【作者】 李蔚；

【导师】 沈世强；

【作者基本信息】 武汉大学 ， 外科学， 2016， 博士

【摘要】 肝细胞癌是一种血供丰富的实体肿瘤,肿瘤血管是肝癌细胞获得营养支持和肿瘤侵袭、迁移的重要途径。抗血管治疗是失去手术机会的晚期肝癌病人的重要治疗方法,近年来多种肿瘤血管阻断治疗方法如经导管动脉化疗栓塞(TACE)已经在临床肝癌治疗中广泛应用并取得了一定的效果。然而,许多肝癌病人经过临床上传统的肿瘤血管阻断治疗或抗肿瘤血管生成治疗后,病情并未得到缓解。因此,我们亟需寻找肝癌血管内皮细胞靶向治疗的有效的靶点,本研究直接以人肝癌血管内皮细胞(hepatocellular carcinoma tumor endothelial cells(HCC TECs)为研究对象并采用流式细胞术检测HCC TECs细胞表面分子CD105、CD31表达来验证HCC TECs细胞纯度,以避免肝癌细胞,巨噬细胞等其他细胞的干扰。微RNA(microRNA (miRNA))在真核生物中广泛存在,虽不直接编码蛋白质,但是其与目的基因结合后可降解nRNA或者抑制翻译,在机体的生长、发育、增殖、分化及凋亡等分子生物学行为中有着重要的作用。miRNAs在基因功能研究,肿瘤早期诊断及基因靶向治疗方面有着很好的应用前景。目前研究的热点主要集中于miRNAs在肿瘤发生、发展中的作用,越来越多的研究发现miRNAs在肝癌细胞中异常表达,这些异常表达的1niRNAs可能参与了肝癌的发生、发展,有关这些异常表达的miRNAs的研究可能有助于肝癌的早期诊断及治疗。：niRNAs与肝癌血管内皮细胞之间联系的相关研究鲜有报道。如果能发现在肝癌血管内皮细胞中异常表达的miRNAs,将其作为靶点来调节其表达,有可能调控肝癌血管内皮细胞的生物学行为,从而调控肝癌肿瘤血管的生成,最终抑制肝癌的生长及转移。本研究筛选前期采用HmiOA v4 Human miRNA OneArray(?)基因芯片表达谱检测出的正常人肝窦血管内皮细胞(hepatic sinusoidal endothelial cells(HSECs))与人肝癌血管内皮细胞中差异表达的miRNAs,选取在HCC TECs中明显低表达(选取标准log2|Fold change|≥0.585 and P< 0.05)且在肝癌相关研究中鲜有报道的miR-3178进行进一步研究,通过合成miR-3178模拟物(miR-3178 mimic)|或 miR-3178抑制物(miR-3178 inhibitor)并转染HCC TECs,实现]niR-3178在HCC TECs中的过表达或低表达,研究miR-3178表达的改变对肝癌血管内皮细胞生物学行为的影响并探讨其分子机制,有望为肝癌的血管内皮细胞靶向治疗提供实验基础及新的靶点。本课题将从以下三个部分展开研究。实验一 筛选并验证HSECs与HCC TECs中差异表达的miRNAs及合成miRNAs mimic,inhibitor转染肝癌血管内皮细胞目的流式细胞术验证HCC TECs细胞纯度。筛选并验证人肝窦血管内皮细胞与人肝癌血管内皮细胞中差异表达的miRNA-3178,合成miRNA-3178 mimic, inhibitor及其对应的阴性序列转染人肝癌血管内皮细胞并验证转染效率。方法通过流式细胞术检测HCC TECs细胞表面CD105,CD31水平从而验证HCC TECs纯度。选取前期研究中在HCC TECs中明显低表达的miR-3178进行进一步研究。通过实时定量聚合酶链反应(Real-time PCR,RT-PCR)验证miR-3178在HSECs和HCC TECs中的差异表达。合成miR-3178 mimic, inhibitor, negative-control mimic (Nc-mimic)及negative-control inhibitor (Nc-inhibitor)并转染HCCTECs。按处理方式的不同分为五个实验组：(1)空白对照组,即未经处理的HCCTECs组(CON组)；(2)miR-3178表达上调组,即转染2’-0修饰的miR-3178 mimic组(mimic组)：(3) miR-3178 mimic阴性对照组,即转染2’-0修饰的miR-3178mimic阴性对照序列组(Nc-mimic组)；(4)miR-3178表达下调组,即转染2’-0修饰的IniR-3178 inhibitor组(inhibitor组)；(5)miR-3178 inhibitor阴性对照组,即转染2’-0修饰的miR-3178 inhibitor阴性对照序列组(Nc-inhibitor组)。RT-PCR检测各实验组miR-3178表达水平,荧光显微镜下观察绿色荧光标记情况并拍照。结果流式细胞术结果显示HCC TECs细胞表面分子CD105表达率为100%,同时CD31表达率为97.9%,排除了肝癌细胞、巨噬细胞和成纤维细胞的污染。选取前期研究中在HCC TECs中明显低表达的miR-3178进一步研究(选取标准为log2|Fold change|≥0.585 and P<0.05)。RT-PCR验证HSECs与HCC TECs中miR-3178差异表达,结果显示：HCC TECs中miR-3178水平(0.31±0.03)明显低于HSECs中miR-3178水平(1.04±0.08),差异有统计学意义(P<0.001),证明了基因芯片结果的准确性；RT-PCR检测各实验组miR-3178表达水平,结果显示CON组(1.04±0.14),Nc-mimic组(0.99±0.09),Nc-inhibitor组(1.02±0.11)niR-3178水平无明显差异(P>0.05), mimic组rniR-3178水平(365.98±49.49)明显高于CON组及Nc-mimic组,差异有统计学意义(P<0.001), inhibitor组(0.19±0.04)miR-3178水平明显低于CON组及Nc-inhibitor组,差异有统计学意义(P<0.001)。荧光显微镜下观察绿色荧光标记情况,CON组无绿色荧光,mimic组,Nc-mimic组,inhibitor组,Nc-inhibitor组可见明显的绿色荧光,转染效率90%以上。结论miR-3178在肝癌血管内皮细胞内明显低表达。转染miR-3178 mimic显著上调了肝癌血管内皮细胞中miR-3178水平,转染]miR-3178 inhibitor显著下调了肝癌血管内皮细胞中miR-3178水平,转染效率均在90%以上。实验二miR-3178表达改变对HCC TECs生物学行为的影响目的研究上调或下调miR-3178表达对HCC TECs增殖、凋亡、细胞周期、侵袭、迁移及血管形成等生物学行为的影响。方法噻唑蓝(MTT)实验检测各实验组HCC TECs细胞增殖能力。流式细胞术检测各实验组HCC TECs细胞凋亡及细胞周期。ranswell侵袭及迁移实验检测各实验组HCC TECs侵袭及迁移能力。血管形成实验检测各实验组HCC TECs血管形成。结果MTT实验结果显示,转染后24,36,48,72h,CON组,Nc-mimic组和Nc-inhi bitor组增殖率无明显差异,mimic组增殖率明显低于其他组,在72h达到最低,inhibitor组增殖率明显高于其他组,在72h达到最高。流式细胞术细胞凋亡结果显示,CON组,Nc-mimic组和Nc-inhibitor组细胞凋亡率无明显差异,mimic组细胞凋亡率明显增高,inhibitor组凋亡率明显降低。流式细胞术细胞周期结果显示,CON组,Nc-mimic组和Nc-inhibitor组G0/G1期及S期细胞比例无明显差异。mimic组G0/G1期细胞比例明显增加。inhibitor组G0/G1期细胞比例明显降低。mimic组S期细胞比例明显降低。inhibitor组S期细胞比例明显增高。Transwell侵袭及迁移实验结果显示,CON组,Nc-mimic组与Nc-inhibitor组侵袭及迁移细胞数无明显差异,mimic组侵袭及迁移细胞数明显减少,inhibitor组侵袭及迁移细胞数明显增加。