摘要
肝细胞癌(Hepatocellular carcinoma,HCC)是世界上最常见的实体恶性肿瘤之一,手术切除和肝移植仍是目前最有效的治疗手段,但疗效不佳,术后复发和转移是其最主要原因。HCC是富血供肿瘤,血管生成被认为与HCC的侵袭转移密切相关,肿瘤细胞与血管内皮细胞之间的相互作用参与了该过程。
Caveolin-1是细胞膜上内陷微区域的主要结构蛋白,在肿瘤侵袭转移和血管生成过程中发挥着重要作用。目前,对caveolin-1在不同肿瘤中的作用尚存在着两种完全不同的观点。Caveolin-1最初被认为是抑癌基因,但最近越来越多的研究发现,caveolin-1过表达参与了肿瘤的侵袭转移和血管生成。在有关HCC的研究中发现,下调caveolin-1能抑制不同淋巴道侵袭能力小鼠HCC的侵袭转移。本研究所前期采用SuperArray基因芯片技术对HCC转移相关基因进行筛选,发现caveolin-1是重要的促转移候选基因之一。这些研究提示,caveolin-1参与了HCC的侵袭转移和血管生成,但其机制有待进一步阐明。已有研究表明,血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)是HCC血管生成中最主要的刺激因子,但多数研究caveolin-1与VEGF诱导血管生成之间的关系都集中在内皮细胞上,而对肿瘤细胞上caveolin-1表达是否与VEGF诱导的血管生成有关尚不清楚。
基于以上研究背景,本研究采用免疫组织化学和细胞化学、qRT-PCR及Western blot技术检测HCC组织标本和肝癌细胞株中caveolin-1的表达,并分析caveolin-1与HCC临床病理学因素、VEGF及肿瘤血管生成相关指标(肿瘤微血管密度(MVD)、非成对动脉(UA))之间的相关性;利用慢病毒表达系统构建caveolin-1 RNA干扰载体,并借助qRT-PCR、ELISA、侵袭实验和划痕实验分析其对人肝细胞癌株SMMC7721 VEGF表达和侵袭、运动能力的影响;利用MTT法和二维凝胶血管生成体外模型探讨下调SMMC7721细胞中caveolin-1表达的培养上清液对人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVEC)增殖和VEGF诱导的血管生成的影响。
主要结果和结论如下:
第一部分:Caveolin-1在HCC中的表达及临床意义
1、26例伴肝内转移的HCC组织标本中,癌组织内caveolin-1 mRNA表达水平高于正常肝组织、肝硬化组织和癌旁组织,差异均具有统计学意义(P=0.046、0.021、0.039)。75例HCC组织标本行免疫组织化学检测显示,量化分析caveolin-1表达强度(P=0.001)、阳性百分比(P=0.002)、计分(P=0.001)均与转移显著相关。Caveolin-1表达强弱与VEGF(r=0.293, P=0.011)、MVD(r=0.361, P=0.001)、UA(r=0.388, P=0.001)均呈显著正相关。生存分析发现,caveolin-1高表达患者预后比低表达者差(P=0.014)。单变量分析发现,肿瘤大小、门静脉癌栓、肝内转移、caveolin-1表达强弱、VEGF表达水平、MVD和UA都是HCC的重要预后因素;但多变量分析(COX回归模型)发现,只有肿瘤大小和VEGF表达水平是其独立预后因素,而caveolin-1表达水平并非其独立预后因素。
2、在HepG2和SMMC7721肝癌细胞株中,免疫细胞化学结果显示,Caveolin-1在SMMC7721呈强阳性表达,而在HepG2中未见明显表达;Western blot检测结果与免疫细胞化学结果一致,且发现在SMMC7721中caveolin-1表达与VEGF高表达呈显著正相关(r=0.888,P<0.01);qRT-PCR检测结果显示,SMMC7721细胞中caveolin-1 mRNA表达水平明显高于HepG2,差异有统计学意义(P<0.01)。侵袭实验分析证实了SMMC7721的侵袭能力高于HepG2(P=0.003)。
第二部分:Caveolin-1 RNAi慢病毒载体构建及其对人肝细胞癌株SMMC7721 VEGF表达和侵袭、运动能力的影响
