lnc-PKD2-2-3, identified by long non-coding RNA expression profiling, is associated with pejorative tumor features and poor prognosis, enhances cancer stemness and may serve as cancer stem-cell marker in cholangiocarcinoma

Abstract

The present study aimed to explore the long non-coding RNA (lncRNA) expression profiles and correlation of lnc-PKD2-2-3 with tumor features and prognosis, and to investigate its effect on regulating cancer-cell stemness and its potential as a cancer stem cell (CSC) marker in cholangiocarcinoma (CCA). lncRNA expression profiles were determined in 3 pairs of CCA tumors and adjacent tissues by microarray analysis, and lnc-PKD2-2-3 expression was then validated in 60 paired samples by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Expression of common CSC markers [(CD44, CD133 and octamer-binding transcription factor 4 (OCT4)], CD44+CD133+ cell proportions, sphere formation efficiency and drug resistance to 5-fluorouracil (5-FU) were measured following ectopic overexpression of lnc-PKD2-2-3 or silencing via small hairpin RNA lentivirus transfection into the TFK-1 and Huh-28 CCA cell lines. Finally, lnc-PKD2-2-3 expression was measured in CCA stem-like cells and normal CCA cells. The results from the microarray analysis identified a total of 4,223 upregulated and 4,596 downregulated lncRNAs between CCA tumor tissue and paired adjacent tissue, which were enriched in regulating cancer-associated pathways. RT-qPCR validation revealed that lnc-PKD2-2-3 was upregulated in CCA and associated with a higher Eastern Cooperative Oncology Group performance score, poor differentiation, advanced TNM stage, increased carcinoembryonic antigen and poor overall survival in CCA patients. In vitro, lnc-PKD2-2-3 increased CD44, CD133 and OCT4 expression as well as the CD44+CD133+ cell proportion, raised the sphere formation efficiency and enhanced drug resistance to 5-FU in TFK-1 and Huh-28 cells. In addition, lnc-PKD2-2-3 was positively correlated with CSC markers in CCA tumor tissues and was markedly upregulated in CCA stem-like cells compared with that in normal CCA cells. In conclusion, lnc-PKD2-2-3, selected by lncRNA expression profiling, was associated with pejorative tumor features and poor prognosis, enhanced cancer stemness and may serve as a CSC marker in CCA.