Long non-coding RNA NNT-AS1 promotes cholangiocarcinoma cells proliferation and epithelial-to-mesenchymal transition through down-regulating miR-203

Abstract

Background: Cholangiocarcinoma (CCA) is a serious malignant tumor. Long non-coding RNA NNT-AS1 (NNT-AS1) takes crucial roles in several tumors. So, we planned to research the roles and underlying mechanism of NNT-AS1 in CCA.

Results: NNT-AS1 overexpression was appeared in CCA tissues and cell lines. Proliferation was promoted by NNT-AS1 overexpression in CCLP1 and TFK1 cells. Besides, NNT-AS1 overexpression reduced E-cadherin level and raised levels of N-cadherin, vimentin, Snail and Slug. However, the opposite trend was occurred by NNT-AS1 knockdown. Further, NNT-AS1 overexpression promoted phosphatidylinositol 3 kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK)1/2 pathways. MiR-203 was sponged by NNT-AS1 and miR-203 mimic reversed the above promoting effects of NNT-AS1. Additionally, insulin-like growth factor type 1 receptor (IGF1R) and zinc finger E-box binding homeobox 1 (ZEB1) were two potential targets of miR-203.

Conclusion: NNT-AS1 promoted proliferation, EMT and PI3K/AKT and ERK1/2 pathways in CCLP1 and TFK1 cells through down-regulating miR-203.

Methods: CCLP1 and TFK1 cells were co-transfected with pcDNA-NNT-AS1 and miR-203 mimic. Bromodeoxyuridine (BrdU), flow cytometry, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot were employed to detect roles and mechanism of NNT-AS1. Interaction between NNT-AS1 and miR-203 or miR-203 and target genes was examined through luciferase activity experiment.

Keywords: cholangiocarcinoma; epithelial-to-mesenchymal transition; long non-coding RNA NNT-AS1; miR-203; proliferation.

Figure 1

NNT-AS1 overexpression was occurred in CCA. NNT-AS1 levels in CCA tissues (A) and cell lines (B) were measured through qRT-PCR. * P < 0.05 and ** P < 0.01 contrasted with indicated group.

Figure 2

Effects of NNT-AS1 on the growth of CCLP1 and TFK1 cells, which were transfected with pcDNA-NNT-AS1 and si-NNT-AS1. (A) NNT-AS1 level was examined via qRT-PCR. (B) Proliferation was examined via BrdU. Expression of cyclinD1 was examined via western blot (C) and analyzed quantitatively (D) in both cells. (E) Apoptosis was examined via flow cytometry. Expression of cleaved-caspase-3 was examined via western blot (F) and analyzed quantitatively (G) in both cells. * P < 0.05, ** P < 0.01 and *** P < 0.001 contrasted with indicated set.

Figure 3

Effect of NNT-AS1 on EMT in CCLP1 and TFK1 cells, which were transfected with pcDNA-NNT-AS1 and si-NNT-AS1. Expression of EMT relative factors was examined via western blot (A, C) and analyzed quantitatively (B, D) in CCLP1 and TFK1 cells. * P < 0.05 and ** P < 0.01 contrasted with indicated set.

Figure 4

The interaction between NNT-AS1 and miR-203 or miR-203 and IGF1R/ZEB1 was examined. (A) The target matching sequence of NNT-AS1 and miR-203. (B) Luciferase activity was measured after co-transfection with NNT-AS1-wt or NNT-AS1-mut and miR-203 mimic or NC mimic. (C) Level of miR-203 was measured via qRT-PCR when cells were transfected with pcDNA-NNT-AS1 and si-NNT-AS1. (D) The target matching sequence of miR-203 and IGF1R/ZEB1. (E–F) Luciferase activities were measured after co-transfection with IGF1R/ZEB1-wt or IGF1R/ZEB1-mut and miR-203 mimic or NC mimic. (G–H) The mRNA and protein levels of IGF1R and ZEB1 were tested through qRT-PCR and western blot after transfection with miR-203 mimic or miR-203 inhibitor and the relative control. * P < 0.05 and ** P < 0.01 contrasted with indicated group.

Figure 5

Mechanism of NNT-AS1 on proliferation and EMT in CCLP1 and TFK1 cells after co-transfection with pcDNA-NNT-AS1 and miR-203 mimic. (A) Standard of miR-203 was examined via qRT-PCR by transfection with miR-203 mimic. (B) Proliferation was examined via BrdU. Expression of cyclinD1 was examined via western blot (C) and analyzed quantitatively (D) in both cells. Expression of EMT related factors was inspected via western blot (E, G) and analyzed quantitatively (F, H) in CCLP1 and TFK1 cells. * P < 0.05 and ** P < 0.01 contrasted with indicated set.

Figure 6

Mechanism of NNT-AS1 on PI3K/AKT and ERK1/2 passageways in CCLP1 and TFK1 cells, which were co-transfected with pcDNA-NNT-AS1 and miR-203 mimic. Expression of PI3K and AKT was measured via western blot (A, C) and analyzed quantitatively (B, D) in both cells. Expression of ERK1/2 was examined via western blot (E, G) and analyzed quantitatively (F, H) in both cells. * P < 0.05, ** P < 0.01 and *** P < 0.001 contrasted with indicated set.

Figure 7

NNT-AS1 regulated other miRNAs in CCA. The expression of miR-363, miR-129-5p and miR-142-3p was examined via qRT-PCR in CCLP1 (A) and TFK1 (B) cells. * P < 0.05 and ** P < 0.01 contrasted with indicated set.