Effects of interference with the expressions of aquaporin 3 and phospholipase D2 by small interfering RNAs on the proliferation and apoptosis of a human cutaneous squamous cell carcinoma cell line A431 / 中华皮肤科杂志

ABSTRACT

Objective To investigate the effects of aquaporin 3 (AQP3) and phospholipase D2 (PLD2) on the proliferation and apoptosis of a human cutaneous squamous cell carcinoma cell line A431.Methods Three small interfering RNAs (siRNAs) were constructed targeting the AQP3 and PLD2 genes separately,and transfected into A431 cells using liposomes.Then,fluorescence quantitative PCR was performed to find the most efficient siRNAs.Western blot was conducted to detect the protein expression levels of AQP3 and PLD2 in A431 cells after transfection with the selected AQP3-siRNA and PLD2-siRNA.Some A431 cells were divided into five groupsnormal control group without any treatment,transfection reagent group treated with the oligofectamine reagent only,negative control group transfected with the negative control siRNA,AQP3-siRNA group transfected with the selected AQP3-siRNA,PLD2-siRNA group transfected with the selected PLD2-siRNA.After additional culture,cell counting kit-8 assay was performed to evaluate the proliferation of A431 cells,flow cytometry to detect the apoptosis of A431 cells after annexin V-fluorescein isocyanate/propidium iodide double-staining.Statistical analysis was carried out by the paired t test.Results The transfection with AQP3-siRNA and PLD2-siRNA induced a significant decrease in the mRNA and protein expressions of AQP3 and PLD2 respectively in A431 cells when compared with the untransfected cells.Compared with the negative control group,the proliferation of A431 cells was significantly decelerated at 24,48 and 72 hours after transfection in the AQP3-siRNA group (t =24.10,11.00,9.54,respectively,all P ＜ 0.01) and PLD2-siRNA group (t =30.47,7.02,8.73,respectively,all P ＜ 0.01).A significant increase was observed in the apoptosis of A431 cells at 48 and 72 hours after transfection with AQP3-siRNA (t =11.36,20.91,respectively,both P ＜ 0.01),and at 72 hours after transfection with PLD2-siRNA (t =4.86,P ＜ 0.05) compared with the negative control group.Conclusion The down-regulation of AQP3 and PLD2 expressions by siRNA can inhibit the proliferation,but induce the apoptosis,of A431 cells.