Construction and assessment of recombinant plasmid pRNAT-U6.1/CFB siRNA and its inhibitory effect on proliferation of human umbilical vein endothelial cells

Background

Choroidal neovascularization (CNV) is one of the causes of blindness in multiple eye diseases. Researches showed that complement system participates in the pathogenesis of CNV.

Objective

This study was to construct the recombinant of complement factor B-small interference RNA (CFB-siRNA) expression vector and to observe its inhibitory effect on human umbilical vein endothelial cells (ECV-304).

Methods

CFB gene primers were designed based on human CFB gene, and an expression vector of CFB-siRNA was constructed by inserting CFB-siRNA into pRNAT-U6. 1/Neo plasmid. Recombinant plasmids were confirmed by the digestion analysis of restriction endonuclease, and all inserted sequences were verified by DNA sequencing. The recombinant pRNAT-U6. 1/CFB-siRNA plasmid and the blank plasmid were transfected into ECV-304 cells in the CFB-siRNA group and blank plasmid group by electroblot, respectively, and non-transfected cells served as the normal control group. The cells were observed under the fluorescence microscope 48 hours after transfection, and the transfective efficiency was calculated. The relative expression of CFB mRNA in the cells of different groups was detected by semi-quantitative reverse transcription PCR (RT-PCR). MTT was employed to calculated the growth inhibitory rates of the cells 24, 48 and 72 hours after transfection. The percentages of the cells in different cell cycles were detected by flow cytometry.

Results

The sequence of the target vector was identical to the designed sequence. The green fluorescence protein (GFP) was seen in both the CFB-siRNA group and the blank plasmid group. The relative expression levels of CFB mRNA were 0. 07±0. 04, 0. 14±0. 02 and 0. 14±0. 03 in the CFB-siRNA group, the blank plasmid group and the normal control group, respectively, a significant difference was obtained among the three groups (F=233. 05, P=0. 00); the expression level of CFB mRNA in the CFB-siRNA group was significantly declined in comparison with the blank plasmid group and the normal control group (both at P<0. 05). The growth inhibitory rates of the cells were (23. 45±0. 01)%, (33. 48±0. 02)% and (45. 49±0. 01)% at 24, 48 and 72 hours after transfection, respectively, a significant difference was obtained among the three groups (F_group=212. 99, P=0. 00); the growth inhibitory rates in CFB-siRNA group were significantly higher than that in the blank plasmid group and normal control group (all at P<0. 05). The percentages of G₁ phase cells were (44. 4±0. 5)%, (25. 8±0. 4)% and (27. 9±0. 6)% in the CFB-siRNA group, the blank plasmid group and the normal control group respectively, a significant difference was obtained among the three groups (F=58. 98, P=0. 00). The percentages of G₁ phase and G₂ phase cells in the CFB-siRNA group were significantly higher than those in the blank plasmid group and the normal control group (all at P<0. 05).

Conclusions

Recombinant pRNAT-U6. 1/CFB siRNA inhibits the proliferation of ECV-304 cells effectively by arresting the cells in G₁ intermediate phase of the growth cycle.

Choroidal neovascularization; Small interfering RNA; Complement factor B; Umbilical vein endothelial cell; Human; ECV-304