【作者】 张军；

【导师】 唐建武；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 背景:癌细胞转移是多步骤、多因素极其复杂的动态、连续的生物学过程,找出其发展中起关键作用的因素和环节,并加以有效地控制,是提高抗癌转移的可靠途径。肝癌转移是攻克肝癌最重要的难关之一,但关于肝癌转移的具体机制,有的问题尚不清楚。淋巴道转移是上皮来源恶性肿瘤转移的早期阶段,近年来我们通过遗传背景相同或相似,但淋巴道转移能力不同的肝癌细胞株(Hca-F细胞和Hca-P细胞),利用基因芯片技术,在体外筛选出了33个在Hca-F细胞和Hca-P细胞之间有显著差异表达的基因;利用定量蛋白质组学技术-荧光差异双向凝胶电泳技术等,得到了差异表达蛋白质点23个。将上述两方面研究结果进行进一步比对发现,共同在mRNA水平和蛋白质水平都有显著差异性表达的基因及蛋白质就包括Ech1。烯酯酰辅酶A水解酶1(Enoyl Coenzyme A hydratase,Ech1)是脂肪酸β氧化的关键酶,存在于过氧化物酶体和线粒体中,近年来研究表明其表达异常与肝癌、胃癌、慢性淋巴细胞白血病等肿瘤有关,但具体作用机制不清楚,未见与肝癌淋巴道转移的相关报道。比较两种细胞之间Ech1的表达,在mRNA检测中的差异为3.0倍,在蛋白质检测中的差异为2.0倍,并通过Western blot和免疫细胞化学检测得到一致的结果。Hca-F细胞/Hca-P细胞是本课题组多年以前通过有限稀释法从同一小鼠肿瘤细胞克隆中筛选分离出的两个不同淋巴道转移能力亚克隆,经局部皮下注射,均特异性地向引流淋巴结转移。Hca-F为高淋巴道转移能力细胞,淋巴结转移率>70%;Hca-P为低淋巴道转移能力细胞,淋巴结转移率<30%。由于二者来源于同一亲本细胞系,具有基本相同的遗传背景,两者的差异主要集中于转移表型上,因此在肿瘤淋巴道转移机制研究中可互为参照,是非常良好、实用的细胞系模型。目的:1.构建pGPU6/GFP/Neo-shRNA-Ech1表达载体,稳定转染至Hca-F细胞,得到Ech1基因表达明显下调的Hca-F细胞株。2.研究Ech1基因表达水平下调后小鼠肝癌细胞株Hca-F细胞在细胞增殖能力、粘附能力、迁移能力、侵袭能力和细胞周期等方面的变化,探讨Ech1基因的表达对小鼠肝癌细胞生物学行为的影响。3.研究Ech1基因表达水平下调后小鼠肝癌细胞株Hca-F细胞中,Annexin A7基因,Clic1基因,Gelsolin基因的表达变化。方法:1:针对Ech1基因设计4个不同靶点的shRNA,分别命名为pGPU6/GFP/Neo-shRNA-Ech1-91、pGPU6/GFP/Neo-shRNA-Ech1-348、pGPU6/ GFP/Neo-shRNA-Ech1-430、pGPU6/GFP/Neo-shRNA-Ech1-460,同时设计无关序列阴性对照。构建好的质粒用BamH I酶,Pst I酶分别酶切鉴定。并且每组选择两个克隆进行测序鉴定。确定所构建质粒正确无误后,将4个不同靶点的pGPU6/GFP/Neo-shRNA-Ech1表达载体和无关序列阴性对照组分别用阳离子聚合物Sofast转染试剂稳定转染至Hca-F细胞,24小时后荧光显微镜下观察转染效率,用含400μg/ml G418的完全培养基筛选21天,获得pGPU6/GFP/Neo-shRNA-Ech1表达载体和无关序列阴性对照稳定转染的Hca-F细胞。提取未处理的Hca-F细胞株、稳定转染无关序列的Hca-F细胞株、稳定转染pGPU6/GFP/Neo-shRNA-Ech1四个不同靶点表达载体的Hca-F细胞株的RNA和蛋白质,应用qRT-PCR和Western blot分别在mRNA和蛋白质水平检测干扰Ech1基因的表达效应。筛选抑制效果最明显的序列用于后续实验。2:抑制效果最明显的转染pGPU6/GFP/Neo-shRNA- Ech1-91的Hca-F细胞,转染无关序列的阴性对照组Hca-F细胞,正常Hca-F细胞三组细胞,应用CCK8试剂盒检测各组细胞增殖能力的变化;应用体外细胞粘附试剂盒检测各组细胞对细胞外基质的五种组分的粘附能力的变化,并检测各组细胞对体内淋巴结粘附能力的变化;应用Transwell小室检测各组细胞迁移能力和侵袭能力的变化;应用流式细胞仪检测各组细胞细胞周期的变化。3:收集稳定转染pGPU6/GFP/Neo-shRNA-Ech1-91的Hca-F细胞,正常Hca-F细胞,冷PBS洗3次后,加入细胞裂解液,提取蛋白并检测蛋白浓度,取各组蛋白50 ug上样,Western blot检测细胞中Annexin A7基因,Clic1基因及Gelsolin基因表达变化。结果:1:重组质粒经酶切和自动基因测序仪测序,结果均与设计序列完全相符,所含目的基因序列准确无误,表明重组质粒构建成功。qRT-PCR检测结果显示,与正常Hca-F细胞相比,pGPU6/GFP/Neo-shRNA-Ech1-91组干扰效率最高为78%,pGPU6/GFP/Neo-shRNA-Ech1-430组干扰效率次之为35%。Western blot检测结果显示,pGPU6/GFP/Neo-shRNA-Ech1-91组在蛋白水平干扰效率最高为43%,pGPU6/GFP/Neo-shRNA-Ech1-348组干扰效率为45%。综合qRT-PCR和Western blot的检测结果,pGPU6/GFP/Neo-shRNA- Ech1-91组抑制效果最明显,为后续实验干扰组细胞。2:CCK8检测细胞增殖能力实验结果显示pGPU6/GFP/Neo-shRNA- Ech1组细胞增殖OD值明显下降,提示Ech1基因表达下调后Hca-F细胞的增殖能力明显下降。对细胞外基质各组分粘附能力的实验结果显示pGPU6/GFP/Neo-shRNA-Ech1组对FN和CollagenⅠ成分的OD值下降,对FN成分的OD值pGPU6/GFP/Neo-shRNA-Ech1组1.01±0.27,无关序列组1.47±0.19,Hca-F组1.42±0.26;对CollagenⅠ成分的OD值pGPU6/GFP/Neo-shRNA-Ech1组0.90±0.09,无关序列组1.26±0.19,Hca-F组1.14±0.07,对其他成分OD值差异无统计学意义(P>0.05),提示下调Ech1基因后Hca-F细胞对FN和CollagenⅠ成分的粘附能力下降;对体内淋巴结粘附能力实验显示pGPU6/GFP/Neo-shRNA-Ech1组的粘附细胞数细胞明显减少,pGPU6/GFP/Neo-shRNA-Ech1组粘附细胞数为58.3±18.6,无关序列组123.0±15.5,Hca-F组101.7±22.8,提示下调Ech1基因后Hca-F细胞的粘附能力下降。对迁移能力实验结果显示pGPU6/GFP/Neo-shRNA-Ech1组细胞迁移数明显下降,pGPU6/GFP/Neo-shRNA-Ech1组细胞迁移数为27.1±17.5;无关序列组细胞迁移数为72.4±18.8;Hca-F组细胞迁移数为59.1±30.3,提示Ech1基因表达下调后Hca-F细胞的迁移能力下降。