Identification of genes related to spcific properties of mammalian cells

为了确定与特定细胞特性功能相关的基因，在生物反应器中培养了不同表型的细胞株，并对其进行了微阵列分析。结合数据过滤和聚类算法对微阵列数据进行归一化处理。根据样本之间的差异表达水平、聚类技术和拟议的功能，识别了几个基因。发现siat7e基因对HeLa细胞的黏附和形态有影响。在非锚定依赖的HeLa细胞中，降低II型糖基化唾液酸转移酶siat7e的表达导致更大的聚集和形态变化。基于上述工作，我们决定专注于MDCK细胞，这些细胞适合生产流感病毒，但不能在悬液中生长，因此不能用于疫苗的商业生产。将人siat7e基因(ST6GalNac V)导入MDCK细胞，使其转化为锚定非依赖细胞。转换后的细胞能够在生物反应器中产生病毒，表明它们有能力取代目前基于鸡蛋的生产过程。作为概念验证，这项工作表明，通过使用适当的策略，可以从悬浮MDCK-SIAT7E细胞的可伸缩培养中产生高产量的生物活性血凝素。 此外，还做了进一步的工作，以了解单基因转染如何将MDCK细胞从锚定依赖转变为锚定非依赖。这项工作表明，一种称为EMT(上皮-间充质转化)的过程参与了这一转变。对亲本MDCK和表达siat7e的MDCK细胞的DNA芯片分析表明，参与EMT的许多基因在这两个细胞系之间的表达存在显著差异。肝细胞生长因子(HGF)基因随后被生物信息学分析鉴定，并被证实在MDCK-siat7e细胞中过表达。这一发现正被用来尝试并改造其他依赖锚定的细胞，使其在悬浮中生长。 改善哺乳动物细胞的细胞特性的另一项尝试是基于识别影响细胞凋亡的microRNA。本研究测定了中国仓鼠卵巢(CHO)细胞暴露在营养耗竭的培养液中诱导细胞凋亡后microRNA表达的变化。微阵列比较CHO细胞暴露在新鲜和耗尽的培养液中，发现在耗尽的培养液中，miR-297-669簇的表达上调。进一步分析发现，miR-466h是该簇中过表达水平最高的成员，并通过qRT-PCR证实了其在耗竭介质中的上调表达。结合生物信息学和实验工具来预测和验证miR-466h抗凋亡靶点。在这项工作的下一阶段，我们能够理解营养耗竭导致的细胞凋亡级联激活的机制。这一点尤其重要，因为microRNA表达的变化与包括癌症在内的各种疾病的发展有关；然而，在不同生理条件下导致这些变化的分子事件尚未得到很好的表征。我们发现，miR-466h-5p、miR-669c和Sfmbt2基因的时间依赖性激活伴随着组蛋白脱乙酰化的抑制，组蛋白脱乙酰化是葡萄糖剥夺诱导的氧化应激的结果。这种氧化应激导致活性氧物种(ROS)的积累和还原型谷胱甘肽(GSH)的耗尽，共同抑制组蛋白去乙酰基酶(HDACs)的活性，降低HDAC2的蛋白水平，增加miR-466h-5p启动子区域的乙酰化，从而导致该miRNA的激活。基于这项研究和前人的工作，我们认为miR-466h-5p(和miR 297-669簇)在有毒代谢产物积累过程中可能在细胞中发挥作用。对导致miR-466h-5p激活的分子事件的表征可能有助于更好地理解细胞环境和miRNA激活之间的关系。

To identify genes functionally related to specific cellular properties, cell lines with different phenotypes were grown in bioreactors and sampled for microarray analysis. A combination of data filtering and clustering algorithms was applied to normalize microarray data. Based on the level of differential expression between samples, clustering techniques, and proposed functionality, several genes were identified. The gene siat7e was found to impact the adhesion and the morphology of HeLa cells. Decreasing the expression of siat7e, a type II membrane glycosylating sialyltransferase, in anchorage-independent HeLa cells resulted in greater aggregation and morphological changes. Based on the above work we decided to concentrate on MDCK cells that are suitable for influenza virus production but cannot grow in suspension and therefore are not being used for commercial production of the vaccine. The anchorage dependent MDCK cells were converted to anchorage independent cells by transfection with the human siat7e gene (ST6GalNac V). The converted cells were able to produce the virus in bioreactors demonstrating their capability to replace the current egg based production process. As a proof-of-concept this work demonstrates that by using a proper strategy, high yields of biologically active hemagglutinin can be produced from scalable cultures of suspension MDCK-siat7e cells. Additional work was done to understand how transfection with a single gene can transform the MDCK cell from anchorage dependent to anchorage independent. This work demonstrated that a process called EMT (Epithelial-mesenchymal transition) is involved in this transformation. DNA microarray analysis on parental MDCK and siat7e-expressing MDCK cells revealed that many of the genes involved in the EMT were significantly differentially expressed between the cell lines. The hepatocyte growth factor (HGF) gene was subsequently identified using bioinformatics analysis and was verified to be over-expressed in MDCK-siat7e cells. The findings are being used to try and transform other anchorage dependent cells to grow in suspension. Additional attempt to improve cellular properties of mammalian cells was based on identifying of microRNA that affect cells apoptosis. This study determined the changes in microRNA expression in Chinese hamster ovary (CHO) cells undergoing apoptosis induced by exposing the cells to nutrient-depleted media. Microarray comparison of microRNAs in CHO cells exposed to fresh or depleted media revealed up-regulation of miR-297-669 cluster in CHO cells subjected to depleted media. miR-466h was chosen for further analysis as the member of this cluster with the highest overexpression and its up-regulation in depleted media was confirmed with qRT-PCR. A combination of bioinformatics and experimental tools was used to predict and verify miR-466h anti-apoptotic targets. In the next phase of this work we were able to understand the mechanism associated with the activation of the apoptosis cascade as a result of the nutrient depletion. This is especially important since changes in microRNA expression have been linked to the development of various diseases including cancer; however, the molecular events leading to these changes at different physiological conditions are not well characterized. We showed that the time-dependent activation of miR-466h-5p, miR-669c and the Sfmbt2 gene followed the inhibition of histone deacetylation which was the result of glucose deprivation-induced oxidative stress. This oxidative stress caused the accumulation of reactive oxygen species (ROS) and depletion of reduced glutathione (GSH) that together inhibited histone deacetylases (HDACs) activity, reduced protein levels of HDAC2, and increased acetylation in miR-466h-5p promoter region which led to the activation of this miRNA. Based on this study and previous work, we suggest a possible role of miR-466h-5p (and miR 297-669 cluster) in the cells during toxic metabolites accumulation. Improved characterization of the molecular events that lead to the activation of miR-466h-5p may provide a better understanding of the relation between cellular environment and miRNA activation.