RNA-Sequencing of Transaldolase and Aldose Reducatase Deficiency in Mouse Liver

Oxidative stress modulates carcinogenesis in the liver; however, direct evidence for metabolic control of oxidative stress during pathogenesis, particularly, of progression from cirrhosis to hepatocellular carcinoma (HCC), has been lacking. Deficiency of transaldolase (TAL), a ratelimiting enzyme of the non-oxidative branch of the pentose phosphate pathway (PPP), elicits growth restriction, and predisposes to cirrhosis and HCC in mice and humans. Here, we show that mitochondrial oxidative stress and disease progression from cirrhosis to HCC and acetaminophen-induced liver necrosis are critically dependent on NADPH depletion by aldose reductase (AR), while this enzyme protects from carbon trapping in the PPP and stunted growth in TAL deficiency. Both TAL and AR are confined to the cytosol, however, their inactivation distorts NADPH production and mitochondrial oxidative stress into opposite directions. Adult mice with heterozygous deletion of transaldolase (TAL+/-) were created and fully backcrossed for >10 generations onto the C57BL/6 strain. TAL+/- and Akr1b3-deficient aldose reductase (AR) knockout (ARKO) strains were crossed to generate double-knockout (DKO) mice lacking both TAL and AR. Both TAL-deficient and DKO mice were breed as heterozygotes for the TAL locus. Wild-type (WT or TAL+/+AR+/+), TAL-deficient (TALKO or TAL-/-AR+/+), AR-deficient (ARKO or TAL+/+AR-/-), and double-knockout (DKO, or TAL-/-AR-/-) mice were matched for age and gender in each experiments. Livers were harvested from adult mice and RNA processed for RNA-Sequencing.氧化应激（oxidative stress）可调控肝脏的癌变进程，但目前仍缺乏在发病过程中，尤其是从肝硬化进展至肝细胞癌（hepatocellular carcinoma，HCC）的阶段，代谢调控氧化应激的直接证据。转醛醇酶（transaldolase，TAL）是磷酸戊糖途径（pentose phosphate pathway，PPP）非氧化分支的限速酶，其缺失会导致生长受限，并使小鼠和人类易患肝硬化及肝细胞癌。本研究证实，线粒体氧化应激、肝硬化向肝细胞癌的疾病进展，以及对乙酰氨基酚诱导的肝坏死，均严重依赖醛糖还原酶（aldose reductase，AR）介导的烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate，NADPH）耗竭；而该酶可对抗磷酸戊糖途径中的碳捕获，并缓解转醛醇酶缺乏引发的生长迟滞。转醛醇酶与醛糖还原酶均定位于细胞质，但二者的失活会以相反方向调控烟酰胺腺嘌呤二核苷酸磷酸的产生与线粒体氧化应激。我们构建了转醛醇酶杂合敲除（TAL+/-）成年小鼠，并将其在C57BL/6品系背景下回交超过10代。将TAL+/-小鼠与Akr1b3缺陷型醛糖还原酶敲除（ARKO）小鼠杂交，获得同时缺失转醛醇酶与醛糖还原酶的双敲除（double-knockout，DKO）小鼠。转醛醇酶缺陷型小鼠与双敲除小鼠均以转醛醇酶位点的杂合子形式繁育。本研究各实验均选取年龄与性别匹配的野生型小鼠（WT或TAL+/+AR+/+）、转醛醇酶缺陷型小鼠（TALKO或TAL-/-AR+/+）、醛糖还原酶缺陷型小鼠（ARKO或TAL+/+AR-/-）及双敲除小鼠（DKO，即TAL-/-AR-/-）。从成年小鼠体内获取肝脏组织，提取RNA并进行RNA测序（RNA-Sequencing）。