The Study On Expression Of CLIC1 On Lymphatic Metastasis Of Mouse Hepatocarcinoma

Background:Hepatocarcinoma (HCC) is a common malignant tumor in China. The mortality of HCC is third of digestive system neoplasm. Currently surgery is the preferred treatment for liver cancer, but 5-years survival rate is still very low. Many studies reported that lymphatic metastasis of liver cancer is the main reason of early metastasis and leading to death. Therefore, it is very important to study the mechanism of lymphatic metastasis and the related molecules. Studies indicate that CLIC1 has a higher expression level in gastric cancer, gallbladder cancer, hepatocellular carcinoma, etc.Hca-F and Hca-p are a pair of syngenetic mouse hepatocarcinoma ascites cell lines which have different potential of lymphatic metastasis. Hca-F is the higher metastatic cell line, with a metastatic rate more than 70%, Hca-P is the lower lymphatic metastasis rate, with a metastatic rate less than 30%. They are the ideal models for the research of mechanism of lymphatic metastasis. Our previous studies indicate that the CLIC1 gene express different between Hca-F and Hca-p. The expressing level of CLIC1 was much higher in Hca-F than that in Hca-P cell lines in both mRNA and protein levels. Subsequently, we used RNA interference technology constructed hepatocarcinoma ascites cells Hca-F, F_Cdown cells in which expression of CLIC1 was significantly reduced and F_control cells which are transfected with unrelated sequence of CLIC1.Method:Western-Blot was used to compare: 1.The different expression of CLIC1, GSN, PKC-αand Annexin A7 between F_control and F_Cdown. 2. The different expression of CLIC1, GSN in F_control, F_Adown, P_control , P_Adown and cells.Result:1. The expression of CLIC1, GSN, PKC-αand Annexin A7 was detected by Western-Blot in vitro experiments in F_control cells and F_Cdown cells . 2. The expression of CLIC1 and PKC-αin F_Cdown cells was lower than that in F_control cells, P <0.05; the expression of GSN和Annexin A7 in F_Cdown cells was higher than that in F_control cells, P <0.05. 3. In vitro experiments, the expression of CLIC1 was higher either Annexin A7 was up regulated or down regulated; the expression of GSN in F_A7down cells was lower than that in F_control cells , and was higher in P_A7up cells than that in P_control cells ,P <0.05. 4. In vivo experiments, the expression of CLIC1 in F_A7down cells was higher than that in F_control cells and was lower in P_A7up cells than that in P_control cells , the expression of GSN was opposite, P <0.05.Conclusion:1.The expression of CLIC1 was lower after inhibition, indicated that the inhibition was successful. 2. The expression of PKC-αwas lower in F_Cdown cells, and GSN and Annexin A7 in F_Cdown cells, indicated that the inhibition of CLIC1 regulated the expression of GSN, PKC-αand Annexin A7. 2. In vitro experiments, the expression of CLIC1 was higher either Annexin A7 was up regulated or down regulated; in vivo experiments, the expression of CLIC1 in F_A7down cells was higher than that in F_control cells and was lower in P_A7up cells than that in P_control cells, indicated that there was a complex regulation mechanism between CLIC1 and Annexin A7. 3. The expression trend of GSN was same as Annexin A7 when Annexin was up or down regulated, indicated that Annexin A7 may regulated the expression of GSN .