Egf17的新作用：通过EGFR活化FAK促进肝细胞癌侵袭转移的研究
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摘要
肝细胞癌(HCC)是当前严重威胁我国人民生命健康的重大恶性肿瘤,而其复发转移是导致死亡率居高不下的最主要原因。究其发生的机制虽有大量研究,但目前仍未完全阐明。因此,深入研究HCC复发转移的分子机制并以此为基础探索防治复发转移的有效措施,对进一步提高HCC的总体治疗水平具有重要意义。
表皮生长因子样结构域7(Egfl7)是近年来新发现的一种分泌型蛋白,已有的研究证明其在胚胎血管发育的成管过程中发挥了重要作用。近来的研究发现Egfl7能够显著增强内皮细胞及成纤维细胞的运动迁移能力,提示Egfl7具有调控细胞运动迁移的生物学功能。更重要的是,Egfl7在胚胎组织中高水平表达,在成熟组织中几乎不表达,而在肿瘤等增殖性组织中呈高度上调,提示Egfl7可能在肿瘤发生、发展中发挥了重要作用。我们前期的研究也发现,Egfl7 mRNA在HCC组织中的表达水平明显上调并与HCC包膜形成密切相关,提示Egfl7可能参与HCC侵袭转移过程,但其作用机制目前尚不清楚。因此,本研究在进一步分析Egfl7在HCC中表达情况的基础上,通过慢病毒介导的小RNA干扰技术抑制Egfl7的表达,通过一系列体外、体内方法研究Egfl7调控HCC侵袭转移的分子机制,同时探讨阻断Egfl7表达对HCC侵袭转移的影响。我们获得了以下研究结果:
1.我们采用半定量RT-PCR、荧光实时定量PCR和Western blot等方法检测了Egfl7 mRNA和蛋白在31例新鲜HCC组织及相应邻近非瘤肝组织(ANLT)中的表达水平,结果发现Egfl7 mRNA和蛋白在HCC组织中均明显高表达,且Egfl7在孤立性大肝癌(SLHCC)中的表达明显低于结节性肝癌(NHCC)。免疫组化法检测112例HCC组织的结果显示Egfl7在90.2%(101/112)的HCC组织中表达,且其表达水平与HCC的肿瘤结节数目、有无静脉浸润及有无包膜形成等临床病理特征密切相关。Egfl7高表达组HCC患者的无瘤生存时间及总体生存时间均明显短于Egfl7低表达组的患者,且单因素与多因素Cox回归模型均显示Egfl7高表达是HCC预后的独立危险因素。同时,夹心ELISA法发现HCC病人血清中Egfl7水平相对于慢性肝病病人、胃肠道恶性肿瘤病人、肝良性肿瘤病人和正常人亦明显上升(详见颜鹏等其他研究生毕业论文)。这些结果提示Egfl7的高表达与HCC的侵袭转移密切相关,并可能因此而影响了HCC患者的预后。
2.双标间接组织免疫荧光法检测Egfl7在HCC组织中的分布情况,结果显示Egfl7绝大多数定位于HCC组织中的肿瘤细胞胞质内。Western blot方法检测Egfl7蛋白在HepG2、MHCC97-L及HCCLM3这三种侵袭转移能力依次升高的肝癌细胞系中的表达并以常氏肝细胞系CCL13作为对照,结果显示Egfl7蛋白在三种肝癌细胞系中的表达水平较之CCL13细胞系均明显升高,且以HCCLM3细胞系的表达水平最高,在MHCC97-L细胞系中的表达水平次之,HepG2最低,提示Egfl7的表达水平与与肝癌细胞的侵袭转移潜能密切相关。
3.我们构建了pLKO.1~(Egfl7RNAi+)siRNA质粒,并建立了稳定表达Egfl7 siRNA片断和对照片断的HCCLM3细胞(HCCLM3~(Egfl7RNAi+)和HCCLM3~(Egfl7RNAi-))。Western blot结果发现HCCLM3~(Egfl7RNAi+)细胞中Egfl7蛋白的表达被明显抑制,干预效率超过70%。划痕实验和Transwell侵袭小室实验结果均显示HCCLM3~(Egfl7RNAi+)细胞较HCCLM3~(Egfl7RNAi-)细胞迁移运动能力明显下降;MTT法结果显示HCCLM3~(Egfl7RNAi+)和HCCLM3~(Egfl7RNAi-)细胞的增殖能力无显著性差异(P>0.05);Annexin V和PI双染法结合流式细胞仪检测结果显示HCCLM3~(Egfl7RNAi+)与HCCLM3~(Egfl7RNAi-)细胞的凋亡的差异无统计学差异(P>0.05)。且Bcl-x_L和BAX在HCCLM3~(Egfl7RNAi+)与HCCLM3~(Egfl7RNAi-)细胞中的表达无明显差异性(P>0.05)。这些研究结果均提示:体外抑制Egfl7的表达可以显著抑制肝癌细胞的迁移运动,而不影响其增殖和凋亡。
