The Inhibitory Effect Of RNA Interference Or Erlotinib By Blocking Epidermal Growth Factor Receptor Pathway In A549Cells And The Involved Mechanisms

ObjectiveThis study was designed to explore the inhibitory effect of RNAi or Erlotinib by blocking epidermal growth factor receptor pathway in A549cells and the involved mechanisms. The morphology changes, EGFR mRNA expression,apoptosis, and EGFR, Bcl-2,Bax protein expression were detected to explain the involved mechanisms with the aim at providing new ways for therapy of lung cancer.MethodsThe A549lung cancer cells were cultured in RPMI-1640in37℃,5%CO2constant temperature incubator, we designed the control group, the RNA interference group, the Erlotinib group. The inverted microscope was used to detect the morphological changes of A549cells by blocking epidermal growth factor receptor pathway.Use RT-PCR to detected the expression of EGFRmRNA, The Apoptosis rate of A549cells were measured by flow cytometry. The expression of EGFR, Bcl-2, Bax was detected by Western blot. All the data above were expressed byx±s,and were recorded and analyzed using t test,χ2 test, analysis of variance,with SPSS11.5software. The statistic significance were defined as P<0.05.Results1. Cellular morphological changes observed under an inverted microscopeA, the control group:Cells grew very well,clinging to the bottle wall with various cell divisions in the control groups.B, the Erlotinib group:The intercellular spaces between cells was broadening effectly, as well as cells rounded, shape changed, adherence was loosened. However,there were still many cells alive.C, the RNA interference group:the inhibitory effect was more marked, cellular morphology could not be recognized.2. The expression of EGFRmRNA/GAPDH in A549cell by RNA interferenceThe ratio of EGFRmRNA/GAPDH were0.76、0.72、0.31、0.70in the blank control group, the negative RNA control group,the experimental group,and the lipidosome2000control group respectively.there was not statistical difference between the negative RNA control group, the lipidosome2000control group and the blank control group.however, there were remarkable.The difference between the experimental group and the blank control group had statistical signification (P<0.05), which indicated that the RNA interference could inhibit the genie expression of EGFR.3,The effect of apoptosis detected by flow cytometry.The apoptosis detected by flow cytometry show:The ratio of apoptosis in the control group, the RNA interference group, the Erlotinib group were23.5%,54.7%,83.2% respectively. Blocking epidermal growth factor receptor pathway could effectively promote the ratio of apoptosis in A549cells, The difference between the control group and the Blocking EGFR groups had statistical signification (P<0.05).5. The expression of EGFR,Bcl-2,Bax determined by western blot.The apoptosis detected by flow cytometry show:the expression of EGFR and Bcl-2was much weaker than the control group,however,the expression of Bax was much Stronger than the control group.the amount of protein show that:The difference between the RNA interference group, the Erlotinib group and the control group had statistical signification (P<0.05).the ratio of Bcl-2/Bax was0.4、0.21in the Erlotinib group and the RNA interference group respectively, The difference between the control group (6.67)and the Blocking EGFR groups had statistical signification (P<0.05).ConclusionsBlocking epidermal growth factor receptor pathway can promote the Apoptosis of A549cells, the mechanism maybe related to down-regulate the ratio of Bcl-2/Bax.