Experimental Study Of Effect On Biological Behavior Of Hepatic Cells By Osteopontin And Anti-sense Osteopontin

Osteopontin (OPN) is a secreted, arginine- glycine- aspartate (RGD)-containing phosphoprotein with cell adhesive and chemotactic properties in vitro and in vivo. It is closely associated with infiltrating macrophage in tumors and it can directly stimulate macrophage migration, which has made it a key target as a molecule likely to be important in mediation tumor metastasis. It has been shown that osteopontin is up-regulated in many kinds of cancer tissues, including hepatocellular carcinoma, breast cancer, prostate cancer, ovarian cancer, brain cancer and lung cancer. Elevated osteopontin (OPN) transcription often correlates with increased metastatic potential of cancers. It is unclear how osteopontin is regulated in HepG2 cells. The aim of this study is to investigate the effect of epidermal growth factor on the expression of osteopontin in HepG2 cells, and to explore the signal transduction pathway mediated this expression. The second aim of this study is to investigate the effect of protein kinase B (Akt) on the expression of osteopontin in HepG2 cells, and to explore the relationship between Akt, a key gene in PI3K signal transduction pathway, and osteopontin expression. In addition, we constructed a plasmid containing anti-sense osteopontin gene in order to use as gene therapy for hepatocellular carcinoma. MethodsOsteopontin expression is detected by RNAase protection assay and western blot. Wortmannin, a specific inhibitors of PI3K, was used to see if PI3K signal transduction involved in the induction of osteopontin gene expression. HepG2 cells were transfected with constitutively active Akt and dominant negative Akt by lipofectmine, and transfectants were confirmed using western blot forAkt. The coding sequence OPI\ fragment was amplified by reverse transcription-PCR from the plasmid pBlueScript-OPN containing full-length human osteopontin. The resultant BamHl-EcoRl cDNA fragment was reversely ligated into the expression vector pcDNAS. 1(+), and confirmed by restricting enzyme digestion and DNA sequencing. Osteopontin expression is detected by Northern blot and western blot. HepG2 cells were transfected with anti-sense osteopontin gene by lipofectmine, and transfectants were confirmed using western blot for Akt. ResultsHepG2 cells constitutively express low levels of osteopontin. Treatment of epidermal growth factor increased osteopontin mRNA and protein level in a dose- and time- dependent manner. Treatment of wortmannin caused dramatic reduction of epidermal growth factor-induced osteopontin expression. HepG2 cells were successfully transfected with Akt genes including constitutively active Akt and dominant negative Akt, and overexpression of exozegenes Akt can be detected in HepG2 cells by Western blot. Using Northern blot and Western blot, we found that Akt gene regulated osteopontin expression in RNA level and protein level. In serum-free condition, HepG2 cells constitutively express low levels of osteopontin. Transfection of constitutively active Akt increased osteopontin mRNA and protein expression. Transfection of dominant negative Akt decreased osteopontin expression. The OPN fragment was successfully cloned to the vector pcDNAS. 1(+). Transfection of HepG2 cells with the plasmid containing anti-sense osteopontin inhibits the colony formation in soft agar. Conclusion(1) Osteopontin gene expression can be induced by treatment of HepG2 cells with epidermal growth factor. Epidermal growth factor may regulate osteopontin gene expression through PI3K signaling pathway. Severalpotential targets in the pathway can be manipulated to block the synthesis of osteopontin and inhibit liver cancer metastasis.(2) Osteopontin gene expression can be regulated by protein kinase B (Akt), It suggested that osteopontin synthesis can be blocked by inactivation of Akt gene, and metastasis of liver cancer can be inhibited by this intervention.(3) The plasmid containing anti-sense osteopontin gene was successfully cloned, and the metastasis of liver cancer cab be inhibited by anti-sense osteopontin g...