The Role Of Testicular Nuclear Receptor 4 In The Developmen,Invasion And Metastasis Of Hepatocellular Carcinoma

Part one Expression of TR4 in hepatocellular carcinoma tissues and para-carcinoma liver tissuesObjectiveThis study was aimed to investigate the expression and distribution of testicular nuclear receptor 4 (TR4) in hepatocellular carcinoma and para-carcinoma liver tissues, and preliminary investigated its role in the development of hepatocellular carcinoma.MethodsThe expression of TR4 was examined in 71 paired hepatocellular carcinoma tissues and their para-carcinoma liver tissues by immunohistochemistry (IHC). The distribution of TR4 in hepatocellular carcinoma tissues and their para-carcinoma liver tissues were analyzed and compared.ResultsIn the cytoplasm of hepatocellular carcinoma, TR4 mainly showed weak positive expression, accounting for 64.8% of the total cases, followed by the moderate positive expression, accounting for 35.2% of the total cases. In the cytoplasm of para-carcinoma liver tissues, TR.4 mostly presented with strong positive and moderate positive expression, accounting for 49.2% and 42.3% of the total cases respectively, followed by weak positive expression, accounting for 8.5% of the total cases,with significant difference between the two groups (P<0.01). Besides,TR4 showed positive expression in the most cell nucleus of hepatocellular carcinoma, including 52.1% cases of weak positive expression,14.1% cases of moderate positive expression and 7.0% cases of strong positive expression, with negative expression accounting for 26.8% of the total cases. TR4 presented with negative expression in most cell nucleus of para-carcinoma liver tissues, accounting for 73.2% of the total cases, followed by weakly positive expression in 26.8% of the total cases. There was significant difference between the two groups (P< 0.01).ConclusionThis study reveals significantly differential distribution of TR4 between hepatocellular carcinoma tissues and para-carcinoma liver tissues, suggesting TR4 may play a role in the development of hepatocellular carcinoma.Part two The role of TR4 in tumor proliferation of hepatocellular carcinomaObjectiveThis study was aimed to investigate the role of TR4 in tumor proliferation of hepatocellular carcinoma.MethodsIn this study, we had established both TR4 knock down and TR4 over-expression stable cell lines in two hepatocellular carcinoma cell lines by lentivirus transfection, including HCC-LM3 and Huh7. In both cell lines, a cell proliferation test was performed to investigate the role of TR4 in tumor proliferation of hepatocellular carcinoma by MTS assay. At the same time, we established an subcutaneous xenotransplanted tumor model of HCC-LM3 in nude mice to exam the influence of both TR4 knock down and over-expression on tumor proliferation of hepatocellular carcinoma in vivo.ResultsSuccessfully, we had established both TR4 knock down and TR4 over-expression stable cell lines in HCC-LM3 and Huh7, which were verified by Real-time PCR and Western Blot. MTS assay demonstrated that neither TR4 knock down nor TR4 over-expression had any influence on the proliferation in both HCC-LM3 and Huh7 cell lines. When TR4 knock down or TR4 over-expression HCC-LM3 cells and their control cells were implanted subcutaneously in nude mice, subcutaneous tumors were found in all nude mice within 42 days, no difference of tumor volume or weight was observed neither in TR4 knock down nor in TR4 over-expression conditions compared with their control groups.ConclusionTR4 does not affect the proliferation ability in hepatocellular carcinoma.Part three The role of TR4 in tumor metastasis of hepatocellular carcinomaObjectiveThis study was aimed to investigate the role of TR4 in tumor metastasis of hepatocellular carcinoma.MethodsIn this study, we had established both TR4 knock down and TR4 over-expression stable cell lines in two hepatocellular carcinoma cell lines by lentivirus transfection, including HCC-LM3 and Huh7. In both cell lines, cell migration/invasion assay was applied to test the role of TR4 on tumor migration and invasion of hepatocellular carcinoma. At the same time, we established a luciferase expressing HCC-LM3 cell line with TR4 knock down (LM3-luc-shTR4), and orthotopic xenografted LM3-luc-shTR4 cells and the control cells (LM3-luc-scr) into the liver of nude mice by surgery to test the influence of TR4 on tumor metastasis of hepatocellular carcinoma in vivo.ResultsSuccessfully, we had established both TR4 knock down and TR4 over-expression stable cell lines in HCC-LM3 and Huh7, which were verified by Real-time PCR and Western Blot. Cell migration/invasion assay demonstrated that tumor migration/invasion ability was significantly enhanced in the condition of TR4 knock down in both HCC-LM3 and Huh7 cell lines, while TR4 over-expression could suppress its migration/invasion ability. When luciferase expressing HCC-LM3 cell line with TR4 knock down (LM3-luc-shTR4) and the control cells (LM3-luc-scr) were orthotopic xenografted in the liver of nude mice, all of the nude mice in LM3-luc-shTR4 and LM3-luc-scr group were observed with hepatocellular carcinoma development in the live 42 days after surgery, while 2 of 3 mice in LM3-luc-shTR4 group with tumor metastasis and none of the 3 mice in LM3-luc-scr group with metastasis.ConclusionTR4 could suppress tumor metastasis of hepatocellular carcinoma.