Merlin/NF2蛋白在原发性肝细胞癌中对肝纤维化调节机制研究
摘要
目的：原发性肝细胞性肝癌(hepatocellular carcinoma, HCC)是我国男性的第二大死亡原因、女性的第五大死亡原因。在世界范围内每年的发病人数约为50万人,其中绝大多数病例出现在发展中国家,尤其是中国、东亚和非洲国家的发病率最高。
肝纤维化是肝脏组织发生持续损伤,从而造成组织发生反复修复反应时,细胞外基质(extracellular matrix, ECM)合成降解以及沉积不平衡而引起的病理过程,是慢性肝病的主要病理特征,也是进一步向肝硬化发展的主要环节。
肝纤维化与肝癌的发生有着密切的关系。肝纤维化发展到晚期即为肝硬化。在我国,肝癌病人80%以上合并有不同程度的肝硬化。在肝硬化中发生肝癌者15%-25%,一般为20%左右,大多数肝癌是在肝炎后肝硬化基础上发生的。
NF2基因是神经纤维瘤病Ⅱ型(neurofibromatosis type2, NF2)的一个关键基因,在很多脑瘤中表达缺失。NF2基因编码的Merlin蛋白属于蛋白4.1超家族成员之一。Merlin是连接细胞膜和皮层细胞骨架的主要分子,它一端结合骨架分子,另一端则和细胞膜的许多受体分子结合；因此,通过这些固定和调节作用,可以精细的调控受体分子复合物在细胞膜上的丰度和分布。Merlin可以控制细胞膜表面可以利用表皮生长因子受体(epidermal growth factor receptor, EGFR)的数量。在生长很密集,相互接触的培养细胞中Merlin可以阻止已经和配体结合的EGFR的内化作用,使EGFR信号通路不能持续激活,即表现为接触性抑制。而在缺失Merlin表达的哺乳动物细胞中,即使细胞密度很高,也可以检测到EGFR内化作用以及EGFR通路的持续激活,即表现为接触性抑制的丧失。
Merlin和HCC的发生具有密切的关系。在最近的研究中,基因剔除小鼠动物模型的建立,证明了Merlin/NF2基因蛋白在HCC的发生中具有关键的抑制作用。NF2(-/-)完全敲除的小鼠胚胎不能成活,说明NF2基因是小鼠胚胎发育过程中所必需的。NF2(+/-)杂合型基因小鼠,即杂合性缺失NF2基因,有自发成瘤,并具有很高的肿瘤转移率。其中,骨肉瘤发生率极高(63%),其次是肝细胞癌(11%)。近期研究显示,在肝脏特异性NF2(-/-)基因敲除鼠中,在发育的基因剔除小鼠会发生肝祖细胞(即肝卵圆细胞,OCs)过度增生,并最终基本全部发生肝细胞性肝癌(HCC)和胆管细胞癌(CC),且转移到肺。在成年小鼠中特异性敲除肝脏NF2(-/-)基因,肝脏的OCs细胞仅有轻度增殖,并且基本不发生HCC；而对这些小鼠肝脏刺激后,比如实行肝叶部分切除后(PHx) OCs细胞增殖明显,并诱发HCC和CC。对NF2(-/-)小鼠使用EGFR抑制剂Erlotinib,可以明显抑制OCs的增殖。
Merlin在肝癌的发生发展中,被认为是一个很强的抑制因素。Merlin对于肝纤维化发生的影响至今尚无人研究。而在我国,肝炎后肝硬化的肝癌发生率很高。那么Merlin是否可以抑制肝纤维化的发生?Merlin是通过何种机制抑制肝纤维化的?因此深入研究了解Merlin对肝癌和肝纤维化的影响关系,对于肝癌、肝纤维化的发生发展机制,以及肝癌的防治方面,都提出了新的指导和新的思路。
方法：我们利用qPCR和Western blot方法检测四种肝癌细胞系：FOCUS、MHCC-97H、HepG2、SMMC-7721以及人正常肝细胞株L-02中TGF-β1的表达情况。采用非接触式共培养技术(Transwell小室共培养)共培养FOCUS细胞和肝星状细胞系LX-2细胞。荧光光定量PCR和Western blot检测肝细胞是否对肝星状细胞有激活刺激作用。利用Western blot方法检测了四种肝癌细胞系：FOCUS、MHCC-97H、 HepG2、SMMC-7721以及人正常肝细胞株L-02中Merlin以及TGF-β1的表达情况。利用瞬时转染、Western blot方法研究Merlin对肝癌细胞株TGF-β1表达的影响,以及Merlin对EGFR的影响；以及对肝星状细胞TGF-β/Smad信号通路和ECM合成的影响。
结果：利用qPCR和Western blot方法检测四种肝癌细胞系：FOCUS、MHCC-97H、HepG2、SMMC-7721以及人正常肝细胞株L-02中TGF-β1的表达情况。结果显示：FOCUS细胞中TGF-β1的表达水平很高,其次为MHCC-97H,再次为SMMC-7721, HepG2最低；但是在人正常肝细胞株L-02中无论在mRNA还是在蛋白水平都很难检测到TGF-β1的表达。通过免疫组织化学检测了20例肝癌组织标本以及10例正常肝组织中TGF-β1的表达水平。在肝细胞癌组织中TGF-β1强阳性率为55%(11例),弱阳性率为40%(8例),阴性表达1例。在肝细胞癌组织中,TGF-β1表达于癌细胞胞浆以及癌组织的间质中。而在所有的正常肝组织中,正常肝细胞都没有检测到TGF-β1的表达,而在肝组织的内皮细胞中可以检测到TGF-β1的强阳性表达。这些结果证明了TGF-β1在肝细胞癌中表达水平明显增高,肝癌细胞可以自身分泌TGF-β1。
