Global transcriptional response to carbonic anhydrase IX deficiency in the mouse stomach

Background Carbonic anhydrases (CAs) are a family of enzymes that regulate pH homeostasis in various tissues. CA IX is an exceptional member of this family because in addition to the basic CA function, it has been implicated in several other physiological and pathological processes. Functions suggested for CA IX include roles in cell adhesion and malignant cell invasion. In addition, CA IX likely regulates cell proliferation and differentiation, which was demonstrated in Car9-/- mice. These mice had gastric pit cell hyperplasia and depletion of chief cells; however, the specific molecular mechanisms behind the observed phenotypes remain unknown. Therefore, we wanted to study the effect of CA IX deficiency on whole-genome gene expression in gastric mucosa. This was done using Illumina Sentrix Mouse-6 Expression BeadChip arrays. The expression of several genes with notable fold-change values was confirmed by QRT-PCR. Results CA IX deficiency caused the induction of 86 genes and repression of 46 genes in the gastric mucosa. There was 92.9% concordance between the results obtained by microarray analysis and QRT-PCR. The differentially expressed genes included those involved in developmental processes and cell differentiation. In addition, CA IX deficiency altered the expression of genes responsible for immune responses and downregulated the expression of several digestive enzymes. Conclusions Microarray analysis identified several potential genes whose altered expression could explain the disturbed cell lineage phenotype in the Car-/-gastric mucosa. The results also indicated a novel role for CA IX in the regulation of immunologic processes and digestion. These findings reinforce the concept that the main role of CA IX is not the regulation of pH in the stomach mucosa. Instead, it is needed for proper function of several physiological processes. C57BL/6 mice were used for the study: 6 Car9 knockout mice (3 males and 3 females) and 6 wildtype mice as their controls (3 males and 3 females). Stomach tissues were analyzed from all these mice.背景：碳酸酐酶（Carbonic anhydrases, CAs）是一类在多种组织中调控pH稳态的酶家族。碳酸酐酶IX（CA IX）是该家族中的特殊成员：除具备基础的碳酸酐酶功能外，它还参与了诸多其他生理及病理生理过程。目前已证实CA IX参与细胞黏附与恶性细胞侵袭过程；此外，研究人员在Car9⁻/⁻小鼠中发现，CA IX可调控细胞增殖与分化。该类小鼠表现为胃腺窝细胞增生与主细胞耗竭，但上述表型背后的具体分子机制仍未明确。因此，本研究旨在探究CA IX缺失对胃黏膜全基因组基因表达的影响，实验采用Illumina Sentrix小鼠表达谱微珠芯片阵列（Illumina Sentrix Mouse-6 Expression BeadChip arrays）进行检测，并通过实时定量逆转录聚合酶链反应（Quantitative Reverse Transcription Polymerase Chain Reaction, QRT-PCR）验证了部分差异倍数显著的基因的表达水平。 结果：在胃黏膜中，CA IX缺失导致86个基因表达上调、46个基因表达下调。微阵列分析与QRT-PCR的检测结果一致性达92.9%。差异表达基因涉及发育过程与细胞分化相关通路；此外，CA IX缺失还改变了免疫应答相关基因的表达，并下调了多种消化酶的编码基因的表达水平。 结论：微阵列分析筛选出多个潜在基因，其表达异常可解释Car9⁻/⁻小鼠胃黏膜中出现的细胞谱系紊乱表型。本研究结果同时揭示了CA IX在调控免疫过程与消化功能中的全新作用，进一步证实CA IX在胃黏膜中的核心功能并非调控pH稳态，而是参与维持多项生理过程的正常运转。 本研究使用C57BL/6小鼠作为实验对象：包括6只Car9基因敲除小鼠（雌雄各3只）与6只野生型对照小鼠（雌雄各3只），并对所有小鼠的胃组织进行了分析。