摘要
肝癌是一种常见的恶性肿瘤，据报道，世界范围内每年新增肝细胞癌患者达到700,000以上。中国是全球肝癌发病率最高的国家，有统计显示，我国肝癌发病人数占全球55%，死亡人数占全球45%，列癌症死亡的第二位，而且发病率不断上升。肝细胞癌的早期发现、早期诊断、早期治疗对肝癌患者的预后和存活时间至关重要。甲胎蛋白是迄今人类发现的第一个具有诊断价值的肿瘤标志物，在肝癌普查、早期诊断、疗效判定和预后评价等方面发挥了重要作用。但临床上约有30%～40％肝癌患者血清甲胎蛋白阴性，且近年来又有增多的趋势。这部分肝癌患者缺乏有效的肿瘤标志物用于辅助诊断、预后判断与疗效评价。因此，寻找新的肝癌的血清标志物，尤其与预后相关的标志物具有重要的意义。我们的研究主要基于利用SELEX（SystematicEvolutionofLigandsbyEXponentialEnrichment）技术发现新的肝癌标志物，并对标志物分子的地位、作用进行了研究，主要分为四个部分：
第一部分：肝癌血清标志物的获得与鉴定
利用液相SELEX技术，我们实验室以伴肝外转移的AFP（Alpha-fetoprotein）阴性HCC（hepatocellularcarcinoma）患者血清为靶标，以正常人血清为消减靶标，经过5轮结合、消减筛选，获得了特异识别肝癌患者血清的富集ssDNA文库；然后，利用凝胶阻滞的方法，从与富集ssDNA（singlestrandedDNA）文库结合的蛋白中鉴定出候选靶蛋白，VASN（vasorin,slitlike2）蛋白。首先对VASN蛋白在血清标本、肿瘤细胞、肿瘤组织中的表达水平进行了研究，对VASN的标志物地位进行了评价。结果发现肝癌患者血清中的VASN蛋白水平高于正常人，且与AFP无明显相关性，提示VASN可以和AFP联合应用来提高辅助诊断的敏感性。随后，我们检测了肝癌细胞和正常肝细胞中VASNmRNA及蛋白水平，结果显示，VASN水平在肝癌细胞株HepG2和SMMC-7721中较高，在正常肝细胞株HL-7702中较低。Real-timePCR检测VASN在肝癌组织、癌旁组织cDNAarray中的表达水平，结果显示，与正常组织相比，肝癌组织的VASNmRNA水平普遍升高。同样，利用间接免疫荧光实验检测VASN蛋白在肝癌组织切片、良性病变肝组织切片中的表达情况，结果显示，VASN蛋白在肝癌组织中呈高表达，主要表达在癌细胞表面和细胞间隙中；而在肝炎组织中VASN表达水平较低。这些数据均提示VASN可能是肝癌细胞表达并分泌的一个重要分子标志物。
第二部分：VASN蛋白作为治疗靶标的初步研究
根据我们的工作基础，相关文献报道和生物信息学分析结果，我们推测VASN蛋白可能在肝癌的发生、发展过程中发挥重要作用，因此，我们利用分子生物学技术对VASN的功能进行了探讨。首先，siRNA沉默肝癌细胞中VASN的表达后，MTT实验证实细胞增殖变慢，细胞划痕和Transwell实验均证实细胞迁移变慢，穿膜细胞减少，流式检测发现凋亡细胞增加，免疫印迹实验提示细胞上皮标志蛋白E-cadherin表达增高，间质标志蛋白N-Catenin、Slug表达降低，说明VASN蛋白对肿瘤细胞的增殖、迁移、上皮-间质转化有促进作用，抑制细胞凋亡；单抗抑制实验发现VASN单抗对细胞的增殖也有抑制作用；以上结果提示，作为膜蛋白和/或游离蛋白，VASN可能成为肝癌生物治疗的有效靶标；同时，我们在正常肝细胞株HL-7702中瞬时过表达了VASN蛋白，Real-timePCR、Westernblot检测证实VASN的mRNA、蛋白水平升高，MTT检测提示细胞增殖变快，Transwell实验表明穿膜细胞增多，进一步证实了VASN蛋白的促癌作用。
第三部分：肝癌中VASN蛋白水平升高机制的初步探讨
生物信息学发现VASN可能是miR-145的靶标。结合我们实验室miR-145的研究基础，即miR-145具有抑癌作用，本论文对miR-145靶向VASN及其生物学作用进行了进一步研究。首先，研究发现在肝癌细胞及正常肝细胞中，VASNmRNA水平与miR-145的水平呈负相关，提示miR-145水平降低可能是导致肝癌中VASN水平升高的原因。通过荧光素酶报告基因，Real-timePCR及Westernblot实验证明了miR-145通过靶向3’UTR（3’UntranslatedRegions）区降低VASN的mRNA水平并导致蛋白水平降低。其次，肝癌细胞中过表达miR-145mimics导致细胞形态发生明显改变，细胞失去伪足样突起同时形状变得不规则，细胞迁移能力下降、增殖变慢，与siRNA沉默VASN后细胞变化一致；共转染miR-145抑制剂能逆转上述变化。因此，我们研究结果表明肝癌细胞株中miR-145低表达是导致VASN高水平的一个重要原因，miR-145通过下调VASN水平，从而发挥其抑癌作用。