采用非接触式共培养技术(Transwell小室共培养)共培养FOCUS细胞和肝星状细胞系LX-2细胞。荧光光定量PCR和Western blot证实：肝癌细胞FOCUS可以分泌TGF-β1,激活LX-2细胞的TGF-β1/Smad通路,促进LX-2细胞合成细胞外基质(ECM)。利用Western blot方法检测了四种肝癌细胞系：FOCUS、MHCC-97H、 HepG2、SMMC-7721以及人正常肝细胞株L-02中Merlin以及TGF-β1的表达情况。两者表达情况呈相反的趋势,即Merlin表达高的细胞株中TGF-β1的表达低甚至缺失,而Merlin表达缺失的细胞株中TGF-β1的表达则很高。
在相同的细胞密度下,分别转染pCDNA-NF2质粒和pCDNA3.1(+)空质粒。在转染处理组中,随着pCDNA-NF2质粒量的依次增加,Merlin的表达依次增加,而TGF-β1表达量则随着Merlin表达的依次增加而逐渐减弱；空载体组中Merlin表达均缺失,TGF-β1表达水平基本一致。这说明Merlin调控了TGF-β1的表达,恢复Merlin的表达可以减低TGF-β1的表达水平。
在相同的细胞密度下,分别转染不同浓度的pCDNA-NF2质粒和pCDNA3.1(+)空质粒至FOCUS细胞。将转染了各质粒的FOCUS细胞与LX-2细胞进行共培养。我们发现随着上室FOCUS细胞中Merlin的表达水平依次增高,下腔LX-2细胞TGF-β1/Smad通路激活依次减弱,如p-Smad2, p-Smad3依次减弱；同时合成细胞外基质(ECM)依次减弱,即Fibronectin, Collagen Ⅰ, Collagen Ⅲ表达依次减弱。这说明在FOCUS细胞中恢复Merlin表达可以减少TGF-β1的表达,而使肝星状细胞合成ECM减少,从而抑制了肝纤维化的发生。
同时Merlin高表达的转染组可以明显抑制表皮生长因子受体(EGFR)的激活,从而抑制TGF-β1的表达；而空载体对照组在EGF刺激后,表皮生长因子受体(EGFR)被激活,TGF-β1的表达随EGF的浓度依次明显增高。结果证实了Merlin可以抑制表皮生长因子受体信号通路,从而下调TGF-β1的表达分泌。随着肝星状细胞LX-2细胞数目的依次增多,Merlin表达依次增高。在这种线性细胞密度下,在TGF-β1刺激下,LX-2细胞TGFBR Ⅰ型受体、Smad2磷酸水平依次减低,TGF-β/Smad信号通路受到抑制；而在同样的条件下,由于肝细胞癌细胞FOCUS缺失Merlin表达,无论细胞数目的多少FOCUS细胞都不表达Merlin。在线性细胞密度下,使用TGF-β1刺激,FOCUS细胞的TGF-β/Smad信号通路始终保持持续激活状态。在肝星状细胞LX-2细胞中,低细胞密度下Merlin表达低；高细胞密度下Merlin表达高。在这两种细胞密度下,使用TGF-β1刺激,低细胞密度的LX-2细胞与高密度的情况相比TGF-β/Smad信号通路被显著激活；其合成ECM能力也大大增加。同时我们在低细胞密度的情况下,转染NF2的真核表达载体,提高Merlin的表达水平,再使用TGF-β1刺激,其TGF-β/Smad信号通路明显受到抑制；ECM合成受到明显抑制。另一方面,在高细胞密度的情况下,转染NF2的shRNA干扰载体,降低Merlin的表达水平,再使用TGF-β1刺激,其TGF-β/Smad信号通路抑制作用解除；ECM合成增加。
结论：综上所述,本研究通过检测各肝癌细胞株以及肝细胞癌患者中的TGF-β1蛋白表达水平,明确了在一些肝癌细胞株中以及肝癌患者中TGF-β1蛋白表达上调；肝癌细胞可以自身分泌TGF-β1,激活肝星状细胞的TGF-β1/Smad通路,促进肝星状细胞合成细胞外基质,促进肝纤维化的发生。Merlin可以抑制表皮生长因子受体信号通路,从而下调TGF-β1的表达分泌。在肝癌细胞中恢复Merlin表达可以减少TGF-β1的表达,而使肝星状细胞合成ECM减少,从而抑制了肝纤维化的发生。同时,Merlin也可以负调控TGF-β/Smad信号通路,从而抑制肝纤维化。
Objective:Hepatocellular carcinoma (HCC), one of the most common fatal malignancies in China and many other countries in Asia and Africa, is the leading cause of cancer mortality in China.
Liver fibrosis is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. It is a critical stage in cirrhosis development which the end stage consequence of fibrosis.
