摘要
肝癌是世界第六大常见恶性肿瘤,其中肝细胞肝癌(Hepatocellular carcinoma,HCC)是最主要的类型,在全球癌症病死率中已上升至第三位,在国内居第二位,严重危害人类的健康。其侵袭力强,易转移,预后差。肝癌的发生和发展机制虽然经过了人们几十年的努力研究,仍然未能阐明。
近年来研究发现,在胚胎发育、组织分化特定过程中调控细胞分化与增殖的信号通路,如Hedgehog、Notch、Wnt和BMP-TGFβ-Activin等,在肿瘤发生发展中发挥着重要作用。尤其是控制脊椎动物内胚层发育分化的Hedgehog信号通路之一的sonichedgehog(SHH)信号转导通路,是一个高度保守的从果蝇到脊椎动物中都存在的中轴器官发育的形态发生通路。SHH信号通路主要由分泌型信号糖蛋白Shh配体、跨膜蛋白受体Ptch和G蛋白偶联磷酸化受体Smo组成的复合物,以及下游转录因子Gli蛋白组成。随后研究发现,SHH信号转导通路在多种来源于内胚层的组织器官肿瘤中异常激活,如皮肤基底细胞癌、肺癌、胰腺癌、前列腺癌、胃癌、结直肠癌、食道癌、乳腺癌和肝癌等。SHH通路保守地存在于肝细胞的整个生命过程,从内胚层肝前体细胞、胚胎干细胞到成体肝细胞等各期均有SHH信号的应答,而作为引起肝细胞肝癌恶性转化的信号通路之一,其所起的具体作用和机制仍有待于阐明。
SHH信号通路中的核转录因子Gli有3种,分别命名为Gli1、Gli2、Gli3,3种Gli蛋白之间氨基序列十分相似,都含有5个高度保守的串联锌指结构以及它们之间的组氨酸一半胱氨酸连接序列。但3种Gli蛋白却发挥不同的核转录激活或抑制功能,Gli1仅有激活功能,而Gli2、Gli3同时具有转录激活与抑制活性。研究显示3种Gli蛋白的C末端均存在激活区;但Gli2、Gli3的N端还存在转录抑制区域。C末端能与其他分子作用将核转录因子定位于细胞质内,而N末端则促使其向核内转移。目前对Gli1的研究多数在其上游如何导致其核转录因子功能状态的变化,而对Gli1是怎样调控下游基因研究甚少。结合生物信息学分析和相关文献线索,发现癌基因c-myc是Gli1调控的下游靶基因之一,由于Gli1蛋白在细胞中的具体定位还没有统一的结论,在HCC中Gli1是否调控c-myc功能及作用,值得进一步探讨。
为此,本课题以SHH信号转导通路为切入点,首先检测SHH通路信号因子在肝细胞肝癌中的分布及激活情况,集中探讨Gli1转录调控下游癌基因c-myc的机制,了解Gli1-c-myc在肝癌细胞增殖、凋亡及功能等方面的作用,最后研究体内体外阻断Gli1引起的生物学行为改变。
首先,我们采用RT-PCR、细胞免疫化学和real-time PCR、免疫化学、Western blot等技术,分别检测HepG2、Hep3B肝癌细胞、人胚肝细胞L02、原代肝细胞PHH和临床肝癌组织及癌旁组织中SHH信号因子(Shh、ptch、Gli1)及hTERT、c-myc的表达。结果提示SHH通路各信号因子在肝癌细胞HepG2和Hep3B均呈高表达,L02呈低表达,PHH则无表达;临床肝细胞肝癌标本中SHH信号通路和c-myc呈激活状态,提示示该信号通路的信号分子Shh,Ptch和Gli1,以及c-myc基因异常激活,可能与肝癌发生发展有关。
在上述工作的基础上,我们构建了含P1和P2的c-myc荧光素酶启动子质粒、Gli1表达质粒和Gli1 RNAi干扰质粒,利用共转染技术和双荧光素酶分析系统,发现原本无或低Gli1表达的HEK293细胞和L02细胞,Gli1质粒呈剂量依赖方式激活c-myc启动子介导的荧光素酶表达,而对原本高表达Gli1的Hep3B细胞c-myc启动子介导的荧光素酶表达则无明显影响。为了解Gli1能否激活内源性c-myc的表达和不同表达水平Gli1对细胞生长的影响,我们筛选出过表达Gli1的稳定L02细胞株和siRNA质粒抑制Gli1基因的Hep3B细胞株。Real-time PCR和Western blot实验表明上述稳定细胞株中Gli1与c-myc表达水平均呈正相关,说明Gli1能够激活内源性c-myc的表达,WST-8细胞增殖实验证实Gli1在肝癌细胞增殖中起正调控作用。
为进一步揭示Gli1在肝癌细胞内发挥生物学功能的可能分子机制,我们通过免疫共沉淀(co-immunoprecipitation,Co-IP)及细胞内免疫荧光共定位实验,证实了Gli1和c-myc两者间存在相互作用。
为揭示Gli1抑制后对肝癌的生物学行为影响,我们采用SHH通路特异性抑制剂cyclopamine作用于Hep3B细胞,发现cyclopamine具有抑制肝癌细胞生长和促进早期凋亡发生的作用,并减少肝细胞癌标志物甲胎蛋白的表达和分泌。我们又建立了Hep3B细胞和稳定抑制Gli1基因的Hep3B细胞裸鼠肝原位移植瘤模型,通过瘤体积测定、HE染色、Western blot和免疫组化技术,观察到cyclopamine对稳定抑制Glil基因的Hep3B细胞裸鼠肝原位移植瘤有协同抑制作用,其机制与抑制Gli1,下调c-myc表达有关。
以上研究结果初步证明,SHH信号转导通路在在肝细胞肝癌中异常激活,该通路中的核转录因子Gli1可调控癌基因c-myc转录和表达,参与肝癌的发生发展。阻断Gli1可以下调c-myc表达,抑制肝癌生长和促进早期凋亡发生。Gli1在肝癌细胞增殖、凋亡等过程中发挥重要作用,可能为肝癌治疗的新靶点。
Liver cancer is the sixth most common malignant tumors worldwide, in which hepatocellular carcinoma (HCC) is the main type and has been ranked the 3rd lethal cancer in the global and the 2nd in China. HCC is a multifocal, high invasive and preferred metastasia with poor prognosis disease. HCC is one of the most seriously endangerous disease to the health of human. Despite of the tremendous efforts, it remains unclear that the mechanisms of carcinogenesis inducing the malignant transformation and the fast progression of HCC.
