[Comparison between lentivirus and plasmid as shRNA vector targeting RhoA gene of ovary cancer cell line HO8910].

ABSTRACT

OBJECTIVE: To compare the different effects of lentivirus and plasmid as shRNA vector targeting RAS homologue gene-family member A (RhoA) of ovary cancer cell line HO8910. METHODS: Lentivirus and plasmid vectors carrying siRNA targeting RhoA gene were respectively transferred into HO8910 cells. The transferred cells were screened with puromycin for the ones stably silencing RhoA gene. As passaging went on, the two types of vectors were compared in the ability of silencing RhoA gene expression by means of fluorescence microscopy, real-time quantitative PCR and Western blotting, and in the inhibitory effects on the invasion and metastasis of cells by scratch wound migration assay and tube formation assay. RESULTS: Two groups of stable RhoA-knockdown cell lines were established with lentivirus and plasmid as RNAi vectors, respectively. Detected by fluorescence microscopy, the expression rate of GFP decreased in the plasmid group as the culture generation increased; it was 70% and 45% at the 15th and 25th generations respectively. The expression rate of GFP in the lentivirus group maintained above 95%. Both real-time quantitative PCR and Western blotting indicated that the expression of RhoA mRNA and protein had no significant difference in the two groups at the 3rd generation; but as the culture generation increased, the expression of RhoA mRNA and protein in the lentivirus group kept at the lower level, while it increased in the plasmid group. The scratch wound migration assay and tube formation assay revealed that lentivirus as RNAi vector suppressed more stably and persistently the invasion and metastasis of ovary cancer cell line HO8910 as compared with the plasmid group (P<0.05). CONCLUSION: Lentivirus as RNAi vector can suppress the RhoA gene expression more stably as compared with the plasmid; the plasmid is more suitable for transient transfection studies.