4.已有研究发现Egfl7敲除的小鼠中,血管内皮细胞中局部粘着斑激酶(FAK)的磷酸化水平明显下降,同时血管内皮细胞的迁移能力明显减弱。我们因而推测,Egfl7可能通过促进FAK磷酸化激活从而调控肝癌细胞的迁移运动能力。因此,我们用Western blot法证明HCCLM3~(Egfl7RNAi+)细胞中总FAK水平没有明显改变,但磷酸化FAK水平明显下降。当我们用全长重组Egfl7蛋白(50ng/ml)刺激HCCLM3~(Egfl7RNAi+)细胞后,Western blot检测结果显示其磷酸化FAK水平明显上调;细胞免疫荧光结果显示其肌动蛋白(F-actin)细胞骨架发生明显的形变;划痕实验和Transwell侵袭小室实验证明其迁移运动能力明显增强。这些结果提示Egfl7可通过调控FAK磷酸化激活从而促进肝癌细胞的迁移运动。
5.鉴于Egfl7拥有两个表皮生长因子样结构域(EGF-likedomain),我们推测其可能通过表皮生长因子受体(EGFR)介导FAK的磷酸化激活。因此,我们利用EGFR的特异性抑制剂(15μM)先行处理HCCLM3~(Egfl7RNAi+)细胞,再施以Egfl7蛋白(50ng/ml)刺激,观察HCCLM3~(Egfl7RNAi+)细胞中FAK磷酸化水平、细胞骨架及迁移运动能力的变化。结果显示,HCCLM3~(Egfl7RNAi+)细胞中FAK磷酸化水平无明显变化,F-actin细胞骨架无明显形变,细胞迁移运动能力亦无明显增强,即EGFR抑制剂可阻断Egfl7蛋白刺激HCCLM3~(Egfl7RNAi+)细胞所产生的后续效应,提示Egfl7通过EGFR介导FAK磷酸化而促进肝癌细胞迁移运动,证明Egfl7/EGFR/FAK这一信号传导通路在肝癌细胞迁移运动的调控中发挥了重要作用。
6.为在体内观察抑制Egfl7对肝癌侵袭转移的影响,我们在HCCLM3转移性人肝癌细胞裸鼠模型的基础上,建立了裸鼠原位肝癌转移模型。免疫组化结果显示,siRNA抑制Egfl7表达后,裸鼠肝脏原位HCCLM3种植瘤中Egfl7及磷酸化FAK的表达水平明显下调;测量肝脏原位种植瘤的大小及周围结节数目,结果显示体内抑制Egfl7的表达能够抑制HCCLM3原发瘤的生长并减少其肝内转移;裸鼠肺组织连续切片计数肺转移的数目,结果显示体内抑制Egfl7能够抑制HCCLM3细胞的肺转移。我们的结果证明体内抑制Egfl7表达可抑制FAK磷酸化并显著抑制了HCC的生长和肝内外转移。
我们的上述研究结果首次证明Egfl7在HCC组织及病人血清中高表达,且其表达水平与HCC肿瘤结节数目、包膜形成及静脉浸润等临床病理特征密切相关,并显著影响了HCC患者的预后。同时,我们首次发现Egfl7主要表达于HCC组织中的肿瘤细胞,且Egfl7可通过EGFR介导FAK磷酸化促进肝癌细胞迁移运动,从而在HCC侵袭转移中发挥重要作用,抑制Egfl7表达可在体内外显著抑制肝癌细胞的侵袭转移。本研究首次揭示了Egfl7在HCC侵袭转移中的新的生物学作用并初步阐明其作用机制,提示Egfl7可作为HCC诊断、复发转移监测和预后评估的新型血清/分子标志物及HCC复发转移治疗的潜在干预靶点,有望为HCC的诊断及复发转移的防治提供新的理论依据和研究思路。
Hepatocellular carcinoma (HCC) is one of the most common malignancies in China. Though the hepatic resection for HCC has evolved into a safe procedure with low operative mortality, the long-term survival remains unsatisfactory because of a high incidence of recurrence and metastasis after the hepatic resection. Thus, the inhibition of invasion and metastasis is of great importance in the HCC therapies.
