【作者】 唐娟；

【导师】 陈志南； 蒋建利；

【作者基本信息】 第四军医大学 ， 生物学， 2010， 博士

【摘要】 肝细胞癌(hepatocellular carcinoma,HCC)是原发性肝癌中最主要的、最普遍的一种,其发病凶险,进展速度快,病死率高,成为医学界所面临的共同难题。90%的肝癌病人死于转移相关的并发症,肝癌的转移是肝癌病人致死的最主要因素,也是临床治疗失败的直接原因。同时,肝癌细胞异常的凋亡途径使肝癌细胞逃避凋亡,进一步促进了肝癌的转移和复发。因此,探讨肝癌侵袭转移的调控因素不仅对深入理解肝癌发生发展的机制具有重要的理论意义,而且可为新型癌标志物及治疗性药靶分子的发现提供现实依据。HAb18G/CD147是我室鉴定的新型肿瘤药靶分子,是CD147家族成员,属于免疫球蛋白超家族类黏附分子。该分子高表达于人肝癌组织和一些转移性肝癌细胞的表面。研究表明HAb18G/CD147可通过刺激成纤维细胞及肿瘤细胞自身产生MMPs促进肿瘤细胞的侵袭转移。然而目前对于HAb18G/CD147的研究多集中于HAb18G/CD147的功能研究,有关HAb18G/CD147分子的作用底物或配体或受体尚未确定,其促进肿瘤转移的具体机制仍不明确。本研究旨在阐明HAb18G/CD147促进肝癌侵袭转移的分子机制和信号转导通路。实验分为四部分:第一部分HAb18G/CD147与integrinα3β1相互作用促进肝癌侵袭转移Integrin广泛表达于脊椎动物细胞表面,可与多种粘附分子相互作用介导细胞的黏附运动。细胞免疫荧光结果显示HAb18G/CD147与integrinα3,β1在人FHCC-98肝癌细胞膜上存在共定位。免疫共沉淀结果显示HAb18G/CD147与integrinα3、β1在人SMMC-7721肝癌细胞中存在共沉淀。将HAb18G/CD147全长真核质粒转染至人SMMC-7721肝癌细胞成功构建HAb18G/CD147稳定高表达的T7721肝癌细胞,采用小干扰RNA(small interfering RNA,siRNA)沉默HAb18G/CD147在人SMMC-7721肝癌细胞中的表达,获得HAb18G/CD147低表达的si-HAb18G/7721肝癌细胞。RT-PCR和Western-blot实验结果显示,在人SMMC-7721肝癌细胞中上调或下调HAb18G/CD147表达,integrinα3、β1表达无明显差异(p﹥0.05)。细胞黏附实验,体外重组基底膜侵袭小室实验以及明胶酶谱实验结果显示,上调HAb18G/CD147表达,可显著促进人SMMC-7721肝癌细胞黏附侵袭和MMP2、MMP9的分泌(p﹤0.01),下调HAb18G/CD147表达,可明显抑制肝癌细胞黏附侵袭和MMP2、MMP9的分泌(p﹤0.01)。抗体阻断实验显示,分别加入integrinα3,β1的抗体能显著逆转HAb18G/CD147介导的细胞黏附侵袭潜能及MMP2、MMP9分泌的上调(p﹤0.01)。以上结果提示HAb18G/CD147与integrinα3β1在人肝癌细胞中存在直接或间接的相互作用。HAb18G/CD147通过与integrinα3β1的相互作用促进肝癌细胞侵袭转移。第二部分HAb18G/CD147通过integrinα3β1促进肝癌细胞侵袭转移的下游信号通路黏着斑激酶FAK是细胞内的一种酪氨酸激酶,在integrin介导的细胞黏附运动中发挥关键作用。Western-blot实验结果显示,上调HAb18G/CD147表达,可促进FAK,Paxillin的表达和活化(p﹤0.01),下调HAb18G/CD147表达,可明显抑制FAK,Paxillin的表达和活化(p﹤0.01)。细胞免疫荧光结果显示,上调HAb18G/CD147表达细胞黏着斑、伪足明显增加,细胞骨架致密,排列整齐;下调HAb18G/CD147表达,肝癌细胞黏着斑明显减少,FAK和Paxillin向细胞边缘分布,细胞骨架稀疏,排列紊乱,以上结果提示FAK,Paxillin参与了HAb18G/CD147促进肝癌细胞侵袭转移的过程。已有研究证实,FAK可直接与PI3K的P85亚基SH2结构域结合而激活PI3K,活化的PI3K可诱导细胞内钙库释放Ca2+及介导胞外钙内流。细胞内Ca2+水平检测显示,上调HAb18G/CD147表达,胞外钙内流显著增加(p<0.01),PI3K的可逆性抑制剂LY294002和不可逆性抑制剂Wortmannin均可显著抑制HAb18G/CD147介导的钙内流(p<0.01),提示HAb18G/CD147可能是通过激活粘着斑激酶FAK,活化PI3K,调控细胞内Ca2+水平。细胞功能实验结果显示LY294002和Wortmannin均可显著抑制HAb18G/CD147介导的人肝癌细胞的黏附、侵袭及MMP2,MMP9的分泌(p﹤0.01),且这种抑制作用呈剂量依赖性。以上结果提示HAb18G/CD147通过integrin介导的FAK-Paxillin和FAK-PI3K-Ca2+通路促进肝癌细胞的侵袭转移。第三部分βig-h3参与HAb18G/CD147促进肝癌细胞侵袭转移的分子机制Western-blot结果显示,采用小干扰RNA沉默HAb18G/CD147在人SMMC-7721肝癌细胞中的表达可显著抑制βig-h3分子的表达(p<0.01),提示βig-h3分子的表达水平与HAb18G/CD147的表达存在正相关。克隆βig-h3基因全长及四个同源重组序列,将βig-h3重组真核质粒转染至人SMMC-7721肝癌细胞构建βig-h3高表达的肝癌细胞。细胞功能实验结果显示,上调βig-h3的表达,可显著促进人SMMC-7721肝癌细胞黏附侵袭和MMP2、MMP9的分泌潜能(p﹤0.01),提示βig-h3可以提高肝癌细胞的侵袭转移能力。免疫共沉淀结果显示βig-h3与integrinα3、β1在人SMMC-7721肝癌细胞中存在共沉淀。抗体阻断实验显示,分别加入integrinα3、β1抗体均能显著逆转βig-h3介导的细胞黏附侵袭潜能及MMP2、MMP9分泌的上调(p﹤0.01)。以上结果提示integrinα3β1参与了βig-h3促进肝癌细胞黏附侵袭过程。细胞免疫荧光结果显示,下调βig-h3表达,人SMMC-7721肝癌细胞中黏着斑明显减少,FAK和Paxillin向细胞边缘分布。细胞骨架稀疏,排列紊乱,伪足减少。以上结果提示,βig-h3通过与integrinα3β1的相互作用,调控FAK-Paxillin信号通路,促进肝癌细胞侵袭转移过程。