第四部分：hnRNPA1特异适配子BC15在肝癌中的初步应用研究
我室利用组织切片-SELEX技术获得了乳腺癌组织特异结合配基BC15，并鉴定出BC15的靶分子为异质性核内核糖核蛋白A1（heterogeneousnuclearribonucleoproteinA1，hnRNPA1），进一步的研究证实hnRNPA1蛋白在多种癌症的表达都高于癌旁组织，我们重点研究了该蛋白及其适配子BC15在肝癌诊断与治疗中的作用。结果表明，在临床肝癌组织标本中，包括血清AFP阴性的肝癌组织，BC15检测到的hnRNPA1蛋白的表达阳性率明显高于癌旁正常组织和良性病变组织。提示hnRNPA1蛋白和BC15可能成为肝癌患者尤其是血清AFP阴性的肝癌的辅助诊断标志物和分子探针。在正常肝细胞中过表达hnRNPA1能够促进细胞的增殖和迁移，而在肝癌细胞中沉默hnRNPA1的表达以及转染BC15能够有效抑制细胞的增殖和迁移，提示该蛋白在肝癌的发展过程中发挥重要作用，并且是治疗肝癌的一个潜在药物靶点。BC15比siRNA能更有效抑制细胞增殖，为开发细胞内靶标的适配子类药物打开了更广阔的前景。
Liver cancer is a common malignancy, it is reported that new patients with hepatocellular carcinoma (HCC) have increased more than700,000each year in the worldwide. China is the country with the highest incidence of liver cancer in the global. The statistics show that the number of liver cancer in China accounted for55percent of the world, the number of deaths in China accounted for45percent of the world which rank the second of cancer deaths with the increasing incidence. Early diagnosis and treatments are essential to HCC patients for improvement of the prognosis and survival time. Alpha-fetoprotein (AFP) is the first discovered tumor biomarker for diagnosis, which have played an important role in cancer screening, early diagnosis, and evaluation of treatment and prognosis. However, AFP has poor sensitivity for the detection of some HCC patients, because about30percent to40percent of patients afflicted with HCC have normal levels of serum AFP, and the number is increasing these years. These patients are lack of effective tumor marker for assisted diagnosis, prognosis, and efficacy evaluation. Clearly, screening for new tumor biomarkers to improve HCC diagnosis and help HCC prognosis evaluation are urgently needed. Our research work mainly involves the discovery of new biomarker of hepatocellular carcinoma by developed SELEX and identification and potentials in HCC.