Epidermal growth factor-like domain 7 (Egfl7) is a recently identified secreted protein and once believed to be specifically expressed in endothelial cells (ECs) and essential in the process of vascular development. Recent studies have demonstrated that Egfl7 can act as a chemoattractant for cell migration and regulate collective migration of ECs, indicating that Egfl7 may be important in cell motility. Interestingly, Egfl7 expression is high during embryonic development, down-regulated in almost all mature tissues, and increases again during tumorigenesis. Moreover, our previous results have shown the overexpression of Egfl7 in HCC tissues, implicating its potential role in HCC. However, its role in HCC remains unknown. Therefore, we carried out the present study to determine the expression of Egfl7 in human HCC tissues as well as cell lines and tried to elucidate the function of Egfl7 in the metastasis of HCC by characterizing its role in cell migration through both in vitro and in vivo models.
1. Semi-quantitative PCR, real-time quantitative PCR and Western blot were employed to detect Egfl7 mRNA and protein in 31 cases of HCC tissues and corresponding adjacent nontumorous liver tissues (ANLTs). Our results showed that both of Egfl7 mRNA and protein expression levels increased significantly in HCC tissues. Meanwhile, Egfl7 mRNA and protein expression levels were significantly higher in nodular hepatocellular carcinoma than those in solitary large hepatocellular carcinoma. Immunohistochemistry was used to detect Egfl7 in 112 cases of HCC tissues and the results showed that Egfl7 expressed in 90.2% of HCC tissues (101/112) and its expression correlates significantly with multiple nodules, without capsular and vein invasion. ELISA determination also showed that serum Egfl7 level in HCC patients increased significantly compared with chronic liver disease patients, gastroenterological cancer patients, benigh liver tumor patients and normal people. Moreover, HCC patients within the high Egfl7 expression group had either poorer disease free survival or poorer overall survival than those within the low expression group. By univariable and multivariable Cox regression analysis, high Egfl7 expression was found to be independent prognostic factors for overall survival.
2. Double-labeled indirect histoimmunofluorescence was performed to detect the distribution of Egfl7 in HCC tissue. HCC cells in tissues, identified by anti-AFP antibody labeling, were positively stained by a special anti-Egfl7 antibody, suggesting that the Egfl7 protein in HCC tissues were mainly expressed in plasma of HCC carcinoma cells, but not ECs. We further confirm the Egfl7 expression in three HCC cell lines: HepG_2, MHCC97-L and HCCLM3, with a liver cell line CCL13 as a control. The results showed a significantly higher expression of Egfl7 in HCC cells than in normal live cells. Among the three cell lines analyzed, HCCLM3 cells have the highest Egfl7 expression, followed by MHCC97-L and HepG2. Of note is that the different expression levels of Egfl7 correlate with the metastatic potential of these HCC cell lines, suggesting a potential role for Egfl7 in HCC metastasis.
