Inhibition Effect Of SiRNA On Aquaporin-1 Gene Expression In Human WISH Cells

Homeostasis of maternal-fetal fluid exchange and amniotic fluid (AF) circulation is critical for normal fetal movement, growth, and development. The placenta and fetal membranes are important organs that maintain the Maternal-fetal fluid homeostasis. Also, in clinical cases, abnormal amniotic fluid volume is a common disease of maternal-fetal fluid exchange obstacle, which includes oligohydramnios and polyhydramnios. Abnormalities amniotic fluid volume is associated with significant perinatal morbidity and mortality. But the underlying molecular mechanisms to regulate the AF balance are still unclear. In recent years, studies have pointed to the importance of the intramembranous (IM) pathway in AF volume balance. However, the underlying molecular and cellular mechanisms for water transfer across the fetal membranes remain unknown. The aquaporins (AQPs) constitute a growing family of water-specific membrane channel proteins which transport water across the cell membrane. Yet, the molecular mechanisms of their role in AF volume homeostasis still unknown. RNA interference is a new method to silencing target gene and analysis gene function with mammalian cells in vitro in recent years. The WISH cell line was esTabellished originally from human amnion epithelia of normal term pregnancy. To research the role of aquaporin in AF volume balance, we studied that the inhibition effect of siRNA on AQP1 expression in human WISH cells. The results are as follows:1. Reverse transcription-polymerase chain reaction (RT-PCR) and Immunofluorescence (IMF) were used to quantify AQP1, 3, 8, 9 mRNA and protein expressionin WISH cells. AQP1, 3, 8, 9 mRNA was found in human WISH cells and the expression level was 0.489±0.103, 0.594±0.093, 0.474±0.136, 0.425±0.150( x±S). AQP1, 3, 8, 9 labeling were observed in cell membrane and intracellular cytosol. The conclusion was that WISH cell lines can be a valuable resource for the in vitro study of the regulation and role of AQP in the intramembranous pathways of amniotic volume regulation.2. WISH cells were transfected with CY3-Negative siRNA. According to the different concentration of siRNA, the transfection protocols were divided into 5 groups: 0 nmol/L, 5nmol/L, 10nmol/L, 20nmol/L and 30nmol/L. Transfection efficiencies of different protocols were evaluated by fluorescence microscope observation and flow cytometer counting after 24 hours. The red fluorescence can be discovered by the fluorescence microscope in different concentration group, no signals in control group. The transfection efficiency were 0, 7.7%, 39.35%, 87.27% and 95.78%; The transfection efficiency of 20 nmol/L and 30 nmol/L CY3-Negative siRNA remarkably higher than 5 nmol/L and 10 nmol/L CY3-Negative siRNA transfection groups(P<0.05). There is no significant difference between 20 nmol/L and 30 nmol/L siRNA transfection groups (P>0.05). To research the influence on apoptosis of WISH cells after siRNA transfection, the Annexin V-FITC/PI Kit was used to detect the apoptosis of WISH cells which were transfected with 20 nmol/L Negative siRNA after 24 hours. The rate of apoptosis was 1.96% and 2.36%,There is no significant difference about apoptosis of WISH cells between transfection group and control group(P>0.05). The conclusion was that 20 nmol/L siRNA can transfect the WISH cells successfully; there was no effect to the apoptosis of WISH cells were chemosynthesis siRNA transfected.3. The WISH cells were transfected with 20 nmol/L three AQP1-siRNA to select the most effective siRNA. The Real-time PCR indicated that the inhibition of AQP1-siRNA-1 was the most effective. To research the AQP1 mRNA was inhibited by AQP1-siRNA-1 of different concentration in WISH cells, the transfection protocols were divided into 4 groups: 20 mol/L, 40 nmol/L, 60 nmol/L and 80 nmol/L. AQP1 mRNA level of different transfection protocols were evaluated by Real-time PCR after 24 hours. The expression of AQP1 mRNA was reduced by 20.47%, 39.86%, 60.34% and 65.81%, respectively. There was no obviously deference between Negative group and blank group.4. The WISH cells were transfected with 60 nmol/L AQP1-siRNA-1. Real-time PCR detected the AQP1 mRNA level after transfection in 24 h, 48 h and 72 h. The results indicated that the AQP1 mRNA was decreased by 60.24% (P<0.05), 90.57% (P<0.01), 87.68% (P<0.01) respectively. It show that the effect of siRNA is time-dependent within 24 h~72 h, and the maximum inhibit effect is at 48 hour.On the whole, in this study, we detected that the expression of AQP1, 3, 8, and 9 in human WISH cells and optimized the condition of siRNA transfected WISH cells; then we chose the effective sequence of AQP1-siRNA; constructed the amniotic epithelial cell model of AQP1 gene silencing.