第四部分HAb18G/CD147通过内质网应激抗凋亡促进肝癌复发转移研究采用N端糖基化抑制剂衣霉素(tunicamycin,Tm)及内质网Ca2+库释放诱导剂( Thapsigargin,Tg )处理人SMMC-7721肝癌细胞,Western-blot结果显示HAb18G/CD147的表达以及内质网应激标志性分子CHOP和Bip的表达均呈时间依赖性增加。上调HAb18G/CD147表达,细胞内Ca2+浓度显著增加。以上结果提示可通过Tm或Tg处理的方法建立肝癌细胞体外内质网应激模型。Western-blot及细胞免疫荧光结果显示,肝癌细胞内质网应激时,上调HAb18G/CD147表达,可显著促进Bip的表达(p﹤0.01),下调HAb18G/CD147表达,可明显抑制Bip的表达(p﹤0.01),提示Bip的表达与HAb18G/CD147呈正相关。上调或下调HAb18G/CD147的表达,内质网应激效应分子IRE1,PERK,ATF6的表达和磷酸化水平均无显著变化(p﹥0.05)。核转录因子TFⅡ-Ⅰ,YY1可以与ATF6形成三聚体,共同参与Bip基因的转录调控。Western-blot结果显示上调HAb18G/CD147的表达,可显著促进TFⅡ-Ⅰ的磷酸化,下调HAb18G/CD147的表达,可明显抑制TFⅡ-Ⅰ的磷酸化(p﹤0.01)。上调或下调HAb18G/CD147的表达,TFⅡ-Ⅰ,YY1表达无显著差异(p﹥0.05)。细胞免疫荧光结果显示,内质网应激条件下,上调HAb18G/CD147的表达,p-TFⅡ-Ⅰ的荧光强度增强,p-TFⅡ-Ⅰ入核增加。下调HAb18G/CD147的表达,p-TFⅡ-Ⅰ荧光强度降低,弥散于胞质。以上结果提示,HAb18G/CD147可能通过磷酸化TFⅡ-Ⅰ并促进其入核调控Bip的表达。先前研究证实,磷酸化FAK可与Src激酶C末端调节性磷酸化Tyr结合而激活Src,激活的Src可磷酸化TFⅡ-Ⅰ第248位酪氨酸而使其活化。Western-blot结果显示,内质网应激条件下,上调HAb18G/CD147的表达,可显著促进FAK、Src的磷酸化,下调HAb18G/CD147的表达,可明显抑制FAK、Src的磷酸化(p﹤0.01),而FAK、Src表达无显著差异(p﹥0.05)。FAK抑制剂能显著抑制肝癌细胞中FAK,Src,TFⅡ-Ⅰ的活化以及Bip表达,此抑制作用在HAb18G/CD147高表达的肝癌细胞中更为显著(p<0.01)。以上实验结果提示,内质网应激条件下,HAb18G/CD147通过激活FAK、活化Src,进而磷酸化TFⅡ-Ⅰ并促使其入核,与YY1,ATF6形成三聚体,促进Bip的表达。内质网应激时,Bip具有明显对抗凋亡作用。Annexin V/PI双染检测细胞凋亡结果显示,内质网应激条件下,上调HAb18G/CD147的表达,可明显抑制肝癌细胞凋亡,下调HAb18G/CD147的表达,可显著促进肝癌细胞凋亡(p﹤0.01)。caspase4活性检测结果显示,上调HAb18G/CD147的表达,caspase4活性明显降低,下调HAb18G/CD147的表达,caspase4活性显著升高(p<0.01),这与细胞凋亡检测结果一致。以上结果提示肝癌细胞内质网应激时,HAb18G/CD147分子通过上调Bip的表达,抑制caspase4活性对抗凋亡。综上所述,在肝癌细胞中,HAb18G/CD147通过与integrinα3β1的相互作用激活FAK-Paxillin和FAK-PI3K-Ca2+信号转导通路,从细胞粘附运动和基质降解两方面促进肝癌细胞的侵袭转移。βig-h3作为协同分子参与了HAb18G/CD147促进肝癌细胞侵袭转移过程。HAb18G/CD147在肝癌细胞内质网应激时通过激活FAK-Src通路,进而磷酸化TFⅡ-Ⅰ并促使其入核,促进抗凋亡分子Bip的表达,并通过抑制caspase4的活性,抑制肝癌细胞的凋亡。本研究首次阐明HAb18G/CD147在促进肝癌细胞侵袭转移过程中的相互作用分子及信号转导通路,并首次发现肝癌细胞内质网应激时HAb18G/CD147抗凋亡导致肝癌的复发转移的分子机制。以上研究为进一步阐明HAb18G/CD147在肝癌发生发展中的作用奠定了基础,并为肝癌的临床诊治提供新的线索和靶点。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is the most common primary hepatic carcinoma. The average live time of HCC patients are only 3 to 6 months. 90% of HCC patients die of complications associated with migration. Migration is the most important lethal factor to HCC patients, and is also the immediate cause of therapeutics failure. Meanwhile, abnormal apotosis pathways help HCC escape from apotosis, thus promoting HCC migration and recurrence. Studies on the regulation factors of HCC invasion and migration will not only broaden our basic knowledge of HCC development, but also provide some promising novel cancer biomarkers and therapeutical targets.HAb18G, a hepatoma-associated antigen, cloned in anti-hepatoma monoclonal antibody HAb18 screening of human hepatocellular carcinoma cDNA library has an identical nucleotide acid and amino acid sequence as CD147. HAb18G/CD147 is a transmembrane glycoprotein of the immunoglobulin superfamily that is broadly expressed in many cell types, especially at high levels in human tumor cells. Our previous studies have demonstrated that HAb18G/CD147 may promote the invasion