Liver fibrosis, also Cirrhosis is very closely related to the occurrence of hepatocellular carcinoma. More than80percent of patients in China who develop liver cancer do so because they have cirrhosis of the liver.15%-25%, usually about20%cirrhosis patients develop liver cancer. The majority of liver cancer occurred on the base of posthepatitic cirrhosis.
Chronic injury leading to fibrosis in liver occurs in response to a variety of insults, including viral hepatitis (especially hepatitis B and C), alcohol abuse, drugs, metabolic diseases due to overload of iron or copper, autoimmune attack of hepatocytes or bile duct epithelium, or congenital abnormalities. The immune system is activated and the repair process swings into gear because of these insults. The injury or death (necrosis) of hepatocytes stimulates inflammatory immune cells to release cytokines, growth factors, and other chemicals. These chemical messengers direct support cells in the liver called hepatic stellate cells (HSCs)to activate and produce collagen, glycoproteins (such as fibronectin), proteoglycans, and other substances. These substances are deposited in the liver, causing the build-up of extracellular matrix (nonfunctional connective tissue). HSCs play a very important role in the process of liver fibrosis. Cytokines are also a critical factor in activated liver fibrosis. They interacted HSCs, ECM, and also interacted themselves, which constituted network-like control networks. TGF-β1is the most effective cytokine to improve hepatic fibrosis, which can inhibit the proliferation of liver cells and stimulate HSC activation, and promote the production of ECM and regulate the apoptosis of hepatocytes.
The tumor suppressor NF2, which is inactivated in the familial cancer syndrome Neurofibromatosis type2, encodes for Merlin, a member of the Ezrin/Radixin/Moesin (ERM) family of proteins. Merlin and the closely related ERM proteins form a subgroup of the Protein4.1superfamily. NF2is a predominantly inherited disorder characterized by the development of schwann cell tumors and other brain tumors. Mutations or the loss of heterozygosity of the NF2locus has been detected in various tumors of the nervous system, such as schwannomas, meningiomas and ependymomas. Merlin is composed of an N-terminal FERM domain, an ensuing a-helical domain and a C-terminal domain that includes an actin-binding module in the ERM proteins but not in Merlin. The FERM domain adopts a cloverleaf structure composed of three interdependent lobes and seems designed to bring multiple proteins together at the membrane. This is well supported by the long list of proteins that have been reported to interact with the Merlin/ERM FERM domain, including transmembrane receptors such as CD43and CD44, and the tandem PDZ-domain-containing adapters Na+/H+exchanger regulatory factor (NHERF)-1and-2, which in turn associate with a variety of membrane receptors. The FERM domain can also associate with regulators of Rho GTPase signaling. The a-helical and C-terminal portions of Merlin can fold back and envelop the FERM domain, masking all known sites of protein interaction in both the FERM and the C-terminal domains, including the ERM actin-binding domain. For Merlin, evidence from studies of mammalian cells indicates that this self-associated'closed'form is the active growth-suppressing conforma-tion. In fact, many tumor-derived missense mutations are predicted to disrupt the closed conformation.
Merlin is a membrane/cytoskeleton-associated protein that can link the membrane proteins to the underlying cortical cytoskeleton and in controlling the distribution of and signaling from membrane receptors. In mammalian cells, Merlin can block the internalization of ligand-bound EGFR specifically in contacting (confluent) cells in culture. In fact, whereas wildtype, Merlin-expressing cells normally downregulate EGFR signaling at high cell density, Merlin-deficient cells fail to do so and also fail to undergo contact-dependent inhibition of proliferation—a phenol-type reversed by pharmacologic inhibition of EGFR.
Merlin and the incidence of HCC have a close relationship. In a recent study, the gene knockout mouse animal model, demonstrated Merlin/NF2protein is the critical inhibition in HCC. NF2homozygous mutant at the mouse leads to embryonic failure immediately before gastrulation, indicating that merlin function is critical at a very early stage in development. Nf2+/-mice developed a variety of malignant tumors later in life.63%developed osteosarcoma, followed by hepatocellular carcinoma (11%). Targeted deletion of NF2in the mouse liver results in massive hepatic enlargement due to OCs hyperproliferation and All mice eventually developed frank liver tumors then metastasized to lung. In adult mice which the liver specific NF2(-/-) gene knockout, liver OCs cell proliferation mildly, and basically does not occur HCC; but after stimulating the liver of these mice, such as partial hepatectomy (PHx), OCs cells was significant proliferation and induce HCC and CC. NF2(-/-) mice injected EGFR inhibitors Erlotinib, can inhibit the proliferation of OCs.
Merlin was considered to be a strong disincentive in the development of liver cancer. Merlin for the occurrence of liver fibrosis research so far no one has. In China, hepatitis, cirrhosis of the liver cancer rate is high. Then Merlin can inhibit hepatic fibrosis? Merlin is the mechanism through which inhibition of liver fibrosis? Therefore, in-depth study to understand Merlin on liver cancer and liver fibrosis in relation to liver cancer, liver fibrosis development mechanism, as well as prevention and treatment of liver cancer, have put forward a new direction and new ideas.