Recently, there is the evidence of aberrant activation of several signaling cascades such as Hedgehog (HH), Notch, Wnt and BMP-TGF-β-Activin in tumorigenesis and development of cancer. These signaling pathways are essential for numerous processes during morphogens, controlling growth and patterning in certain tissues of a range of different organisms at different developmental stages. Sonic hedgehog (SHH), one of the Hedgehog pathways, is an evolutionarily conserved signalling pathway for the regulation of patterning and cell fates of endoderm-derived axial organs in Drosophila and vertebrae embryonic development. SHH pathway plays important roles in control many essential tissues and cellular properties such as patterning fields of cells or regulating cell differentiation and proliferation in human embryo development. SHH pathway mainly included four factors: the secreted glycoprotein ligand Shh, the transmembrane protein receptor Ptch, the G-protein coupled phosphoprotein receptor Smo and nuclear transcription factor Gli. Homever, it has been reported that there was an abnormal activation of SHH in an increasing number of solid tumours originate from endoderm, including basal cell carcinoma, small-cell lung carcinoma, pancreatic adenocarcinoma, oesophageal, stomach, biliary tract cancers, prostate cancer, breast cancer, colon cancer and liver cancer. SHH signaling pathway is also responded in the vital process of hepatocyte, from endoderm precursor cell and embryonic stem cell to adult hepatic cell as well as HCC. But as a signaling pathway to promote hepatic cell malignant transformation, the molecular mechanisms of SHH in the carcinogenesis and progression of HCC still remain to be clarified.
The Gli homologue family of transcription factors in SHH signaling pathway, Gli1, Gli2 and Gli3, mediate the HH morphogenetic signal by regulating the expression of HH target genes. They have similarly amino acid motifs of highly conserved five tandem zinc fingers and a consensus cysteine-histidine link. This zinc finger motif shows significant sequence to be important in the development process. The Gli proteins family has different functions depending on the difference of construction. The Gli proteins all have C-terminal that is transactivation domain. But Gli2 and Gli3 undergo cleavage generating an N-terminal protein that preferentially accumulates in the nucleus and acts as a repressor of HH target genes, so that Gli2 and Gli3 show strong dominant-negative regulation function in C-terminaHy truncated forms. In particular, Gli1 appears to be a constitutive activator and of various gene expression. Current knowledge has revealed that Gli1 is an upstream factor modulating the expression regulation function of various downstream genes, but there are few studies on how Gli1 regulate downstream gene.
