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, 13 (2), 1563-9

Interleukin‑6 Induces Epithelial‑mesenchymal Transition in Human Intrahepatic Biliary Epithelial Cells

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Interleukin‑6 Induces Epithelial‑mesenchymal Transition in Human Intrahepatic Biliary Epithelial Cells

Gui-Xing Jiang et al. Mol Med Rep.

Abstract

The aim of the present study was to determine the role of interleukin-6 (IL-6) in the epithelial-mesenchymal transition (EMT) of human intrahepatic biliary epithelial cell (HIBEC) lines in vitro. HIBECs were stimulated with IL-6 at concentrations of 0, 10, 20, 50 and 100 µg/l for 24 h. A wound healing and Transwell assay were performed to determine the migratory and invasive capacity of HIBECs, respectively. Following 24 h of incubation, IL-6 at 10 and 20 µg/l significantly increased the number of migrated and invaded cells (P<0.05), while stimulation with 50 and 100 µg/l IL-6 resulted in a further increase of the migratory and invasive capacity compared to that in all other groups (P<0.05). Furthermore, reverse-transcription quantitative polymerase chain reaction and western blot analyses were used to detect the mRNA and protein expression of EMT markers E-cadherin and vimentin in HIBECs. Decreased mRNA levels of E-cadherin accompanied by higher mRNA levels of vimentin were observed in the 10, 20, 50, 100 µg/l IL-6 groups compared to those in the 0 µg/l group (all P<0.05). Furthermore, the protein expression of E-cadherin was decreased, while that of vimentin was increased in the 50 and 100 µg/l IL-6 groups compared to those in the 0, 10 and 20 µg/l IL-6 groups (all P<0.05). The present study therefore indicated that IL-6 promoted the process of EMT in HIBECs as characterized by increased migration and invasion of HIBECs and the typical changes in mRNA and protein expression of the EMT markers E-cadherin and vimentin.

Figures

Figure 1
Figure 1
Morphology of HIBECs observed under an optical microscope prior to stimulation with interleukin-6. (A) Light microscopy image (magnification, ×200); (B) hematoxylin-eosin-stained HIBECs (magnification, ×200). HIBEC, human intrahepatic biliary epithelial cell.
Figure 2
Figure 2
Morphology of human intrahepatic biliary epithelial cells exposed to interleukin-6 at concentrations of (A) 0, (B) 10, (C) 20, (D) 50 and (E) 100 µg/l for 24 h observed under an inverted phase contrast microscope (magnification, ×200).
Figure 3
Figure 3
Migratory potential of human intrahepatic biliary epithelial cells after stimulation with IL-6. Inverted microscopy images of scratched cell monolayers subjected to the wound assay (magnification, ×40). (A–E) cells at 0 h in the presence of 0, 10, 20, 50 and 100 µg/l IL-6, respectively; (F–J) cells following incubation with 0, 10, 20, 50 and 100 µg/l IL-6, respectively, for 24 h. IL, interleukin-6.
Figure 4
Figure 4
Number of cells migrated into the scratched region following incubation with interleukin-6 (0, 10, 20, 50 or 100 µg/l) for 24 h. Values are expressed as the mean ± standard deviation. *P<0.05 vs. 0 µg/l; #P<0.05, vs. 10 and 20 µg/l.
Figure 5
Figure 5
Effects of IL-6 on the invasive capacity of human intrahepatic biliary epithelial cells were assessed using a Transwell assay. Fluorescence microscopy images of cells which had transgressed to the lower side of the Transwell membranes (magnification, ×200). Nuclei were stained with DAPI. Representative images of three experiments are shown. (A) 0 µg/l IL-6 group; (B) 10 µg/l IL-6 group; (C) 20 µg/l IL-6 group; (D) 50 µg/l IL-6 group; (E) 100 µg/l IL-6 group. IL, interleukin.
Figure 6
Figure 6
Number of cells transgressed through the Transwell membrane at 24 h following incubation with interleukin-6 (0, 10, 20, 50 and 100 µg/l). Values are expressed as the mean ± standard deviation (n=3). *P<0.05 vs. 0 µg/l; #P<0.05 vs. 10 and 20 µg/l.
Figure 7
Figure 7
Relative mRNA expression of (A) E-cad and (B) vimentin in the human intrahepatic biliary epithelial cells incubation with interleukin-6 (0, 10, 20, 50 and 100 µg/l) for 24 h detected by reverse-transcription quantitative polymerase chain reaction analysis. Values are expressed as the mean ± standard deviation (n=3). *P<0.05 vs. 0 µg/l; #P<0.05 vs. 20 µg/l. E-cad, E-cadherin.
Figure 8
Figure 8
Effects of IL-6 on the protein expression of E-cad and vimentin in human intrahepatic biliary epithelial cells. (A) Representative western blots showing the protein levels of E-cad and vimentin following incubation with IL-6 for 24 h. Lanes, 1, 0; 2, 10; 3, 20; 4, 50; 5, 100 µg/l IL-6. Densitometric analysis was used for the quantification of the protein expression of (B) E-cad and (C) vimentin. Values are expressed as the mean ± standard deviation. *P<0.05 vs. 0 µg/l.; #P<0.05. vs. 10 and 20 µg/l. IL, interleukin; E-cad, E-cadherin.

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