Method and Result:We used qPCR and Western blot detect the expression level of TGF-β1in four liver cancer cell lines:FOCUS、 MHCC-97H、HepG2、SMMC-7721and human normal liver cell line L-02. The results showed that:in FOCUS cells, the expression level of TGF-β1is the highest, and followed by the MHCC-97H, SMMC-7721, HepG2is the minimum; However, in normal human liver cell line L-02in both the mRNA or protein levels are difficult to detect the expression of TGF-β1. We detected by immunohistochemistry20cases of HCC specimens and10normal liver tissue levels of TGF-β1expression. TGF-β1in hepatocellular carcinoma strongly positive rate was55%(11cases), weak positive rate was40%(8cases), and negative in1case. TGF-β1expressed in the cytoplasm of cancer cells and stroma in hepatocellular carcinoma tissues. In all of the normal liver tissue, normal liver cells are not detected the expression of TGF-β1, while in liver endothelial cells can detect the strong TGF-β1expression. These results demonstrate that TGF-β1expression in HCC was significantly higher liver cancer cells themselves could secrete TGF-β1.
Non-contact co-culture technique (Transwell chamber co-culture) was used to culture FOCUS cells and liver stellate cell line LX-2cells. Fluorescence quantitative PCR and Western blot confirmed that FOCUS hepatocellular carcinoma cells can secrete TGF-β1, activate TGF-β1/Smad pathway and promote synthesis of extracellular matrix (ECM) in LX-2cells.
We used Western blot to detect the expression of Merlin and TGF-β1in four liver cancer cell lines:FOCUS, MHCC-97H, HepG2, SMMC-7721and human normal liver cell line L-02. The levels of expression of the two proteins are totally opposite. The cell lines with high expression of Merlin expressed low or absent TGF-β1, and Merlin missing cell lines expressed very high TGF-β1.
In the same cell density, FOCUS cells were transfected with pCDNA-NF2plasmid and pCDNA3.1(+) plasmid. In the treatment group that transfected with pCDNA-NF2plasmid, Merlin gradually increased by improved the amount of plasmid, contrast to TGF-β1; but in the vector group, Merlin was always absent and TGF-β1was the same levels. It shows that Merlin controls the expression of TGF-β1, and recovery Merlin can reduce the expression of TGF-β1.
In the same cell density, FOCUS cells were transfected with different concentrations of pCDNA-NF2plasmid and pCDNA3.1(+) plasmid. The FOCUS cells which were transfected with the plasmid and LX-2cells were co-cultured. We found that Merlin in FOCUS cells on the up room was gradually increased depend on the concentration of plasmid, and TGF-β1/Smad pathway of LX-2cells in the inferior well was gradual deactivation, such as p-Smad2, p-Smad3were declined; at the same time the synthesis of extracellular matrix (ECM) was reduced, such as the Fibronectin, Collagen Ⅰ, Collagen Ⅲ were also declined. This shows that resumption of Merlin in FOCUS cells can reduce the TGF-β1expression, promote to reduce the synthesis of ECM in hepatic stellate cell and inhibit liver fibrosis.
The high expression of Merlin transfected with plasmid can significantly inhibit the epidermal growth factor receptor (EGFR) activation, thereby inhibit the expression of TGF-β1; and in the vector control under the EGF stimulation, the epidermal growth factor receptor (EGFR) is activated, TGF-β1expression along with the concentration of EGF was significantly increased in turn. The results confirmed that Merlin can inhibit the epidermal growth factor receptor signaling pathway, which reduced the secretion of TGF-β1.
With the LX-2hepatic stellate cells gradually increased the number of cells, Merlin expression also gradually increased. In this linear cell density, in TGF-β1stimulation, the level of phosphated TGFBR Ⅰ receptor and Smad2in LX-2cells gradually reduced, TGF-β/Smad signaling pathway was inhibited. In the same conditions, due to Merlin in cancer cells FOCUS was loss, no matter how much the number of cells, FOCUS cells do not express Merlin. In the different cell density, stimulated by TGF-β1, TGF-β/Smad signaling pathway was activated continuously in FOCUS cells. In low cell density, hepatic stellate cell line LX-2cells expressed low Merlin, and high cell density with high expression of Merlin. In both cell density, stimulated by TGF-β1, low cell density LX-2cells compared with the high density TGF-β/Smad signaling pathway was significantly activated; its ability to greatly increase the synthesis of ECM. Meanwhile, in the low cell density, we transfected pCDNA-NF2to improve the expression level of Merlin, and then stimulated with TGF-β1and its TGF-β/Smad signaling pathway was significantly inhibited, ECM synthesis was significantly inhibited. On the other hand, in the high cell density, transfected shRNA to interfere Merlin, reducing the expression level of Merlin, and then stimulated with TGF-β1and the inhibitions of TGF-β/Smad signaling pathway and ECM synthesis were rescinded.
Conclusion:In summary, this study was to detect TGF-β1protein levels in the HCC cell lines and hepatocellular carcinoma patients, and identified in hepatocellular carcinoma and liver cancer cell lines the TGF-β1protein expression were up-regulated; HCC cells can secrete TGF-β1themselves, and activated hepatic stellate cells TGF-β1/Smad pathway, promote hepatic stellate cells to synthesize extracellular matrix, and promote liver fibrosis. Merlin can inhibit the epidermal growth factor receptor signaling pathway, which reduced the expression of TGF-β1secretion. Restored Merlin in HCC cells can reduce the expression of TGF-β1, inhibit the hepatic stellate cell synthesize ECM, thereby inhibit the occurrence of liver fibrosis. Meanwhile, Merlin can negatively regulate TGF-β/Smad signaling pathway, and suppress liver fibrosis.