侵袭能力实验结果显示各组细胞穿膜细胞数的差异无统计学意义(P>0.05),提示Ech1基因可能不是影响Hca-F细胞侵袭能力的主要因素。流式细胞技术检测细胞周期的变化实验显示Ech1基因表达下调后Hca-F细胞细胞周期S期延长,细胞周期G1期缩短,pGPU6/GFP/Neo-shRNA- Ech1组G1期细胞占9.4%,S期细胞占86.6% ;无关序列组G1期细胞占24.2% ,S期细胞占75.8%;Hca-F组G1期细胞占30.3%,S期细胞占66.2%。3:Ech1基因表达下调后,Western blot检测Hca-F细胞中Annexin A7蛋白表达增加11%,Clic1蛋白表达增加69%,Gelsolin蛋白表达增加13%。结论:1:成功构建了四个不同靶点pGPU6/GFP/Neo-shRNA-Ech1表达载体,并将该载体稳定转染至Hca-F细胞,获得Ech1基因表达稳定下调的Hca-F细胞株,经验证pGPU6/GFP/Neo-shRNA-Ech1-91对Hca-F细胞中Ech1干扰效率最明显,为后续研究Ech1功能实验的细胞株。2:Ech1基因表达下调后,Hca-F细胞的细胞增殖能力下降、粘附能力下降和迁移能力下降,细胞S期延长,G1期缩短。Ech1可能在促进肿瘤淋巴道转移中发挥作用。3:Ech1基因表达下调后,Hca-F细胞中Annexin A7蛋白、Clic1蛋白和Gelsolin蛋白表达增加,尤其是Clic1蛋白表达增加十分显著。更多还原

【Abstract】 Background: The high mortality rates associated with cancers are mainly attributable to their strong tendency to metastasize into surrounding or distant tissues. Lymphatic metastases are usually an early step, with lymph nodes often the first organs to develop metastases. It is believed that the presence of lymph node metastases (LNM) is associated with badly prognosis in head and neck cancer. Despite the importance of this in malignancies, a detailed understanding of the mechanism of lymphatic metastasis is still lacking. Immunocytochemistry (ICC) analysis and Western blot (WB) analysis indicated that the protein levels of Ech1 in Hca-F were 1.5-fold and 1.2-fold of that in Hca-P , which was consistent with our previous genomic (3.0 fold) and proteomic (2.7 fold) results.Hca-F with a high lymphatic metastasis rate of 75%, while Hca-P with a low lymphatic metastasis rate of 30%, which has been proved to be an ideal cell model for studying the lymphatic metastasis.Ech1 is the second enzyme in fatty acid degradation viaβ-oxidation pathway. A variety of studies already demonstrated that Ech1 was associated with tumor progression. The abnormal expressions of Ech1 were associated with putative preneoplasia and neoplastia and hepatocellular carcinoma infected by HCV and the pathogenesis of gastric cancer. And the down-regulation of Ech1 at the gene level was found to be associated with the DNA damage-induced apoptosis resistantance of B cell chronic lymphoid leukemia (B-CLL). However, no study has been addressed on the relevance of Ech1 with tumor lymphatic metastasis. To gain an insight into the role that Ech1 is playing in a high LNM potential tumor cell line, an RNA interference (RNAi) study was performed to downregulate Ech1 expression in the Hca-F cell line with observation of the subsequent effects. Objective: Short hairpin RNA (shRNA)-Ech1 expression plasmids were constructed and got the stably transfected cells. To study the effect to cell proliferation, adhesion, migration, invasion, and cell cycle status when down-regulation of Ech1 gene expression in mouse hepatocarcinoma cell. And to study the expression of Annexin A7, Clic1 and Gelsolin when downregulation of Ech1.Methods: 1 Four shRNAs that targeted different sites of the mouse Ech1 gene (NM-016772)(Ech1-430)5-CTCATCAGCAAGTACCAGA-3,(Ech1-91)5-GCCAGCTGTACTTCAACATCA-3, (Ech1-348)5-GCAGGAAA GATGT TCACTTCA-3,(Ech1 -460)5-GCAAGTACCAGAAGACCTTCA-3 were designed, synthesized and inserted into the pGPU6/GFP/Neo expression vector. pGPU6/GFP/Neo-shRNA-Ech1 were constructed,and all the expression plasmids were confirmed by DNA sequence analysis. Hca-F cells were added into a 24-well plate prior to transfection, and transfected with 2μg of pGPU6/GFP/Neo plasmid using sofastTM transfection reagent (Sunma Corp., China) according to the instructions of the manufacturer. Transfection efficiencies were determined by fluorescence microscopy at 24 h and 48 h. The stably transfected cells were obtained by growing them in neomycin (400μg/ml) for 3 weeks. The levels of expression of Ech1 mRNA and protein were detected by real time quantitative polymerase chain reaction (qRT-PCR) and WB analysis, respectively. The shRNA-Ech1-91- Hca-F cells were chosen for further experiments. 2. Cells were divided into three groups: (a) shRNA-Ech1-91-Hca-F; (b) control shRNA-Hca-F; (c) unmanipulated Hca-F cells were used as control. Cell proliferation was assessed by Cell counting kit-8 (CCK8) assay according to the protocol of the manufacturer. Cell adhesion in vitro was measured using the CytoSelectTM 48-well extracellular matrix (ECM) array, with kits that contained collagen I, collagen IV, fibrinogen, fibronectin and laminin. While Cell adhesion in vivo was measured using cell suspension (1×106 cells/ml) placed onto the surface of frozen slices of lymph nodes and stained with hematoxylin and eosin (HE), then adherent cells were counted. Cell migration assay and invasion assay were measured using the Boyden-transwell assay (8-μm pore size). The cell cycle status was measured by fluorescence activated cell sorting, to obtain the distribution and percentage of cells in the G1, S, and G2/M phases. 3. Equal amounts of protein from each group were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). These were incubated with a primary antibody against Annexin A7 (Sigma; 1:1500), Clic1 (Santa Cruz; 1:200), Gelsolin (BD Biosciences; 1:1000) and a monoclonal antibody against GAPDH (Kangcheng; 1:7500),β-actin (Santa Cruz; 1:300) in 5% dried milk for 1 h, then incubated with horseradish peroxidase-linked second antibodies (1:5000 dilution) for 1 h at room temperature. Enhanced chemiluminescence (ECL; GE Healthcare, USA) was performed to visualize the protein bands.Results: 1: Endonuclease activity assays and DNA sequence analysis confirmed the recombinant plasmids of shRNA-Ech1. The levels of mRNA expression of Ech1 in shRNA-Ech1-91-Hca-F and shRNA-Ech1-430-Hca-F were decreased by 78% and 35%, respectively, quantified by qRT-PCR analysis compared to Hca-F cells. WB results indicated shRNA-Ech1-91-Hca-F and shRNA-Ech1-430-Hca-F reduced the Ech1 protein levels by 46% and 35% compared to Hca-F cells, but nonspecific-sequence control shRNA transfected Hca-F cells showed no difference compared to Hca-F cells. shRNA-Ech1-91-Hca-F was more efficient than others, so this was chosen as the shRNA-Ech1-Hca-F for further experiments. 