and metastasis potential of human hepatoma cells by stimulating fibroblast cells to produce elevated levels of MMPs. Although published data support a crucial role for CD147 on migration and metastasis potential of tumor progression, but the detailed investigation on the precise mechanism is required. Now it is becoming increasingly apparent that full understanding of HAb18G /CD147 functional activities may require understanding of its associations with other molecules and its signal transduction pathway. The aim of this study is to identify the exact mechanism of HAb18G/CD147 promoting human hepatoma cells invasion and migration that has eluded investigators for many years. The study is composed of four parts.Part 1. HAb18G/CD147 interacts with integrinα3β1 to promote HCC invasion and migrationIntegrin which is widly expressed in vertebrate cell membrane, may interact with adhesion molecures to promote cell adhesion and movement. Immunofluorescent double staining showed co-localizations of HAb18G/CD147 with integrinα3 andβ1 on the human hepatoma FHCC-98 cells membrane. integrinα3β1 was then found co-immunoprecipitated with endogenous HAb18G/CD147 in 7721 cell lysates. RT-PCR and western-blot analysis showed that there were no significant expression modifications of integrinα3 andβ1 subunits in HAb18G/CD147 upregulated HCC cells and HAb18G/CD147 downregulated HCC cells. In-vitro adhesion, invasion and gelatinase zymography assay showed that upregulation of HAb18G/CD147 in HCC cells could significanty promote cell adhesion,invasion and MMPs secretion (p<0.01), downregulation of HAb18G/CD147 in HCC cells could significanty inhibit cell adhesion, invasion and MMPs secretion (p<0.01). Antibodies toα3 andβ1 integrins completely blocked HAb18G/CD147 enhanced cell adhesion,invasion potential and MMPs secretion (p<0.01). These results demonstrate that HAb18G/CD147 and integrinα3β1 are both required and functionally dependent for HCC adhesion, invasion potential and MMPs secretion.Part 2. Involvement of integrin signal pathways in HAb18G/CD147-mediated HCC invasion and migration processFocal adhesion kinese FAK is a kind of tyrosine kinese, which has protical role in integrin promoting HCC adhesion and movement process. Upregulation of HAb18G/CD147 in HCC cells could significantly increase the expression and phosphorylation of FAK and Paxiilin (p<0.01), and downregulation of HAb18G/CD147 could significantly diminish them (p<0.01). Downregulation of HAb18G/CD147 in HCC cells could decrease focal adhesions and F-actin stress fibers, and also redistribute FAK and paxillin to the periphery. Upregulation of HAb18G/CD147 in HCC cells could increase focal adhesions and filopodial protrusions.The addition of PI3K inhibitor wortmannin or LY294002 could significantly block HAb18G/CD147 enhanced intracellular free calcium ([Ca2+]i) concentration (p<0.01). In-vitro adhesion, invasion and gelatinase zymography assay showed that wortmannin or LY294002 could inhibit HAb18G/CD147-elevated cells adhesion, invasion and MMPs secretion in a dose-dependent manner in both cell lines (p<0.01). These results suggest that HAb18G/CD147 promotes HCC adhesion, invasion and MMPs secretion potential via integrinα3β1 mediated FAK-Paxillin and FAK-PI3K-Ca2+ singnal pathways.Part 3.βig-h3 is involed in HAb18G/CD147-mediated HCC adhesion and metastasis processWestern-blot analysis showed that the expression ofβig-h3 markedly reduced in si-HAb18G transfected 7721 cells (p<0.01). The four homologous recombination sequences and the full length ofβig-h3 were correctly cloned and transiently transfected to 7721 cells. In-vitro adhesion, invasion and gelatinase zymography assay showed that upregulation ofβig-h3 in HCC cells could significanty promote cell adhesion,invasion and MMPs secretion (p<0.01). This finding suggests thatβig-h3 may enhance invasion and metastasis potential of human hepatoma cells.Integrinα3β1 was found to co-immunoprecipitate with endogenousβig-h3 in 7721 cell lysates. The presence of antibodies forα3 andβ1 integrins completely blockedβig-h3-enhanced cell adhesion and adhesion potential (p<0.01). These results suggest that integrinα3β1 is involved inβig-h3 induced invasion and metastasis potential of human hepatoma cells.Downregulation ofβig-h3 in HCC cells could significantly decrease focal adhesions and F-actin stress fibers, and redistribute both FAK and paxillin to the periphery. These results indicate thatβig-h3 may promote HCC invasion and migration by integrinα3β1 and its downstream signal molecules FAK and paxillin.Part 4. HAb18G/CD147 promotes HCC recurrence by ERS induced anti-apotosisThe expression of HAb18G/CD147 increased as time-dependent manner in tunicamycin (Tm) or Thapsigargin (Tg) incubated 7721 cells. The expression levels of ERS markers-CHOP and Bip were also increased in the time-dependent manner. Upregulation of HAb18G/CD147 in HCC cells could markedly increase the Ca2+ level. Western-blot analysis and immunofluorescence analysis showed that in ERS condition, upregulation of HAb18G/CD147 in HCC cells could markedly increase Bip expression, and downregulation of HAb18G/CD147 could significantly decrease Bip expression. These results suggest that the expression of Bip is in positive correlation with HAb18G/CD147.No significant expression and phosphorylation modifications of IRE1, PERK and ATF6 were found in HAb18G/CD147 upregulated HCC cells and HAb18G/CD147 downregulated HCC cells. It is known that TFⅡ-Ⅰand YY1 can form trimer with ATF6 to regulate Bip expression. Western-blot analysis showed that upregulation of HAb18G/CD147 in HCC cells could markedly increase TFⅡ-Ⅰphosphorylation level (p﹤0.01), downregulation of HAb18G/CD147 