3. To define the correlation of Egfl7 expression and HCC metastasis, we employed siRNA approach to inhibit expression of Egfl7 in HCCLM3 cells. We identified three putative candidate sequences and one control sequence and cloned them into the pLKO.1.puro lentiviral expression vector. After selection with puromycin, HCCLM3 cell lines stably expressed siRNA-expressing sequence and control sequences, were obtained and named as HCCLM3~( Egfl7RNAi+) and HCCLM3 ~( Egfl7RNAi-) respectively. The expression of Egfl7 protein was decreased dramatically in HCCLM3~( Egfl7RNAi+) compared with HCCLM3~( Egfl7RNAi-), which showed a more than 70% inhibitory efficiency. Wound-healing assay and Transwell invasion assay were employed to determine the migration of HCCLM3 cells and the results showed the decreased migration of HCCLM3~( Egfl7RNAi+) cells compared with HCCLM3~( Egfl7RNAi-) cells. Proliferation rates of HCCLM3~( Egfl7RNAi+) and HCCLM3~( Egfl7RNAi-) cells were compared by MTT method and the results showed no significant deference between these two cells. At the same time, apoptosis in HCCLM3~( Egfl7RNAi+) and HCCLM3~( Egfl7RNAi-) cells, as determined using FASC, showed no significant difference (P>0.05). Bcl-x_L and BAX were also detected by Western blot and their expression levels were not significantly changed between HCCLM3~( Egfl7RNAi+) and HCCLM3~( Egfl7RNAi-) cells. These results suggest that Egfl7 contributes to metastasis without significantly effecting proliferation and apoptosis of HCCLM3 cell.
4. In Egfl7 deficient mice, the focal adhesion kinase (FAK) phosphrylation of EC was significantly reduced, implicating the potential property of Egfl7 in regulation of cell motility. Based on this, we presumed that Egfl7 might enhance cell motility by activating FAK. Therefore, we documented the decreased phosphrylated FAK in HCCLM3~( Egfl7RNAi+) cells without significant change of total FAK level by Western blot. Then we treated HCCLM3~( Egfl7RNAi+) cells with recombinant Egfl7 protein and found the phosphorylated FAK level significantly elevated after this stimulation. Meantime, F-actin was reorganized in HCCLM3~( Egfl7RNAi+) cells and the enhanced migration also was evidenced by wound healing assay and Transwell assay, implicating an important role of Egfl7 in controlling cell motility through mediating FAK phosphrylation.
5. Based on two EGF-like domains included in the structure of Egfl7, we presumed Egfl7 may mediate FAK phosphrylation through EGFR just like EGF. Then we treated HCCLM3~( Egfl7RNAi+)cells with EGFR inhibitor (15μM) before recombinant Egfl7 protein (50ng/ml) stimulation and found that the FAK phosphrylation, F-actin reorganization level, and cell migration did not change siginificantly. The effects of Egfl7 protein on HCCLM3~( Egfl7RNAi+)cells were all blocked by EGFR inhibitor, which implicated that FAK activation by Egfl7 was EGFR-dependent and the Egfl7/EGFR/FAK pathway may be critical in controlling HCC cell motility.
6. We examined the in vivo relevance of the potential role for Egfl7 in HCC tumorigenesis by using a mouse implantation model. The expression of Egfl7 and phosphorylated FAK in HCCLM3 tumor was determined by immunohistochemistry and the results showed that the expression of Egfl7 and phosphorylated FAK was all significantly decreased in HCCLM3~( Egfl7RNAi+) tumor than that in HCCLM3~( Egfl7RNAi-) tumor. At the 35th day after implantation, we found that the average size of liver tumors in HCCLM3~( Egfl7RNAi+) group was dramatically smaller than that of HCCLM3~( Egfl7RNAi-) group. Two of eight mice in the HCCLM3~( Egfl7RNAi+) group showed intrahepatic metastasis (25%), which is significantly lower than that in the HCCLM3~( Egfl7RNAi-) group (seven of eight, 88%). The pulmonary metastasis was observed in the lung tissue sections of only one mouse in HCCLM3~( Egfl7RNAi+) group (one of eight, 13%), much less than the ratio of pulmonary metastasis in HCCLM3~( Egfl7RNAi-) group (four of eight, 50%). Together, these data support an important role for Egfl7 in HCC metastasis.
In conclusion, our study has shown for the first time that Egfl7 expresses in HCC carcinoma cells and its overexpression in HCC tissues and serum significantly correlates to poor prognosis of HCC. Furthermore, we have demonstrated the novel role of Egfl7 in metastasis of HCC by enhancing cell motility through EGFR- dependent FAK phosphorylation, implicating Egfl7 as a novel serum/ molecular marker for diagnosis, monitoring after hepatectomy and prognosis of HCC and a potential therapeutic target for metastasis of HCC.
