[Functions of NET-1 gene in skin squamous cell carcinoma cell line (A431): a siRNA study]

Li Chen, Jian-Li Wang, Hui Li, Jing Qin, Yuan-Yuan Wu
Zhonghua Bing Li Xue za Zhi Chinese Journal of Pathology 2009, 38 (10): 691-6

OBJECTIVE: To investigate the effect of NET-1 siRNA on NET-1 expression and on the proliferation and infiltration of skin squamous cell carcinoma cell line A431.

METHODS: Four recombinant vectors of pU6H1-GFP-siRNAs NET-1 were transfected into A431 cells. The levels of NET-1 mRNA expression were measured by semi-quantitative RT-PCR for selecting the most effective one in the four kind vectors of pU6H1-GFP-siRNA-NET-1. The controls consisted of pU6H1-GFP-siRNA-target off with random double-stranded RNA, pcDNA3.1 sense and other controls including the empty vectors (pU6H1-GFP and pcDNA3.1), NET-specific siRNA alone, lipofection reagent alone, target off siRNA alone, or untreated cells. After transfection, levels of NET-1 mRNA and protein expression were detected using semi-quantitive RT-PCR and Western blotting, respectively. The intracellular location of NET-1 protein was documented by immunofluorescence staining followed by laser scanning confocal microscopy. Proliferation rates of A431 cells were determined by MTT assay and flow cytometry. Abilities of migration and infiltration of A431 cells were determined by wound healing effect and transwell migration assay, respectively.

RESULTS: siRNAs coding NET-1 sequences (19-23nt) were confirmed between H1 and U6 promoters of pU6H1-GFP vector by sequencing. The transfection efficiency of pU6H1-GFP in A431 cells was about 80% as the percentage of GFP expression cells in total cells. Transfection with either pU6H1-GFP-siRNA NET-1 or pcDNA3.1 antisense NET-1 gave rise to an obvious reduction in the expression of NET-1 mRNA and protein in A431 cells. The abilities of proliferation, migration and infiltration of A431 cells were significantly reduced 48 hours after transfection with either pU6H1-GFP-siRNA NET-1 or pcDNA3.1 antisense NET-1, compared with the vector controls including pU6H1-GFP or pcDNA3.1 (P < 0.01, respectively). pU6H1-GFP-siRNA NET-1 showed a stronger inhibition effect on proliferation, migration and infiltration of A431 cells over pcDNA3.1 antisense NET-1 (P < 0.05). Transfection with pcDNA3.1 sense NET-1 resulted in an obvious increase in the expression level of NET-1 mRNA and protein, but without inhibition effect to proliferation, migration and infiltration of A431 cells.No obviously inhibition effects were observed when transfected with pU6H1-GFP-siRNA-target or the controls.

CONCLUSIONS: RNAi targeting NET-1 gene effectively down-regulates the expression of NET-1 in A431 cells, leading to an inhibition of proliferation, migration and infiltration. RNAi technique appears to be superior to antisense oligonucleotide technique in the suppression of gene expression.

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