膜—细胞骨架联接蛋白ezrin对肝癌细胞HGF-c-met介导的侵袭转移的影响
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摘要
肝癌是我国常见的恶性肿瘤之一,不仅起病隐匿,而且转移早,是影响肝癌患者预后的主要原因之一。众所周知,肿瘤转移是恶性肿瘤最基本的生物学特征之一,也是影响肿瘤患者生存和预后的重要因素之一。因此对肝癌转移的研究不仅有利于进一步了解阐明肝癌的生物学行为,更有益于探索针对性的治疗途径,为提高肝癌的治疗疗效提供理论依据。
肿瘤细胞信号转导异常与肿瘤转移密切相关,SF/HGF-c-Met是近年来得到学者们重视的一条信号通路。肝细胞生长因子及其受体c-met的激活对肝癌的发生、生长和转移起着关键性的作用。阻断HGF-c-met信号通路对抑制肝癌的复发转移有着重要的意义。膜-细胞骨架联接蛋白ezrin是HGF/c-met信号通路的下游分子之一,通过ezrin的连接作用,将来源于细胞外或细胞膜上的信号传递到骨架蛋白等效应分子,调节细胞形态和运动。
我们前期通过对组织芯片和不同转移潜能的肝癌细胞进行检测,发现在低分化的肝组织及高转移潜能的肝癌细胞中ezrin的表达增高。针对ezrin不同位点设计合成小干扰RNA,可显著下调ezrin表达,并降低细胞运动侵袭能力。证明ezrin在肝癌侵袭转移中的重要地位。但ezrin影响肝癌细胞侵袭、转移的具体机制,及ezrin在HGF介导的细胞信号通路中所起的作用,还不甚清楚。在本研究中我们将对这些问题进行更进一步的探讨。
第一部分携带ezrin小发夹RNA腺病毒载体的构建及应用
目的:构建携带ezrin小发夹RNA的腺病毒载体,使ezrin在细胞内获得稳定、持续的下调,从而深入研究膜-骨架联接蛋白ezrin在肝癌细胞运动转移方面的作用以及相关的分子信号转导。
方法:利用前期研究筛选出的针对ezrin的有效小干扰RNA,构建小发夹RNA(short hairpin RNA,shRNA)腺病毒质粒载体,转染293包装细胞,获得携带ezrin小发夹RNA的重组腺病毒ezrin-shRNA/Ad;感染肝癌细胞系MHCC97-H和SF/SMMC7721,Western-blot检测重组腺病毒ezrin-shRNA/Ad对ezrin表达的下调作用,同时肝癌细胞系经连续传代,观察重组腺病毒介导的RNA抑制能否持续发生。
结果:重组腺病毒质粒转染293包装细胞,获得的病毒原液经过滤纯化,得到滴度为2×10~8和5×10~8的2种重组腺病毒ezrin-shRNA/Ad。10,50,100m.o.i.的ezrin-shRNA/Ad作用于MHCC97-H和SF/SMMC7721细胞,感染后3天Westernblot测细胞ezrin表达。浓度为50和100m.o.i.的ezrin-shRNA/Ad对ezrin表达有明显抑制作用;细胞经连续传代,SF/SMMC7721细胞在连续传代4代后ezrin表达仍能得到有效抑制,MHCC97-H细胞传代2次后ezrin表达仍能得到有效抑制。
结论:携带ezrin小发夹RNA的重组腺病毒能够有效下调ezrin的表达,细胞经连续传代,对ezrin的下调作用仍能持续发生。
第二部分腺病毒小发夹RNA下调ezrin表达对肝癌细胞转移潜能的影响
目的:探讨腺病毒小发夹RNA下调ezrin表达后对肝癌细胞系运动、侵袭转移潜能的影响以及相应机制。
方法:50m.o.i.的重组腺病毒ezyin-shRNA/Ad作用于MHCC97-H,SF/SMMC7721细胞,MTT实验测定细胞增殖能力;Transwell实验测定细胞运动侵袭能力;扫描电子显微镜观察细胞伪足;钙黄绿素AM标记的荧光信号测定细胞与细胞之间以及细胞与基质之间的粘附能力;免疫荧光检测ezrin和粘附分子E-cadherin和CD44在细胞内的表达;RT-PCR检测ezrin和骨架蛋白actin,E-cadherin,CD44的mRNA表达。重组腺病毒感染后的MHCC97-H经抗生素筛选,以MHCC97-H作对照接种裸鼠进行体内实验,每周3天测肿瘤生长及大小,6周后处死,病理切片检测裸鼠肺及淋巴结转移灶。
结果:体外实验:ezrin-shRNA/Ad对MHCC97-H,SF/SMMC7721的生长增殖均有不同程度的抑制作用,对SF/SMMC7721增殖的抑制作用从第4天开始慢慢显现,而对MHCC97-H的增殖的抑制作用则更为明显,从第2天开始,细胞的增殖速度明显放缓;Transwell实验测细胞穿膜细胞数,2种腺病毒ezrin-shRNA感染MHCC97-H后穿膜细胞数与感染前相比显著减少(ezrin-shRNA1/Ad:44.00±7.06,ezrin-shRNA2/Ad:35.50±3.70,前:63.80±6.26,,P＜0.05),而对照病毒则没有显著变化(56.50±7.01,P＞0.05),ezrin-shRNA/Ad作用于SF/SMMC7721得到相似的结果;电子显微镜观察细胞伪足,ezrin-shRNA/Ad感染后细胞的伪足数目比感染前减少,对照病毒没有影响;钙黄绿素AM标记的荧光信号测定细胞与细胞之间以及细胞与基质之间的粘附能力,MHCC97-H经ezrin-shRNA/Ad感染后,细胞-细胞之间的粘附增强,细胞-基质之间的粘附作用减弱,而SF/SMMC7721细胞-基质之间的粘附作用减弱,细胞-细胞之间的粘附作用没有显著变化;免疫荧光结果显示,细胞经ezrin-shRNA/Ad感染后ezrin表达下降,而E-cadherin表达增高,且主要呈膜分布。而CD44的表达与E-cadherin相反,随ezrin表达下调而下降。RT-PCR结果显示,细胞经ezrin-shRNA/Ad感染后,骨架蛋白γ-actin的表达随ezrin的下调而降低,β-actin的表达没有明显变化,粘附分子CD44的表达随ezrin下调而下降,E-cadherin及下游分子B-catenin的表达增高。
体内实验:实验组裸鼠肿瘤生长速度明显慢于实验组,处死时瘤重约为对照组的2/3(实验组4.946±1.926mg vs对照组7.358±1.592mg)。病理切片显示实验组肿瘤肺转移率为50%(5/10),低于对照组77.8%(7/9);实验组的淋巴结转移率为30.0%(3/10)低于对照组55.6%(5/9)(P＜0.05)。
结论:Ezrin在肝癌细胞生长,运动和侵袭转移过程中发挥重要作用;腺病毒小发夹RNA下调ezrin的表达,或通过调节骨架蛋白,粘附分子的表达和分布而引起细胞运动、侵袭等转移相关潜能的改变。
第三部分Ezrin在HGF介导的细胞信号通路中的作用
目的:探讨ezrin在HGF介导的细胞信号通路中的作用
方法:HGF作用于细胞,Western-blot检测细胞磷酸化ezrin的表达;腺病毒小发夹RNA下调ezrin的表达,HGF作用于细胞,划痕实验检测细胞运动能力,扫描电镜检测细胞伪足形成,RT-PCR检测E-cadherin,CD44等基因的mRNA表达,观察在HGF刺激下细胞运动能力,伪足形成,及粘附分子表达的变化。
结果:HGF刺激细胞引起细胞ezrin的磷酸化,磷酸化的ezrin在HGF作用细胞30min后即可以检测到;腺病毒小发夹RNA敲除ezrin的表达,细胞失去对HGF介导的形态发生和细胞运动的反应能力,其伪足形成和细胞运动能力在HGF作用前后都没有发生显著改变。HGF对于细胞E-cadherin和CD44的表达没有显著影响。下游信号分子磷酸化MAPK和磷酸化Akt的表达在重组病毒感染后消失,HGF刺激细胞,激活HGF/SF通路后,磷酸化MAPK p44/42的表达重新出现。
结论:Ezrin是HGF/c-met信号通路的下游效应分子,ezrin对于HGF介导的细胞变形和细胞运动是必需的。
总结
本研究成功构建携带ezrin小发夹RNA的重组腺病毒ezrin-shRNA/Ad,应用于肝癌细胞系能够有效下调ezrin的表达,细胞经连续传代,对ezrin的下调作用仍能持续发生。腺病毒小发夹RNA下调ezrin的表达,引起肝癌细胞系增殖速度减慢,细胞运动侵袭能力下降,细胞-细胞粘附增强,细胞-基质粘附减弱,说明ezrin在肝癌细胞运动,侵袭中的重要作用。细胞运动侵袭能力下降,同时伴有骨架蛋白γ-actin,粘附分子CD44表达下降,粘附分子E-cadherin,β-catenin的表达增高,提示ezrin通过调节骨架蛋白,粘附分子的表达和分布而影响细胞运动、侵袭等转移相关潜能。体内实验,重组腺病毒感染后的肝癌细胞成瘤减慢,肿瘤的肺及淋巴结转移率下降,进一步证明ezrin在肝癌生长转移中的作用。HGF刺激细胞后可导致ezrin磷酸化,重组腺病毒介导的ezrin抑制使细胞丧失对HGF的反应,说明ezrin是HGF/c-met信号通路的下游效应分子,对于HGF介导的细胞变形和细胞运动是必需的。我们的研究提示ezrin是HGF-c-Met介导的肝癌细胞侵袭转移信号通路中的关键分子之一,或可成为治疗肝癌转移复发的新靶点。
Hepatocellular carcinoma (HCC) is a malignant tumor with high relapse and metastasis. Despite recent improvement in long-term survival, the prognosis of HCC is still poor. As we all known, metastasis is one of the characteristics of malignant tumor. To investigate the mechanism of metastasis is helpful for illustrating biological activities of malignant tumor; it is also useful for finding new therapeutic molecular targets and ameliorating the poor outcome of malignant tumor.
