The Impact Of RNA Interference On AFP Gene Expression Of HepG2 Cells

Objective: To study the impacts of RNA interference on AFP geneexpression of HepG2 cells and the effects on the proliferation and apoptosis ofHepG2 cells after suppressing the expression of AFP gene in vitro. Methods: PartⅠ: The method of RNAi suppressing the expression of AFPgene was built. Choosing target gene sequences and constructing the plasmidvectors. BLAST (Basic Local Alignment Search Tool) search candidates,eliminating those with significant homology to other coding sequences. TwoAFP target gene sequences were singled out –AFP1 and AFP2. Constructingplasmid vectors pGenesil-1-AFP1(containing GFP coding sequence)andpGenesil-2-AFP2. We observed the rate of siRNA transfection by usingfluorescence microscope, and detected the AFP level of supernatant, singling outthe optimized ratio of META/pGenesil-1-AFP1. PartⅡ: The impact of RNAinterference on AFP gene expression of HepG2 cells .The cultured supernatantwas collected for measurement of AFP with MLFA. The suppression wasobserved on the cell level by immunocytochemistry. Part Ⅲ: The effect on theproliferation of HepG2 cells after suppressing the expression of AFP gene. Cellviability and cell apoptosis were determined by the MTT colorimetric assay ,nuclear DNA fragmentation and the level of the cAMP within HepG2 cellsrespectively. Results: 1:The optimized ratio of META/PGenesil-1-AFP1 wasselected .That is 8μl : 2.5μg . 2:The levels of AFP was lower inpGenesil-1-siAFP1 and pGenesil-2-siAFP2 groups than controls, and thesuppressing rate of the pGenesil-1-siAFP1 was about 50%.Compared withpGenesil-2-siAFP2 group, the level of AFP pGenesil-1-siAFP1 group declinedtoo. 3.With DNA Fragmentation, apoptosis was not detected inpGenesil-1-siAFP1 group. The result of MTT colorimetric assay demonstratedthe suppressed AFP expression had distinct influence on the proliferation ofHepG2 cells. And the suppressed AFP expression could reduce the cAMPconcentration within HepG2 cells. Conclusion: RNAi can efficiently suppress the expression of AFP genein HepG2 cells in vitro, and the method will become a tool for further anti-tumorresearch.