表皮生长因子样结构域7(Egfl7)是近年来新发现的一种分泌型蛋白,已有的研究证明其在胚胎血管发育的成管过程中发挥了重要作用。近来的研究发现Egfl7能够显著增强内皮细胞及成纤维细胞的运动迁移能力,提示Egfl7具有调控细胞运动迁移的生物学功能。更重要的是,Egfl7在胚胎组织中高水平表达,在成熟组织中几乎不表达,而在肿瘤等增殖性组织中呈高度上调,提示Egfl7可能在肿瘤发生、发展中发挥了重要作用。我们前期的研究也发现,Egfl7 mRNA在HCC组织中的表达水平明显上调并与HCC包膜形成密切相关,提示Egfl7可能参与HCC侵袭转移过程,但其作用机制目前尚不清楚。因此,本研究在进一步分析Egfl7在HCC中表达情况的基础上,通过慢病毒介导的小RNA干扰技术抑制Egfl7的表达,通过一系列体外、体内方法研究Egfl7调控HCC侵袭转移的分子机制,同时探讨阻断Egfl7表达对HCC侵袭转移的影响。我们获得了以下研究结果:
1.我们采用半定量RT-PCR、荧光实时定量PCR和Western blot等方法检测了Egfl7 mRNA和蛋白在31例新鲜HCC组织及相应邻近非瘤肝组织(ANLT)中的表达水平,结果发现Egfl7 mRNA和蛋白在HCC组织中均明显高表达,且Egfl7在孤立性大肝癌(SLHCC)中的表达明显低于结节性肝癌(NHCC)。免疫组化法检测112例HCC组织的结果显示Egfl7在90.2%(101/112)的HCC组织中表达,且其表达水平与HCC的肿瘤结节数目、有无静脉浸润及有无包膜形成等临床病理特征密切相关。Egfl7高表达组HCC患者的无瘤生存时间及总体生存时间均明显短于Egfl7低表达组的患者,且单因素与多因素Cox回归模型均显示Egfl7高表达是HCC预后的独立危险因素。同时,夹心ELISA法发现HCC病人血清中Egfl7水平相对于慢性肝病病人、胃肠道恶性肿瘤病人、肝良性肿瘤病人和正常人亦明显上升(详见颜鹏等其他研究生毕业论文)。这些结果提示Egfl7的高表达与HCC的侵袭转移密切相关,并可能因此而影响了HCC患者的预后。
2.双标间接组织免疫荧光法检测Egfl7在HCC组织中的分布情况,结果显示Egfl7绝大多数定位于HCC组织中的肿瘤细胞胞质内。Western blot方法检测Egfl7蛋白在HepG2、MHCC97-L及HCCLM3这三种侵袭转移能力依次升高的肝癌细胞系中的表达并以常氏肝细胞系CCL13作为对照,结果显示Egfl7蛋白在三种肝癌细胞系中的表达水平较之CCL13细胞系均明显升高,且以HCCLM3细胞系的表达水平最高,在MHCC97-L细胞系中的表达水平次之,HepG2最低,提示Egfl7的表达水平与与肝癌细胞的侵袭转移潜能密切相关。
3.我们构建了pLKO.1~(Egfl7RNAi+)siRNA质粒,并建立了稳定表达Egfl7 siRNA片断和对照片断的HCCLM3细胞(HCCLM3~(Egfl7RNAi+)和HCCLM3~(Egfl7RNAi-))。Western blot结果发现HCCLM3~(Egfl7RNAi+)细胞中Egfl7蛋白的表达被明显抑制,干预效率超过70%。划痕实验和Transwell侵袭小室实验结果均显示HCCLM3~(Egfl7RNAi+)细胞较HCCLM3~(Egfl7RNAi-)细胞迁移运动能力明显下降;MTT法结果显示HCCLM3~(Egfl7RNAi+)和HCCLM3~(Egfl7RNAi-)细胞的增殖能力无显著性差异(P>0.05);Annexin V和PI双染法结合流式细胞仪检测结果显示HCCLM3~(Egfl7RNAi+)与HCCLM3~(Egfl7RNAi-)细胞的凋亡的差异无统计学差异(P>0.05)。且Bcl-x_L和BAX在HCCLM3~(Egfl7RNAi+)与HCCLM3~(Egfl7RNAi-)细胞中的表达无明显差异性(P>0.05)。这些研究结果均提示:体外抑制Egfl7的表达可以显著抑制肝癌细胞的迁移运动,而不影响其增殖和凋亡。