Hepatocyte growth factor/scatter factor-Met signaling has been implicated in tumor growth, invasion, and metastasis. Activation of hepatocyte growth factor and its receptor Met may enhance tumor growth, invasion, and metastasis, while block Met activation can suppress tumor metastasis. The membrane-cytoskeleton organizer ezrin has been reported to be an effecter of hepatocyte growth factor. As a membrane-cytoskeleton organizer, ezrin can transfer signaling from membrane to the effecter—cytoskeleton F-actin, and mediate migration and morphogenesis.
In our previous study, hepatocelluar carcinoma cell lines with different metastasis potential and 200 points HCC tissues array were used to explore ezrin expression in HCC. It came that over expression of ezrin was found in low differential tumor tissues and HCC cell lines with high metastasis potential. Small interference RNA (siRNA) sequences directed against ezrin can downregulate expression levels of ezrin, they also suppress tumor growth, invasion, and metastasis as well. But the mechanism involved in this process is not well understood. In this study, by means of adenovirus mediated RNA interference (RNAi) we try to explore the mechanism by which ezrin influence tumor invasion and metastasis.
Part One: The construction and application of adenovirus vectors carrying short-hairpin RNA against ezrin
Objective To establish adenovirus vectors carrying short-hairpin RNA (shRNA) against ezrin. By means of these constructions, which may result in a steady-going downregulation of ezrin, we can investigate the crucial role of ezrin on metastasis of heptocellular carcinoma cells, and we may also explore the mechanism involved in this process as well.
Methods Short-hairpin RNA which designed according to effective small-interfering RNA was cloned into a shuttle vector to establish recombined adenovirus constructs. After transfecting 293 packaging cell lines, reconstructed adenovirus containing short-hairpin RNA against ezrin (ezrin-shRNA/Ad) were harvested. Human heptocellular carcinoma cells: MHCC97-H and SF/SMMC7721 were infected with ezrin-shRNA/Ad at m.o.i. of 10, 50 and 100, and ezrin expression was determined by Western blotting. Next, we asked how long the RNAi effect continues after cell passage. MHCC97-H and SF/SMMC7721 were infected with ezrin-shRNA/Ad at m.o.i. of 100, and ezrin expression was determined at 3-day intervals for up to 9 days after cell passage.
Results Human heptocellular carcinoma cells MHCC97-H and SF/SMMC7721 were infected with ezrin-shRNA/Ad at m.o.i. of 10, 50 and 100. Ezrin expression was determined on day 3 after infection by Western blot. The expression of ezrin protein was dramatically suppressed by ezrin-shRNA/Ad at m.o.i. 50 and 100; Control Ad virus infected cells showed no effect on ezrin expression. Ezrin expression was still strongly suppressed even undergoing cell passage. On MHCC97-H, ezrin expression was still suppressed after a second passage, while a strong suppression was still observed after a forth passage on SF/SMMC7721 cell lines.
Conclusion Reconstructed adenovirus containing short-hairpin RNA against ezrin can downregulate ezrin expression. Ezrin expression was still strongly suppressed even after cell passage.
Part two: The effect of Adenovirus mediated RNAi on the invasiveness and metastasis of hepatocelluar carcinoma cell lines
Objective To explore the effect of Adenovirus mediated ezrin suppression on growth, migration, invasiveness and metastasis of heptocellular carcinoma cells, and to study the mechanism involved in this process as well.
Methods Human heptocellular carcinoma cells MHCC97-H and SF/SMMC7721 were infected with ezrin-shRNA/Ad at m.o.i. of 50, the proliferation were determined by MTT assay. Scanning electron microscopy (SEM) and Matrigel Invasion Assay (Transwell assay) was used to study cellular morphology and invasion. Cell-cell interaction and cell -matrix adhesion was estimated by a Calcein AM labeled fluorescence OD. Immunofluorescence was used to observe expression and location of ezrin, E-cadherin and CD44 protein. RT-PCR was performed for ezrin, actin, E-cadherin, CD44 andβ-catenin expression. MHCC97-H infected with ezrin-shRNA/Ad was injected into nude mice, MHCC97-H as control, the proliferation and metastasis were observed in vivo nude mice experiment.
Results In vitro: After infected with reconstructed adenovirus ezrin-shRNA/Ad, the proliferation rate of MHCC97-H and SF/SMMC7721 were distinctly decreased. Proliferation curve show the proliferation suppression of SF/SMMC7721 occurred at the day4 after infection, while the proliferation suppression of MHCC97-H appeared only at the second day. Scanning electron microscopy performed with MHCC97-H and SF/SMMC7721 revealed rather rounded cellular morphology with reduced pseudopods number after infection with ezrin-shRNA/Ad. The decreased invasion were also confirmed by Matrigel Invasion Assay, compared to control Ad virus, MHCC97-H and SF/SMMC7721 infected with ezrin-shRNA/Ad resulted in significant reduction in the number of Matrigel invading cells (number of Matrigel invading MHCC97-H cells :ezrin-shRNAl/Ad 44.00±7.06; ezrin-shRNA2/Ad 35.50±3.70; control Ad virus 56.50±7.01; uninfected 63.80±6.26). Enhanced cell-cell interaction and reduced cell -matrix adhesion were observed with MHCC97-H and SF/SMMC7721 cells after ezrin-shRNA/Ad infection, as indicated by Calcein AM labeled fluorescence OD. Immunofluorescence showed weak ezrin expression, concomitant with increased E-cadherin expression, mainly located on membrane, and decreased expression of CD44, after ezrin-shRNA/Ad infection. RT-PCR was performed for ezrin, actin, E-cadherin, CD44 andβ-catenin expression. Expression of E-cadherin andβ-catenin showed a moderate upregulation in response to ezrin-shRNA/Ad treatment, while CD44 andγ- actin was found to be downregulated. Ezrin-shRNA/Ad treatment seemed to have no effect onβ-actin expression. In vivo: The tumor formation in nude mice began in 2 weeks after cell injection. Tumors of MHCC-97H infected with ezrin-shRNA/Ad grew rapidly than tumors of control MHCC-97H. Infected MHCC97-H also had lower lung and lymphoid node metastasis rates than control (Infected MHCC97-H of 50% and 30.0% vs MHCC97-H of 77.8% and 55.6%).