Part One:The discovery and identification of HCC serum biomarker
Our research group has gained an enriched single stranded DNA pool that specifically recognized serum components of hepatic carcinoma after five rounds of positive and subtractive selections through a developed subtractive EMSA-SELEX using the AFP negative serum of HCC patient with extra-hepatic metastasis as targets and the normal serum as counter target. One of the targets, VASN protein, of the enriched pool was identified by EMSA assay using serum and labeled pool and then characterized by mass spectrometry from the band with retarded migration. The expression of VASN in serum of HCC patients, tumor cells and tumor tissues was detected. Results showed that the level of VASN in HCC patients'serum is higher than that in normal serum and has no relationship with the level of AFP, which indicated the potential of combining use of VASN and AFP to improve the sensitivity of auxiliary diagnosis. Real-time quantitative PCR and Western blot results showed that the VASN is higher expressed in HepG2and SMMC-7721than in normal liver cell (HL-7702). VASN mRNA is generally highly expressed in cancerous tissues compared to normal tissues detected by real-time quantitative PCR using cDNA microarrays. Consistently, VASN was further verified to be specifically and highly expressed in cancerous tissues of liver with a main localization at cell surface and in the intercellular substances, and lowly expressed in benign liver tissues by VASN antibody immunostaining. These data indicates that VASN probably is an important molecular biomarker which is expressed and secreted by hepatoma carcinoma cells.
Part Two:The preliminary study on VASN as a therapeutic target
Based on results of our previous work, related literature report and bioinformatics analysis, we suspected that VASN may play an important role in the development and progression of liver cancer. We therefore focused on the functions of VASN using molecular biology techniques in this part. Firstly, The knockdown of VASN by RNA interference assay in hepatoma cells lead to decreased proliferation, repressed migration, increased apoptosis and inhibited epithelial-mesenchymal transition (EMT) of the cell with elevated expression of epithelial E-cadherin and down expression of N-catenin and Slug, supporting the carcinogenesis of VASN. The proliferation of hepatoma cell was also repressed by anti-VASN antibody, further suggesting the potential therapeutic target of VASN as a membrane and secreted protein. On the other hand, we over expressed VASN protein in normal hepatic cells, and the results showed that the growth of cells was increased by MTT assay and so as the migration of cells detected by transwell experiments, further confirmed the carcinogenesis of VASN.
Part Three:The preliminary investigation on mechanism of VASN overexpression in liver cancer
Bioinformatics analysis suggests that VASN may be regulated by miR-145, which was proved to be down regulated in many cancer cell lines. Our research focused on the regulation of VASN level by miR-145and thus the changes of cell biology. Firstly, our research found that there was a negative correlation between level of VASN mRNA and that of miR-145in liver cancer cell lines and normal cell lines, indicating that decreased expression of miR-145is one of the main causes of VASN over expression. Then we confirmed that VASN mRNA is the target of miR-145via the luciferase reporter gene assay and the real-time quantitative RT-PCR experiment and Western blot experiment. Secondly, we showed that over expression of miR-145in hepatoma cells resulted in obvious changes in cell morphology with an irregular shapes and disappeared pseudopodia apophysis, companied by decreased migration and slowdown of proliferation of the cell, which are consistent with the changes caused by VASN siRNA. The changes can be recovered by a cotransfection of miR-145inhibitor. Therefore, our research elucidate that miR-145down regulate VASN level and developed its carcinogenesis.
Part Four:The primary applied research of BC15as hnRNPAl-specific aptamer in hepatocarcinoma
In a previous study, we gain a BC15aptamer that specifically differentiate breast cancerous tissues from normal ones through recognizing heterogenous nuclear ribonucleoprotein Al (hnRNP Al) by a tissue-slide based SELEX. Moreover, our group has also shown that the hnRNP Al was high expressed in a variety of cancerous tissues. The status of hnRNP Al and BC15were highlighted in this research on the diagnosis and treatment of liver cancer. We elucidated that hnRNP Al was over expressed in clinical liver cancer tissues including AFP-negative ones compared to para-cancerous normal tissues and benign tissues by histological examination with BC15aptamer, suggesting that hnRNP Al may be a valuable diagnostic marker for liver cancer especially for those with negative serum AFP level and BC15be a molecular probe due to its properties of easy synthesis and direct modification and thus a simpler procedure of in situ examination than antibody. Further experiments showed that upregulation of hnRNP Al can promote the proliferation and migration of liver cells in vitro, while knockdown of hnRNP Al by RNAi and BC15inhibited the proliferation and the migration of cancerous cells, strongly suggesting that hnRNP Al is involved in the development of liver cancer, and thus may be a potential target for biological therapy. BC15has been found to have stronger inhibition effect compared with the siRNA hnRNP Al, laying a foundation for the preferential application of aptamer than siRNA against intracellular target in nucleic acid based therapeutic area.
引文
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