4.已有研究发现Egfl7敲除的小鼠中,血管内皮细胞中局部粘着斑激酶(FAK)的磷酸化水平明显下降,同时血管内皮细胞的迁移能力明显减弱。我们因而推测,Egfl7可能通过促进FAK磷酸化激活从而调控肝癌细胞的迁移运动能力。因此,我们用Western blot法证明HCCLM3~(Egfl7RNAi+)细胞中总FAK水平没有明显改变,但磷酸化FAK水平明显下降。当我们用全长重组Egfl7蛋白(50ng/ml)刺激HCCLM3~(Egfl7RNAi+)细胞后,Western blot检测结果显示其磷酸化FAK水平明显上调;细胞免疫荧光结果显示其肌动蛋白(F-actin)细胞骨架发生明显的形变;划痕实验和Transwell侵袭小室实验证明其迁移运动能力明显增强。这些结果提示Egfl7可通过调控FAK磷酸化激活从而促进肝癌细胞的迁移运动。
5.鉴于Egfl7拥有两个表皮生长因子样结构域(EGF-likedomain),我们推测其可能通过表皮生长因子受体(EGFR)介导FAK的磷酸化激活。因此,我们利用EGFR的特异性抑制剂(15μM)先行处理HCCLM3~(Egfl7RNAi+)细胞,再施以Egfl7蛋白(50ng/ml)刺激,观察HCCLM3~(Egfl7RNAi+)细胞中FAK磷酸化水平、细胞骨架及迁移运动能力的变化。结果显示,HCCLM3~(Egfl7RNAi+)细胞中FAK磷酸化水平无明显变化,F-actin细胞骨架无明显形变,细胞迁移运动能力亦无明显增强,即EGFR抑制剂可阻断Egfl7蛋白刺激HCCLM3~(Egfl7RNAi+)细胞所产生的后续效应,提示Egfl7通过EGFR介导FAK磷酸化而促进肝癌细胞迁移运动,证明Egfl7/EGFR/FAK这一信号传导通路在肝癌细胞迁移运动的调控中发挥了重要作用。
6.为在体内观察抑制Egfl7对肝癌侵袭转移的影响,我们在HCCLM3转移性人肝癌细胞裸鼠模型的基础上,建立了裸鼠原位肝癌转移模型。免疫组化结果显示,siRNA抑制Egfl7表达后,裸鼠肝脏原位HCCLM3种植瘤中Egfl7及磷酸化FAK的表达水平明显下调;测量肝脏原位种植瘤的大小及周围结节数目,结果显示体内抑制Egfl7的表达能够抑制HCCLM3原发瘤的生长并减少其肝内转移;裸鼠肺组织连续切片计数肺转移的数目,结果显示体内抑制Egfl7能够抑制HCCLM3细胞的肺转移。我们的结果证明体内抑制Egfl7表达可抑制FAK磷酸化并显著抑制了HCC的生长和肝内外转移。
我们的上述研究结果首次证明Egfl7在HCC组织及病人血清中高表达,且其表达水平与HCC肿瘤结节数目、包膜形成及静脉浸润等临床病理特征密切相关,并显著影响了HCC患者的预后。同时,我们首次发现Egfl7主要表达于HCC组织中的肿瘤细胞,且Egfl7可通过EGFR介导FAK磷酸化促进肝癌细胞迁移运动,从而在HCC侵袭转移中发挥重要作用,抑制Egfl7表达可在体内外显著抑制肝癌细胞的侵袭转移。本研究首次揭示了Egfl7在HCC侵袭转移中的新的生物学作用并初步阐明其作用机制,提示Egfl7可作为HCC诊断、复发转移监测和预后评估的新型血清/分子标志物及HCC复发转移治疗的潜在干预靶点,有望为HCC的诊断及复发转移的防治提供新的理论依据和研究思路。
Hepatocellular carcinoma (HCC) is one of the most common malignancies in China. Though the hepatic resection for HCC has evolved into a safe procedure with low operative mortality, the long-term survival remains unsatisfactory because of a high incidence of recurrence and metastasis after the hepatic resection. Thus, the inhibition of invasion and metastasis is of great importance in the HCC therapies.