Conclusion Cell proliferation and invasiveness, as well as metastasis potential of HCC cell lines were depressed by ezrin-shRNA/Ad mediated RNA interference, which indicated ezrin is involved in hepatocellular carcinoma proliferation, invasion and metastasis.
Part three: The role of Ezrin played in HGF/met cell signaling
Objective To explore the mechanism that ezrin involved in HGF mediated tumorgrowth, invasion, and metastasis.
Methods Human heptocellular carcinoma cells SMMC77211, SF/SMMC7721 andSF/SMMC7721 infected with ezrin-shRNA/Ad reconstruction were treated with orwithout HGF stimulation. Western-blot was performed for phosphorylated ezrin.Cellular migration was determined by Wound healing assay. Scanning electronmicroscopy (SEM) was used to study cellular morphology. RT-PCR was performedfor E-cadherin and CD44 expression.
Results Ezrin is a substrate for HGF/met cell signaling. Upon HGF treatment,phosphorylated ezrin appeared in 30 minutes. Knocking down of ezrin byezrin-shRNA/ Ad mediated RNAi, impairs motility and morphogenesis response toHGF, as measured by the slower wound healing and less pseudopods number. HGFshows no effect on migration and morphogenesis of ezrin-shRNA/Ad infected celllines. Phosphorylated p44/42 MAP kinase and phosphorylated Akt disappeared afterinfection. Phosphorylated p44/42 MAP kinase reappeared upon exposure of cells toHGF.
Conclusion Ezrin is an effecter of HGF mediated migration and morphogenesis inhepatocellular carcinoma cell lines. Summarization
In this study, we have successfully obtained reconstructed adenovirus whichcontaining short-hairpin RNA against ezrin (ezrin-shRNA/Ad). Upon treated withezrin-shRNA/Ad, the expression levels of ezrin protein was dramaticallydownregulated and ezrin expression was still strongly suppressed even after cellpassage.
Cell proliferation and invasiveness of hepatocellular carcinoma (HCC) cell linesMHCC97-H and SF/SMMC7721 were depressed by ezrin-shRNA/Ad mediated RNAinterference, which indicated ezrin is involved in HCC proliferation and invasion. Theexpression and location of actin, E-cadherin, CD44 may be involved in this process aswell. In vivo nude mice experiment confirmed that ezrin played an important role onHCC growth and metastasis.
Upon HGF treatment, phosphorylated ezrin appeared. Knocking down of ezrin byezrin-shRNA/ Ad mediated RNAi, impairs cellular motility and morphogenesisresponse to HGF, which indicated ezrin is an effecter of HGF mediated migration andmorphogenesis in hepatocellular carcinoma cell lines.
The whole study suggests that ezrin play an important role on HCC growth, migrationinvasion and metastasis, and may become a new therapeutic target in HCC therapy.
肿瘤细胞信号转导异常与肿瘤转移密切相关,SF/HGF-c-Met是近年来得到学者们重视的一条信号通路。肝细胞生长因子及其受体c-met的激活对肝癌的发生、生长和转移起着关键性的作用。阻断HGF-c-met信号通路对抑制肝癌的复发转移有着重要的意义。膜-细胞骨架联接蛋白ezrin是HGF/c-met信号通路的下游分子之一,通过ezrin的连接作用,将来源于细胞外或细胞膜上的信号传递到骨架蛋白等效应分子,调节细胞形态和运动。
我们前期通过对组织芯片和不同转移潜能的肝癌细胞进行检测,发现在低分化的肝组织及高转移潜能的肝癌细胞中ezrin的表达增高。针对ezrin不同位点设计合成小干扰RNA,可显著下调ezrin表达,并降低细胞运动侵袭能力。证明ezrin在肝癌侵袭转移中的重要地位。但ezrin影响肝癌细胞侵袭、转移的具体机制,及ezrin在HGF介导的细胞信号通路中所起的作用,还不甚清楚。在本研究中我们将对这些问题进行更进一步的探讨。
第一部分携带ezrin小发夹RNA腺病毒载体的构建及应用
目的:构建携带ezrin小发夹RNA的腺病毒载体,使ezrin在细胞内获得稳定、持续的下调,从而深入研究膜-骨架联接蛋白ezrin在肝癌细胞运动转移方面的作用以及相关的分子信号转导。
方法:利用前期研究筛选出的针对ezrin的有效小干扰RNA,构建小发夹RNA(short hairpin RNA,shRNA)腺病毒质粒载体,转染293包装细胞,获得携带ezrin小发夹RNA的重组腺病毒ezrin-shRNA/Ad;感染肝癌细胞系MHCC97-H和SF/SMMC7721,Western-blot检测重组腺病毒ezrin-shRNA/Ad对ezrin表达的下调作用,同时肝癌细胞系经连续传代,观察重组腺病毒介导的RNA抑制能否持续发生。
结果:重组腺病毒质粒转染293包装细胞,获得的病毒原液经过滤纯化,得到滴度为2×10~8和5×10~8的2种重组腺病毒ezrin-shRNA/Ad。10,50,100m.o.i.的ezrin-shRNA/Ad作用于MHCC97-H和SF/SMMC7721细胞,感染后3天Westernblot测细胞ezrin表达。浓度为50和100m.o.i.的ezrin-shRNA/Ad对ezrin表达有明显抑制作用;细胞经连续传代,SF/SMMC7721细胞在连续传代4代后ezrin表达仍能得到有效抑制,MHCC97-H细胞传代2次后ezrin表达仍能得到有效抑制。
结论:携带ezrin小发夹RNA的重组腺病毒能够有效下调ezrin的表达,细胞经连续传代,对ezrin的下调作用仍能持续发生。
第二部分腺病毒小发夹RNA下调ezrin表达对肝癌细胞转移潜能的影响
目的:探讨腺病毒小发夹RNA下调ezrin表达后对肝癌细胞系运动、侵袭转移潜能的影响以及相应机制。
方法:50m.o.i.的重组腺病毒ezyin-shRNA/Ad作用于MHCC97-H,SF/SMMC7721细胞,MTT实验测定细胞增殖能力;Transwell实验测定细胞运动侵袭能力;扫描电子显微镜观察细胞伪足;钙黄绿素AM标记的荧光信号测定细胞与细胞之间以及细胞与基质之间的粘附能力;免疫荧光检测ezrin和粘附分子E-cadherin和CD44在细胞内的表达;RT-PCR检测ezrin和骨架蛋白actin,E-cadherin,CD44的mRNA表达。重组腺病毒感染后的MHCC97-H经抗生素筛选,以MHCC97-H作对照接种裸鼠进行体内实验,每周3天测肿瘤生长及大小,6周后处死,病理切片检测裸鼠肺及淋巴结转移灶。
结果:体外实验:ezrin-shRNA/Ad对MHCC97-H,SF/SMMC7721的生长增殖均有不同程度的抑制作用,对SF/SMMC7721增殖的抑制作用从第4天开始慢慢显现,而对MHCC97-H的增殖的抑制作用则更为明显,从第2天开始,细胞的增殖速度明显放缓;Transwell实验测细胞穿膜细胞数,2种腺病毒ezrin-shRNA感染MHCC97-H后穿膜细胞数与感染前相比显著减少(ezrin-shRNA1/Ad:44.00±7.06,ezrin-shRNA2/Ad:35.50±3.70,前:63.80±6.26,,P＜0.05),而对照病毒则没有显著变化(56.50±7.01,P＞0.05),ezrin-shRNA/Ad作用于SF/SMMC7721得到相似的结果;电子显微镜观察细胞伪足,ezrin-shRNA/Ad感染后细胞的伪足数目比感染前减少,对照病毒没有影响;钙黄绿素AM标记的荧光信号测定细胞与细胞之间以及细胞与基质之间的粘附能力,MHCC97-H经ezrin-shRNA/Ad感染后,细胞-细胞之间的粘附增强,细胞-基质之间的粘附作用减弱,而SF/SMMC7721细胞-基质之间的粘附作用减弱,细胞-细胞之间的粘附作用没有显著变化;免疫荧光结果显示,细胞经ezrin-shRNA/Ad感染后ezrin表达下降,而E-cadherin表达增高,且主要呈膜分布。而CD44的表达与E-cadherin相反,随ezrin表达下调而下降。RT-PCR结果显示,细胞经ezrin-shRNA/Ad感染后,骨架蛋白γ-actin的表达随ezrin的下调而降低,β-actin的表达没有明显变化,粘附分子CD44的表达随ezrin下调而下降,E-cadherin及下游分子B-catenin的表达增高。
