transfected with LV-PVT1-GFP. Protein expression levels of (C) MRP1, (D) P-gp, (E) mTOR and (F) Bcl-2 in the HCT-8/5-FU cells and HCT-116/5-FU cells transfected with LV-PVT1-GFP. GAPDH was used as a loading control. All values are presented as the mean ± standard deviation. **P<0.01 and ***P<0.001 vs. respective NC. PVT1, plasmacytoma variant translocation 1; 5-FU, 5-fluorouracil; LV-PVT1-GFP, lentiviral vector-PVT1-green fluorescent protein; MDR, multidrug resistance; NC, negative control; MRP1, multidrug resistance protein 1; P-gp, P-glycoprotein; mTOR, serine/threonine-protein kinase mTOR; Bcl-2, apoptosis regulator Bcl2; oePVT1, overexpression of PVT1.