Transcription profiling of mouse PKCa -/- mutant vs. wild type small intestine

Members of the PKC family of serine / threonine kinases play key regulatory roles in numerous cellular processes including differentiation and proliferation. Of the eleven mammalian PKC isoforms known several have been implicated in tumor development and progression. However, in most cases isotype specificity is poorly defined and even contrary functions for a single PKC have been reported, mostly because appropriate molecular and genetic tools were missing to specifically assess the contribution of single PKC isoforms in vivo. In this report we therefore used PKC genetic targeting to study the role of PKCa and PKCz in colorectal cancer. Both isoforms were found to be strongly down-regulated in intestinal tumors of ApcMin/+ mice. A deletion of PKCz did not affect tumorigenesis in this animal model. In contrast, PKCa deficient ApcMin/+ mice developed more aggressive tumors and died significantly earlier than their PKCa proficient littermates. Even without an additional Apc mutation PKCa knock out mice showed an elevated tendency to develop spontaneous intestinal tumors. Transcriptional profiling revealed a role for this kinase in regulating EGFR signaling and proposed a synergistic mechanism for EGFR / AP-1 and WNT / APC pathways in mediating intestinal tumor development. Experiment Overall Design: Adult male animals (PKCa -/- and congenic wt controls; 50-60 days old) were sacrificed by cervical dislocation. Small intestines were dissected and total RNA (~5µg per sample) was extracted using the QIAGEN RNeasy kit. Experiment Overall Design: Sample processing (performed at the RZPD, Deutsches Ressourcenzentrum fur Genomforschung GmbH, Heubnerweg 6, 14059 Berlin) included cRNA generation and labeling with biotin. cRNA samples were hybridized to the GeneChips and chips stained with a streptavidine-phycoerythrine conjugate, Experiment Overall Design: washed and scanned. Hybridization images were analyzed using Experiment Overall Design: GCOS 1.1.丝氨酸/苏氨酸激酶（serine/threonine kinases）的PKC家族成员，在细胞分化、增殖等诸多细胞过程中发挥关键调控作用。目前已鉴定出11种哺乳动物PKC同工型（isoforms），其中多种被证实与肿瘤发生及进展密切相关。然而，绝大多数情况下PKC同工型的功能特异性尚未明确，甚至有研究报道单个PKC同工型存在截然相反的生物学功能，这主要是由于此前缺乏可在体内特异性评估单一PKC同工型贡献的合适分子及遗传学工具。本研究采用PKC基因靶向技术，探究PKCα（PKCa）与PKCζ（PKCz）在结直肠癌中的功能。实验发现，两种同工型在ApcMin/+小鼠的肠道肿瘤组织中均显著下调。剔除PKCζ并不会对该动物模型的肿瘤发生过程产生影响；与之相反，PKCα缺陷型ApcMin/+小鼠会形成侵袭性更强的肿瘤，且死亡时间显著早于PKCα正常的同窝对照小鼠。即便不存在额外的Apc突变，PKCα敲除小鼠也表现出更高的自发性肠道肿瘤发生倾向。转录组分析（transcriptional profiling）揭示了该激酶在调控EGFR信号通路中的关键作用，并提出EGFR/AP-1与WNT/APC通路协同介导肠道肿瘤发生的潜在机制。实验总体设计：选取50~60日龄的成年雄性动物（PKCα敲除[PKCa -/-]及同基因野生型对照），通过颈椎脱臼法实施安乐死。解剖分离小肠组织，采用QIAGEN RNeasy试剂盒提取总RNA（每份样本约5μg）。实验总体设计：样本处理工作由德国基因组研究资源中心（Deutsches Ressourcenzentrum fur Genomforschung GmbH, RZPD，地址：Heubnerweg 6, 14059 柏林）完成，流程包括cRNA合成与生物素标记。将制备好的cRNA样本与基因芯片（GeneChips）进行杂交，随后使用链霉亲和素-藻红蛋白偶联物（streptavidine-phycoerythrine conjugate）对芯片进行染色。实验总体设计：洗涤芯片后进行扫描成像，杂交图像采用GCOS 1.1软件完成分析。