Functional Studies Of Gene Tiam1 In Hepatocellular Carcinoma Invasion And Metastasis

BACKGROUND & OBJECTIVEHepatocellular carcinoma (HCC) is one of the common cancers in the world. The incidence rate of HCC in china is increasing fast during the past decades. Metastasis is one of the basic characteristic of malignant tumors and is the main cause which affects the therapeutic efficacy and leads to the death of cancer patients. It is an urgent task to work out the metastasis-associated factors and find out the preventive and therapeutic methods.Tiam1(T lymphoma invasion and metastasis 1)was originally identified as the invasion-and metastasis-inducing gene by proviral tagging in combination with in vitro selection for invasiveness in T lymphoma cells. Tiam1 is one of guanine nucleotide exchange factors (GEFs), which activate GTPases by promoting the exchange of their inactive GDP-bound forms to their active GTP-bound forms. Whereas Tiam1 displays GEF activity towards all three Rho-like GTPases Rac1, Cdc42 and RhoA in vitro, Tiam1 specifically activates Rac in vivo.Recent evidence suggests that Tiaml could influence Rac GTPases signaling specificity in addition to promoting their activation.Tiam1 has been implicated to directly bind to many different cytoplasmic and membrane-associated proteins, which couples Tiam1-Rac activity to specific signaling pathways.The role of Tiaml in cellular migration, invasion and metastasis may not be limited to T lymphoma. It was reported to be important in promoting the tumor progression in a variety of cancers such as breast cancer, lung cancer and Ras-induced skin tumors. The possible role of Tiaml in the metastasis of colorectal cancer is unclear.In our previous study, by using tissue chip of normal tissues and common malignant tumors which was designed and produced by ourselves and immunohistochemistry, Tiaml protein expressions were detected in human normal tissues and common malignant tumors,213 cases of hepatocellular carcinoma tissues, 9 human hepatocellular carcinoma cell lines and 1 human normal hepatic cell. Tiaml protein was highly expressed in many tumors and it can be acted as one important tumor maker for tumorigenesis and development. Tiaml protein was up-regulated in hepatocellular carcinoma tissues, expressed lowly in liver sclerotic tissues and had no expression in normal hepatic tissue, indicating that Tiaml may be regarded as the important marker for clinical detection of HCC.Combined evaluation of Tiaml protein high expression and clinical follow-up data analysis showed that Tiam1 had close relationship with prognosis and metastasis of hepatocellular carcinoma patients.Tiam1 may act as the important marker for judging prognosis of HCC.In this study, we aim to clarify the possible role of Tiaml gene in the proliferation, invasion and metastasis of HCC.It will be helpful to understand the molecular basis of HCC,and establish Tiaml as a new target for early metastatic diagnostic markers and novel therapeutic strategies.METHODS1.Expression of Tiaml gene/protein in human normal liver cells and liver cancer cells.Real-time PCR, Western blot, Immunocytochemistry, Cellular immunofluoresce-nce and Cellular immunofluorescence confocal were used to examine the expressions of Tiaml in 2 hepatocellular carcinoma cell lines named M6,97L and 1 normal named HL-7702.2.Effect of Tiaml overexpression on the biological behaviors of human HCCLentiviral vector pCDF1-Tiam1-copGFP was constructed by gene cloning. Recombinant lentivirus was harvested from 293FT cells cotransfected with the pPACKF1 Packaging Plasmid Mix.Tiam1/C1199HA cDNA was transfected into the 97L cell,which had lower Tiaml expression. Single transfectant cells clone were established 97L/copGFP/Tiaml+line by copGFP, Single mock cells clone were also established 97L/copGFP/mock line by copGFP. The Tiaml expression in transfectant was determined by RT-PCR, immunohistochemistry and Western blot. The biological behaviors of tranfectant were investigated by MTT assay, plate colony formation assay, flow cytometry, cell wounding heal assay, invasion assays in vitro, subcutaneous tumor model in nude mice, metastasis assay in vivo through the lateral tail-vein injection through LT-9MACIMSYSPULS system which were used to assess the functional effects of Tiaml silencing 97L cells in vitro or in vivo respectively.3.Lentivirus-mediated silencing of Tiaml gene in human HCC M6 cell line.Lentiviral expression vectors containing enhanced green fluorescence protein (EGFP) and Tiaml small interfering RNA (Lenti-Tiamlsi), or the control siRNA (Lenti-NC) gene were constructed. Human HCC M6 cell line was transfected with a different multiplicity of infection (MOI) of Lenti-Tiaml-si-A, B,C,D or Lenti-NC, and cultured to obtain stably-transfected M6/EGFP/Tiam1- and M6/EGFP/mock cells. The expression of Tiam1 mRNA/protein was determined by real-time PCR, western blot and confocal immunofluorescence.The biological behaviors of tranfectant were investigated by MTT assay, plate colony formation assay, flow cytometry, cell wounding heal assay, invasion assays in vitro, subcutaneous tumor model in nude mice, metastasis assay in vivo through the lateral tail-vein injection through LT-9MACIMSYSPULS system which were used to assess the functional effects of Tiam1 silencing M6 cells in vitro or in vivo, respectively.4.Statistical analysisStatistical analysis was done using SPSS 16.0. MTT assay, cell wounding heal assay, subcutaneous tumor model in nude mice were analyzed by repeated measure square analyze.Real-time PCR, plate colony formation assay, flow cytometry, invasion assays in vitro were analyzed one-factor analysis of variance.RESULTSThe main