体内实验:实验组裸鼠肿瘤生长速度明显慢于实验组,处死时瘤重约为对照组的2/3(实验组4.946±1.926mg vs对照组7.358±1.592mg)。病理切片显示实验组肿瘤肺转移率为50%(5/10),低于对照组77.8%(7/9);实验组的淋巴结转移率为30.0%(3/10)低于对照组55.6%(5/9)(P＜0.05)。
结论:Ezrin在肝癌细胞生长,运动和侵袭转移过程中发挥重要作用;腺病毒小发夹RNA下调ezrin的表达,或通过调节骨架蛋白,粘附分子的表达和分布而引起细胞运动、侵袭等转移相关潜能的改变。
第三部分Ezrin在HGF介导的细胞信号通路中的作用
目的:探讨ezrin在HGF介导的细胞信号通路中的作用
方法:HGF作用于细胞,Western-blot检测细胞磷酸化ezrin的表达;腺病毒小发夹RNA下调ezrin的表达,HGF作用于细胞,划痕实验检测细胞运动能力,扫描电镜检测细胞伪足形成,RT-PCR检测E-cadherin,CD44等基因的mRNA表达,观察在HGF刺激下细胞运动能力,伪足形成,及粘附分子表达的变化。
结果:HGF刺激细胞引起细胞ezrin的磷酸化,磷酸化的ezrin在HGF作用细胞30min后即可以检测到;腺病毒小发夹RNA敲除ezrin的表达,细胞失去对HGF介导的形态发生和细胞运动的反应能力,其伪足形成和细胞运动能力在HGF作用前后都没有发生显著改变。HGF对于细胞E-cadherin和CD44的表达没有显著影响。下游信号分子磷酸化MAPK和磷酸化Akt的表达在重组病毒感染后消失,HGF刺激细胞,激活HGF/SF通路后,磷酸化MAPK p44/42的表达重新出现。
结论:Ezrin是HGF/c-met信号通路的下游效应分子,ezrin对于HGF介导的细胞变形和细胞运动是必需的。
总结
本研究成功构建携带ezrin小发夹RNA的重组腺病毒ezrin-shRNA/Ad,应用于肝癌细胞系能够有效下调ezrin的表达,细胞经连续传代,对ezrin的下调作用仍能持续发生。腺病毒小发夹RNA下调ezrin的表达,引起肝癌细胞系增殖速度减慢,细胞运动侵袭能力下降,细胞-细胞粘附增强,细胞-基质粘附减弱,说明ezrin在肝癌细胞运动,侵袭中的重要作用。细胞运动侵袭能力下降,同时伴有骨架蛋白γ-actin,粘附分子CD44表达下降,粘附分子E-cadherin,β-catenin的表达增高,提示ezrin通过调节骨架蛋白,粘附分子的表达和分布而影响细胞运动、侵袭等转移相关潜能。体内实验,重组腺病毒感染后的肝癌细胞成瘤减慢,肿瘤的肺及淋巴结转移率下降,进一步证明ezrin在肝癌生长转移中的作用。HGF刺激细胞后可导致ezrin磷酸化,重组腺病毒介导的ezrin抑制使细胞丧失对HGF的反应,说明ezrin是HGF/c-met信号通路的下游效应分子,对于HGF介导的细胞变形和细胞运动是必需的。我们的研究提示ezrin是HGF-c-Met介导的肝癌细胞侵袭转移信号通路中的关键分子之一,或可成为治疗肝癌转移复发的新靶点。
Hepatocellular carcinoma (HCC) is a malignant tumor with high relapse and metastasis. Despite recent improvement in long-term survival, the prognosis of HCC is still poor. As we all known, metastasis is one of the characteristics of malignant tumor. To investigate the mechanism of metastasis is helpful for illustrating biological activities of malignant tumor; it is also useful for finding new therapeutic molecular targets and ameliorating the poor outcome of malignant tumor.
Hepatocyte growth factor/scatter factor-Met signaling has been implicated in tumor growth, invasion, and metastasis. Activation of hepatocyte growth factor and its receptor Met may enhance tumor growth, invasion, and metastasis, while block Met activation can suppress tumor metastasis. The membrane-cytoskeleton organizer ezrin has been reported to be an effecter of hepatocyte growth factor. As a membrane-cytoskeleton organizer, ezrin can transfer signaling from membrane to the effecter—cytoskeleton F-actin, and mediate migration and morphogenesis.
In our previous study, hepatocelluar carcinoma cell lines with different metastasis potential and 200 points HCC tissues array were used to explore ezrin expression in HCC. It came that over expression of ezrin was found in low differential tumor tissues and HCC cell lines with high metastasis potential. Small interference RNA (siRNA) sequences directed against ezrin can downregulate expression levels of ezrin, they also suppress tumor growth, invasion, and metastasis as well. But the mechanism involved in this process is not well understood. In this study, by means of adenovirus mediated RNA interference (RNAi) we try to explore the mechanism by which ezrin influence tumor invasion and metastasis.
Part One: The construction and application of adenovirus vectors carrying short-hairpin RNA against ezrin
Objective To establish adenovirus vectors carrying short-hairpin RNA (shRNA) against ezrin. By means of these constructions, which may result in a steady-going downregulation of ezrin, we can investigate the crucial role of ezrin on metastasis of heptocellular carcinoma cells, and we may also explore the mechanism involved in this process as well.
Methods Short-hairpin RNA which designed according to effective small-interfering RNA was cloned into a shuttle vector to establish recombined adenovirus constructs. After transfecting 293 packaging cell lines, reconstructed adenovirus containing short-hairpin RNA against ezrin (ezrin-shRNA/Ad) were harvested. Human heptocellular carcinoma cells: MHCC97-H and SF/SMMC7721 were infected with ezrin-shRNA/Ad at m.o.i. of 10, 50 and 100, and ezrin expression was determined by Western blotting. Next, we asked how long the RNAi effect continues after cell passage. MHCC97-H and SF/SMMC7721 were infected with ezrin-shRNA/Ad at m.o.i. of 100, and ezrin expression was determined at 3-day intervals for up to 9 days after cell passage.
Results Human heptocellular carcinoma cells MHCC97-H and SF/SMMC7721 were infected with ezrin-shRNA/Ad at m.o.i. of 10, 50 and 100. Ezrin expression was determined on day 3 after infection by Western blot. The expression of ezrin protein was dramatically suppressed by ezrin-shRNA/Ad at m.o.i. 50 and 100; Control Ad virus infected cells showed no effect on ezrin expression. Ezrin expression was still strongly suppressed even undergoing cell passage. On MHCC97-H, ezrin expression was still suppressed after a second passage, while a strong suppression was still observed after a forth passage on SF/SMMC7721 cell lines.
Conclusion Reconstructed adenovirus containing short-hairpin RNA against ezrin can downregulate ezrin expression. Ezrin expression was still strongly suppressed even after cell passage.
