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Hypoxia-inducible factor 1α participates in hypoxia-induced epithelial-mesenchymal transition via response gene to complement 32.

ABSTRACT: The aim of the present study was to explore the function of response gene to complement 32 (RGC-32) in hypoxia-induced epithelial-mesenchymal transition (EMT) in pancreatic cancer. Three kinds of hypoxia-inducible factor 1α (HIF-1α) small interfering (si)RNA were synthesized and the different effects on the expression of HIF-1α were detected by western blotting. In human pancreatic cancer BxPC-3 cells, HIF-1α levels were diminished using siRNA transfection or HIF-1α inhibitor pretreatment, and the expression levels of RGC-32 and EMT-associated proteins were analyzed using reverse transcription-quantitative polymerase chain reaction and western blotting. Subsequently, the protein levels of epithelial marker, E-cadherin, and mesenchymal marker, vimentin, were determined by western blotting. Results demonstrated that HIF-1α-Homo-488 siRNA and HIF-1α-Homo-1216 siRNA diminished the protein level of HIF-1α. Compared with normoxia, hypoxia induced the levels of HIF-1α, RGC-32, N-cadherin and vimentin, but suppressed the expression of E-cadherin and cytokeratins. The inhibition of HIF-1α by HIF-1α-Homo-1216 siRNA transfection or HIF-1α inhibitor repressed hypoxia-induced HIF-1α, RGC-32, N-cadherin and vimentin, but increased the expression of E-cadherin and cytokeratins. When RGC-32 was knocked down, hypoxia-induced vimentin was suppressed; however, hypoxia-suppressed N-cadherin was released. In conclusion, the present results demonstrated that hypoxia induced the expression of HIF-1α to activate the levels of RGC-32, in turn to regulate the expression EMT-associated proteins for EMT. These findings revealed the function of RGC-32 in hypoxia-induced EMT and may have identified a novel link between HIF-1α and EMT for pancreatic cancer therapy.

SUBMITTER: Zhu L

PROVIDER: S-EPMC5526130 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Project description:PurposeThe purpose of this study was to identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins (fibronectin and alpha smooth muscle actin, α-SMA) without sacrificing the synthesis and accumulation of the prosurvival protein vascular endothelial growth factor (VEGF) in cultured virally transformed human lens epithelial (HLE) cells.MethodsHLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3β (GSK-3β) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of β-catenin, as well as the total lysate content of fibronectin and α-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1α (HIF-1α) translation inhibitor and an HIF-2α translation inhibitor were independently employed to evaluate the effect of hypoxia inducible factor inhibition on EMT marker protein and VEGF expression. XAV932 was used to assess the suppression of nuclear β-catenin and its downstream effect on EMT marker proteins and VEGF expression.ResultsSB216763-treated HLE-B3 cells caused marked inhibition of GSK-3β activity prompting a significant increase in the translocation of cytoplasmic β-catenin to the nucleus. The enhancement of nuclear β-catenin looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and α-SMA. In conjunction with SB216763, coadministration of an HIF-1α translation inhibitor, but not an HIF-2α translation inhibitor, markedly suppressed the expression of fibronectin and α-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear β-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed.ConclusionsRecently, we reported that nuclear β-catenin, but not HIF-2α, regulates the expression of fibronectin and α-SMA in atmospheric oxygen. In marked contrast, data from the hypoxic condition clearly establish that nuclear β-catenin plays little apparent role in the expression of EMT marker proteins. Instead, the loss of HIF-1α (but not HIF-2α) decreases the expression of the EMT marker proteins without sacrificing the levels of the prosurvival protein VEGF. These findings support the development of a potentially relevant therapeutic strategy to undermine the progression of normal cells to the mesenchymal phenotype in the naturally hypoxic lens without subverting cell viability.

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Hypoxia-inducible factor 1α participates in hypoxia-induced epithelial-mesenchymal transition via response gene to complement 32.

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