Epidermal growth factor-like domain 7 (Egfl7) is a recently identified secreted protein and once believed to be specifically expressed in endothelial cells (ECs) and essential in the process of vascular development. Recent studies have demonstrated that Egfl7 can act as a chemoattractant for cell migration and regulate collective migration of ECs, indicating that Egfl7 may be important in cell motility. Interestingly, Egfl7 expression is high during embryonic development, down-regulated in almost all mature tissues, and increases again during tumorigenesis. Moreover, our previous results have shown the overexpression of Egfl7 in HCC tissues, implicating its potential role in HCC. However, its role in HCC remains unknown. Therefore, we carried out the present study to determine the expression of Egfl7 in human HCC tissues as well as cell lines and tried to elucidate the function of Egfl7 in the metastasis of HCC by characterizing its role in cell migration through both in vitro and in vivo models.
1. Semi-quantitative PCR, real-time quantitative PCR and Western blot were employed to detect Egfl7 mRNA and protein in 31 cases of HCC tissues and corresponding adjacent nontumorous liver tissues (ANLTs). Our results showed that both of Egfl7 mRNA and protein expression levels increased significantly in HCC tissues. Meanwhile, Egfl7 mRNA and protein expression levels were significantly higher in nodular hepatocellular carcinoma than those in solitary large hepatocellular carcinoma. Immunohistochemistry was used to detect Egfl7 in 112 cases of HCC tissues and the results showed that Egfl7 expressed in 90.2% of HCC tissues (101/112) and its expression correlates significantly with multiple nodules, without capsular and vein invasion. ELISA determination also showed that serum Egfl7 level in HCC patients increased significantly compared with chronic liver disease patients, gastroenterological cancer patients, benigh liver tumor patients and normal people. Moreover, HCC patients within the high Egfl7 expression group had either poorer disease free survival or poorer overall survival than those within the low expression group. By univariable and multivariable Cox regression analysis, high Egfl7 expression was found to be independent prognostic factors for overall survival.
2. Double-labeled indirect histoimmunofluorescence was performed to detect the distribution of Egfl7 in HCC tissue. HCC cells in tissues, identified by anti-AFP antibody labeling, were positively stained by a special anti-Egfl7 antibody, suggesting that the Egfl7 protein in HCC tissues were mainly expressed in plasma of HCC carcinoma cells, but not ECs. We further confirm the Egfl7 expression in three HCC cell lines: HepG_2, MHCC97-L and HCCLM3, with a liver cell line CCL13 as a control. The results showed a significantly higher expression of Egfl7 in HCC cells than in normal live cells. Among the three cell lines analyzed, HCCLM3 cells have the highest Egfl7 expression, followed by MHCC97-L and HepG2. Of note is that the different expression levels of Egfl7 correlate with the metastatic potential of these HCC cell lines, suggesting a potential role for Egfl7 in HCC metastasis.
