Effects of silencing KLF4 expression on biological behaviors of HSC-LX2 in vitro

doi:10.3969/j.issn.1002-7386.2017.07.005

Abstract：Objective To screen the siRNA which can effectively silence KLF 4 expressions , and to observe the effects of KLF4 gene silence on biological behaviors of LX 2 in human hepatic stellate cells ( HSC-LX2 ) .Methods The three pairs of siRNAs (siRNA1,siRNA2,siRNA3)targeting at human KLF4 mRNA were designed and chemically synthesized , then which were transfected into HSC-LX2.The expression levels of KLF4 mRNA and protein were detected by Real-time fluorescent quantitative PCR ( Real-time RT-PCR) and Western Blot ,respectively ,with the nonspecific siRNA transfected into HSC-LX2 being regarded as negative control group .One of the siRNAs with the best effects of silencing KLF 4 expression was selected,then which was transfected into HSC-LX2, the effects of KLF4 silence on cell proliferation of HSCs were measured by MTT.Moreover the expression levels of transforming growth factor ( TGF-1 ) , interleukin-1 ( IL-1 ) , tumor necrosis factor-α( TNF-α) in supernatant of cell culture were detected by ELISA .Results The examination results by Real-time RT-PCR and Western Blot showed that the silencing effect of siRNA 3 was the best,thus,which was used in the subsequent experiments . MTT assay suggested that the cell vitalities were significantly decreased at 12h, 24h,48h after siRNA3 transfection, as compared with those in negative control group .ELISA assay showed the expression levels of TGF-1, TNF-α, IL-1 were obviously decreased at 48 h after transfection , as compared with those in negative control group .Conclusion The siRNA3 can effectively silence KLF4 expression,and KLF4 gene silence can inhibit the cell proliferation of HSC-LX2 and can affect its biological behaviors to decrease the expression levels of TGF-1, TNF-αand IL-1.