The Effect Of HINT1on The Expression Of Nf-κB And IKB-a And Clinical Correlation In Hepatocellular Carcinoma

Primary Hepatocellular carcinoma(HCC) is one of the most common malignant tumors in China, It is a synergy results by multiple factors. The process includes virus infection, the role of carcinogen, the activation of oncogene and inactivation of tumor suppressor gene, cell apoptosis and out of control proliferation and so on. The molecular basis of cancer is that the activation of oncogene and the mutation of tumor suppressor gene. The histidine triad (HIT) nucleotide-binding protein1(HINT1) is a novel tumor suppressor, its expression level are closely related to the occurrence,development and metastasis of liver cancer. There is accumulating evidence that the HINT1protein plays an inhibitory role in several pathways that control gene transcription. However, the mechanism of action of HINT1with respect to tumor suppression is not known. Sustained activation of nuclear transcription factor kappa B(NF-kB) can regulate cell proliferation, inhibit of apoptosis, promote tumor formation. Its transcriptional activities can be inhibited by blinding to its suppressor protein IKB-a. The research focus of this experiment is that the roles of Inhibition of tumor of HINT1whether it is related to IKB-a and NF-kB signaling pathway and to explore its possible mechanism of tumor suppression. Objective:1. Investigate the expression of NF-KBp50and IKB-a protein in human HCC tissues and its clinical expression correlation.2. To study the inhibition of the expression of IKB-a and nuclear transcription factor-KB activity by Hintl gene over expression in human hepatoma cells. To explore the possible tumor suppressor mechanism of Hintl.Methods:1. Immunohistochemical method was performed to detect the expressions of NF-KBp50and IKB-a protein in HCC tissues, paracarcinoma tissues and normal liver tissues. To investigate clinic significance of them in hepatocellular liver cancer.2. The Hint1gene was amplified, and then was inserted into the pcDNA3.1/HA eukaryotic expression plasmid.The PHA-Hint1was transfected into the human hepatoma7701cells by Lipofectamine2000. There were three groups include Hintl transducted group, lipofecting group and pcDNA3.1(+) group. Parental cells were used as blank control.The cells were cultured for24h,48h,72h,96h,120h. Using an optical microscope to observe the morphology of the cells, we use MTT assay to measure the OD yield in each group and draw the growth curve of cells. Western blot was used to detect the protein expression of Hintl and IKB-a and NF-kB in cells which transfection with pHA-Hintl plasmid.Result:1. The experiment results showed that, The positive expression rate of NF-KBp50in HCC tissues was86%,which was significantly higher than47%in paracarcinoma tissues and13%in normal liver tissues (P<0.05); The positive expression rate of IKB-a in HCC tissues was18%,which was significantly lower than62%in paracarcinoma tissues and100%in normal liver tissues (P<0.05).There was a negative correlation between the expression of NF-KBp50and IKB-a (rs=-0.411,P<0.05) in HCC tissues. The expression of NF-kBp50and IKB-a was correlated with HBsAg(+), tumor differentiation, the level of AFP and TNM stage (P<0.05), but not with sex, age and tumor size (P>0.05).2. The cells were cultured in the same nutritional conditions before Hintl transfected,The morphology and vitality of cells was no significant difference in four groups. The proliferation of transfected cells was significant slow down than other cell growth; The results of the growth curve of cells showed that the cell proliferation was significantly inhibited after transducted72h in Hint] group; The results of Western blot showed that the expression of1KB protein was significantly higher than before and the expression of NF-kB significantly decreased after cells transfection with pHA-Hintl plasmid.Conclusion:1. The high expression of NF-kBp50and the low expression of IKB-a in hepatocellular carcinoma tissues; The expression of NF-KBp50was related to that of IKB-a.They may exist regulating relations in the development of HCC.2.(1) Hintl gene can inhibit the proliferation of hepatoma cells;(2) Over-expression of Hint1can enhance the expression of IKB-a protein and inhibit the nuclear transcription factor-KB activitv in hepatoma cells,which may be one of the mechanisms to play the inhibition of tumor function and provide a theoretical basis for Hintl is a tumor suppressor.