3. To define the correlation of Egfl7 expression and HCC metastasis, we employed siRNA approach to inhibit expression of Egfl7 in HCCLM3 cells. We identified three putative candidate sequences and one control sequence and cloned them into the pLKO.1.puro lentiviral expression vector. After selection with puromycin, HCCLM3 cell lines stably expressed siRNA-expressing sequence and control sequences, were obtained and named as HCCLM3~( Egfl7RNAi+) and HCCLM3 ~( Egfl7RNAi-) respectively. The expression of Egfl7 protein was decreased dramatically in HCCLM3~( Egfl7RNAi+) compared with HCCLM3~( Egfl7RNAi-), which showed a more than 70% inhibitory efficiency. Wound-healing assay and Transwell invasion assay were employed to determine the migration of HCCLM3 cells and the results showed the decreased migration of HCCLM3~( Egfl7RNAi+) cells compared with HCCLM3~( Egfl7RNAi-) cells. Proliferation rates of HCCLM3~( Egfl7RNAi+) and HCCLM3~( Egfl7RNAi-) cells were compared by MTT method and the results showed no significant deference between these two cells. At the same time, apoptosis in HCCLM3~( Egfl7RNAi+) and HCCLM3~( Egfl7RNAi-) cells, as determined using FASC, showed no significant difference (P>0.05). Bcl-x_L and BAX were also detected by Western blot and their expression levels were not significantly changed between HCCLM3~( Egfl7RNAi+) and HCCLM3~( Egfl7RNAi-) cells. These results suggest that Egfl7 contributes to metastasis without significantly effecting proliferation and apoptosis of HCCLM3 cell.
4. In Egfl7 deficient mice, the focal adhesion kinase (FAK) phosphrylation of EC was significantly reduced, implicating the potential property of Egfl7 in regulation of cell motility. Based on this, we presumed that Egfl7 might enhance cell motility by activating FAK. Therefore, we documented the decreased phosphrylated FAK in HCCLM3~( Egfl7RNAi+) cells without significant change of total FAK level by Western blot. Then we treated HCCLM3~( Egfl7RNAi+) cells with recombinant Egfl7 protein and found the phosphorylated FAK level significantly elevated after this stimulation. Meantime, F-actin was reorganized in HCCLM3~( Egfl7RNAi+) cells and the enhanced migration also was evidenced by wound healing assay and Transwell assay, implicating an important role of Egfl7 in controlling cell motility through mediating FAK phosphrylation.
5. Based on two EGF-like domains included in the structure of Egfl7, we presumed Egfl7 may mediate FAK phosphrylation through EGFR just like EGF. Then we treated HCCLM3~( Egfl7RNAi+)cells with EGFR inhibitor (15μM) before recombinant Egfl7 protein (50ng/ml) stimulation and found that the FAK phosphrylation, F-actin reorganization level, and cell migration did not change siginificantly. The effects of Egfl7 protein on HCCLM3~( Egfl7RNAi+)cells were all blocked by EGFR inhibitor, which implicated that FAK activation by Egfl7 was EGFR-dependent and the Egfl7/EGFR/FAK pathway may be critical in controlling HCC cell motility.
6. We examined the in vivo relevance of the potential role for Egfl7 in HCC tumorigenesis by using a mouse implantation model. The expression of Egfl7 and phosphorylated FAK in HCCLM3 tumor was determined by immunohistochemistry and the results showed that the expression of Egfl7 and phosphorylated FAK was all significantly decreased in HCCLM3~( Egfl7RNAi+) tumor than that in HCCLM3~( Egfl7RNAi-) tumor. At the 35th day after implantation, we found that the average size of liver tumors in HCCLM3~( Egfl7RNAi+) group was dramatically smaller than that of HCCLM3~( Egfl7RNAi-) group. Two of eight mice in the HCCLM3~( Egfl7RNAi+) group showed intrahepatic metastasis (25%), which is significantly lower than that in the HCCLM3~( Egfl7RNAi-) group (seven of eight, 88%). The pulmonary metastasis was observed in the lung tissue sections of only one mouse in HCCLM3~( Egfl7RNAi+) group (one of eight, 13%), much less than the ratio of pulmonary metastasis in HCCLM3~( Egfl7RNAi-) group (four of eight, 50%). Together, these data support an important role for Egfl7 in HCC metastasis.
In conclusion, our study has shown for the first time that Egfl7 expresses in HCC carcinoma cells and its overexpression in HCC tissues and serum significantly correlates to poor prognosis of HCC. Furthermore, we have demonstrated the novel role of Egfl7 in metastasis of HCC by enhancing cell motility through EGFR- dependent FAK phosphorylation, implicating Egfl7 as a novel serum/ molecular marker for diagnosis, monitoring after hepatectomy and prognosis of HCC and a potential therapeutic target for metastasis of HCC.
引文
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