RESUMEN
Pannexin 1 (PANX1) has been implicated in cancer emergence and progression. However, its roles in gastric cancer remain unclear. In the present study, the function and molecular mechanisms of PANX1 in gastric cancer were investigated in vitro. Two gastric cancer cell lines exhibiting low and high PANX1 expression (SNU-16 and HCG-27, respectively) were transfected using a PANX1-containing plasmid or PANX1 transcript-targeting short hairpin (sh)RNA. In addition, HCG-27 cells and PANX1-overexpressing SNU-16 cells were subjected to short interfering (si)RNA-mediated aquaporin 5 (AQP5) knockdown. In vitro cell migration (scratch) and transwell invasion assays were performed to evaluate the cell migratory and invasive abilities. Real-time fluorescence quantitative PCR was used to detect transcripts encoding epithelial-mesenchymal transition markers. Immunofluorescence and Western blotting were conducted to quantify corresponding proteins. In SNU-16 cells, PANX1 overexpression induced conversion from round (cobblestone-like) to elongated (spindle-like) morphologies and enhanced the cell migratory and invasive abilities. PANX1 knockdown had the opposite effect in HGC-27 cells. In PANX1-overexpressing SNU-16 cells, expression of SLUG, vimentin, and AQP5 was significantly upregulated, whereas expression of E-cadherin was downregulated. In HGC-27 cells, PANX1 knockdown showed the opposite effect. In both PANX1-overexpressing SNU-16 cells and untransfected HGC-27 cells, silencing of AQP5 expression significantly inhibited PANX1-induced upregulation of SLUG and vimentin expression, as well as downregulation of E-cadherin expression and enhanced migratory and invasive abilities. In summary, elevated PANX1 expression induces gastric cancer cell epithelial-mesenchymal transition and the associated promotion of migratory and invasive abilities by inducing expression of AQP5, which facilitates SLUG-mediated regulation of vimentin and E-cadherin expression.
Asunto(s)
Acuaporina 5/metabolismo , Conexinas/metabolismo , Células Epiteliales , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , Neoplasias Gástricas/metabolismo , Estómago , Antígenos CD/metabolismo , Acuaporina 5/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Conexinas/genética , Humanos , Invasividad Neoplásica , Proteínas del Tejido Nervioso/genética , ARN Interferente Pequeño , Factores de Transcripción de la Familia Snail/metabolismo , Estómago/patología , Transfección , Vimentina/metabolismoRESUMEN
The present study aimed to investigate the roles of Notch1 in the biological processes of bladder cancer cells (BCCs) in vitro. Short hairpin (sh)RNA targeting Notch1 was designed and constructed, and the T24 and 5637 BCCs were selected for transfection. The cells were classified into two groups: shRNA negative control (NC) and Notch1 shRNA. MTT and Transwell assays, and flow cytometry were performed to examine the changes in cell proliferation, invasiveness, and apoptosis, respectively. In addition, reverse transcription-quantitative PCR and western blot analysis was used to detect the mRNA and protein expression levels of apoptosis-related proteins (Bax, Bid and Bcl2) and epithelial-mesenchymal transition factors (vimentin and E- and N-cadherin). Compared with that in the shRNA NC group, the Notch1 shRNA group showed significantly decreased cell proliferation rate and invasiveness; increased apoptotic rate; elevated mRNA expression levels of Bad, Bid and E-cadherin; and reduced mRNA expression levels of Bcl2, N-cadherin and vimentin. The trends for protein expression levels were the same as those for mRNA levels. Notch1 silencing inhibited invasion and promoted apoptosis of BCCs.