Part two: The effect of Adenovirus mediated RNAi on the invasiveness and metastasis of hepatocelluar carcinoma cell lines
Objective To explore the effect of Adenovirus mediated ezrin suppression on growth, migration, invasiveness and metastasis of heptocellular carcinoma cells, and to study the mechanism involved in this process as well.
Methods Human heptocellular carcinoma cells MHCC97-H and SF/SMMC7721 were infected with ezrin-shRNA/Ad at m.o.i. of 50, the proliferation were determined by MTT assay. Scanning electron microscopy (SEM) and Matrigel Invasion Assay (Transwell assay) was used to study cellular morphology and invasion. Cell-cell interaction and cell -matrix adhesion was estimated by a Calcein AM labeled fluorescence OD. Immunofluorescence was used to observe expression and location of ezrin, E-cadherin and CD44 protein. RT-PCR was performed for ezrin, actin, E-cadherin, CD44 andβ-catenin expression. MHCC97-H infected with ezrin-shRNA/Ad was injected into nude mice, MHCC97-H as control, the proliferation and metastasis were observed in vivo nude mice experiment.
Results In vitro: After infected with reconstructed adenovirus ezrin-shRNA/Ad, the proliferation rate of MHCC97-H and SF/SMMC7721 were distinctly decreased. Proliferation curve show the proliferation suppression of SF/SMMC7721 occurred at the day4 after infection, while the proliferation suppression of MHCC97-H appeared only at the second day. Scanning electron microscopy performed with MHCC97-H and SF/SMMC7721 revealed rather rounded cellular morphology with reduced pseudopods number after infection with ezrin-shRNA/Ad. The decreased invasion were also confirmed by Matrigel Invasion Assay, compared to control Ad virus, MHCC97-H and SF/SMMC7721 infected with ezrin-shRNA/Ad resulted in significant reduction in the number of Matrigel invading cells (number of Matrigel invading MHCC97-H cells :ezrin-shRNAl/Ad 44.00±7.06; ezrin-shRNA2/Ad 35.50±3.70; control Ad virus 56.50±7.01; uninfected 63.80±6.26). Enhanced cell-cell interaction and reduced cell -matrix adhesion were observed with MHCC97-H and SF/SMMC7721 cells after ezrin-shRNA/Ad infection, as indicated by Calcein AM labeled fluorescence OD. Immunofluorescence showed weak ezrin expression, concomitant with increased E-cadherin expression, mainly located on membrane, and decreased expression of CD44, after ezrin-shRNA/Ad infection. RT-PCR was performed for ezrin, actin, E-cadherin, CD44 andβ-catenin expression. Expression of E-cadherin andβ-catenin showed a moderate upregulation in response to ezrin-shRNA/Ad treatment, while CD44 andγ- actin was found to be downregulated. Ezrin-shRNA/Ad treatment seemed to have no effect onβ-actin expression. In vivo: The tumor formation in nude mice began in 2 weeks after cell injection. Tumors of MHCC-97H infected with ezrin-shRNA/Ad grew rapidly than tumors of control MHCC-97H. Infected MHCC97-H also had lower lung and lymphoid node metastasis rates than control (Infected MHCC97-H of 50% and 30.0% vs MHCC97-H of 77.8% and 55.6%).
Conclusion Cell proliferation and invasiveness, as well as metastasis potential of HCC cell lines were depressed by ezrin-shRNA/Ad mediated RNA interference, which indicated ezrin is involved in hepatocellular carcinoma proliferation, invasion and metastasis.
Part three: The role of Ezrin played in HGF/met cell signaling
Objective To explore the mechanism that ezrin involved in HGF mediated tumorgrowth, invasion, and metastasis.
Methods Human heptocellular carcinoma cells SMMC77211, SF/SMMC7721 andSF/SMMC7721 infected with ezrin-shRNA/Ad reconstruction were treated with orwithout HGF stimulation. Western-blot was performed for phosphorylated ezrin.Cellular migration was determined by Wound healing assay. Scanning electronmicroscopy (SEM) was used to study cellular morphology. RT-PCR was performedfor E-cadherin and CD44 expression.
Results Ezrin is a substrate for HGF/met cell signaling. Upon HGF treatment,phosphorylated ezrin appeared in 30 minutes. Knocking down of ezrin byezrin-shRNA/ Ad mediated RNAi, impairs motility and morphogenesis response toHGF, as measured by the slower wound healing and less pseudopods number. HGFshows no effect on migration and morphogenesis of ezrin-shRNA/Ad infected celllines. Phosphorylated p44/42 MAP kinase and phosphorylated Akt disappeared afterinfection. Phosphorylated p44/42 MAP kinase reappeared upon exposure of cells toHGF.
Conclusion Ezrin is an effecter of HGF mediated migration and morphogenesis inhepatocellular carcinoma cell lines. Summarization
In this study, we have successfully obtained reconstructed adenovirus whichcontaining short-hairpin RNA against ezrin (ezrin-shRNA/Ad). Upon treated withezrin-shRNA/Ad, the expression levels of ezrin protein was dramaticallydownregulated and ezrin expression was still strongly suppressed even after cellpassage.
Cell proliferation and invasiveness of hepatocellular carcinoma (HCC) cell linesMHCC97-H and SF/SMMC7721 were depressed by ezrin-shRNA/Ad mediated RNAinterference, which indicated ezrin is involved in HCC proliferation and invasion. Theexpression and location of actin, E-cadherin, CD44 may be involved in this process aswell. In vivo nude mice experiment confirmed that ezrin played an important role onHCC growth and metastasis.
Upon HGF treatment, phosphorylated ezrin appeared. Knocking down of ezrin byezrin-shRNA/ Ad mediated RNAi, impairs cellular motility and morphogenesisresponse to HGF, which indicated ezrin is an effecter of HGF mediated migration andmorphogenesis in hepatocellular carcinoma cell lines.
The whole study suggests that ezrin play an important role on HCC growth, migrationinvasion and metastasis, and may become a new therapeutic target in HCC therapy.
引文
1. Xie Q, Liu KD, Hu MY, et al. SF/HGF-c-Met autocrine and paracrine promote metastasis of hepatocellular carcinoma. World J Gastroenterol. 2001, 7:816-20.
2. Hasuike S, Ido A, Uto H, et al. Hepatocyte growth factor accelerates the proliferation of hepatic oval cells and possibly promotes the differentiation in a 2-acetylaminofiuorene/partial hepatectomy model in rats. J Gastroenterol Hepatol.2005,20(11): 1753-1761.
3. Bretscher A. Regulation of cortical structure by the ezrin-radixin-moesin protein family. Curr Opin Cell Biol. 1999, 11(1): 109-116.
4. Pawlak G, Helfman DM. Cytoskeletal changes in cell transformation and tumourigenesis. Curr Opin Genet Dev. 2000, 11(1):41-47.
5. Hiscox S, Jiang WG. Ezrin regulates cell- cell and cell-matrix adhesion, a possible role with E-cadherin/beta-catenin. J Cell Sci. 1999, 112(18):3081- 3090.
6. Andreoli C, Martin M, Le Borgne R, et al. Ezrin has properties to self-associate at the plasma membrane. J Cell Sci. 1994,107(9):2509- 2512.
7. Bruce B, Khanna G, Ren L, et al. Expression of the cytoskeleton linker protein ezrin in human cancers. Clin Exp Metastasis. 2007, 24(2):69-78. Epub 2007 Mar 17
8. Sizemore S, Cicek M, Sizemore N, et al. Podocalyxin increases the aggressive phenotype of breast and prostate cancer cells in vitro through its interaction with ezrin. Cancer Res. 2007, 67(13):6183-91.
9. Li Q, Wu M, Wang H, et al. Ezrin silencing by small hairpin RNA reverses metastatic behaviors of human breast cancer cells. Cancer Lett. 2008, 261(1):55-63. Epub 2007 Dec 26.