血管形成实验结果显示,CON组,Nc-mimic组和Nc-inhibitor组管腔样结构数量无明显差异,mimic组管腔样结构数量明显减少,inhibitor组管腔样结构数量明显增加。结论miR-3178可能作为一种抑癌基因参与对肝癌血管内皮细胞生物学行为的调控,上调miR-3178表达可抑制肝癌血管内皮细胞增殖,侵袭,迁移,血管形成,促进其凋亡及G0/G1期阻滞。实验三miR-3178调控HCC TECs生物学行为的机制研究目的通过靶基因预测软件预测miR-3178调控HCC TECs生物学行为的候选靶基因,观察调节miR-3178表达水平对候选靶基因mRNA及蛋白表达的影响以验证靶基因。方法miRNAs主要作用于目的基因的3’UTR区域,从而影响该基因转录后的表达。我们使用TargetScan、PicTar、Miranda、RNAhybrid等靶基因预测软件预测miR-3178的候选靶基因。Western Blot检测HCC TECs中靶基因蛋白表达情况。RT-PCR检测HCC TECs中靶基因mRNA表达情况。结果TargetScan、PicTar、Miranda、RNAhybrid等靶基因预测软件预测miR-3178调控HCC TECs生物学行为的候选靶基因,结果显示EGR3可能是miR-3178调控HCC TECs生物学行为的靶基因之一。Western Blot检测EGR3蛋白表达,结果显示CON组(0.77±0.08),Nc-mimic组(0.73±0.06)及N c-inhibitor组(0.79±0.07)EGR3蛋白表达水平无明显差异(P>0.05),mimic组(0.51±0.04)EGR3蛋白表达水平明显低于CON组及Nc-mimic组,差异有统计学意义(P<0.01),inhibitor组(1.27±0.12)EGR3蛋白水平明显高于CON组及Nc-inhibitor组,差异有统计学意义(P<0.01)。RT-PCR检测各实验组EGR3基因表达,实验结果显示：CON组(1.02±0.10),Nc-mimic组(1.01±0.09)及Nc-inhibitor组(0.99±0.07)EGR3基因表达水平无明显差异(P>0.05),mimic组(0.69±0.05)EGR3基因表达水平明显低于CON组及Nc-mimic组,差异有统计学意义(P<0.01),inhibitor组(1.31±0.11)EGR3基因表达水平明显高于CON组及Nc-inhibitor组,差异有统计学意义(P<0.05)。结论EGR3可能是miR-3178调控HCC TECs生物学行为的靶基因之一,上调miR-3178表达可能通过抑制EGR3基因及蛋白表达从而抑制HCC TECs的增殖,侵袭,迁移及血管形成,促进其凋亡及G0/G1期阻滞。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is a kind of solid tumors with rich blood supply.Tumor vessels is an important source of nutritional support,tumor invasion and migration of hepatocellular carcinoma. Antiangiogenesis therapy has become importa nt treatment of patients with advanced hepatocellular carcinoma which has lost the best operation opportunity.In recent years, tumor blood vessel blocking treatment such as transcatheter arterial chemoembolization (TACE) had been widely used in clinic in the treatment of HCC and and benefits.However,after traditional clinical blood vessel blocking treatment or anti-tumor angiogenesis treatment,the disease of some HCC patients had not been relieved.To avoid the interference of liver cancer cells, macrophages and other cells, this study directly choose hepatocellular carcinoma tumor endothelial cells (HCC TECs) as the research objecct, and flow cytometry was used to detect the expression of cell-surface molecules CD 105 and CD31 to determine purity of HCC TECs.Small RNA (microRNAs (miRNA)) widely exists in eukaryotic organisms, although not directly encoded proteins,but when combination with the target gene, miRNAs can degrade mRNA or inhibit translation and plays an important role in the molecular biology behavior such as groth,development,proliferation,differentiation and cell apoptosis.miRNAs has a good application prospectin in gene function research, early diagnosis and target gene therapy of tumor. At present the hot topics of research mainly concentrated in the role of miRNAs on tumor occurrence and development,a growing number of studies have found abnormal expression of miRNAs in HCC,the abnormal expression of miRNAs may be involved in the occurrence and development of HCC.Studies of these abnormal expression of miRNAs may help early diagnosis and treatment of HCC.However,the research of communication between miRNAs and hepatocellular carcinoma tumor endothelial cells rarely reported.If we can find the abnormal expression of miRNAs in hepatocellular carcinoma tumor endothelial cells and regulate its expression,then we can regulate generation and