TWIST Regulates TNF-α-Induced Epithelial-Mesenchymal Transition In Human Hepatoma Cell Lines

BackgroundHepatocellular carcinoma (HCC) is one of the most serious health problems worldwide. In the most common malignant tumors, the incidence of HCC in men and women are the sixth and ninth, respectively. The incidence of HCC has been increasing in recent years. The main reasons are that the early symptoms of HCC are not obvious and it progresses quickly, the majority has progressed to middle-late when diagnosed where there have been the distant spread and metastasis. The clinical challenge is to identify the metastatic potential of patients in order to choose a more suitable therapeutic schedule. Therefore, it is of great clinical significance to explore the etiology and pathogenesis of HCC, to study the molecular mechanisms involved in HCC recurrence, metastasis and metastasis, to look for new diagnostic markers. It can also provide a strong theoretical basis for the development of new therapeutic strategies.Epithelial-mesenchymal transition (EMT) refers to the phenomenon of epithelial cells in embryogenesis and pathological state transforming into mesenchymal-like cells with mesenchymal features, which plays a key role in embryonic development. More and more evidence shows that: in tumors, EMT is a key step to promote cell migration, tumor invasion and metastasis. The role of multiple transcription factors including TWIST is crucial in the process of regulation of EMT. Recent studies have shown that as a key regulator of EMT, TWIST plays an important role in tumor invasion and metastasis. EMT typically occurs in tumor invasion front (tumor-stromal boundary), activating by cell signaling in the microenvironment. While infiltrating inflammatory cells are the main factors that effect on EMT in the tumor microenvironment. Infiltrating inflammatory cells including macrophages are able to produce a variety of growth factors and cytokines, and promote tumor angiogenesis, extracellular matrix degradation, invasion and metastasis through a variety of mechanisms. TNF-a is an important proinflammatory cytokines which plays an important role in tumor progression. The constitutive expression of TNF-α in the tumor microenvironment is a common feature of many malignant tumors, the expression of which is closely related to the poor prognosis of tumor patients. This study was designed to investigate the role of TWIST protein in inflammatory cytokine TNF-a-mediated hepatocellular carcinoma cell migration, invasion, and metastasis. This will help us understand the molecular mechanisms of tumor progression and metastasis promoted by inflammatory microenvironment, and provides experimental evidence of improving the understanding of tumor biology and the development of new tumor therapeutic intervention.ObjectiveTo investigate the role of TWIST protein in inflammatory cytokineTNF-α-mediated hepatocellular carcinoma cell migration, invasion, and metastasis. This will help us understand the molecular mechanisms of tumor progression and metastasis promoted by inflammatory microenvironment, and provides experimental evidence of improving the understanding of tumor biology and the development of new tumor therapeutic intervention.Methods(1) qRT-PCR analyses were used to detect and GeNorm, NormFinder, and BestKeeper was used to analyze stable reference genes in human hepatoma cell lines after TNF-a treatment. To choose the reliable reference gene for normalizing qRT-PCR data in human hepatoma cell lines following TNF-a treatment.(2) Western blotting was used to detect the expression level of TWIST protein of human hepatoma cell line BEL-7402in different concentrations and time points after TNF-a treatment. The change of TWIST mRNA was detected by qRT-PCR in different time points. Transwell invasion and migration test was used for the detection of TNF-a on migration and invasion of BEL-7402cells. Western blotting was applied to test the effects of TNF-a on BEL-7402cells EMT. To study the effects of TNF-a on TWIST protein expression, invasion and metastasis and EMT in BEL-7402cells.(3) Western blotting was used to detect the effect of TNF-a on the signal transduction pathway of TWIST protein expression in BEL-7402cells. The expression of TWIST protein was detected by Western blotting after interfered with the use of RelA-siRNA. To determine if the expression of TWIST was regulated by TNF-a through NF-кB signaling pathway.(4) pGPU6-TWIST targeted TWIST and pGPU6-Scrambled vector as negative control was constructed and transfected into the invasive human hepatoma cell line BEL-7402. The TWIST inhibition efficiency was examined by Western blotting and qRT-PCR, respectively. Select the pGPU6-TWIST (si-TWIST) hepatoma cell model which has the highest TWIST inhibition efficiency through G418screening. The expression of EMT-associated protein (E-cadherins vimentin) in pGPU6-TWIST (si-TWIST)、pGPU6-Scrambled(si-Scrambled)and BEL-7402was detected by Western blotting. The invasion and migration was detected by Transwell invasion and migration test in pGPU6-TWIST (si-TWIST)> pGPU6-Scrambled (si-Scrambled) and BEL-7402. To confirm that the transcription factor TWIST is a key molecule, with which TNF-a can promote the occurrence of EMT, invasion and migration in human hepatoma cells.(5) Animal studies:Nude mice HCC tumors and metastases model was first established by cell suspension inoculation. The nude mice were divided into4groups (Subcutaneous BEL-7402group, Subcutaneous pGPU6-TWIST group, Intraperitoneal BEL-7402group, Intraperitoneal pGPU6-TWIST group), given TNF-a by subcutaneous or intraperitoneal injection respectively every other day. We regularly observed the mice and tumor growth, measured the tumor long diameter (a), the short diameter (b), and calculated the tumor volume according to the formula V=ab2/2. Mice were killed38days later. In vivo, we further confirmed that the transcription factor TWIST is a key molecule, with which TNF-a can promote the occurrence of EMT, invasion and migration in human hepatoma cells.Results (1) TBP can be used as a reference gene to normalize the experimental data for normalizing qRT-PCR data in human hepatoma cell lines following TNF-a treatment analyzed by three independent algorithms:geNorm, NormFinder, and BestKeeper.(2) The expression of TWIST protein and TNF-a showed a dose-and time-dependent manners. The expression of TWIST mRNA was not significantly changed in human hepatoma cell line BEL-7402following lOng/ml TNF-a treatment in different time points. TNF-a promote the occurrence of invasion, migration and EMT in BEL-7402cells.(3) TNF-a induce the expression of TWIST protein in human hepatoma cells by regulating NF-кB signaling transduction pathways.(4) si-TWIST inhibits cell invasion, migration and EMT in human hepatoma cells following TNF-a treatment.(5) The rate of tumor growth and volume of tumor in subcutaneous pGPU6-TWIST group was significantly lower than that of subcutaneous BEL-7402group. The dissemination of tumor and ascites formation in intraperitoneal pGPU6-TWIST group was significantly lower than that of Intraperitoneal BEL-7402group.Conclusions(1) It is necessary to screen the candidate reference gene in specific experimental conditions. We recommend TBP as a reference gene to normalize the experimental data under the experimental conditions. Our results also provide a theoretical basis for researchers who are interested in studying the role of different cytokines in different tumors.(2) Inflammatory cytokine TNF-a might promote the invasion, metastasis and EMT of human hepatoma cells by activating TWIST via the NF-кB signal transduction pathway.Figure41, table8, reference84.