10. Deng X, Tannehill-Gregg SH, Nadella MV, et al. Parathyroid hormone-related protein and ezrin are up-regulated in human lung cancer bonemetastases. Clin Exp Metastasis. 2007, 24(2): 107-19. Epub 2007 Mar 17.
11. Elliott BE, Meens JA, SenGupta SK, et al. The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells. Breast Cancer Res. 2005, 7(3):R365-73.
12. Yu Y, Khan J, Khanna C, et al. Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators. Nat Med. 2004, 10(2): 175-81.
13. Khanna C, Wan X, Bose S, et al. The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis.Nat Med.2004,10(2):182-6.
14.Crepaldi T,Gautreau A,Paolo M,et al.Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogrnesis in epithelial cell.J of Cell Bio.1997,138(2):423-434.
15.Harrison GM,Davies G,Martin TA,et al.Distribution and expression of CD44 isoforms and Ezrin during prostate cancer-endothelium interaction.Int J Oncol.2002,21(5):935-40.
16.Tracey A,Martin,Gregory H,The role of the CD44/ezrin complex in cancer metastasis.Crit Rev Oncol Hematol.2003,46(2):165-86.Review.
17.Pujuguet P,Del Maestro L,Gautreau A,et al.Ezrin regulates E-cadherin-dependent adherens junction assembly through Racl activation.Mol Biol Cell.2003,14(5):2181-91
18.张岩,胡美玉,陈碧华,等.体外RNA干扰下调ezrin表达对肝癌细胞生长和运动能力的影响.中华医学杂志.2006,86:530-535.
19.Zhang Y,Hu MY,Chen BH,et al.The membrane-cytoskeleton organizer ezrin is necessary for hepatocellular carcinoma cell growth and invasiveness.J Cancer Res Clin Oncol.2006,132:685-97.
20.Hunter KW.Ezrin,a key component in tumormetastasis.Trends Mol Med.2004,10:201-204.
21.田健,汤钊猷,叶胜龙,等.具有高转移潜能的人肝癌细胞系的建立及其生物学特征.中华肿瘤杂志.1998,20:405-407
22.Sui G,Soohoo C,Affar el B,et al.A DNA vector- based RNAi technology to supress gene expression in mammalian cells.Proc Natl Acad Sci U S A.2002,99:5515-5520.
23.AdEasyTM Adenoviral Vector System Instruction Manual:Stratagene Campany
24.Naba A,Reverdy C,Louvard D,et al.Spatial recruitment and activation of the Fes kinase by ezrin promotes HGF-induced cell scattering.EMBO J.2008,27(1):38-50.Epub 2007 Nov 29.
25.Poullet P,Gautreau A,Kadare G,et al.Ezrin interacts with focal adhesion kinase and induces its activation independently of cell-matrix adhesion.J Biol Chem.2001,276(40):37686-91.Epub 2001 Jul
26.Orian Rousseau V,Morrison H,Matzke A,et al.Hepatocyte growth factor-induced Ras activation requires ERM proteins linked to both.CD44v6 and F-actin. Mol Biol Cell. 2007, 18(1):76-83. Epub 2006 Oct.
27. Orian Rousseau V, Chen L, Sleeman JP, et al. CD44 is required for two consecutive steps in HGF/c-Met signaling. Genes Dev. 2002,16(23):3074-86.
28. Elliott BE, Qiao H, Louvard D, et al. Co-operative effect of c-Src and ezrin in deregulation of cell-cell contacts and scattering of mammary carcinoma cells. J Cell Biochem. 2004, 92(1): 16-28.
29. Kreimann EL, Morales FC, de Orbeta-Cruz J, et al. Cortical stabilization of beta-catenin contributes to NHERF1/EBP50 tumor suppressor function. Oncogene.2007,26(36):5290-9. Epub 2007 Feb.
30. Johnson MH, McConnell JM. Lineage allocation and cell polarity during mouse embryogenesis. Semin Cell Dev Biol. 2004,15(5):583-97.
31. Dard N, Louvet-Vallee S, Santa-Maria A, Maro B. Phosphorylation of ezrin on threonine T567 plays a crucial role during compaction in the mouse early embryo.Dev Biol. 2004, 271(1):87-97.
1. Bretscher A, Reczek D, Berryman M. Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures [J]. J Cell Sci, 1997, 110(24): 3011.
2. Yonemura S, Tsukita S, Tsukita S. Direct involvement of ezrin/ radixin/moesin (ERM)-binding membrane proteins in the organization of microvilli in collaboration with activated ERM proteins[J]. J Cell Biol, 1999, 145(7): 1497.
3. Granes F, Urena JM, Rocamora N, et al. Ezrin links syndecan-2 to the cytoskeleton[J]. J Cell Sci, 2000, (7): 1267.
4. Bretscher A. Regulation of cortical structure by the ezrin-radixin-moesin protein family[J]. Curr Opin Cell Biol, 1999, 11(1): 109.
5. Akisawa N, Nishimori I, Iwamura T, et al. High levels of ezrin expressed by human pancreatic adenocarcinoma cell lines with high metastatic potential[J].Biochem Biophys Res Commun, 1999, 258(2): 395.
6. Hiscox S, Jiang WG. Ezrin regulates cell_ cell and cell_matrix adhesion, a possible role with E-cadherin/beta-catenin[J]. J Cell Sci, 1999, 112(18): 3081.
7. Pawlak G, Helfman DM. Cytoskeletal changes in cell transformation and tumourigenesis[J]. Curr Opin Genet Dev, 2000, 11(1): 41.
8. Bruce B, Khanna G, Ren L, et al. Expression of the cytoskeleton linker protein ezrin in human cancers. Clin Exp Metastasis. 2007, 24(2):69-78. Epub 2007 Mar 17.
9. Sizemore S, Cicek M, Sizemore N, et al. Podocalyxin increases the aggressive phenotype of breast and prostate cancer cells in vitro through its interaction with ezrin. Cancer Res. 2007, 67(13):6183-91.
10. Li Q, Wu M, Wang H, et al. Ezrin silencing by small hairpin RNA reverses metastatic behaviors of human breast cancer cells. Cancer Lett. 2008, 261(1):55-63. Epub 2007 Dec 26.
11. Deng X, Tannehill-Gregg SH, Nadella MV, et al. Parathyroid hoimone-related protein and ezrin are up-regulated in human lung cancer bone metastases. Clin Exp Metastasis. 2007, 24(2):107-19. Epub 2007 Mar 17.
12. Elliott BE, Meens JA, SenGupta SK, et al. The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells. Breast Cancer Res. 2005, 7(3):R365-73.
13. Yu Y, Khan J, Khanna C, et al. Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators. Nat Med. 2004, 10(2): 175-81.
14. Khanna C, Wan X, Bose S, et al. The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis. Nat Med. 2004,10(2): 182-6.
15. Crepaldi T, Gautreau A, Paolo M, et al. Ezrin is an effector of hepatocyte growth factor -mediated migration and morphogrnesis in epithelial cell. J of Cell Bio.1997,138(2): 423-434.
16. Hayashida Y, Honda K, Idogawa M, et al. E-cadherin regulates the association between beta-catenin and actinin-4. Cancer Res. 2005, 65(19):8836-45.
17. Pelosi G, Scarpa A, Puppa G, et al. Alteration of the E-cadherin/beta-catenin cell adhesion system is common in pulmonary neuroendocrine tumors and is an independent predictor of lymph node metastasis in atypical carcinoids. Cancer.2005, 103(6): 1154-64.
18. Marhaba R, Zoller M. CD44 in cancer progression: adhesion, migration and growth regulation. J Mol Histol. 2004, 35(3):211-31.
19. Gong Y, Sun X, Huo L, et al. Expression of cell adhesion molecules, CD44s and E-cadherin, and microvessel density in invasive micropapillary carcinoma of the breast. Histopathology. 2005, 46(l):24-30.