metastasis of hepatocellular carcinoma tumor endothelial cells,thereby regulating angiogenesis of HCC,eventually inhibit the growth and metastasis of hepatocellular carcinoma.This study screening the result of differential expression of miRNAs between hepatic sinusoidal endothelial cells (HSECs) and HCC TECs in our previous HmiOA v4 Human miRNA OneArray(?) Chip expression spectrum,miR-3178 was lower expression in HCC TECs (option standard log2|a Fold change|acuity 0.585 and P< 0.05) and was not reported in other studies,thus,miR-3178 was selected for further research.Explore the malignant biological behaviors of HCC TECs and its molecular mechanism caused by miR-3178 is expected to provide experimental basis and new targets for HCC TECs targeted therapy. This study was divided into three parts as follow.Part Ⅰ Screening and validation of differentially expression of miRNAs between hepatic sinusoidal endothelial cells and hepatocellular carcinoma tumor endothelial cells and synthetic miRNAs mimic or inhibitor and transfection hepatocellular carcinoma tumor endothelial cellsObjective Screening and validation of differentially expression of miR-3178 between hepatic sinusoidal endothelial cells and hepatocellular carcinoma tumor endothelial cells and hepatocellular carcinoma tumor endothelial cells were transfected with miRNA-3178 mimic/inhibitor or their respective negative sequence.Methods Flow cytometry was used to detect cell surface molecules CD105, CD31 expression levels of HCC TECs to validate purity of HCC TECs. Screening the results of our previous HmiOA v4 Human miRNA OneArray(?) Chip,miR-3178 was significantly lower expression in HCC TECs and was selected for further research. Real-time polymerase chain reaction (RT-PCR) was performed to detect the differential expression of miR-3178 in HSECs and HCC TECs.Hepatocellular carcinoma tumor endothelial cells were transfected with miRNA-3178 mimic/ inhibitor, (negative-control mimic) Nc-mimic or (negative-control mimic) Nc- inhib itor.According to the different methods of processing, HCC TECs were divided into five experimental groups:(1) the blank control group, namely the untreated HCC TECs group (CON group);(2) the miR-3178 up-regulation group, namely the group transfected with 2’-O modified miR-3178 mimic group (mimic group);(3)the miR-3178 negative-control mimic group, namely the group transfected with 2’-O modified sequence of negative-control mimic(Nc-mimic group);(4)the miR-3178 down-reg ulation group,namely the group transfected with 2’-O modified miR-3178 inhibitor (inhibitor group); (5) the miR-3178 negative-control inhibitor group, namely the group transfected with 2’-O modified sequence of negative-control inhibitor (Nc-inhibitor group).Real-time quantitative polymerase chain reaction was used to detect expression of miR-3178.Green fluorescent tags was observed and photographed under fluorescence microscopy.Results The results of flow cytometry showed that the HCC TECs cell surface molecule CD105 expression rate was 100% and the CD31 expression rate was 97.9%, excluding the contamination of hepatoma cells,macrophages and fibroblasts.miR-3178 was significantly lower expression in HCC TECs and was selected for further research(the standard to choose miR-3178:log2|Fold change|≥0.585 and P<0.05).RT-PCR was used to validation differentially expressioin of miR-3178 