| Objective: Raf/MEK/ERK pathway played an important regulatory role in cell cycle regulation, cell proliferation, cell apoptosis, cellular growth inhibiting and differentiation. This cascade was an essential oncogenic progression. Cyclin D1 was a cell cycle associated protein that affected on the G1 phase of the cell cycle. It could be overexpressed when it played coorperation with lots of proteins or oncogenes. After the Raf/MEK/ERK pathway was actived, Cyclin D1 was promoted to form an active enzyme complex with cyclin dependent kinase 4/6 (CDK4/6) that phosphorylated and activated CDK and leaded to G1/S transition. Thus, it enhanced cell proliferation and promoted tumorigenesis. There were studies about the activation of Raf/MEK/ERK pathway and the Cyclin D1 overexpression in human hepatocellular carcinoma. To identify whether different Raf-1, MEK-1, ERK-1 and Cyclin D1 expressional levels predicts different clinical outcome of hepatocellular carcinoma (HCC), we perform immunostaining of these oncogenes in cancerous, non-cancerous and normal hepatic tissues. Our research indicates new clinical target for tumor theraphy. Methods: 1 Samples collection and patients follow up: 50 specimens of HCC, 17 specimens of adjacent non-cancerous cirrhosis hepatic tissue and 14 specimens of normal hepatic tissue obtained from the fourth hospital of Hebei Medical University without clinical treatment before operation. All patients were followed up for 2 years.2 Imunostaining: The samples were fixed by 10% formaldehyde, embeded in paraffin after excision. Raf-1, MEK-1, ERK-1 and Cyclin D1 were stained, and two pathologist without backgroud on these samples graded these oncogenes expression and proliferation on each tissue.Resluts: 1 Survival analysis of the clinical and pathological data in postoperative HCC patients: Age, the portal vein tumor thrombosis, the size of tumor, HBsAg, HBeAb, the number of tumor foci and the integrity of the tumor's envelope were no statistical difference in postoperative HCC patients. The clinical stage (χ~2=7.636, P=0.006), the pathological classification (χ~2=6.296, P=0.043) and AFP (χ~2=4.07, P=0.044) were the significant prognostic factors in postoperative HCC patients.2 Raf-1 mostly expressed in cytoplasm and cellular membrane. The Raf-1 expressed not differently refered to sex, the number of tumor foci, AFP, HBeAb, HBsAg and the integrity of the tumor's envelope based on our statistical analysis. In the 50 HCC samples, Raf-1 weakly expressed in 17 samples, moderately expressed in 20 samples and strongly expressed 9 samples, repectively. Whereas it weakly expressed in 5/17 of adjacent non-cancerous cirrhosis hepatic tissues and 2/14 of normal hepatic tissues. Raf-1 expression frequency in HCC was significantly higher than that in adjacent non-cancerous hepatic tissue and in normal hepatic tissue (Z=-5.079 and Z=-5.082, P=0.000), and no statistical difference existed bwteen in adjacent non-cancerous hepatic tissue and in normal hepatic tissue. Our data demonstrated that Raf-1 expression was positively correlated with hepatic carcinogenesis. Raf-1 expression in early clinical stage in HCC was stronger than that in late clinical stage (χ~2=13.496, P=0.009. r=-0.452, P=0.001). Raf-1 expression in the well-differentiated HCC about the pathological classification was stronger than that in poorly differentiated tissue (χ~2=14.904, P=0.001. r=-0.547, P=0.000). Raf-1 expression in older than or equal to 50 years old of HCC patients was stronger than that in younger than 50 years old (Z=-2.109, P=0.035. r=0.301, P=0.034). Raf-1 expression in less than 5cm about the size of tumor was stronger than that in larger than or equal to 5cm (Z=-2.502, P=0.012. r=-0.357, P=0.011). Raf-1 expression in the tissue without the portal vein tumor thrombosis was stronger than that with the portal vein tumor thrombosis (Z=-3.246, P=0.001. r=-0.464, P=0.001). The