The Effects Of ARNT On The Growth And Metastasis Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC) is one of the most common cancers in China in terms of tumor cases,and tumor invasion and metastasis are the leading cause of HCC death,so it is of great clinical importance to investigate molecular mechanisms of invasion and metastasis for an effectual therapeutic way of anti-metastasis and relapse of HCC.Although the roles of HIF-1 signaling pathway in tumor evolution and progression have attracted more attentions,especially on its tumor biological functions,the effects of ARNT,which is also called HIF-1β,still kept known unclearly.Therefore we first observed the relationship of the intratumor and peritumor expression of ARNT with the evolution,progression and prognosis of HCC. Then we study the effect of ARNT knock-down on the growth,invasion and mestatasis of HCC.Finally we further investigate its possible molecular mechanisms of how ARNT play its roles in the growth and metastasis of HCC.Part one Protein levels,its correlation and clinical significance of ARNT in hepatocellular carcinomaObjective:To compare the differential expression of ARNT between the intratumor and peritumor tissue,and its relationship with carcinogenesis of HCC.To study the correlation and clinical significance of ARNT with HCC.Methods:ARNT protein expression in 105 patients with HCC proven by surgery and pathology during 1999 to 2006 was analyzed retrospectively by high-throghput tissue microarray technology and histoimunochemistry.The overall survival and recurrence rates of the groups with differential ARNT expression were calculated by Kaplan-Meire method,and statistical differences were tested by log-rank method. Univariate and multivariate study with COX's proportional hazard model were used to evaluate their relative effects on the overall survival and recurrence rates.Result:ARNT mainly expressed in the nucleus of cancerous cells and normal liver cells.The positive expression rate of ARNT in intratumor tissue and in peritumor tissue of HCC was 53.3%and 71.4%respectively,and there was significant correlation(p=0.007).The expression of ARNT was related to tumor size(p=0.009), but not to other clinicopathologic factors such as age,sex,AFP,intrahepatic metastasis,TNM stage,vascular invasion and HBV infection(p>0.05).The expression of ARNT was significantly associated with overall survival and recurrence of HCC patients.The median survival time of patients with lower intratumor ARNT expression was 26.3 months,significantly shorter than that of the patients with higher intratumor ARNT expression,which was 84.0 months(p=0.000).The 5-year recurrence rate after resection for the group with lower intratumor expression of ARNT was 46.9%,which was significantly higher than that of the group with higher intratumor expression of ARNT(p=0.016).The univariate log-rank analysis found a significant relationship between tumor size,intrahepatic metastasis,TNM stage, vascular invasion,AFP,HBV infection,intratumor ARNT expression and the overall survival,but only a significant relationship between tumor size,intrahepatic metastasis,TNM stage,vascular invasion,intratumor ARNT expression and the recurrence was found.A multivariate analysis indicated a significant association between AFP,TNM stage,intratumor ARNT expression and the overall survival,but only TNM stage was significantly associated with recurrence.Conclusion:The low-intratumor-expression of ARNT may be related to the carcinogenesis of HCC and it seems to be a new independent predictor of prognosis for HCC.Part two Effects of ARNT on the growth,invasion and metastasis of experimental hepatocellular carcinomaObjective:To observe the effects of ARNT on tumor growth,invasion and metastasis in experimental hepatocellular carcinoma.Methods:Two recombicant lentiviral vectors,namely pLentiARNTi-1 and pLentiARNTi-2 against Arnt gene,were constructed and then tranfected into HCCLM6 cells.The expression of ARNT both in mRNA and protein level was determined by RT-PCR and western blot in stable tranfection HCCLM6 cell lines. Cell proliferation,invasion and migration was tested in vitro by cell fluorescent quantitation,FACS,transwell and scratch analyses using stable transfection HCCLM6 cells and their parental cell lines,respectively.Tumor growth and metastasis in vivo were also observed.Result:RT-PCR and??ern blot showed that ARNT was significantly knocked down in two lentivirus transfection HCCLM6 cell lines.The ability of proliferation,the percentage of S-phase cells,the ability of invasion and migration in ARNT knocked down HCC cells were significantly increased.Tumor growth in ARNT knocked down HCC xenograft models was remarkably accelerated,and metastasis foci in mouse lung and abdomen of those groups were sigificantly increased.Conclusion:ARNT is a suppressive factor on tumor growth and metastasis of HCC.Part three The mechanism for ARNT of inhibiting the growth and metastasis of hepatocellular carcinomaObjective:To investigate the molecular mechanism of ARNT inhibitory effects on HCC growth and metastasis.Method:Realtime PCR and western blot were used to test the expression of possible targeting genes and proteins regulating cell proliferation,invasion and metastasis in stable transfection HCCLM6 cells and their parental cell lines.Then the above expression was verified in its xenograft in nude mice by immunohistochemical Staining.Result:Realtime PCR and western blot showed that the expression of cyclinE and CDK2 which was associated with proliferation upregulated,and the expression of MMP7 which was related to invasion and metastasis increased too,while the expression of TIMP-1 and TIMP-2,which was also related to invasion and metastasis, decreased.All molecular siganitures in vivo was verified by immunohistochemical Staining.Conclusion:ARNT can inhibit the growth and metastasis of HCC by down-regulating the expression of cyclinE,CDK2 and MMP7,and up-regulating the expression of TIMP-1 and TIMP-2.