2: Cell proliferation: After 72 h, the number of shRNA-Ech1-Hca-F cells was 84% of the number of unmanipulated Hca-F cells and 87% of the control shRNA-Hca-F cells. The cell number of shRNA-Ech1-Hca-F was 71% that of the Hca-F and 86% of the control shRNA-Hca-F at 96 h. The downregulation of Ech1 may inhibit to some extent the proliferative capacity of the Hca-F cells. Cell adhesion assay in vitro: The absorbance of shRNA-Ech1-Hca-F cells adhered to fibronectin was 1.0±0.3, which was 71% that of the Hca-F cells and 69% of the control shRNA-Hca-F cells. The data for shRNA-Ech1-Hca-F adherence to collagenⅠwas 0.9±0.1, which was 79% of the results for the Hca-F cells and 71% of the control shRNA-Hca-F cells. The adherence of the transfected Hca-F cells to the other tested components of the ECM showed no differences. Cell adhesion assay in vivo: The number of Hca-F and control shRNA-Hca-F cells adhered to lymph nodes were 107±23 and 118±16, respectively. However, the number of shRNA-Ech1-Hca-F cells adhered to lymph nodes was only 58±19, which was only 54% of the number for Hca-F cells and 49% of the control shRNA-Hca-F cells (p<0.05). Cell migration assay: showed that Hca-F and control shRNA had a similar ability to pass through the filter, the numbers of shRNA-Ech1 cell (27±18) passing through the filter were markedly lower than the numbers of Hca-F (59±30) and control shRNA (72±19), and there was no significant difference between control shRNA and Hca-F cells. Cell invasion assay: No differences were observed in invasion ability between shRNA-Ech1-Hca-F and Hca-F cells. Flow cytometric analysis of cell cycle status: The S phase cells were 86.06% in shRNA-Ech1 and 75.75% in negative control and 66.15% in Hca-F cell; The G1 phase cells were 9.42% in shRNA-Ech1 and 24.21% in negative control and 30.29% in Hca-F cell, respectively, the result showed that the ratio of cells was significantly increased at S phase and decreased at G1 phase after Ech1 downregulated when compared with Hca-F cells, and there was no significant difference between control shRNA and Hca-F cells. 3: The expression of Annexin A7 were upregulated 11%, the expression of Clic1 were upregulated 69%, the expression of Gelsolin were upregulated 13%, especially the expression of Clic1 were upregulated obviously.Conclusion: The stably transfected pGPU6/GFP/Neo-shRNA-Ech1- Hca-F cell line was obtained; The down-regulation of Ech1 was proved to inhibit the adhesion ability of Hca-F cell, inhibit the cell proliferation of Hca-F cells, decrease the migration capacities of Hca-F cells, increased the ratio of Hca-F cells in S phase and decreased the ratio of G1 phase. when the expression of Ech1 was downregulated, the expression of Annexin A7, Clic1, Gelsolin were upregulation, especially the expression of Clic1.更多还原