20. Karmakar S, Das C. Modulation of ezrin and E-cadherin expression by IL-1beta and TGF-betal in human trophoblasts。 J Reprod Immunol. 2004, 64(1-2):9-29.
21. Harrison GM, Davies G, Martin TA, et al. Distribution and expression of CD44 isoforms and Ezrin during prostate cancer-endothelium interaction[J]. Int J Oncol,2002,21:935-940.
22. Wang HS, Hung Y, Su CH, et al. CD44 cross-linking induces integrin-mediated adhesion and transendothelial migration in breast cancer cell line by up-regulation of LFA-1 (alpha L beta2) and VLA-4 (alpha4betal).Exp Cell Res.2005, 304(1): 116-26. Epub 2004 Nov 26.
23. Brown KL, Birkenhead D, Lai JC, et al. Regulation of hyaluronan binding by F-actin and colocalization of CD44 and phosphorylated ezrin/radixin/moesin (ERM) proteins in myeloid cells. Exp Cell Res. 2005, 303(2):400-14.
24. Pujuguet P. Del Maestro L, Gautreau A, et al. Ezrin regulates E-cadherin-dependent adherens junction assembly through Racl activation[J].Mol Biol Cell, 2003, 14: 2181-2191.
25. Poullet P, Gautreau A, Kadare G, Ezrin interacts with focal adhesion kinase and induces its activation independently of cell-matrix adhesion. J Biol Chem. 2001,276(40):37686-91. Epub 2001 Jul.
26. Defacque H. Egeberg M, Habermann A, et al. Involvement of Ezrin/Moesin in de novo actin assembly on phagosomal membranes. EMBO J, 2000,19: 199.
27. Lugini L. Lozupone F, Matarrese P, et al. Potent phagocytic activity discriminate metastatic and primary human malignant melanoma: a key role of ezrin[J]. Lab Invest, 2003, 83: 1555.
28. Moilanen J, Lassus H, Leminen A, et al. Ezrin immunoreactivity in relation to survival in serous ovarian carcinoma patients[J]. Gynecologic Oncology, 2003,90: 273.
29. Makitie T, Carpen O, Vaheri A, et al. Ezrin as a prognostic indicator and its relationship to tumor characteristics in uveal malignant melanoma[J]. Invest Ophthalmol Vis Sci, 2001, 42: 2442.
30. Clark EA, Golub TR, Lander ES, et al. Genomic analysis of metastasis reveals an essential role for RhoC. Nature, 2000, 406: 532.
31. Fiorentini C, Falzano L, Fabbri A, et al. Activation of Rho GTPases by cytotoxic necrotizing factor 1 induces macropinocytosis and scavenging activity in epithelial cells[J]. Mol Biol Cell, 2001, 12: 206.
32. Dard N, Louvet-Vallee S, Santa-Maria A, Maro B. Phosphorylation of ezrin on threonine T567 plays a crucial role during compaction in the mouse early embryo.Dev Biol. 2004 Jul 1;271(1):87-97.
33. Orian Rousseau V, Morrison H, Matzke A, et al. Hepatocyte growth factor-induced Ras activation requires ERM proteins linked to both CD44v6 and F-actin. Mol Biol Cell. 2007, 18(1):76-83. Epub 2006 Oct.
2. Hasuike S, Ido A, Uto H, et al. Hepatocyte growth factor accelerates the proliferation of hepatic oval cells and possibly promotes the differentiation in a 2-acetylaminofiuorene/partial hepatectomy model in rats. J Gastroenterol Hepatol.2005,20(11): 1753-1761.
3. Bretscher A. Regulation of cortical structure by the ezrin-radixin-moesin protein family. Curr Opin Cell Biol. 1999, 11(1): 109-116.
4. Pawlak G, Helfman DM. Cytoskeletal changes in cell transformation and tumourigenesis. Curr Opin Genet Dev. 2000, 11(1):41-47.
5. Hiscox S, Jiang WG. Ezrin regulates cell- cell and cell-matrix adhesion, a possible role with E-cadherin/beta-catenin. J Cell Sci. 1999, 112(18):3081- 3090.
6. Andreoli C, Martin M, Le Borgne R, et al. Ezrin has properties to self-associate at the plasma membrane. J Cell Sci. 1994,107(9):2509- 2512.
7. Bruce B, Khanna G, Ren L, et al. Expression of the cytoskeleton linker protein ezrin in human cancers. Clin Exp Metastasis. 2007, 24(2):69-78. Epub 2007 Mar 17
8. Sizemore S, Cicek M, Sizemore N, et al. Podocalyxin increases the aggressive phenotype of breast and prostate cancer cells in vitro through its interaction with ezrin. Cancer Res. 2007, 67(13):6183-91.
9. Li Q, Wu M, Wang H, et al. Ezrin silencing by small hairpin RNA reverses metastatic behaviors of human breast cancer cells. Cancer Lett. 2008, 261(1):55-63. Epub 2007 Dec 26.
10. Deng X, Tannehill-Gregg SH, Nadella MV, et al. Parathyroid hormone-related protein and ezrin are up-regulated in human lung cancer bonemetastases. Clin Exp Metastasis. 2007, 24(2): 107-19. Epub 2007 Mar 17.
11. Elliott BE, Meens JA, SenGupta SK, et al. The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells. Breast Cancer Res. 2005, 7(3):R365-73.
12. Yu Y, Khan J, Khanna C, et al. Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators. Nat Med. 2004, 10(2): 175-81.
13. Khanna C, Wan X, Bose S, et al. The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis.Nat Med.2004,10(2):182-6.
14.Crepaldi T,Gautreau A,Paolo M,et al.Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogrnesis in epithelial cell.J of Cell Bio.1997,138(2):423-434.
15.Harrison GM,Davies G,Martin TA,et al.Distribution and expression of CD44 isoforms and Ezrin during prostate cancer-endothelium interaction.Int J Oncol.2002,21(5):935-40.
16.Tracey A,Martin,Gregory H,The role of the CD44/ezrin complex in cancer metastasis.Crit Rev Oncol Hematol.2003,46(2):165-86.Review.
17.Pujuguet P,Del Maestro L,Gautreau A,et al.Ezrin regulates E-cadherin-dependent adherens junction assembly through Racl activation.Mol Biol Cell.2003,14(5):2181-91
18.张岩,胡美玉,陈碧华,等.体外RNA干扰下调ezrin表达对肝癌细胞生长和运动能力的影响.中华医学杂志.2006,86:530-535.
19.Zhang Y,Hu MY,Chen BH,et al.The membrane-cytoskeleton organizer ezrin is necessary for hepatocellular carcinoma cell growth and invasiveness.J Cancer Res Clin Oncol.2006,132:685-97.
20.Hunter KW.Ezrin,a key component in tumormetastasis.Trends Mol Med.2004,10:201-204.
21.田健,汤钊猷,叶胜龙,等.具有高转移潜能的人肝癌细胞系的建立及其生物学特征.中华肿瘤杂志.1998,20:405-407
22.Sui G,Soohoo C,Affar el B,et al.A DNA vector- based RNAi technology to supress gene expression in mammalian cells.Proc Natl Acad Sci U S A.2002,99:5515-5520.
23.AdEasyTM Adenoviral Vector System Instruction Manual:Stratagene Campany
24.Naba A,Reverdy C,Louvard D,et al.Spatial recruitment and activation of the Fes kinase by ezrin promotes HGF-induced cell scattering.EMBO J.2008,27(1):38-50.Epub 2007 Nov 29.
25.Poullet P,Gautreau A,Kadare G,et al.Ezrin interacts with focal adhesion kinase and induces its activation independently of cell-matrix adhesion.J Biol Chem.2001,276(40):37686-91.Epub 2001 Jul
26.Orian Rousseau V,Morrison H,Matzke A,et al.Hepatocyte growth factor-induced Ras activation requires ERM proteins linked to both.CD44v6 and F-actin. Mol Biol Cell. 2007, 18(1):76-83. Epub 2006 Oct.
