Long non-coding RNA LINC00665 promotes gemcitabine resistance of Cholangiocarcinoma cells via regulating EMT and stemness properties through miR-424-5p/BCL9L axis

Min Lu,Yajun Zhou,Haodi Yue,Gang Li,Xiwen Geng,Xinglei Qin,Zhaoyang Liu

doi:10.1038/s41419-020-03346-4

Abstract

Gemcitabine is the first-line chemotherapy drug for cholangiocarcinoma (CCA), but acquired resistance has been frequently observed in CCA patients. To search for potential long noncoding RNAs (lncRNAs) involved in gemcitabine resistance, two gemcitabine resistant CCA cell lines were established and dysregulated lncRNAs were identified by lncRNA microarray. Long intergenic non-protein coding RNA 665 (LINC00665) were found to rank the top 10 upregulated lncRNAs in our study, and high LINC00665 expression was closely associated with poor prognosis and chemoresistance of CCA patients. Silencing LINC00665 in gemcitabine resistant CCA cells impaired gemcitabine tolerance, while enforced LINC00665 expression increased gemcitabine resistance of sensitive CCA cells. The gemcitabine resistant CCA cells showed increased EMT and stemness properties, and silencing LINC00665 suppressed sphere formation, migration, invasion and expression of EMT and stemness markers. In addition, Wnt/β-Catenin signaling was activated in gemcitabine resistant CCA cells, but LINC00665 knockdown suppressed Wnt/β-Catenin activation. B-cell CLL/lymphoma 9-like (BCL9L), the nucleus transcriptional regulators of Wnt/β-Catenin signaling, plays a key role in the nucleus translocation of β-Catenin and promotes β-Catenin-dependent transcription. In our study, we found that LINC00665 regulated BCL9L expression by acting as a molecular sponge for miR-424-5p. Moreover, silencing BCL9L or miR-424-5p overexpression suppressed gemcitabine resistance, EMT, stemness and Wnt/β-Catenin activation in resistant CCA cells. In conclusion, our results disclosed the important role of LINC00665 in gemcitabine resistance of CCA cells, and provided a new biomarker or therapeutic target for CCA treament.

Highlights

Cholangiocarcinoma (CCA) is a group of epithelial tumors arising from different locations within the biliary tree with features of cholangiocyte differentiation.It is the second most common primary hepatic malignancy, accounting for 15–20% new diagnosed cases[1]
To screen for long noncoding RNAs (lncRNAs) that are involved in gemcitabine resistance of CCA cells, lncRNA microarrays were conducted to evaluate the dysregulated lncRNAs (|Log[2] fold change | ≥2) in the two pairs of CCA cell lines (HuCCT1 vs. HuCCT1-Gem, and SNU-245 vs. SNU-245-Gem)
A total of 108 aberrant expressed lncRNAs were found both in HuCCT1-Gem and SNU-245-Gem cells, indicating their potential role in gemcitabine resistance of CCA cells (Supplementary Table 4)

Summary

Introduction

Cholangiocarcinoma (CCA) is a group of epithelial tumors arising from different locations within the biliary tree with features of cholangiocyte differentiation. It is the second most common primary hepatic malignancy, accounting for 15–20% new diagnosed cases[1]. According to the anatomical location, cholangiocarcinoma can be classified into intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA) subtypes. PCCA (50%) and dCCA (40%) represent the majority of cholangiocarcinoma cases, while iCCA is less than 10% of total[2]. Surgical treatment is the preferred cure method for this disease, only a small subset (~35%) of patients with early-stage CCA are amendable to surgical resection, and the 5-year survival rates for iCCA, pCAA, and dCCA patients are only 63%, 30%, and 23%, respectively[2].

Methods

Results

Conclusion