coule significantly decrease TFⅡ-Ⅰphosphorylation level (p﹤0.01). There were no significant expression modifications of TFⅡ-Ⅰand YY1 in HAb18G/CD147 upregulated HCC cells and HAb18G/CD147 downregulated HCC cells. The results of immunofluorescence staining showed that upregulation of HAb18G/CD147 in HCC cells could obviously increase the fluorenscence indensity representing p-TFⅡ-Ⅰand accumulate p-TFⅡ-Ⅰto the nucleus, downregulation of HAb18G/CD147 in HCC cells could decrease p-TFⅡ-Ⅰand diffuse p-TFⅡ-Ⅰin the cytoplasm. These results indicate that HAb18G/CD147 may promote the expression of Bip by activiting TFⅡ-Ⅰ.Western-blot analysis showed that upregulation of HAb18G/CD147 in HCC cells obviously enhance c-Src and FAK phosphorylation levels (p﹤0.01), and downregulation of HAb18G/CD147 significantly decrease c-Src and FAK phosphorylation levels (p﹤0.01). There were no significant expression modifications of c-Src and FAK in HAb18G/CD147 upregulated HCC cells and HAb18G/CD147 downregulated HCC cells (p﹥0.05). The addition of FAK inhibitor could significantly decrease the phosphorylation levels of FAK, Src, TFⅡ-Ⅰand expression level of Bip in HAb18G/CD147 upregulated HCC cells (p<0.01). There results suggest that by activiting c-Src and TFⅡ-Ⅰ, HAb18G/CD147 may promote Bip expression.AnnexinV/PI double staining showed that upregulation of HAb18G/CD147 in HCC cells could obviously inhibit apoptosis, and downregulation of HAb18G/CD147 could significantly promote HCC apoptosis. The results of caspase4 activity test showed that upregulation of HAb18G/CD147 in HCC cells could obviously dercease caspase4 activity (p<0.01),and downregulation of HAb18G/CD147 could significantly increase caspase4 activity (p<0.01). This result was coincidence with the AnnexinV/PI double staining analysis results. These results demonstrate that in ERS condition, by derceasing caspase4 activity, HAb18G/CD147 may inhibit apoptosis in HCC cells.All the above results suggest that HAb18G/CD147 may enhance invasion and metastatic potential of human hepatoma cells via integrinα3β1 mediated FAK-paxillin and FAK-PI3K-Ca2+ signal pathways.βig-h3 is cooperated with HAb18G/CD147 in this process. In ERS condition, by FAK-Src signal pathways, HAb18G/CD147 may phosphorylate TFⅡ-Ⅰand help p-TFⅡ-Ⅰaccumulate to the nucleus, thus promoting Bip expression. By promoting Bip expression and decreasing the activity of caspase4, HAb18G/CD147 may inhibit HCC apoptosis. This is the first time to determine the signal transduction pathway of HAb18G/CD147 in promoting HCC invasion and migration, and to examine the mechanism of HAb18G/CD147 anti-apoptosis in ERS condition. The present study founds a basis for the further demonstration of the function of HAb18G/CD147 in HCC generation and development, and provides some new evidence for its further application in clinical.更多还原