Effects Of Nephroblastoma Overexpressed Gene On Biological Behaviors Of Rat Hepatic Stellate Cells

【Objective】To construct recombinant plasmids that can express small interfering RNA (siRNA) targeting nephroblastoma overexpressed gene (NOV) and investigate the effects of NOV on the biological behaviors of hepatic stellate cells (HSCs).【Methods】Hairpin siRNA templates targeting NOV gene were synthesized and cloned into plasmid vector psiRNA-H1neo. Three vector-derived siRNAs (denoted psiRNA1, 2 and 3) and one mocking pconsiRNA (as control) were constructed. The recombinant NOV siRNA plasmids were constructed and identified by using restrictive enzyme analysis and DNA sequencing, and then were transfected into HSCs by lipofectamine. HSCs were divided into group psiRNA1, psiRNA2, psiRNA3 and pconsiRNA according to the recombinant plasmid transfected. Blank group consisted of HSCs contaning no plasmids. The expression of NOV andα-SMA was detected by semi-quantitative RT-PCR and western blot. The expression of type I collagen and type III collagen were detected by semi-quantitative RT-PCR. The cell proliferation was assayed by MTT method and the cell apoptosis by flow cytometry.【Results】Restrictive enzyme analysis and DNA sequencing revealed the successful construction of siRNA expression plasmids. Compared with negative control group, extrogenous recombinant plasmid psiRNA2 could reduce the level of NOV mRNA (73.0% vs 23.2%, P <0.05) and type I collagen and type III collagen mRNA (59.8% vs 17.0%,37.1% vs 6.6%, all P<0.05), and inhibit the expression ofα-SMA at mRNA level (51.4% vs 15.1%, P<0.05)and protein level. Compared with non-transfection group, extrogenous recombinant plasmid psiRNA2 significantly decreased the proliferating activity of HSC (24h: 0.172±0.005 vs 0.318±0.018, P<0.05; 48h: 0.296±0.004 vs 0.472±0.029, P<0.05; 72h: 0.432±0.024 vs 0.672±0.050, P<0.05). No obvious changes were found in psiRNA1 group and psiRNA3 group (all P>0.05). Cell apoptosis was not significant in HSCs transfected with recombinant plasmids compared with blank group.【Conclusion】The NOV siRNA expressing plasmids were successfully constructed and the plasmid psiRNA2 could reduce the expression of NOV and the secrection of ECM, inhibit the proliferation and activation of HSC. The NOV may be a novel target for gene therapy of hepatic fibrosis.