lncRNA NEAT1 抑制通过 miRNA-338-3p 介导的谷氨酰胺代谢调控诱导类风湿关节炎成纤维样滑膜细胞功能障碍。
Inhibition of lncRNA NEAT1 induces dysfunction of fibroblast-like synoviocytes in rheumatoid arthritis via miRNA-338-3p-mediated regulation of glutamine metabolism.
机构信息
Department of Rheumatology and Immunology, Tianjin Medical University General Hospital, 154 An-Shan Road, Heping, Tianjin, 300052, People's Republic of China.
Department of Breast Medical Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin, 300060, People's Republic of China.
出版信息
J Orthop Surg Res. 2022 Sep 1;17(1):401. doi: 10.1186/s13018-022-03295-y.
BACKGROUND
Rheumatoid arthritis (RA) is a systemic chronic autoimmune disease; cellular glutamine metabolism in fibroblast-like synoviocytes (FLSs) of RA was known to be essential for RA pathogenesis and progression. NEAT1, a long non-coding RNA, functions as an oncogene in diverse cancers. The exact roles and molecular mechanisms of NEAT1 in fibroblast-like synoviocytes (FLSs) of RA patients are unknown.
METHODS
Expression of NEAT1 and miR-338-3p was measured by qRT-PCR. lncRNA-miRNA and miRNA-mRNA interactions were predicted from starBase and validated by RNA pull-down and luciferase assay. The glutamine metabolism of FLSs was evaluated by glutamine uptake and glutaminase activity. Cell death in FLSs in response to HO was assessed by MTT and Annexin V assays.
RESULTS
NEAT1 was significantly upregulated, and miR-338-3p was significantly downregulated in FLSs from RA patients compared to normal FLSs. Silencing of NEAT1 and overexpression of miR-338-3p suppressed glutamine metabolism in FLSs-RA and promoted HO-induced apoptosis. Bioinformatics analysis showed that NEAT1 sponges miR-338-3p to form competing endogenous RNA (ceRNAs), which was verified by RNA pull-down assay and luciferase assay FLSs-RA had an increased rate of glutamine metabolism compared to normal FLSs increased compared to normal FLSs. The results confirmed that GLS (Glutaminase), a key enzyme in glutamine metabolism, is a direct target of miR-338-3p in FLSs-RA. miR-338-3p inhibition of glutamine metabolism was verified by rescue experiments verified. Finally, restoration of miR-338-3p in FLSs-RA expressing NEAT1 overcomes NEAT1-promoted glutamine metabolism and resistance to apoptosis.
CONCLUSIONS
This study reveals the essential role and molecular targets of NEAT1-regulated glutamine metabolism and FLSs-RA dysfunction in fibroblast-like synoviocytes of RA and indicates that blocking the molecular pathway via non-coding RNAs may be beneficial for RA patients.
背景
类风湿关节炎（RA）是一种系统性慢性自身免疫性疾病；已知 RA 成纤维样滑膜细胞（FLS）中的细胞谷氨酰胺代谢对于 RA 的发病机制和进展至关重要。NEAT1 是一种长链非编码 RNA，在多种癌症中作为癌基因发挥作用。NEAT1 在 RA 患者成纤维样滑膜细胞（FLS）中的确切作用和分子机制尚不清楚。
方法
通过 qRT-PCR 测量 NEAT1 和 miR-338-3p 的表达。通过 starBase 预测 lncRNA-miRNA 和 miRNA-mRNA 的相互作用，并通过 RNA 下拉和荧光素酶测定进行验证。通过谷氨酰胺摄取和谷氨酰胺酶活性评估 FLS 中的谷氨酰胺代谢。通过 MTT 和 Annexin V 测定评估 FLS 对 HO 的细胞死亡。
结果
与正常 FLS 相比，RA 患者的 FLS 中 NEAT1 显著上调，miR-338-3p 显著下调。沉默 NEAT1 和过表达 miR-338-3p 抑制 FLS-RA 中的谷氨酰胺代谢并促进 HO 诱导的细胞凋亡。生物信息学分析表明，NEAT1 海绵 miR-338-3p 形成竞争性内源 RNA（ceRNA），这通过 RNA 下拉测定和荧光素酶测定在 FLS-RA 中得到验证。与正常 FLS 相比，FLS-RA 中的谷氨酰胺代谢增加，而 GLN 代谢的关键酶 GLS（谷氨酰胺酶）是 FLS-RA 中 miR-338-3p 的直接靶标。通过挽救实验验证了 miR-338-3p 抑制谷氨酰胺代谢。最后，在表达 NEAT1 的 FLS-RA 中恢复 miR-338-3p 克服了 NEAT1 促进的谷氨酰胺代谢和抗细胞凋亡。
结论
这项研究揭示了 NEAT1 调节的谷氨酰胺代谢和 RA 成纤维样滑膜细胞功能障碍在 RA 中的重要作用和分子靶点，并表明通过非编码 RNA 阻断分子途径可能对 RA 患者有益。
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