肝纤维化是肝脏组织发生持续损伤,从而造成组织发生反复修复反应时,细胞外基质(extracellular matrix, ECM)合成降解以及沉积不平衡而引起的病理过程,是慢性肝病的主要病理特征,也是进一步向肝硬化发展的主要环节。
肝纤维化与肝癌的发生有着密切的关系。肝纤维化发展到晚期即为肝硬化。在我国,肝癌病人80%以上合并有不同程度的肝硬化。在肝硬化中发生肝癌者15%-25%,一般为20%左右,大多数肝癌是在肝炎后肝硬化基础上发生的。
NF2基因是神经纤维瘤病Ⅱ型(neurofibromatosis type2, NF2)的一个关键基因,在很多脑瘤中表达缺失。NF2基因编码的Merlin蛋白属于蛋白4.1超家族成员之一。Merlin是连接细胞膜和皮层细胞骨架的主要分子,它一端结合骨架分子,另一端则和细胞膜的许多受体分子结合；因此,通过这些固定和调节作用,可以精细的调控受体分子复合物在细胞膜上的丰度和分布。Merlin可以控制细胞膜表面可以利用表皮生长因子受体(epidermal growth factor receptor, EGFR)的数量。在生长很密集,相互接触的培养细胞中Merlin可以阻止已经和配体结合的EGFR的内化作用,使EGFR信号通路不能持续激活,即表现为接触性抑制。而在缺失Merlin表达的哺乳动物细胞中,即使细胞密度很高,也可以检测到EGFR内化作用以及EGFR通路的持续激活,即表现为接触性抑制的丧失。
Merlin和HCC的发生具有密切的关系。在最近的研究中,基因剔除小鼠动物模型的建立,证明了Merlin/NF2基因蛋白在HCC的发生中具有关键的抑制作用。NF2(-/-)完全敲除的小鼠胚胎不能成活,说明NF2基因是小鼠胚胎发育过程中所必需的。NF2(+/-)杂合型基因小鼠,即杂合性缺失NF2基因,有自发成瘤,并具有很高的肿瘤转移率。其中,骨肉瘤发生率极高(63%),其次是肝细胞癌(11%)。近期研究显示,在肝脏特异性NF2(-/-)基因敲除鼠中,在发育的基因剔除小鼠会发生肝祖细胞(即肝卵圆细胞,OCs)过度增生,并最终基本全部发生肝细胞性肝癌(HCC)和胆管细胞癌(CC),且转移到肺。在成年小鼠中特异性敲除肝脏NF2(-/-)基因,肝脏的OCs细胞仅有轻度增殖,并且基本不发生HCC；而对这些小鼠肝脏刺激后,比如实行肝叶部分切除后(PHx) OCs细胞增殖明显,并诱发HCC和CC。对NF2(-/-)小鼠使用EGFR抑制剂Erlotinib,可以明显抑制OCs的增殖。
Merlin在肝癌的发生发展中,被认为是一个很强的抑制因素。Merlin对于肝纤维化发生的影响至今尚无人研究。而在我国,肝炎后肝硬化的肝癌发生率很高。那么Merlin是否可以抑制肝纤维化的发生?Merlin是通过何种机制抑制肝纤维化的?因此深入研究了解Merlin对肝癌和肝纤维化的影响关系,对于肝癌、肝纤维化的发生发展机制,以及肝癌的防治方面,都提出了新的指导和新的思路。
方法：我们利用qPCR和Western blot方法检测四种肝癌细胞系：FOCUS、MHCC-97H、HepG2、SMMC-7721以及人正常肝细胞株L-02中TGF-β1的表达情况。采用非接触式共培养技术(Transwell小室共培养)共培养FOCUS细胞和肝星状细胞系LX-2细胞。荧光光定量PCR和Western blot检测肝细胞是否对肝星状细胞有激活刺激作用。利用Western blot方法检测了四种肝癌细胞系：FOCUS、MHCC-97H、 HepG2、SMMC-7721以及人正常肝细胞株L-02中Merlin以及TGF-β1的表达情况。利用瞬时转染、Western blot方法研究Merlin对肝癌细胞株TGF-β1表达的影响,以及Merlin对EGFR的影响；以及对肝星状细胞TGF-β/Smad信号通路和ECM合成的影响。
结果：利用qPCR和Western blot方法检测四种肝癌细胞系：FOCUS、MHCC-97H、HepG2、SMMC-7721以及人正常肝细胞株L-02中TGF-β1的表达情况。结果显示：FOCUS细胞中TGF-β1的表达水平很高,其次为MHCC-97H,再次为SMMC-7721, HepG2最低；但是在人正常肝细胞株L-02中无论在mRNA还是在蛋白水平都很难检测到TGF-β1的表达。通过免疫组织化学检测了20例肝癌组织标本以及10例正常肝组织中TGF-β1的表达水平。在肝细胞癌组织中TGF-β1强阳性率为55%(11例),弱阳性率为40%(8例),阴性表达1例。在肝细胞癌组织中,TGF-β1表达于癌细胞胞浆以及癌组织的间质中。而在所有的正常肝组织中,正常肝细胞都没有检测到TGF-β1的表达,而在肝组织的内皮细胞中可以检测到TGF-β1的强阳性表达。这些结果证明了TGF-β1在肝细胞癌中表达水平明显增高,肝癌细胞可以自身分泌TGF-β1。
采用非接触式共培养技术(Transwell小室共培养)共培养FOCUS细胞和肝星状细胞系LX-2细胞。荧光光定量PCR和Western blot证实：肝癌细胞FOCUS可以分泌TGF-β1,激活LX-2细胞的TGF-β1/Smad通路,促进LX-2细胞合成细胞外基质(ECM)。利用Western blot方法检测了四种肝癌细胞系：FOCUS、MHCC-97H、 HepG2、SMMC-7721以及人正常肝细胞株L-02中Merlin以及TGF-β1的表达情况。两者表达情况呈相反的趋势,即Merlin表达高的细胞株中TGF-β1的表达低甚至缺失,而Merlin表达缺失的细胞株中TGF-β1的表达则很高。