27. Orian Rousseau V, Chen L, Sleeman JP, et al. CD44 is required for two consecutive steps in HGF/c-Met signaling. Genes Dev. 2002,16(23):3074-86.
28. Elliott BE, Qiao H, Louvard D, et al. Co-operative effect of c-Src and ezrin in deregulation of cell-cell contacts and scattering of mammary carcinoma cells. J Cell Biochem. 2004, 92(1): 16-28.
29. Kreimann EL, Morales FC, de Orbeta-Cruz J, et al. Cortical stabilization of beta-catenin contributes to NHERF1/EBP50 tumor suppressor function. Oncogene.2007,26(36):5290-9. Epub 2007 Feb.
30. Johnson MH, McConnell JM. Lineage allocation and cell polarity during mouse embryogenesis. Semin Cell Dev Biol. 2004,15(5):583-97.
31. Dard N, Louvet-Vallee S, Santa-Maria A, Maro B. Phosphorylation of ezrin on threonine T567 plays a crucial role during compaction in the mouse early embryo.Dev Biol. 2004, 271(1):87-97.
1. Bretscher A, Reczek D, Berryman M. Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures [J]. J Cell Sci, 1997, 110(24): 3011.
2. Yonemura S, Tsukita S, Tsukita S. Direct involvement of ezrin/ radixin/moesin (ERM)-binding membrane proteins in the organization of microvilli in collaboration with activated ERM proteins[J]. J Cell Biol, 1999, 145(7): 1497.
3. Granes F, Urena JM, Rocamora N, et al. Ezrin links syndecan-2 to the cytoskeleton[J]. J Cell Sci, 2000, (7): 1267.
4. Bretscher A. Regulation of cortical structure by the ezrin-radixin-moesin protein family[J]. Curr Opin Cell Biol, 1999, 11(1): 109.
5. Akisawa N, Nishimori I, Iwamura T, et al. High levels of ezrin expressed by human pancreatic adenocarcinoma cell lines with high metastatic potential[J].Biochem Biophys Res Commun, 1999, 258(2): 395.
6. Hiscox S, Jiang WG. Ezrin regulates cell_ cell and cell_matrix adhesion, a possible role with E-cadherin/beta-catenin[J]. J Cell Sci, 1999, 112(18): 3081.
7. Pawlak G, Helfman DM. Cytoskeletal changes in cell transformation and tumourigenesis[J]. Curr Opin Genet Dev, 2000, 11(1): 41.
8. Bruce B, Khanna G, Ren L, et al. Expression of the cytoskeleton linker protein ezrin in human cancers. Clin Exp Metastasis. 2007, 24(2):69-78. Epub 2007 Mar 17.
9. Sizemore S, Cicek M, Sizemore N, et al. Podocalyxin increases the aggressive phenotype of breast and prostate cancer cells in vitro through its interaction with ezrin. Cancer Res. 2007, 67(13):6183-91.
10. Li Q, Wu M, Wang H, et al. Ezrin silencing by small hairpin RNA reverses metastatic behaviors of human breast cancer cells. Cancer Lett. 2008, 261(1):55-63. Epub 2007 Dec 26.
11. Deng X, Tannehill-Gregg SH, Nadella MV, et al. Parathyroid hoimone-related protein and ezrin are up-regulated in human lung cancer bone metastases. Clin Exp Metastasis. 2007, 24(2):107-19. Epub 2007 Mar 17.
12. Elliott BE, Meens JA, SenGupta SK, et al. The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells. Breast Cancer Res. 2005, 7(3):R365-73.
13. Yu Y, Khan J, Khanna C, et al. Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators. Nat Med. 2004, 10(2): 175-81.
14. Khanna C, Wan X, Bose S, et al. The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis. Nat Med. 2004,10(2): 182-6.
15. Crepaldi T, Gautreau A, Paolo M, et al. Ezrin is an effector of hepatocyte growth factor -mediated migration and morphogrnesis in epithelial cell. J of Cell Bio.1997,138(2): 423-434.
16. Hayashida Y, Honda K, Idogawa M, et al. E-cadherin regulates the association between beta-catenin and actinin-4. Cancer Res. 2005, 65(19):8836-45.
17. Pelosi G, Scarpa A, Puppa G, et al. Alteration of the E-cadherin/beta-catenin cell adhesion system is common in pulmonary neuroendocrine tumors and is an independent predictor of lymph node metastasis in atypical carcinoids. Cancer.2005, 103(6): 1154-64.
18. Marhaba R, Zoller M. CD44 in cancer progression: adhesion, migration and growth regulation. J Mol Histol. 2004, 35(3):211-31.
19. Gong Y, Sun X, Huo L, et al. Expression of cell adhesion molecules, CD44s and E-cadherin, and microvessel density in invasive micropapillary carcinoma of the breast. Histopathology. 2005, 46(l):24-30.
20. Karmakar S, Das C. Modulation of ezrin and E-cadherin expression by IL-1beta and TGF-betal in human trophoblasts。 J Reprod Immunol. 2004, 64(1-2):9-29.
21. Harrison GM, Davies G, Martin TA, et al. Distribution and expression of CD44 isoforms and Ezrin during prostate cancer-endothelium interaction[J]. Int J Oncol,2002,21:935-940.
22. Wang HS, Hung Y, Su CH, et al. CD44 cross-linking induces integrin-mediated adhesion and transendothelial migration in breast cancer cell line by up-regulation of LFA-1 (alpha L beta2) and VLA-4 (alpha4betal).Exp Cell Res.2005, 304(1): 116-26. Epub 2004 Nov 26.
23. Brown KL, Birkenhead D, Lai JC, et al. Regulation of hyaluronan binding by F-actin and colocalization of CD44 and phosphorylated ezrin/radixin/moesin (ERM) proteins in myeloid cells. Exp Cell Res. 2005, 303(2):400-14.
24. Pujuguet P. Del Maestro L, Gautreau A, et al. Ezrin regulates E-cadherin-dependent adherens junction assembly through Racl activation[J].Mol Biol Cell, 2003, 14: 2181-2191.
25. Poullet P, Gautreau A, Kadare G, Ezrin interacts with focal adhesion kinase and induces its activation independently of cell-matrix adhesion. J Biol Chem. 2001,276(40):37686-91. Epub 2001 Jul.
26. Defacque H. Egeberg M, Habermann A, et al. Involvement of Ezrin/Moesin in de novo actin assembly on phagosomal membranes. EMBO J, 2000,19: 199.
27. Lugini L. Lozupone F, Matarrese P, et al. Potent phagocytic activity discriminate metastatic and primary human malignant melanoma: a key role of ezrin[J]. Lab Invest, 2003, 83: 1555.
28. Moilanen J, Lassus H, Leminen A, et al. Ezrin immunoreactivity in relation to survival in serous ovarian carcinoma patients[J]. Gynecologic Oncology, 2003,90: 273.
29. Makitie T, Carpen O, Vaheri A, et al. Ezrin as a prognostic indicator and its relationship to tumor characteristics in uveal malignant melanoma[J]. Invest Ophthalmol Vis Sci, 2001, 42: 2442.
30. Clark EA, Golub TR, Lander ES, et al. Genomic analysis of metastasis reveals an essential role for RhoC. Nature, 2000, 406: 532.
31. Fiorentini C, Falzano L, Fabbri A, et al. Activation of Rho GTPases by cytotoxic necrotizing factor 1 induces macropinocytosis and scavenging activity in epithelial cells[J]. Mol Biol Cell, 2001, 12: 206.
32. Dard N, Louvet-Vallee S, Santa-Maria A, Maro B. Phosphorylation of ezrin on threonine T567 plays a crucial role during compaction in the mouse early embryo.Dev Biol. 2004 Jul 1;271(1):87-97.
33. Orian Rousseau V, Morrison H, Matzke A, et al. Hepatocyte growth factor-induced Ras activation requires ERM proteins linked to both CD44v6 and F-actin. Mol Biol Cell. 2007, 18(1):76-83. Epub 2006 Oct.