patients in whom Raf-1 negatively and weakly expressed had 12.93 months'mean survival time, and the cumulative survival rate was 14.3%. The patients in whom Raf-1 moderately and strongly expressed had 28.23 months' mean survival time, and the cumulative survival rate was 54.3%. Raf-1 expression was stronger, the survival rate was higher, and the prognosis was better (By single variable Log rank test,χ~2=6.793, P=0.009).3 MEK-1 mostly expressed in cytoplasm and cellular membrane. The MEK-1 expressed not differently refered to sex, the number of tumor foci, AFP, HBeAb, HBsAg and the integrity of the tumor's envelope based on our statistical analysis. In the 50 HCC samples, MEK-1 weakly expressed in 14 samples, moderately expressed in 24 samples and strongly expressed 7 samples, repectively. Whereas it weakly expressed in 6/17 of adjacent non-cancerous cirrhosis hepatic tissues and 3/14 normal hepatic tissues. MEK-1 expression frequency in HCC was significantly higher than that in adjacent non-cancerous hepatic tissue and in normal hepatic tissue (Z=-4.909 and Z=-4.873, P=0.000), and no statistical difference existed bwteen in adjacent non-cancerous hepatic tissue and in normal hepatic tissue. Our data demonstrated that MEK-1 expression was positively correlated with hepatic carcinogenesis. MEK-1 expression in early clinical stage in HCC was stronger than that in late clinical stage (χ~2=9.869, P=0.043. r=-0.433, P=0.002). MEK-1 expression in the well-differentiated HCC about the pathological classification was stronger than that in poorly differentiated tissue (χ~2=17.569, P=0.000. r=-0.599, P=0.000). MEK-1 expression in older than or equal to 50 years old of HCC patients was stronger than that in younger than 50 years old (Z=-2.375, P=0.018. r=0.339, P=0.016). MEK-1 expression in less than 5cm about the size of tumor was stronger than that in larger than or equal to 5cm (Z=-2.635, P=0.008. r=-0.376, P=0.007). MEK-1 expression in the tissue without the portal vein tumor thrombosis was stronger than that with the portal vein tumor thrombosis (Z=-2.891, P=0.004. r=-0.413, P=0.003). The patients in whom MEK-1 negatively and weakly expressed had 13.77 months'mean survival time, and the cumulative survival rate was 15.4%. The patients in whom MEK-1 moderately and strongly expressed had 27.64 months'mean survival time, and the cumulative survival rate was 51.5%. MEK-1 expression was stronger, the survival rate was higher, and the prognosis was better (By single variable Log rank test,χ~2=6.581, P=0.01).4 ERK-1 mostly expressed in cytoplasm and cellular nucleus. The ERK-1 expressed not differently refered to sex, the number of tumor foci, AFP, HBeAb, HBsAg and the integrity of the tumor's envelope based on our statistical analysis. In the 50 HCC samples, ERK-1 weakly expressed in 13 samples, moderately expressed in 22 samples and strongly expressed 7 samples, repectively. Whereas it weakly expressed in 7/17 of adjacent non-cancerous cirrhosis hepatic tissues and 2/14 normal hepatic tissues. ERK-1 expression frequency in HCC was significantly higher than that in adjacent non-cancerous hepatic tissue and in normal hepatic tissue (Z=-4.264 and Z=-4.635, P=0.000), and no statistical difference existed bwteen in adjacent non-cancerous hepatic tissue and in normal hepatic tissue. Our data demonstrated that ERK-1 expression was positively correlated with hepatic carcinogenesis. ERK-1 expression in early clinical stage in HCC was stronger than that in late clinical stage (χ~2=10.227, P=0.03. r=-0.422, P=0.002). ERK-1 expression in the well-differentiated HCC about the pathological classification was stronger than that in poorly differentiated tissue (χ~2=18.946, P=0.000. r=-0.615, P=0.000). ERK-1 expression in older than or equal to 50 years old of HCC patients was stronger than that in younger than 50 years old (Z=-2.266, P=0.023. r=0.324, P=0.022). ERK-1 expression in less