between HSECs and HCC TECs,the result showed that the expression of miR-3178 was significantly lower in HCC TECs (0.31±0.03) than in HSECs (1.04±0.08) (P<0.001),the result proves the accuracy of the result of gene chip.RT-PCR was used to detect the expressioin of miR-3178 in each experimental group,the result showed that no significant difference in miR-3178 expression was detected between the CON group (1.04±0.14) and the Nc-mimic group(0.99±0.09) or the Nc-inhibitor group (1.02± 0.11),the mimic group (365.98±49.49) showed significantly higher miR-3178 express ion than the other groups (P<0.001),whereas the inhibitor group (0.19±0.04) showed significantly lower miR-3178 expression than the other groups (P<0.001).Green fluorescent tags was observed and photographed under fluorescence microscopy,no green fluorescent was observed in CON group, obvious green fluores cence was observed in mimic group,Nc-mimic group,inhibitor groups and Nc-inhibitor group,the transfection efficiency in HCC TECs were higher than 90%.Conculsion miR-3178 expression is significantly reduced in HCC TECs.Transfection of miR-3178 mimic significantly up-regulated miR-3178 expression, and transfection of miR-3178 inhibitor significantly down-regulated miR-3178 expression,the transfect ion efficiency in HCC TECs were higher than 90%.Part II The effect of changes in miR-3178 expression on biological behavior of HCC TECsObjective Study the effect of up-regulate or down-regulate the expression of miR-3178 on malignant biological behavior such as proliferation,apoptosis,cell cycle, invasion, migration and tube formation of HCC TECs.Methods MTT(Methyl thiazol tetrazolium) assay was used to detect HCC TECs proliferation in each experimental group.Flow cytometry was used for detecting HCC TECs cell apoptosis and cell cycle in each experimental group. Trans well invasion and migration assay was used to detect invasion and migration ability of HCC TECs in each experimental group.Tube formation assay was used to detect HCC TECs tube fomation in each experimental group.Results MTT assay showed that transfection after 24,36,48 and 72 h, no significant difference in the proliferation rate of HCC TECs was observed between the CON group and the Nc-mimic or the Nc-inhibitor group.The proliferation rate of HCC TECs was lower in the mimic group than in the other groups at corresponding time points after transfection and achieved the lowest proliferation rate at 72 h.The proliferation rate of HCC TECs was higher in the inhibitor group than in the other groups at corresponding time points after transfection and achieved the highest proliferation rate at 72 h.The result of flow cytometry showed that no significant difference in the apoptotic rate of HCC TECs was detected between the CON group and the Nc-mimic group or the Nc-inhibitor group.The mimic group showed significantly higher apoptotic rate than the other groups, whereas the inhibitor group showed significantly lower apoptotic rate than the other groups.The result of flow cytometry cell cycle assay showed that no significant difference in the cell population at the G0/G1 phase and S phase was observed between the CON group and the Nc-mimic or Nc-inhibitor group.The mimic group showed significantly higher G0/G1 phase cell population than the other groups.The inhibitor group showed significantly lower G0/G1 cell population