Effects Of Shikonin On Hippo Signaling Pathway In Liver-specific Nf2 Knockout Mice

Background and Objective:Liver cancer,a major health challenge,is becoming one of the most common types of cancer nationally.The Hippo signaling pathway acts a pivotal part in the occurrence and growth of most tumors by regulating cell proliferation and apoptosis.Shikonin exhibited remarkable anti-tumor activities on various kinds of cancer cells.However,the detailed molecular mechanism of shikonin against cancer has not been fully elucidated.Liver-specific Nf2 knockout mouse is a genetically modified mouse model of the Hippo signaling pathway.The purpose of this study was to investigate whether shikonin plays an anti-tumor role in vivo.Methods:Homozygous Nf2^fl/flmice were crossed with Alb-Cre mice to obtain liver-specific Nf2 knockout mice.PCR,q RT-PCR and Western Blot were carried out to identify and verify liver-specific Nf2 knockout mice.H&E staining,Sirius-red staining,Masson staining and immunohistochemical staining were performed to evaluate the histological characteristics in different ages.Pregnant mothers bearing Nf2^fl/fl:Alb-Cre⁺embryos received shikonin injections every other day starting at embryonic day 9（E9）of the Nf2^fl/fl:Alb-Cre⁺embryos until birth.Bile duct epithelial cells（BECs）were visualized by H&E staining and CK19 staining at E18.5.1-month-old Nf2^fl/fl:Alb-Cre⁺mice were injected with shikonin every other day throughout 14 d.Histopathological changes were detected by H&E staining.Fibrosis was evaluated by Sirius-Red staining and Masson staining.Proliferating cholangiocytes were identified by Ki67 staining.Apoptotic cells were detected by Tunel staining and Caspase-3 staining.Hepatocyte damage was revealed by serum ALT,AST and TBIL activities.Western blot and q RT-PCR were used to analyze the m RNA levels and protein expressions in the Hippo signaling pathway.Results:Liver-specific deletion of Nf2 led to marked abdominal enlargement due to massive hepatomegaly.The adult liver-specific Nf2 knockout mice suffered cirrhosis and cholestasis-related liver damage.Histological analysis showed that liver damage resulted from cholangiocyte expansion.All mice survived eventually developed into cholangiocarcinoma.The number of CK19-positive BECs was significantly reduced in shikonin-treated Nf2-deficient livers compared with control-treated Nf2-deficient livers at E18.5.Moreover,this cholangiocyte overproliferation was greatly suppressed by shikonin in a dose-dependent manner at 1 month of age.In the meantime,the liver/body ratio was significantly reduced in shikonin-treated Nf2-deficient mice.Histological analysis of shikonin-treated Nf2-deficient livers revealed less lesions and collagen deposition,which was supported by descending serum ALT,AST and TBIL.Shikonin-treated Nf2-deficient livers showed reduced proliferation and enhanced apoptosis compared with control-treated Nf2-deficient livers.Results from the Western blot and q RT-PCR assays demonstrated that shikonin altered the m RNA levels and protein expression of the Hippo pathway.The up-regulation of P-LATS1and P-YAP and the down-regulation of YAP were observed in shikonin-treated Nf2-deficient livers.Shikonin treatment also reduced Ctgf,Cyr61,Epcam and Opn m RNA expression.Conclusion:The liver-specific Nf2 knockout mouse model generated in this study closely recapitulated the histopathologic progression of liver cancer.Shikonin suppresses cholangiocyte overproliferation induced by Nf2 deficiency via regulating the Hippo signaling pathway.These results suggested that shikonin may be a promising anticancer strategy for the treatment of liver cancer patients.