在相同的细胞密度下,分别转染pCDNA-NF2质粒和pCDNA3.1(+)空质粒。在转染处理组中,随着pCDNA-NF2质粒量的依次增加,Merlin的表达依次增加,而TGF-β1表达量则随着Merlin表达的依次增加而逐渐减弱；空载体组中Merlin表达均缺失,TGF-β1表达水平基本一致。这说明Merlin调控了TGF-β1的表达,恢复Merlin的表达可以减低TGF-β1的表达水平。
在相同的细胞密度下,分别转染不同浓度的pCDNA-NF2质粒和pCDNA3.1(+)空质粒至FOCUS细胞。将转染了各质粒的FOCUS细胞与LX-2细胞进行共培养。我们发现随着上室FOCUS细胞中Merlin的表达水平依次增高,下腔LX-2细胞TGF-β1/Smad通路激活依次减弱,如p-Smad2, p-Smad3依次减弱；同时合成细胞外基质(ECM)依次减弱,即Fibronectin, Collagen Ⅰ, Collagen Ⅲ表达依次减弱。这说明在FOCUS细胞中恢复Merlin表达可以减少TGF-β1的表达,而使肝星状细胞合成ECM减少,从而抑制了肝纤维化的发生。
同时Merlin高表达的转染组可以明显抑制表皮生长因子受体(EGFR)的激活,从而抑制TGF-β1的表达；而空载体对照组在EGF刺激后,表皮生长因子受体(EGFR)被激活,TGF-β1的表达随EGF的浓度依次明显增高。结果证实了Merlin可以抑制表皮生长因子受体信号通路,从而下调TGF-β1的表达分泌。随着肝星状细胞LX-2细胞数目的依次增多,Merlin表达依次增高。在这种线性细胞密度下,在TGF-β1刺激下,LX-2细胞TGFBR Ⅰ型受体、Smad2磷酸水平依次减低,TGF-β/Smad信号通路受到抑制；而在同样的条件下,由于肝细胞癌细胞FOCUS缺失Merlin表达,无论细胞数目的多少FOCUS细胞都不表达Merlin。在线性细胞密度下,使用TGF-β1刺激,FOCUS细胞的TGF-β/Smad信号通路始终保持持续激活状态。在肝星状细胞LX-2细胞中,低细胞密度下Merlin表达低；高细胞密度下Merlin表达高。在这两种细胞密度下,使用TGF-β1刺激,低细胞密度的LX-2细胞与高密度的情况相比TGF-β/Smad信号通路被显著激活；其合成ECM能力也大大增加。同时我们在低细胞密度的情况下,转染NF2的真核表达载体,提高Merlin的表达水平,再使用TGF-β1刺激,其TGF-β/Smad信号通路明显受到抑制；ECM合成受到明显抑制。另一方面,在高细胞密度的情况下,转染NF2的shRNA干扰载体,降低Merlin的表达水平,再使用TGF-β1刺激,其TGF-β/Smad信号通路抑制作用解除；ECM合成增加。
结论：综上所述,本研究通过检测各肝癌细胞株以及肝细胞癌患者中的TGF-β1蛋白表达水平,明确了在一些肝癌细胞株中以及肝癌患者中TGF-β1蛋白表达上调；肝癌细胞可以自身分泌TGF-β1,激活肝星状细胞的TGF-β1/Smad通路,促进肝星状细胞合成细胞外基质,促进肝纤维化的发生。Merlin可以抑制表皮生长因子受体信号通路,从而下调TGF-β1的表达分泌。在肝癌细胞中恢复Merlin表达可以减少TGF-β1的表达,而使肝星状细胞合成ECM减少,从而抑制了肝纤维化的发生。同时,Merlin也可以负调控TGF-β/Smad信号通路,从而抑制肝纤维化。
Objective:Hepatocellular carcinoma (HCC), one of the most common fatal malignancies in China and many other countries in Asia and Africa, is the leading cause of cancer mortality in China.
Liver fibrosis is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. It is a critical stage in cirrhosis development which the end stage consequence of fibrosis.
Liver fibrosis, also Cirrhosis is very closely related to the occurrence of hepatocellular carcinoma. More than80percent of patients in China who develop liver cancer do so because they have cirrhosis of the liver.15%-25%, usually about20%cirrhosis patients develop liver cancer. The majority of liver cancer occurred on the base of posthepatitic cirrhosis.
