Effects Of Silencing Alpha-fetoprotein Gene By RNAi On Proliferation And Apoptosis In Hepatocellular Cancer Cell

Effects of Silencing Alpha-fetoprotein Gene by RNAi on Proliferation and Apoptosis in Hepatocellular Cancer Cell PhD Candidate:Xiao Jun Yang Mentor: You Cheng ZhangBackground:Discovered in 1963, forty years ago, Alpha-fetoprotein (AFP) still is an important tumor marker used in diagnosis, therapy and prognosis for hepatocellular carcinoma (HCC) now. However, it is demonstrated from recent studies that AFP play a critical role in proliferation and apoptosis of HCC, not only as a tumor-associated protein. Although there have been considerable researches indicating that AFP can modulate apoptotic signals, the precise mechanism of AFP-mediated cell growth regulation and apoptosis resistance remains obscure.Objective:To study the growth promotion and apoptosis-resistant effects of AFP on hepatocellular cancer cells using RNAi, also explore the possible molecular mechanisms that underlie the apoptosis induction by silencing AFP in Huh7 cells.Methods:1.Detecting the expression levels of AFP in HepG2, Huh7, SMMC7721 and Changliver cells using ECLIA(electro-chemiluminescence immunoassay), RIA(Radioimmunoassay) and ELISA(Enzyme-Linked ImmunoSorbent Assay).2.According to the siRNA design guidelines, three stealth RNAi targeting AFP were customarily synthesized by Invitrogen and transfected into Huh7 cells using the lipofectamine RNAi Max, then detecting AFP mRNA and protein by real time RT-PCR, ELISA and Western blot to confirm the best RNAi sequences and transfection conditions.3.Determining cell proliferation rates of all groups by Dimethylthiazolyl-2,5-diphenyl-tetrazolium bromide (MTT) assay after silencing AFP gene in Huh7 cell using the best stealth RNAi targeting AFP; We also detected cell apoptosis by Hochest staining and transmission electron microscope (TEM), the apoptosis rates were determined by flow cytometry(FCM).4. Detecting the proteins levels involved in apoptosis pathway using Western blot, such as P53, Bax, Bcl-2, Cytochrome c, Caspase-3, to explore the possible molecular mechanisms that underlie the apoptosis induction after silencing AFP in Huh7 cells.Results: 1.It was found that the expression of AFP protein in Huh7 and HepG2 cells was remarkably higher than that in SMMC7721 cell. But we didn't find the expression of AFP protein in Changliver cell.2.The results obtained from real time RT-PCR, Western blot and ELISA assay demonstrated that AFP expression was significantly inhibited both at mRNA and protein levels in Huh7 cells after transfection of the stealth RNAi (HSS303, HSS304, HSS305) and the RNAi cocktail for 48h and 72h; Among them, AFP-HSS304 and the cocktail had the most potent effects, The reduction rates were 97.4% and 98.5% at mRNA level(P<0.01),94.57% and 94.84% at protein level(P<0.01) at 72h after transfection respectively.3. Compared to the blank control, the results came from MTT assay demonstrated that the cell viability was reduced significantly by AFP-HSS304 and RNAi cocktail at 24h,48h and 72h after transfection, the inhibition rates of cell proliferation were 65.63% and 67.72%(P< 0.05) at 72h after transfection respectively. AFP-HSS304 and RNAi cocktail also induced apoptosis in Huh7 cells, the apoptotic bodies can be observed in the two groups by Hochest staining and TEM, FCM assay demonstrated that the apoptotic rate in early stage were 60.73% and 69.23% (P< 0.05) at 72h after transfection respectively.4.The results obtained from Western blot showed that the levels of P53, Bcl-2, Procaspase-3 and Cytochrome C in mitochondrial intermembrane space were reduced distinctly(P<0.05) in Huh7 cells after AFP silenced, however, the levels of Bax and Cytochrome C in cytoplasm was elevated markedly (P<0.05). So the effect of inducing apoptosis after AFP silenced in Huh7 cell may mediated by P53/Bax/Cytochrome C/Caspase-3 pathway.Conclusions: 1.The expression of AFP gene in Huh7 can be inhibited obviously both at mRNA and protein levels by the StealthTM RNAi that targeting AFP.2. Target-silencing AFP gene can inhibited cell proliferation and induced apoptosis in Huh7 cell, the apoptotic cells were mainly at the early apoptotic stage.3. The effect of inducing apoptosis in Huh7 cell after AFP silenced may mediated by P53/Bax/Cytochrome C/caspase-3 apoptotic signaling pathway.4. AFP can be further studied as an important target for hepatocellular cancer gene therapy.