1、成功构建慢病毒系统介导的caveolin-1 RNAi载体,其可较长时间、稳定表达于SMMC7721,感染5天后转导效率为(96.0±1.2)%,并能显著敲除靶细胞SMMC7721中目的基因caveolin-1的mRNA(敲除率约90%)和蛋白表达。
2、沉默Caveolin-1能显著降低SMMC7721细胞内VEGF mRNA表达水平,相对阴性感染对照组下降水平约50%(P<0.05);ELISA法检测显示分泌至胞外的VEGF蛋白水平表达约下降20%(P<0.05)。
3、Caveolin-1下调能显著抑制SMMC7721的侵袭和运动能力,各个时相点相对对照组差异均有统计学意义(P均小于0.01);给予外源性重组人VEGF后能显著恢复其侵袭能力(P<0.05),证实caveolin-1可通过调控VEGF参与HCC的侵袭转移。
第三部分:SMMC7721 Caveolin-1沉默培养上清液对HUVEC eNOS表达和内皮细胞增殖、成管的影响
1、采用原代培养的HUVEC可较好的保持其纯度和降低变异程度。Caveolin-1 RNAi后SMMC7721培养上清液使HUVEC细胞活力显著下降,增殖能力受到抑制,差异有统计学意义(P<0.01)。
2、Caveolin-1 RNAi后SMMC7721细胞上清液能显著抑制HUVEC管腔结构的形成(P<0.05),给予外源性重组人VEGF后可恢复HUVEC管腔结构的形成(P<0.05),CD34标记的免疫组化检测结果证实了该结果。
3、SMMC7721细胞caveolin-1沉默后上清液能显著抑制HUVEC细胞上eNOS ser-1177位点的磷酸化(P<0.05),于24h抑制最为明显,而eNOS总蛋白表达没有发生变化。
综上所述,本研究主要结论如下:
1、人HCC组织中caveolin-1表达水平升高与肿瘤转移、预后差和肿瘤血管生成显著正相关;高侵袭潜能人肝细胞癌株SMMC7721中caveolin-1表达较高,并与VEGF表达升高呈显著正相关。Caveolin-1可能是预测HCC侵袭转移潜能和预后的指标之一。
2、利用慢病毒系统成功构建的caveolin-1 RNAi载体可较长时间、稳定表达于SMMC7721,并能显著下调其caveolin-1的表达;沉默caveolin-1能显著降低SMMC7721细胞内VEGF mRNA表达及其分泌至胞外的VEGF蛋白表达水平;Caveolin-1下调能显著抑制SMMC7721的侵袭和运动能力,给予外源性重组人VEGF后能恢复其侵袭能力,证实caveolin-1可通过调控VEGF参与HCC的侵袭转移。
3、人肝细胞癌株SMMC7721中caveolin-1沉默引起的分泌型VEGF表达下调能显著抑制HUVEC的增殖能力;利用基质胶构建的二维凝胶血管生成体外模型发现,caveolin-1沉默的人肝细胞癌株SMMC7721条件培养基能显著抑制HUVEC管腔结构的形成和eNOS的活化,而给予外源性重组人VEGF后则能恢复其管腔结构;这表明肝癌细胞中caveolin-1沉默引起的分泌型VEGF表达下调通过抑制肿瘤血管生成中VEGF介导的eNOS活化而阻遏了HUVEC管腔结构的形成,即肝癌细胞中caveolin-1表达可能通过诱导VEGF介导的肿瘤血管生成,进而促进HCC的侵袭转移,这可能是干预HCC侵袭转移的有效靶点。
BACKGROUNDS and OBJECTIVES
Hepatocellular carcinoma (HCC) is one of the most common solid tumors throughout the world. Though surgical ablation and liver transplantation are the effective measures for treating HCC, the effect is dissatisfactory. Metastasis and recurrence of HCC are the maximal barrier influencing therapeutic effect. As HCC is characterized by hypervascularization, angiogenesis is involved in its invasion and migration through the interaction between tumor cells and vascular endothelial cells.
