Z Gastroenterol 2006; 44 - P258
DOI: 10.1055/s-2006-950855

SiRNA – mediated gene silencing of midkine affects Bcl-2 in primary murine hepatocytes

U Rüdrich 1, M Iken 1, A Deep Sharma 1, MP Manns 1, M Ott 1, A Jochheim-Richter 1
  • 1Hannover Medical School, Department of Gastroenterology, Hepatology and Endocrinology, Hannover, Germany

Introduction: Recently, we could show that a subset of developmentally regulated fetal liver genes are also expressed during adult liver regeneration. One of these genes, Midkine, was found to be strongly expressed in the early fetal liver and was also regulated in a 2/3 hepatectomy model with peak expression at 24h after resection. For further functional analysis we sucessfuly silenced the gene by targeted siRNA's in primary murine hepatocytes as demonstrated by “real-time“ PCR. We then investigated whether the inhibition of Midkine expression affects the expression of downstream genes. Since Midkine is known to exert antiapoptotic activity we induced apotosis by treatment with the anti-FAS antibody in primary hepatocytes.

Methods: Hepatocytes were isolated by liver perfusion of BALB/C mice and seeded on 6 well plates. Two days after plating, the cells were transfected with 100nM siRNA following standard protocols. Gene siliencing of the Midkine transcript was determined in the cells after 24 and 48h of transfection by “real-time“ PCR. We further studied the effect of experimental silencing of Midkine gene expression in untreated and anti – FAS treated hepatocytes (BD Pharmingen™, 1µg antibody/2ml Media) on expression of the Bcl-2 gene.

Results: In three independent siRNA transfection experiments of primary hepatocytes, the Midkine mRNA inhibition effect was ≥70%. Midkine inhibition led to a down-regulation of the Bcl-2 mRNA level ranging from 50–80%. By combining gene-silencing of the Midkine gene with anti-FAS antibody treatment, the effect of Midkine inhibition was intensified in a way that the down-regulation of the Bcl-2 mRNA level was increased.

Conclusion: Our studies show, that inhibition of Midkine expression resulted in down-regulation of Bcl-2, which is known to have anti-apototic properties. Further investigations like immune precipitation will follow to analyze the underlying molecular interaction between these genes in more details.