than the other groups.The mimic group showed significantly lower S phase cell population than the other groups,whereas the inhibitor group showed significantly higher S phase cell population than the other groups.The result of transwell invasion and migration assay showed that no significant difference in the number of invaded and migrated cells was detected between the CON group and the Nc-mimic group or the Nc-inhibitor group.The mimic group showed significantly lesser number of invaded and migrated cells than the other groups, whereas the inhibitor group showed significantly greater number of invaded and migrated cells than the other groups. The result of tube formation assay showed that no significant difference in the number of capillary-like structures was found between the CON group and the Nc-mimic group or the Nc-inhibitor group.The mimic group showed significantly lesser number of capillary-like structures than the other groups, whereas the inhibitor group showed significantly greater number of capillary-like structures than the other groups.Conculsion miR-3178 may be act as a tumor suppressor gene involved in the regulation of biological behavior of HCC TECs,up-regulation of miR-3178 expression can inhibit HCC TECs proliferation,invasion,migration,tube formation and promote apoptosis and cell cycle G0/G1 phase arrest of HCC TECs.Part III Mechanism of the effect of miR-3178 on biological behavior of HCC TECsObjective To further investigate the mechanism of the effect of miR-3178 on biological behavior such as proliferation,apoptosis,cell cycle,invasion,migration and tube formation of HCC TECs.Observe the effect of regulating miR-3178 expression on mRNA expression and protein expression of potential target genes to verify the target gene.Methods Because the important role of the 3’UTR region in miRNA target genes, which could affect post-transcriptional expression of this gene.TargetScan,PicTar,Miranda and RNAhybrid target gene prediction software were used to predict the target genes of miR-3178.Western Blot was used to detect target gene protein expression in HCC TECs. RT-PCR was used to detect target gene mRNA expression in HCC TECs.Results Target gene prediction software showed EGR3 as a possible candidate target. Western blot showed that no significant difference in EGR3 protein expression was detected between the CON group(0.77±0.08)and the Nc-mimic group(0.73±0.06) or Nc-inhibitor group(0.79±0.07)(P>0.05).The mimic group(0.51±0.04) showed signi ficantly lower EGR3 protein expression than the other groups(P<0.01),whereas the inhibitor group(1.27±0.12) showed significantly higher EGR3 protein expression than the other groups(P<0.01).RT-PCT showed that no significant difference in EGR3 mRNA expression were detected between the CON group (1.02±0.10)and the Nc-mimic (1.01 ±0.09) group or Nc-inhibitor group(0.99±0.07)(P>0.05).The mimic group (0.69±0.05) showed significantly lower EGR3 mRNA expression than the other groups(P<0.01), whereas the inhibitor group(1.31±0.11) showed significantly higher EGR3 mRNA expression than the other groups(P<0.01).Conculsion EGR3 may be one of the target genes of miR-3178.The up-regulation of miR-3178 expression could inhibit EGR3 gene and protein expression in inhibiting cell proliferation,invasion,migration, angiogenesis and promoting apoptosis, G0/G1 phase arrest of HCC TECs.更多还原