| Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third cause of cancer associated death which presents gradual upward trend in recent years. Most radical resection of liver cancer can get with the development of science and technology and surgical techniques, but the overall 5-year survival rate still remains low. The most important factor is that HCC extremely had the ability of invasion and metastasis. Tumor recurrence and distant metastasis is very prone to happen in 5 years after radical resection of hepatocellular. So, HCC recurrence and metastasis after operation are our clinical problems.Tumor microenvironment is a special circumstance during the progress of tumor cells Growth, which is formed after the interaction of tumor cells and extracellular matrix, including tumor cells, a variety of mesenchymal and inflammatory cells, soluble cytokines, and extracellular matrix and so on. It has played an important role in the process of tumor development and metastasis. Epithelial-mesenchymal transition (EMT) refers to the biological process that epithelial cells transfer into mesenchymal phenotype cells and have their characteristics. Currently, most scholars regarded as EMT is an important way of tumor invasion and metastasis.Cytokines which is a component of the tumor microenvironment, also involve in the tumorigenesis and development of various tumors. Some papers already proved that cytokines could induce tumor cells to promote the invasion and metastasis of tumor cells in EMT way. TGF-?1, which is a hot topic in recent years, has been proven to play a very important role in tumor invasion and metastasis. Meanwhile, it also plays similar importa-nt role in HCC.Based on the above background, we hypothesized whether cytokines TGF-?1 in HCC microenvironment might promote HCC cells invasion and metastasis in an EMT way. So far, there was no similar report and then in order to verify our hypothesis, the study mainly included in the following aspects:1.To verify that TGF-?1 can induce EMT-like changes of SMMC-7721 and effect its biological behaviors including cell proliferarion, migration and invasion, the possible molecular mechanisms need to be explored; 2. We design siRNAs for possible signal pathway protein and transfected SMMC-7721 in order to detect the effect on TGF-?1-induced biological behaviors changes including cell proliferarion, migration and invasion; 3. Western blot assay was used to detect the expression of EMT-related proteins and signaling pathway related proteins in order to explore possible molecular mechanism; 4. Collection of clinical HCC tissues and detected the expression of TGF-?1, signaling pathway related proteins and EMT-related proteins by IHC; 5. Collection of clinical pathological data and obtain the relationship between TGF-?1 and HCC clinical covariates and EMT-related markers by statistical analysis.This study is divided into three parts.Part I TGF-?1 promotes the ability of invasion and metastasis in SMMC-7721 by inducing EMT-like changes.Objective To clarify TGF-?1 can induce EMT-like changes of SMMC-7721 and promote cell proliferation, invasion and metastasis.Methods First, the different concentrations of cytokines TGF-?1 (5,10,20 ng/mL) was added to the complete medium and SMMC-7721 hepatoma cells were co-cultured; morphological changes were observed by using an optical microscope, MTT assay was used to detect proliferation index changes, wound-healing assay and transwell chamber assay were used to detect the influence of TGF-?1 on liver cancer cell migration and invasion ability, western blot assay was used to detect the effect of TGF-?1 expression of EMT markers associated protein.Results Different concentrations of cytokine TGF-?1 (5,10,20 ng/mL) and liver cancer cells SMMC-7721 were co-cultured for 48 h. After that hepatoma cell morphology changed significantly, the cells gradually become loose from the tight space, the cells from cubic gradually out feelers to fibroblast-like transformation results suggest that TGF-?1 can induce liver cancer cells EMT-like change in a concentration dependent manner. MTT results showed that:TGF-?1 can significantly promote hepatoma SMMC-7721 cell proliferation, and having a time- and dose-dependent manner. Wound-healing assay and transwell chamber assay results showed that:TGF-?1 can significantly enhance the hepatoma cell SMMC-7721 in vitro migration and invasion. Western blot assay detected EMT associated marker protein expression, the results show:the expression of epithelial cell marker protein E-cadherin of hepatoma cells SMMC-7721 was down-regulated by TGF-?1, the expression of mesenchymal cell marker protein Vimentin and signaling pathway related protein p-catenin were up-regulated, these results suggest that we may be TGF-?1 promote EMT-like change of liver cancer cells through Wnt/?