than 5cm about the size of tumor was stronger than that in larger than or equal to 5cm (Z=-2.769, P=0.006. r=-0.396, P=0.004). MEK-1 expression in the tissue without the portal vein tumor thrombosis was stronger than that with the portal vein tumor thrombosis (Z=-2.953, P=0.003. r=-0.422, P=0.002). The patients in whom ERK-1 negatively and weakly expressed had 11.71 months'mean survival time, and the cumulative survival rate was 9.5%. The patients in whom ERK-1 moderately and strongly expressed had 28.92 months'mean survival time, and the cumulative survival rate was 55%. ERK-1 expression was stronger, the survival rate was higher, and the prognosis was better (By single variable Log rank test,χ~2=9.869, P=0.002).5 Cyclin D1 mostly expressed in cellular nucleus and cytoplasm. The Cyclin D1 expressed not differently refered to clinical stage, sex, age, the size of tumor, the number of tumor foci, AFP, HBeAb, HbsAg, the portal vein tumor thrombosis and the integrity of the tumor's envelope based on our statistical analysis. In the 50 HCC samples, Cyclin D1 weakly expressed in 14 samples, moderately expressed in 17 samples and strongly expressed 8 samples, repectively. Whereas it weakly expressed in 6/17 of adjacent non-cancerous cirrhosis hepatic tissues and 3/14 normal hepatic tissues. Cyclin D1 expression frequency in HCC was significantly higher than that in adjacent non-cancerous hepatic tissue and in normal hepatic tissue (Z=-3.853 and Z=-3.987, P=0.000), and no statistical difference existed bwteen in adjacent non-cancerous hepatic tissue and in normal hepatic tissue. Our data demonstrated that Cyclin D1 expression was positively correlated with hepatic carcinogenesis. Cyclin D1 expression in the well-differentiated HCC about the pathological classification was stronger than that in poorly differentiated tissue (χ~2=6.227, P=0.044. r=-0.352, P=0.012). The patients in whom Cyclin D1 negatively and weakly expressed had 15.55 months'mean survival time, and the cumulative survival rate was 14.3%. The patients in whom Cyclin D1 moderately and strongly expressed had 27.26 months'mean survival time, and the cumulative survival rate was 55.6%. Cyclin D1 expression was stronger, the survival rate was higher, and the prognosis was better (By single variable Log rank test,χ~2=4.219, P=0.04).6 The correlation among Raf-1, MEK-1, ERK-1 and Cyclin D1 expression in HCC: Raf-1 expressional level was positively correlated to MEK-1 expressional level in HCC (r=0.747, P=0.000), and was positively correlated ERK-1 expressional level in HCC (r=0.691, P=0.000), and was positively correlated to Cyclin D1 expressional level in HCC (r=0.341, P=0.015). MEK-1 expressional level was positively correlated to ERK-1 expressional level in HCC (r=0.858, P=0.000), and was positively correlated to Cyclin D1 expressional level in HCC (r=0.429, P=0.002). ERK-1 expressional level was positively correlated to Cyclin D1 expressional level in HCC (r=0.378, P=0.007).Conclusions: 1 The clinical stage, the pathological classification and AFP were the significant prognostic factors in postoperative HCC patients. In the patients in early clinical stage with low level of AFP whose hepatic tissue was well-differentiated, the prognosis was better.2 Raf-1, MEK-1, ERK-1 expression was positively correlated with hepatic carcinogenesis. Raf-1, MEK-1, ERK-1 weakly expressed in the younger patients, late clinical stage, poorly differentiated tissue, the big size of tumor and with the portal vein tumor thrombosis. While the survival rate was shorter, the prognosis was poorer.3 Cyclin D1 expression was positively correlated with hepatic carcinogenesis. Cyclin D1 strongly expressed in well-differentiated tissue. While the survival rate was higher, the prognosis was better. 4 There were positively correlations among Raf-1, MEK-1, ERK-1 and Cyclin D1 expression. |
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