Combination the bioinformatics and pertinent literature, we find that oncongene c-myc is extensively involed in the carcinogenesis and progression of various cancers including HCC, and c-myc is one of Gli1 target genes in some kinds of cancer other than HCC. However, there is no specific sub-localization of Gli1 in different cells. It is interesting whether the regulation of Gli1 on c-myc expression plays some role in the development of HCC.
Subjecting to the signaling transduction molecule Gli1 as a focal point, we investigated the expression status of molecules of SHH pathway in hepatoma carcinoma cell lines and clinical HCC tissues, and the mechanism of transcriptional regulation of Gli1 to the target gene of c-myc, explored the effects of Gli1 regulating to c-myc on the proliferation, apoptosis and cell function of hepatoma carcinoma cell lines, and finally investigate the change of the biological behaviours of hepatic cancer cells with over- and down- regulation of Gli1 protein expression in vitro and in vivo respectively.
In the first part of this study, the expression patterns of Shh, Ptch, Gli1, hTERT and c-myc in HepG2, Hep3B hepatoma carcinoma cells and L02 human normal embry liver cells, and primary human hepatocytes (PHH) were investigated by reverse transcription PCR (RT-PCR), immunocytochemistry, and by real-time PCR, immunohiso-hemistry and Western blot in clinical HCC. The results showed that the SHH signaling was activated in both HCC cell lines and clinical HCC tissues, indicated by the much higher levels of mRNA and protein of SHH pathway constitute monculers, c-myc, hTERT than these in L02, PHH lines, and adjacent non neoplastic live tissues and normal liver tissues.
According to above results, it has been proposed that Gli1 is involved in the regulation of c-myc expression. In the second part, a luciferase reporter vector c-myc-luc including its P1 and P2 promoter, Gli1 expression vector and siRNA target Gli1 vector were constructed respectively. Double luciferase reporter system and cotransfection were used. The results showed that in HEK293 and L02 cells that were original no or low expression c-myc, Gli1 expression vector could significantly activate c-myc expression in a dose-dependent manner, but in hep3B cells with high expression level of endogenous Gli1 and c-myc, Gli1 expression vector did not significantly induce the expression of c-myc cotransfection of pGL3-c-mycp plasmid and various doses of Gli1 RNAi vector showed a dose-dependent inhibition of c-myc expression in Hep3B cells. To prove that Gli1 can activate endogenous c-myc and increase cell proliferation, stably express Gli1 cell lines of L02 and siRNA stably inhibited Gli1 cell lines of Hep3B were established, real-time PCR and Western blot were performed to determine the effect of Gli1 on endogenous expression level of c-myc, and WST-8 method was applied to test the cell proliferation. The results showed that endogenous c-myc was activated by Gli1 in L02 cells, and Gli1 has a powerful positive regulation effect on Hep3B cell proliferation.
To further elucidate the molecule mechanism of the interaction between Gli1 and c-myc in Hep3B cells, immunoprecipitation and double immunofluorescent staining analysis were used. The results indicated that Gli1 and c-myc colocalize and interact with each other in Hep3B cells.
To investigate the influence and mechanism of down-regulation of the Gli1 with SHH signaling specific inhibitor cyclopamine and siRNA on biological behaviours of HCC, serum starvation culture Hep3B cells were treated with cyclopamine, and nude orthotropic transplant tumor model was established and treated with cyclopamine. It was found that cyclopamine inhibited HCC cell proliferation and caused mitochondrial membrane potential collapse, an indicator of early stage of apoptosis, and down-regulation the expression of HCC markerα-fato-protein (AFP). In vivo, inhibition of Gli1 with siRNA and cyclopamine decreased the growth of transplanting HCC tumor, and down regulated the expression of Gli1 and c-myc synergically.
Above evidences have suggested, SHH signaling is abnormally activated in HCC. As the nuclear activating factor of SHH pathway, Gli1 could regulate the transcription and translation of c-myc, which is involved in the carcinogenesis and growth of HCC. Blocking Gli1 would induce that down-regulation of c-myc, inhibit the growth of HCC, and prompt the apoptosis of HCC. Gli1 plays some roles in the carcinogenesis, growth and apoptosis of HCC and it may be a new target of hepatocellular carcinoma treatment.
引文
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