Excess Cholesterol Inhibits The Development Of Liver Tumors In Hras12V Transgenic Mice

Objective:To investigate the effects of cholesterol on hepatic tumorigenesis and development in Hras12V transgenic mice.Methods:1.The liver tissues of wild type C57BL/6J mice(Wt),the hepatic tumor tissues(T)and peri-tumoral tissues(P)of Hras12V transgenic mice at 8-month-age were collected.HE staining was used for pathological diagnosis.2.Cholesterol content in liver tissues and serums were detected by enzyme coupling colorimetric method.3.The expression and activation levels of signal molecules were detected by Western blot.The mRNA levels of genes were detected by RT-qPCR.4.The Hras12V transgenic mice were treated with cholesterol by intragastric administration to investigate the effects of cholesterol on the hepatic tumorigenesis and development.Results:1.The expression of Hras12V transgene was significantly and gradually increased in P and T of transgenic mice.The liver tumors of Hras12V transgenic mice were yellowish white.HE staining showed macrovesicular steatosis in the hepatic tumor cells and suggests the disorders of lipid metabolism.2.Cholesterol contents in P,T,and serum of Hras12V transgenic mice were significantly increased comparing with that in Wt(p<0.05).Interestingly,although the expression of Hras12V oncogene in T was significantly higher than that in P(p<0.001),the cholesterol level in T was significantly lower than P(p<0.01).3.Bile acid contents in P and T of Hras12V transgenic mice were significantly reduced comparing with that in Wt(p<0.05).The mRNA and protein levels of bile acid biosynthesis rate-limiting enzymes CYP7A1/Cyp7al,CYP8B1/Cyp8b1,and CYP27A1/Cyp27al were also significantly reduced.4.Cholesterol treatment of Hras12 V transgenic mice significantly increased the cholesterol levels in serum and liver tumors(p<0.05)comparing with the control group.There was no significant difference between the treated and control mice in the total number of liver tumors,the number of liver nodules(<2 mm)and liver adenoma(2-5 mm)(p>0.05),but the number of liver carcinoma(>5,mm)was significantly lower in treated mice comparing with the control mice(p<,0.05).Consistently,the liver weight and liver/body weight ratio of the treated mice were significantly lower than the control group(p<0.05).5.RT-qPCR results of cholesterol treatment of Hr as 12V transgenic mice showed that the levels of Albumin and Hr as 12V did not been significantly affected by cholesterol treatment.Western blot analysis showed that,compared with the control group,in T of treated mice:the protein levels of cysteine aspartic acid proteinase 3(35KDa)were markedly reduced and the protein levels of cleaved-Caspase 3(17KDa)was significantly increased;the protein level of B lymphocyte tumor-2(Bcl2)was obviously decreased;the protein level of GSK-3 beta,beta-Catenin and p-ERK have not been changed.Conclusions:1.The expression of ras oncogene effectively inhibits the expression of cholesterol metabolism related key enzymes CYP7A1,CYP8B1 and CYP27A1 in both hepatoma cells and’ hepatocytes,which results in the elevated cholesterol levels and the reduced bile acids levels.2.The excessive cholesterol inhibits the hepatic tumor development but not tumorigenesis.