Chronic injury leading to fibrosis in liver occurs in response to a variety of insults, including viral hepatitis (especially hepatitis B and C), alcohol abuse, drugs, metabolic diseases due to overload of iron or copper, autoimmune attack of hepatocytes or bile duct epithelium, or congenital abnormalities. The immune system is activated and the repair process swings into gear because of these insults. The injury or death (necrosis) of hepatocytes stimulates inflammatory immune cells to release cytokines, growth factors, and other chemicals. These chemical messengers direct support cells in the liver called hepatic stellate cells (HSCs)to activate and produce collagen, glycoproteins (such as fibronectin), proteoglycans, and other substances. These substances are deposited in the liver, causing the build-up of extracellular matrix (nonfunctional connective tissue). HSCs play a very important role in the process of liver fibrosis. Cytokines are also a critical factor in activated liver fibrosis. They interacted HSCs, ECM, and also interacted themselves, which constituted network-like control networks. TGF-β1is the most effective cytokine to improve hepatic fibrosis, which can inhibit the proliferation of liver cells and stimulate HSC activation, and promote the production of ECM and regulate the apoptosis of hepatocytes.
The tumor suppressor NF2, which is inactivated in the familial cancer syndrome Neurofibromatosis type2, encodes for Merlin, a member of the Ezrin/Radixin/Moesin (ERM) family of proteins. Merlin and the closely related ERM proteins form a subgroup of the Protein4.1superfamily. NF2is a predominantly inherited disorder characterized by the development of schwann cell tumors and other brain tumors. Mutations or the loss of heterozygosity of the NF2locus has been detected in various tumors of the nervous system, such as schwannomas, meningiomas and ependymomas. Merlin is composed of an N-terminal FERM domain, an ensuing a-helical domain and a C-terminal domain that includes an actin-binding module in the ERM proteins but not in Merlin. The FERM domain adopts a cloverleaf structure composed of three interdependent lobes and seems designed to bring multiple proteins together at the membrane. This is well supported by the long list of proteins that have been reported to interact with the Merlin/ERM FERM domain, including transmembrane receptors such as CD43and CD44, and the tandem PDZ-domain-containing adapters Na+/H+exchanger regulatory factor (NHERF)-1and-2, which in turn associate with a variety of membrane receptors. The FERM domain can also associate with regulators of Rho GTPase signaling. The a-helical and C-terminal portions of Merlin can fold back and envelop the FERM domain, masking all known sites of protein interaction in both the FERM and the C-terminal domains, including the ERM actin-binding domain. For Merlin, evidence from studies of mammalian cells indicates that this self-associated'closed'form is the active growth-suppressing conforma-tion. In fact, many tumor-derived missense mutations are predicted to disrupt the closed conformation.
Merlin is a membrane/cytoskeleton-associated protein that can link the membrane proteins to the underlying cortical cytoskeleton and in controlling the distribution of and signaling from membrane receptors. In mammalian cells, Merlin can block the internalization of ligand-bound EGFR specifically in contacting (confluent) cells in culture. In fact, whereas wildtype, Merlin-expressing cells normally downregulate EGFR signaling at high cell density, Merlin-deficient cells fail to do so and also fail to undergo contact-dependent inhibition of proliferation—a phenol-type reversed by pharmacologic inhibition of EGFR.
Merlin and the incidence of HCC have a close relationship. In a recent study, the gene knockout mouse animal model, demonstrated Merlin/NF2protein is the critical inhibition in HCC. NF2homozygous mutant at the mouse leads to embryonic failure immediately before gastrulation, indicating that merlin function is critical at a very early stage in development. Nf2+/-mice developed a variety of malignant tumors later in life.63%developed osteosarcoma, followed by hepatocellular carcinoma (11%). Targeted deletion of NF2in the mouse liver results in massive hepatic enlargement due to OCs hyperproliferation and All mice eventually developed frank liver tumors then metastasized to lung. In adult mice which the liver specific NF2(-/-) gene knockout, liver OCs cell proliferation mildly, and basically does not occur HCC; but after stimulating the liver of these mice, such as partial hepatectomy (PHx), OCs cells was significant proliferation and induce HCC and CC. NF2(-/-) mice injected EGFR inhibitors Erlotinib, can inhibit the proliferation of OCs.
Merlin was considered to be a strong disincentive in the development of liver cancer. Merlin for the occurrence of liver fibrosis research so far no one has. In China, hepatitis, cirrhosis of the liver cancer rate is high. Then Merlin can inhibit hepatic fibrosis? Merlin is the mechanism through which inhibition of liver fibrosis? Therefore, in-depth study to understand Merlin on liver cancer and liver fibrosis in relation to liver cancer, liver fibrosis development mechanism, as well as prevention and treatment of liver cancer, have put forward a new direction and new ideas.