-catenin signaling pathway, thereby promoting change in the biological behavior of hepatocellular carcinoma cell proliferation, migration and invasion.Conclusions TGF-?1 could promote cancer cell SMMC-7721 proliferation, invasion and metastasis through inducing EMT-like changes.Part II Down-regulated P-catenin could inhibit the proliferation, invasion and metastasis of liver cancer cells SMMC-7721 induced by TGF-?1.Objective To clarify whether siRNA-?-catenin could inhibit the proliferation, invasion and metastasis SMMC-7721 induced by TGF-?1.Methods First Western blot experiments were used to detect the interference effect of siRNA-?-catenin; hepatoma cells were transfected with control plasmid and siRNA-?-catenin when added cytokine TGF-?1 were co-cultured, and. then used MTT, wound-healing assay, transwell experiments used to detect cell proliferation, migration, invasion changes; western blot assays were used to detect EMT-related markers after transfection of siRNA-?-catenin meanwhile added cytokine TGF-?1.Results siRNA-?-catenin significantly down-regulated expression of ?-catenin protein in liver cancer cell SMMC-7721; MTT results showed that siRNA-?-catenin transfected hepatoma cells can significantly inhibit TGF-?1-induced promotion of liver cancer cell proliferation; cell scratch experiment and Transwell experiments also confirmed transfected with siRNA-?-catenin in hepatoma cells can be partially resistant to TGF-?1-induced promote cell migration and invasion; after Western blot results showed that siRNA-?-catenin transfected hepatoma cells, can inhibition of TGF-?1-induced EMT-like changes, showing the expression of EMT-related marker proteins. These results suggest that ?-catenin might be a key protein in TGF-?1-induced liver cancer cells EMT-like changes.Conclusions Down-regulated?-catenin could inhibit the proliferation, invasion and metastasis of liver cancer cells and the possible mechanism was Wnt/?-catenin signal pathway.Part III Highly expression of TGF-?1 might be a potential indicator for HCC.Objective To detecting the expression of TGF-?1, ?-catenin and EMT-related protein in HCC and determine the relationship with their clinicopathological data.Methods Using Immunohistochemistry test to identify the expression of TGF-?1, P-catenin, E-cadherin and vimentin in the liver cancer tissue and then according to the stainning degree of TGF-?1, we divided 62 patients into high and low expression group and then analyzed their date with ?-catenin and EMT-related protein and clinicopathological data.Results TGF-?1 expression mainly loated in liver cancer cell cytoplasm and high expressed in 48.39%(30/62) HCC samples. We also found that (?) expression was accompanied with decreased expression of E-cadherin and increase expression of P-catenin and vimentin.62 patients were divided into high TGF-?1 expression group (30/62) and low expression group (32/62) and high (?)-expression was negatively correlated with the expression of E-cadherin (4/30,13.33%, P< 0.001) and positively correlated with the expression of ?-catenin and vimentin (29/30,96.67%; 27/30,90%, P<0.001). Further study with the patient's pathology and clinical data, we inferred that the TGF-?1 expression was significantly correlated with tumor size (P= 0.000), vascular invasion (P= 0.014), AFP level (P= 0.043), tumor number (P= 0.008) and differentiation degree (P= 0.007).Conclusions TGF-?1 expressed was associated with increased expression of ?-catenin and Vimentin, decreased expression of E-cadherin. These results suggest that TGF-?1 might induce liver cancer cells present EMT-like changes by Wnt/?-catenin signaling pathway, TGF-?1 may become an new target of HCC clinical intervention in future.Summary TGF-?1 could induce EMT-like changes of liver cancer cell SMMC-7721 and effect its biological behavior including proliferation, migration, invasion. The mechanism may be through the Wnt/?-catenin signaling pathway. Clinical trials further demonstrated, TGF-?1 expression in hepatocellular carcinoma and correlation with ?-catenin signaling pathway proteins and EMT markers associated protein. TGF-?1 expression and tumor size, number of tumors, AFP level, degree of differentiation and whether vascular invasion of liver cancer clinicopathological parameters and other obvious correlation. |
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