摘要
背景
肝细胞肝癌(hepatocellular carcinoma, HCC)是全世界最严重的健康问题之一。在最常见的恶性肿瘤中,HCC的发病率在男性和女性中分别为第六位和第九位。近几年HCC的发病率却一直在增加。其中主要原因是HCC的早期症状不明显,病情进展快,确诊时多数已进展至中晚期,已发生了远处的扩散和转移。临床的挑战是能够提前识别患者转移的潜能,从而选择更为合适的治疗方案。因此,探讨HCC的病因、发病机制,深入了解参与HCC复发、转移和转移的相关分子机制,寻找新的诊断标志物具有重大的临床意义,并为发展新的治疗策略提供有力的理论依据。上皮间质转化(Epithelial-mesenchymal transition, EMT)是指上皮细胞在胚胎发生及病理状态下,转化为具有间充质特点的间叶样细胞的现象,在胚胎发育中起着关键的作用。越来越多的证据表明:在肿瘤中,EMT是促进细胞迁移、肿瘤侵袭和转移的关键步骤。而包括TWIST在内的多个转录因子在EMT的调节中所起的作用至关重要。研究表明,TWIST作为EMT的关键调控因子,对肿瘤的侵袭和转移有重要作用。EMT通常发生在肿瘤的侵袭前沿(肿瘤-基质边界),由来自微环境中的细胞信号启动,而浸润炎性细胞是肿瘤微环境中对EMT起作用的主要因素。包括巨噬细胞在内的所有的浸润炎性细胞能够产生多种生长因子和细胞因子,并可通过多种机制促进肿瘤的血管生成、细胞外基质降解、侵袭和转移。TNF-α是重要的炎性细胞因子,在肿瘤进展中发挥重要作用。肿瘤微环境中TNF-α的组成性表达是许多恶性肿瘤的共同特征,其表达与肿瘤患者的预后不良密切相关。本研究拟探讨TWIST蛋白在炎症因子TNF-α介导的肝癌细胞的迁移、侵袭、转移中的作用。这将为我们深入理解炎症微环境促进肿瘤进展及转移的分子机制,完善对肿瘤生物学的认识和发展新的肿瘤治疗干预手段提供实验依据。
目的
探讨TWIST蛋白在炎症因子TNF-α介导的肝癌细胞的迁移、侵袭、转移中的作用,为我们深入理解炎症微环境促进肿瘤进展及转移的分子机制,完善对肿瘤生物学的认识和发展新的肿瘤治疗干预手段提供理论基础和实验依据。
方法
1.采用荧光定量PCR (qRT-PCR)方法检测和GeNorm、 NormFinder、BestKeeper软件分析在TNF-α作用下,肝癌细胞中稳定表达的内参基因,以推荐在细胞因子TNF-α处理肝癌细胞这个特定的实验条件下对实验数据进行标准化的内参基因。
2.通过Western blotting方法检测肝癌细胞BEL-7402在不同浓度及时间点TNF-a作用后TWIST蛋白表达水平的差异;利用qRT-PCR检测10ng/mlTNF-α作用后不同时间点TWIST mRNA的变化;利用Transwell侵袭和迁移试验检测TNF-α对肝癌细胞侵袭迁移能力的影响;通过Western blotting方法检测TNF-α对肝癌细胞EMT的影响;研究TNF-α对肝癌细胞BEL-7402TWIST蛋白的表达、侵袭转移及EMT的影响。
3.运用Western blotting方法检测TNF-α影响肝癌细胞BEL-7402中TWIST蛋白表达的信号转导通路;利用针对NF-кB的RelA-siRNA特异性的干扰其作用,Western blotting方法检测干扰后TWIST蛋白的表达;以确定TNF-α是否通过NF-кB信号通路调控TWIST的表达。
4.构建针对TWIST的RNAi质粒(pGPU6-TWIST-782. pGPU6-TWIST-812)和阴性对照质粒(pGPU6-Scrambled)并转染具有侵袭能力的人肝癌细胞系BEL-7402细胞,采用Western blotting和qRT-PCR检测TWIST的抑制效率,选择TWIST抑制效率最高的经G418筛选得到稳定TWIST表达抑制的肝癌细胞模型pGPU6-TWIST (si-TWIST)。采用Western blotting检测转染组pGPU6-TWIST (si-TWIST)、对照组pGPU6-Scrambled (si-Scrambled)及BEL-7402未转染组三组细胞中EMT相关蛋白(E-cadherin、vimentin)的表达;采用Transwell侵袭和迁移试验检测转染组pGPU6-TWIST (si-TWIST)、对照组pGPU6-Scrambled (si-Scrambled)及BEL-7402未转染组三组细胞侵袭迁移能力的影响,以证实转录因子TWIST是TNF-α能够促进肝癌细胞发生EMT和侵袭迁移的关键分子。
5.动物实验研究:采用细胞悬液接种法,将pGPU6-TWIST和BEL-7402细胞种植于裸鼠皮下及腹腔内,建立裸鼠肝癌细胞移植瘤和转移瘤模型。裸鼠分为4组,分别为:皮下BEL-7402组、皮下pGPU6-TWIST组、腹腔BEL-7402组、腹腔pGPU6-TWIST组。分别于皮下及腹腔给予TNF-α,隔日腹腔注射,定期观察裸鼠及肿瘤生长情况,测量肿瘤的长径(a)、短径(b),按公式V=ab2/2计算肿瘤的近似体积,38天后处死裸鼠,病理观察肿瘤的生长、浸润及转移情况,在体内进一步证实转录因子TWIST是TNF-α能够促进肝癌细胞发生EMT和侵袭迁移的关键分子。
结果
1.经GeNorm、NormFinder、BestKeeper软件分析在细胞因子TNF-α处理肝癌细胞特定的实验条件下,TBP可以作为对实验数据进行标准化的内参基因。
2. TWIST蛋白的表达与TNF-α呈现出剂量和时间的依赖性。而在10ng/ml的TNF-α作用不同的时间后,BEL-7402细胞中TWIST的mRNA的表达并没有明显的改变。TNF-α促进肝癌细胞侵袭和迁移及EMT的发生。
3. TNF-α通过调节NF-кB信号转导通路进而诱导人肝癌细胞TWIST蛋白的表达。
4. si-TWIST抑制TNF-α诱导的肝癌细胞侵袭迁移能力和EMT的发生。
5.裸鼠成瘤结果显示:皮下pGPU6-TWIST组在肿瘤生长速度、体积均明显低于皮下BEL-7402组;腹腔pGPU6-TWIST组在肿瘤播散及腹水形成也均明显低于腹腔BEL-7402组。
结论
1.在特定的实验条件下对候选的内参基因进行筛选是非常必要的。本实验我们推荐TBP作为内参基因对实验数据进行标准化。我们的实验结果也为那些有兴趣研究不同的细胞因子在不同的肿瘤中所起的作用的研究人员提供了理论基础。
2.炎症细胞因子TNF-α可能通过依赖NF-кB信号转导通路激活转录因子TWIST从而促进肝癌细胞发生EMT和侵袭迁移。
图41幅,表8个,参考文献84篇
Background
Hepatocellular 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.
Objective
To 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.
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