Method and Result:We used qPCR and Western blot detect the expression level of TGF-β1in four liver cancer cell lines:FOCUS、 MHCC-97H、HepG2、SMMC-7721and human normal liver cell line L-02. The results showed that:in FOCUS cells, the expression level of TGF-β1is the highest, and followed by the MHCC-97H, SMMC-7721, HepG2is the minimum; However, in normal human liver cell line L-02in both the mRNA or protein levels are difficult to detect the expression of TGF-β1. We detected by immunohistochemistry20cases of HCC specimens and10normal liver tissue levels of TGF-β1expression. TGF-β1in hepatocellular carcinoma strongly positive rate was55%(11cases), weak positive rate was40%(8cases), and negative in1case. TGF-β1expressed in the cytoplasm of cancer cells and stroma in hepatocellular carcinoma tissues. In all of the normal liver tissue, normal liver cells are not detected the expression of TGF-β1, while in liver endothelial cells can detect the strong TGF-β1expression. These results demonstrate that TGF-β1expression in HCC was significantly higher liver cancer cells themselves could secrete TGF-β1.
Non-contact co-culture technique (Transwell chamber co-culture) was used to culture FOCUS cells and liver stellate cell line LX-2cells. Fluorescence quantitative PCR and Western blot confirmed that FOCUS hepatocellular carcinoma cells can secrete TGF-β1, activate TGF-β1/Smad pathway and promote synthesis of extracellular matrix (ECM) in LX-2cells.
We used Western blot to detect the expression of Merlin and TGF-β1in four liver cancer cell lines:FOCUS, MHCC-97H, HepG2, SMMC-7721and human normal liver cell line L-02. The levels of expression of the two proteins are totally opposite. The cell lines with high expression of Merlin expressed low or absent TGF-β1, and Merlin missing cell lines expressed very high TGF-β1.
In the same cell density, FOCUS cells were transfected with pCDNA-NF2plasmid and pCDNA3.1(+) plasmid. In the treatment group that transfected with pCDNA-NF2plasmid, Merlin gradually increased by improved the amount of plasmid, contrast to TGF-β1; but in the vector group, Merlin was always absent and TGF-β1was the same levels. It shows that Merlin controls the expression of TGF-β1, and recovery Merlin can reduce the expression of TGF-β1.
In the same cell density, FOCUS cells were transfected with different concentrations of pCDNA-NF2plasmid and pCDNA3.1(+) plasmid. The FOCUS cells which were transfected with the plasmid and LX-2cells were co-cultured. We found that Merlin in FOCUS cells on the up room was gradually increased depend on the concentration of plasmid, and TGF-β1/Smad pathway of LX-2cells in the inferior well was gradual deactivation, such as p-Smad2, p-Smad3were declined; at the same time the synthesis of extracellular matrix (ECM) was reduced, such as the Fibronectin, Collagen Ⅰ, Collagen Ⅲ were also declined. This shows that resumption of Merlin in FOCUS cells can reduce the TGF-β1expression, promote to reduce the synthesis of ECM in hepatic stellate cell and inhibit liver fibrosis.
The high expression of Merlin transfected with plasmid can significantly inhibit the epidermal growth factor receptor (EGFR) activation, thereby inhibit the expression of TGF-β1; and in the vector control under the EGF stimulation, the epidermal growth factor receptor (EGFR) is activated, TGF-β1expression along with the concentration of EGF was significantly increased in turn. The results confirmed that Merlin can inhibit the epidermal growth factor receptor signaling pathway, which reduced the secretion of TGF-β1.
With the LX-2hepatic stellate cells gradually increased the number of cells, Merlin expression also gradually increased. In this linear cell density, in TGF-β1stimulation, the level of phosphated TGFBR Ⅰ receptor and Smad2in LX-2cells gradually reduced, TGF-β/Smad signaling pathway was inhibited. In the same conditions, due to Merlin in cancer cells FOCUS was loss, no matter how much the number of cells, FOCUS cells do not express Merlin. In the different cell density, stimulated by TGF-β1, TGF-β/Smad signaling pathway was activated continuously in FOCUS cells. In low cell density, hepatic stellate cell line LX-2cells expressed low Merlin, and high cell density with high expression of Merlin. In both cell density, stimulated by TGF-β1, low cell density LX-2cells compared with the high density TGF-β/Smad signaling pathway was significantly activated; its ability to greatly increase the synthesis of ECM. Meanwhile, in the low cell density, we transfected pCDNA-NF2to improve the expression level of Merlin, and then stimulated with TGF-β1and its TGF-β/Smad signaling pathway was significantly inhibited, ECM synthesis was significantly inhibited. On the other hand, in the high cell density, transfected shRNA to interfere Merlin, reducing the expression level of Merlin, and then stimulated with TGF-β1and the inhibitions of TGF-β/Smad signaling pathway and ECM synthesis were rescinded.
Conclusion:In summary, this study was to detect TGF-β1protein levels in the HCC cell lines and hepatocellular carcinoma patients, and identified in hepatocellular carcinoma and liver cancer cell lines the TGF-β1protein expression were up-regulated; HCC cells can secrete TGF-β1themselves, and activated hepatic stellate cells TGF-β1/Smad pathway, promote hepatic stellate cells to synthesize extracellular matrix, and promote liver fibrosis. Merlin can inhibit the epidermal growth factor receptor signaling pathway, which reduced the expression of TGF-β1secretion. Restored Merlin in HCC cells can reduce the expression of TGF-β1, inhibit the hepatic stellate cell synthesize ECM, thereby inhibit the occurrence of liver fibrosis. Meanwhile, Merlin can negatively regulate TGF-β/Smad signaling pathway, and suppress liver fibrosis.
引文
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