RNA sequencing analysis for comparing gene expression of the corneal endothelium of Slc4a11-/- vs Slc4a11+/+ mice

Purpose: Slc4a11 KO mice show significant edema and altered corneal endothelial morphology at an early age with concomitant increased mitochondrial ROS and oxidative damage relative to wild type. Here we used RNA-Seq with the goal of finding pathways related to corneal endothelial metabolic, pump and barrier function alterations. Methods: Corneal endothelium-Descemet's membrane (CEDM) samples were from WT and Slc4a11 KO mice at 12 weeks of age. RNA sequencing data was subjected to Ingenuity Pathway Analysis and QPCR for validation. Results: The RNA sequencing IPA analysis predicted activation, inhibition or differential regulation of several pathways. We validated downregulation of Glycolytic enzymes, Mitochondrial complex components and Ion transporters. We observed upregulation of genes of cholesterol biosynthesis, GSH metabolism and tight and adherens junctions. Conclusions: Slc4a11 KO induces a coordinated decrease in glycolysis, glutaminolysis, oxygen consumption, lactate transporters and Na-K-ATPase. These changes together with the altered barrier function cause an accumulation of stromal lactate in Slc4a11 KO mice leading to chronic corneal edema. Corneal endothelium mRNA from Slc4a11-/- vs Slc4a11+/+ mice at 12 weeks of age.目的：与野生型（wild type, WT）小鼠相比，Slc4a11敲除（Slc4a11 KO）小鼠在幼年阶段即可出现显著角膜水肿与角膜内皮形态异常，同时伴随线粒体活性氧（mitochondrial ROS）水平升高及氧化损伤。本研究采用RNA测序（RNA-Seq）技术，旨在筛选与角膜内皮代谢、泵功能及屏障功能异常相关的信号通路。 方法：实验样本取自12周龄野生型与Slc4a11敲除小鼠的角膜内皮-后弹力膜（Corneal endothelium-Descemet's membrane, CEDM）组织。对RNA测序数据开展Ingenuity通路分析（IPA），并通过实时定量PCR（QPCR）进行验证。 结果：RNA测序联合IPA分析预测了多条通路的激活、抑制或差异调控。本研究验证发现糖酵解酶、线粒体复合物组分及离子转运体的表达均出现下调；同时观察到胆固醇生物合成、谷胱甘肽（glutathione, GSH）代谢相关基因，以及紧密连接与黏着连接相关基因的表达上调。 结论：Slc4a11敲除可协同下调糖酵解、谷氨酰胺分解、耗氧量、乳酸转运体及钠钾ATP酶的表达。上述改变与屏障功能异常共同导致Slc4a11敲除小鼠基质乳酸蓄积，进而引发慢性角膜水肿。本数据集的样本为12周龄Slc4a11纯合敲除（Slc4a11-/-）与野生型（Slc4a11+/+）小鼠的角膜内皮mRNA。