results and findings are as follows:1.The expressions of Tiaml gene in human normal liver cell line and liver cancer cell lines.Tiam1 gene expressions in 2 human hepatocellular carcinoma cell lines and 1 human normal hepatic cell line were detected by real time PCR. The results of one-factor analysis of variance showed that the expressions of Tiam1 in 3 cell lines had significant difference (F=155.975,P=0.000).The expresions of Tiaml in M6 was higher than 97L and normal hepatic cell line HL-7702 with significant difference.2.Effect of Tiam1 overexpression on the biological behaviors of human HCCRecombinant pCDFl-Tiaml-copGFP lentivirus vector was constructed and identified by restriction endonuclease analysis and DNA sequencing.6771bp vector segment and 3800bp Tiam1 were got after EcoRI digested.After 60 hours of transferring pCDF1-Tiam1-copGFP and packaging plasmid into 293FT cell,the green fluorescence could be observed in some of the transferring cells.97L cells were infected with recombinant lentivirus or mock lentivirus. A higher level of Tiaml expression was seen in the Tiaml transfected clone 2, we chose 97L/T2 clone (named 97L/copGFP/Tiam1+)cells and mock clone (named 97L/copGFP/mock) as function experiments in vitro or vivo.The result showed a significantly enhanced proliferation features compared with the 97L and 97L/copGFP/mock cells as determined by Real-time PCR, Western blot, Immunofluorescence confocal. The results showed that Tiaml overexpression promoted proliferation:MTT assay (F=24.658,P=0.000); plate colony formation assay(P=0.028,P=0.044); flow cytometry (P=0.000,P=0.000);cell wounding heal assay(F=93.331,P=0.000) and invasive capabilities(P=0.000,P=0.000)in 97L/copGFP/Tiaml+cells compared with 97L or 97L/copGFP/mock in vitro. Next, the effect of Tiaml on tumor growth was assessed by subcutaneous injection of 97L/copGFP/Tiam1+ and 97L/copGFP/mock cells for thirty days in vivo. Compared with 97L/copGFP/mock cells, the expression of Tiaml led to a pronounced increase in 97L/copGFP/Tiaml+cell growth starting from day 15,up to 2.1-fold increase in mice of tumor volume at day 30 after cell injection(F=192.294,P=0.000).To unambiguously elucidate the enhanced effect of Tiaml on HCC metastasis, we performed and metastasis assay in vivo through the lateral tail-vein injection. Mice were sacrificed 2 months later because the mice of 97L/copGFP/Tiaml+ group were moribund. The metastasis in the liver, lung, bone and other organs were determined the metastatic ability by whole-body visualizing orthotopic animal model and histological examination;in 97L/copGFP/Tiam1+ group,50%(3/6) of mice developed lung metastasis that appeared as numerous green nodules on the lung, 50%(3/6) of mice developed liver metastasis,34%(2/6) developed bone metastasis. In 97L/copGFP/mock group, only 29%(1/6) of animal had lung metastasis. These data indicated that Tiaml expression in liver 97L cell was associated with enhanced migratory and invasive ability.3.Effect of Tiaml silencing on the biological behaviors of human HCC M6 cells.To require prolonged suppression of Tiaml protein in M6 cells, we constructed 4 vshRNA (pGC-LV recombination vector) containing Tiam1 interfere sequens A, B, C and D.Real-time PCR analysis showed that the mRNA levels of Tiaml in four Tiaml-si cells were all suppressed, especially in Tiaml-si-B cells (84.0%).Then, we reinfected M6 cells.with si-B for further identifycation.M6 cells were infected with recombinant lentivirus or mock lentivirus. A lower level of Tiaml expression was seen in the Tiaml transfected clone 2(90.4%), we chose M6/T2 clone (named M6/EGFP/Tiaml-)cells and mock clone (named M6/EGFP/mock) as function experiments in vitro or vivo.The result showed a significantly declined proliferation features of M6/EGFP/Tiam1- compared with the M6 and M6/EGFP/mock cells as determined by Real-time PCR, Western blot, Immunofluorescence confocal. The results showed that Tiam1 overexpression promoted proliferation:MTT assay (F=59.732,P=0.000);plate colony formation assay(P=0.028, P=0.044); flow cytometry (P=0.000, P=0.000); cell wounding heal assay(F=248.998,P=0.000) and invasion assays in vitro (P=0.000, P=0.000) in M6/EGFP/Tiam1- cells compared with M6 or M6/EGFP/mock in vitro.Next, the effect of Tiaml on tumor growth was assessed by subcutaneous injection of M6/EGFP/mock and M6/EGFP/Tiam1- cells for thirty days in vivo.Compared with M6/EGFP/mock cells, the silencing of Tiaml led to a pronounced decrease in M6/EGFP/Tiam1- cell growth starting from day 15,up to 2.2-fold decrease in mice of tumor volume at day 30 after cell injection(F=192.294,P=0.000). To unambiguously elucidate the declined effect of Tiaml on HCC metastasis, we performed and metastasis assay in vivo through the lateral tail-vein injection.Mice were sacrificed 2 months later because the mice of M6/EGFP/Tiam1- group were moribund.The metastasis in the liver, lung, bone and other organs were determined by the metastatic ability evaluated by whole-body visualizing orthotopic animal model and histological examination. In M6/EGFP/Tiam1- group, only 17%(1/6) of animal had lung metastasis,17%(1/6) had bone metastasis. But in M6/EGFP/mock group,100%(6/6) of mice developed lung metastasis that appeared as numerous green nodules on the lung,83%(5/6) of mice developed liver metastasis,100%(6/6) developed bone metastasis.These data indicated that Tiam1 silencing in liver M6 cell was associated with decreasing migratory and invasive ability.CONCLUSIONTiaml gene plays an important role in proliferation, invasion and metastasis of HCC and is a metastasis-related gene.Clinically it may be a useful indicator of the tumor progression and metastasis in HCC.