Study On The Expression Of CXCR4Gene Silenced By CXCR4-siRNA Recombinant Adenoviral In Human Ovarian Cancer Cells

PART ONE CONSTRUCTION OF CXCR4-SIRNARECOMBINANT ADENOVIRALVECTOR BY ADMAX SYSTEMObjective: To construct recombinant adenoviral vector carrying shortinterfering RNA(siRNA) by AdMax vector system in order to study theexpression of CXCR4gene in SKOV3cells.Methods: The short interfering RNA(siRNA) of CXCR4gene wascloned into the shuttle plasmid PAVsi1.1.The PCR and sequence analysiswere used to detect interfering fragment.The positive recombinant shuttleplasmid, adenovirus helper plasmid pBHGlox(delta)E1and3Cre weretransfected into HEK293cells to construct CXCR4-siRNA recombinantadenoviral vector. CXCR4-siRNA recombinant adenoviral vector wasrepeatedly packaged and amplified in HEK293cells and virus titer wasdetermined.Results: PCR analysis and sequencing confirmed that the short interfering RNA(siRNA) was successfully inserted into the adenovirusvector. After the package and amplification of HEK293cells,the greenfluorescent protein expressed highly in HEK293cells under fluorescentmicroscope. The viral titers were6×108PFU/ml.Conclusion: The CXCR4-siRNA recombinant adenoviral vector canbe successfully constructed by AdMax vector system. PART TWO EXPERIMENTAL STUDY OF CXCR4-SIRNARECOMBINANT ADENOVIRAL VECTORIN SKOV3CELLSObjective: To study the effect of the CXCR4-siRNA recombinantadenoviral vector on the expression of CXCR4gene in SKOV3cells.Methods: CXCR4-siRNA recombinant adenoviral vector weretransfected into SKOV3cells. The SKOV3cells were divided into threegroups: siRNA group with adenovirus carrying RNA interference, negativegroup with vacant adenovirus vector, and control group without anyinfection. The expression level of CXCR4gene in SKOV3cells wasmeasured by RT-PCR, Western blot and immunocytochemistry analysis.Results: The green fluorescent protein expressed highly in SKOV3cells under fluorescent microscope. The CXCR4gene was greatly inhibitedafter infection with CXCR4-siRNA recombinant adenoviral vector.Conclusion: The CXCR4-siRNA recombinant adenoviral vector canefficiently transfect targeted cells and silence the expression of CXCR4gene, which would lay a foundation for the further study on clinical genetherapy of RNAi technology in ovarian cancer.