Caveolin-1, an essential constituent of the plasma membrane invaginations named as caveolae, is known to be involved in cancer invasion and angiogenesis. Numerous studies indicated that the role of caveolin-1 expression depends on the cell types and cancer stages. Whereas in some cases caveolin-1 acts as a tumor suppressor, several more recent studies found that caveolin-1 overexpression was involved in invasion and angiogenesis. During study on HCC, it showed that down-regulation of caveolin-1 reduced lymphangiogenic factor variables, vascular endothelial growth factor (VEGF) expression and prevent lymphatic metastasis in mouse hepatocarcinoma cells. On our early research, we found that caveolin-1 is one of the upregulated genes by SuperArray microarrays on screening of HCC metastasis related genes. However, the connection between the expression of caveolin-1 and cancer invasion and angiogenesis in HCC remains a puzzle and its mechanism is still not clear. The majority of studies have revealed that VEGF was one of the key positive regulators during angiogenesis in HCC. However, the relevance of caveolin-1 to VEGF induced angiogenesis was mainly focused on endothelial cells. The connection between the expression of caveolin-1 in tumor cell and angiogenesis induced by VEGF remains uncertain. Based on these previous findings, we supposed that caveolin-1 could induce VEGF expression in HCC, enhanceing tumor angiogenesis and promoting the invasion of HCC.
METHODS
To confirm our hypothesis, we proposed following experiments. Firstly, the caveolin-1 protein and mRNA expression in tissue specimens of HCC and human hepatocarcinoma cell lines were determined by using immunohistochemistry, Western blot, and quantitative Real-Time PCR (qRT-PCR). The levels of caveolin-1 were correlated with the clinicopathologic variables, VEGF, microvessel density (MVD) and unpaired artery (UA). Secondly, used the lentiviral expression vector of human caveolin-1 with RNA interference approach, down-regulation of caveolin-1 was correlated with VEGF expression, invasiveness and migration in SMMC7721 by qRT-PCR, ELISA, invasion assay and wound healing scratch assay. Lastly, the effect of conditioned medium from supernatant of SMMC7721 treated with caveolin-1 RNAi on eNOS expression, proliferation and capillary-like tubule formation of human umbilical vein endothelial cells (HUVEC) was screened by Western blot, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and angiogenesis two-dimensional gel model in vitro.
RESULTS
Part 1: The characteristics of caveolin-1 expression in HCC and its relationship with the clinicopathologic variables and angiogenesis
1. In 26 tissue specimens of HCC with intrahepatic metastases, caveolin-1 expression of tumor tissues was significantly higher than that of nomal (P=0.046), cirrhosis (P=0.021) or adjacent tumor tissues (P=0.039). In all 75 tissue specimens of HCC, the caveolin-1 expression intensity (P=0.001), proportion (P=0.002) or score (P=0.001) was significantly correlated with metastasis. Levels of caveolin-1 correlated positively with VEGF expression (r=0.293, P=0.011), MVD (r=0.361, P=0.001) and UA (r=0.388, P=0.001). A Kaplan-Meier survival curve showed that the survival rate of patients with higher caveolin-1 expression was considerably worse than that of patients with lower caveolin-1 expression. Univariate analyses identified tumor size, portal vein tumor thrombus, metastasis, caveolin-1 intensity, caveolin-1 score, caveolin-1 expression, VEGF expression, MVD and UA as significant prognostic factors for cancer-specific survival. Multivariate analyses indicated that only tumor size and VEGF levels were independent prognostic factors for cancer-specific survival.
