Department of Pathology, The Ohio State University College of Medicine, Columbus, OH, USA; Comprehensive Cancer Center, The Ohio State University College of Medicine, Columbus, OH, USA.
Department of Biomedical Informatics, The Ohio State University College of Medicine, Columbus, OH, USA.
Mol Metab. 2020 Apr;34:174-186. doi: 10.1016/j.molmet.2020.01.003. Epub 2020 Jan 9.
It is well established that the liver-specific miR-122, a bona fide tumor suppressor, plays a critical role in lipid homeostasis. However, its role, if any, in amino acid metabolism has not been explored. Since glutamine (Gln) is a critical energy and anaplerotic source for mammalian cells, we assessed Gln metabolism in control wild type (WT) mice and miR-122 knockout (KO) mice by stable isotope resolved metabolomics (SIRM) studies.
Six-to eight-week-old WT and KO mice and 12- to 15-month-old liver tumor-bearing mice were injected with [U-C,N]-L-Gln, and polar metabolites from the liver tissues were analyzed by nuclear magnetic resonance (NMR) imaging and ion chromatography-mass spectrometry (IC-MS). Gln-metabolism was also assessed in a Gln-dependent hepatocellular carcinoma (HCC) cell line (EC4). Expressions of glutaminases (Gls and Gls2) were analyzed in mouse livers and human primary HCC samples.
The results showed that loss of miR-122 promoted glutaminolysis but suppressed gluconeogenesis in mouse livers as evident from the buildup of C- and/or N-Glu and decrease in glucose-6-phosphate (G6P) levels, respectively, in KO livers. Enhanced glutaminolysis is consistent with the upregulation of expressions of Gls (kidney-type glutaminase) and Slc1a5, a neutral amino acid transporter in KO livers. Both Gls and Slc1a5 were confirmed as direct miR-122 targets by the respective 3'-UTR-driven luciferase assays. Importantly, expressions of Gls and Slc1a5 as well as glutaminase activity were suppressed in a Gln-dependent HCC (EC4) cell line transfected with miR-122 mimic that resulted in decreased C-Gln, C-á-ketoglutarate, C-isocitrate, and C-citrate levels. In contrast, C-phosphoenolpyruvate and C-G6P levels were elevated in cells expressing ectopic miR-122, suggesting enhanced gluconeogenesis. Finally, The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database analysis showed that expression of GLS is negatively correlated with miR-122 in primary human HCCs, and the upregulation of GLS RNA is associated with higher tumor grade. More importantly, patients with higher expressions of GLS or SLC1A5 in tumors exhibited poor survival compared with those expressing lower levels of these proteins.
Collectively, these results show that miR-122 modulates Gln metabolism both in vitro and in vivo, implicating the therapeutic potential of miR-122 in HCCs that exhibit relatively high GLS levels.
已有研究证实，肝脏特异性 miR-122 是一种真正的肿瘤抑制因子，在脂质稳态中发挥关键作用。然而，其在氨基酸代谢中的作用尚未被探索。由于谷氨酰胺（Gln）是哺乳动物细胞的关键能量和氨补充来源，我们通过稳定同位素分辨代谢组学（SIRM）研究评估了对照野生型（WT）小鼠和 miR-122 敲除（KO）小鼠中的 Gln 代谢。
将 6-8 周龄的 WT 和 KO 小鼠和 12-15 月龄的肝肿瘤荷瘤小鼠注射[U-C，N]-L-Gln，并用核磁共振（NMR）成像和离子色谱-质谱（IC-MS）分析肝组织中的极性代谢物。还在 Gln 依赖性肝细胞癌（HCC）细胞系（EC4）中评估了 Gln 代谢。分析了小鼠肝脏和人原发性 HCC 样本中的谷氨酰胺酶（Gls 和 Gls2）表达。
结果表明，miR-122 的缺失促进了小鼠肝脏中的谷氨酰胺分解代谢，但抑制了糖异生，这从 KO 肝脏中 C-和/或 N-Glu 的积累和葡萄糖-6-磷酸（G6P）水平的降低可以看出。增强的谷氨酰胺分解代谢与 KO 肝脏中 Gls（肾型谷氨酰胺酶）和 Slc1a5 的上调一致，Slc1a5 是一种中性氨基酸转运蛋白。通过各自的 3'-UTR 驱动的荧光素酶测定，均证实 Gls 和 Slc1a5 是 miR-122 的直接靶标。重要的是，在转染 miR-122 模拟物的 Gln 依赖性 HCC（EC4）细胞系中，Gls 和 Slc1a5 的表达以及谷氨酰胺酶活性被抑制，导致 C-Gln、C-α-酮戊二酸、C-异柠檬酸和 C-柠檬酸水平降低。相比之下，在表达外源性 miR-122 的细胞中，C-磷酸烯醇丙酮酸和 C-G6P 水平升高，表明糖异生增强。最后，癌症基因组图谱-肝肝细胞癌（TCGA-LIHC）数据库分析表明，在原发性人 HCC 中，GLS 的表达与 miR-122 呈负相关，并且 GLS RNA 的上调与更高的肿瘤分级相关。更重要的是，与表达这些蛋白质水平较低的患者相比，在肿瘤中表达较高水平的 GLS 或 SLC1A5 的患者的生存情况较差。
总之，这些结果表明，miR-122 在体外和体内调节 Gln 代谢，提示 miR-122 在表现出相对较高 GLS 水平的 HCC 中的治疗潜力。
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