2. The caveolin-1 protein and mRNA were loss in HepG2 with low invasive ability, and then significantly increasing in SMMC7721 with high invasive ability (P<0.01). Moreover, the expression of caveolin-1 in SMMC7721 was significantly associated with VEGF (r=0.888, P<0.01).
Part 2: Construction of recombinant lentivirol expression vector carring human caveolin-1 with RNA interference and its correlation with VEGF expression, invasiveness and migration of SMMC7721
1. Construction of recombinant lentivirol expression vector carring human caveolin-1 with RNA interference could express in SMMC7721 presistandtly and stablely. The transduction efficiency of SMMC7721 was (96.0±1.2)%. The caveolin-1 mRNA level was knocked down as 90%. The protein expression of caveolin-1 was significantly declined.
2. Caveolin-1 silenced in SMMC7721 resulted in the mRNA level of VEGF in SMMC7721 declining about 50% compared with negative infection (P<0.05). Meanwhile, the secreted VEGF expression were decreased about 20% by ELISA (P<0.05).
3. After caveolin-1 was knocked down, the invasiveness and migration of SMMC7721 were prevented at every phase (P<0.01), and dramaticly restored again by supplemented with recombinant human VEGF (P<0.05). The results implied that the invasion of SMMC7721 were positively regulated by caveolin-1 expression through inducing VEGF expression.
Part 3: The effect of conditioned medium from SMMC7721 with caveolin-1 silenced on eNOS expression, proliferation and capillary-like tubule formation of HUVEC
1. The cell purity was maintained and variation was reduced by primary cultured HUVEC. It indicated that conditioned medium from SMMC7721 with caveolin-1 silenced could significantly hampered HUVEC proliferation (P<0.01).
2. Capillary-like tubules developed in conditioned medium from SMMC7721 with caveolin-1 silenced were reduced (P<0.05), and dramatically reorganized by supplemented with recombinant human VEGF (P<0.05). MVD by Immunocytochemistry (CD34 labled) was performed at the twenty-fourth hour to further confirm the results.
3. Conditioned medium from SMMC7721 with caveolin-1 silenced could significantly reduce the phosphorylation of eNOS on the serine 1177 (P<0.05). However, the total protein of eNOS was unchanged.
CONCLUSI ON
1. Overexpression of caveolin-1 in HCC was positively correlated with metastasis, poor prognosis and angiogenesis. The caveolin-1 expression in human hepatocarcinoma cell lines SMMC7721 with high invasiveness was increased, correlating positively with VEGF. Caveolin-1 was one of hallmarker for predicting the invasion and prognosis of HCC.
2. Construction of recombinant lentivirol expression vector carring human caveolin-1 with RNA interference could express in SMMC7721 presistandtly and stablely. Caveolin-1 silenced in SMMC7721 reduced the mRNA level of VEGF in SMMC7721 and the secreted VEGF expression in supernatant. Following by caveolin-1 down-regulated, the invasiveness and migration of SMMC7721 were prevented, and dramaticly restored again by supplemented with recombinant human VEGF. The results implied that the invasion of SMMC7721 were positively regulated by caveolin-1 expression through inducing VEGF expression.
3. Conditioned medium from SMMC7721 with caveolin-1 silenced could significantly hampered HUVEC proliferation. Capillary-like tubules developed in conditioned medium from SMMC7721 with caveolin-1 silenced were reduced, and dramatically reorganized by supplemented with recombinant human VEGF. The activation of eNOS was prevented too. It indicated that VEGF down-regulation induced by caveolin-1 silenced hampered capillary-like tubule formation of HUVEC through reducing the activation of eNOS induced by VEGF during angiogenesis. These data highlights the fact that overexpression of caveolin-1 in HCC promotes invasion and metastasis in HCC through up-regulating VEGF induced angiogenesis. It may open new insights about the possibility of novel therapeutic strategies able to block the tumor’s blood supply and to decrease invasion and metastasis in HCC.
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