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中国癌症防治杂志

中国癌症防治杂志 ›› 2022, Vol. 14 ›› Issue (4): 370-377.doi: 10.3969/j.issn.1674-5671.2022.04.03

• 基础研究 • 上一篇    下一篇

lncRNA BCYRN1在结外NK/T细胞淋巴瘤糖酵解激活中的作用及其机制研究

  

  1. 首都医科大学附属北京同仁医院血液内科 
  • 出版日期:2022-08-25 发布日期:2022-09-02
  • 通讯作者: 王亮 E-mail：wangliangtrhos@126.com
  • 基金资助:
    国家自然科学基金项目（81873450；82170181）；北京市医院管理局“青苗”计划专项（QML20200201）

The role of lncRNA BCYRN1 in activation of glycolysis in extranodal NK/T⁃cell lymphoma and its mechanism

  • Online:2022-08-25 Published:2022-09-02

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摘要： 目的 探讨长链非编码RNA（long non-coding RNA，lncRNA）BCYRN1在结外NK/T细胞淋巴瘤（extranodal NK/T- cell lymphoma，ENKTCL）糖酵解激活中的作用及其机制。方法 收集2010—2021年于首都医科大学附属北京同仁医院诊治的236例ENKTCL患者信息，分析空腹血糖（fasting blood glucose，FBG）与无疾病进展生存（progression-free survival，PFS）的相关性；采用qRT-PCR检测32例初诊ENKTCL患者组织中的BCYRN1含量并分析其与PFS的相关性。利用质粒转染法构建过表达BCYRN1（OE-BCYRN1组）和干扰BCYRN1（shBCYRN1组）的ENKTCL SNK-6细胞株，采用Screen QuestTM比色法检测葡萄糖摄取能力，用乳酸检测试剂盒检测乳酸的生成能力；采用qRT-PCR和Western blot法检测糖酵解关键分子PKM2、HIF-1α、SLC2A1、LDHA和PDK1的表达水平；分别添加蛋白合成抑制剂、溶酶体抑制剂或激活剂后观察BCYRN1对PKM2稳定性的影响。采用RNA pull-down和RNA结合蛋白免疫沉淀（RIP）实验判断BCYRN1与PKM2相互作用的模式。结果 236例ENKTCL患者中，高血糖组（FBG>5.6 mmol/L）49例，正常血糖组（FBG≤5.6 mmol/L）187例，高血糖组5年PFS率低于低血糖组（32.1% vs 63.7%，P<0.001）；BCYRN1高表达组3年PFS率低于低表达组（26.1% vs 82.5%，P=0.014）。干扰BCYRN1后，ENKTCL SNK-6细胞对葡萄糖的摄取量和乳酸生成水平均显著降低（均P<0.05）；过表达BCYRN1后，糖酵解关键分子PKM2、HIF-1α、SLC2A1、LDHA和PDK1的表达量均显著升高（均P<0.05）。应用蛋白合成抑制剂（放线菌酮）分别处理SNK-6细胞3 h和6 h后，OE-BCYRN1组中PKM2的降解比例均显著低于OE-CTRL组（均P<0.05），而shBCYRN1组中PKM2的降解比例均显著高于shCTRL组（均P<0.05）。经溶酶体抑制剂（Leupeptin）处理后shBCYRN1+Leupeptin组的PKM2降解比例明显下降（P<0.01）；经溶酶体激活剂（6-Aminonicotinamide，6-AN）处理后OE-BCYRN1+6-AN组的PKM2降解比例显著增加（P<0.001）。RNA pull-down和RIP实验显示BCYRN1基因可与PKM2蛋白发生直接相互作用。结论 BCYRN1可能通过溶酶体途径上调PKM2表达而激活ENKTCL糖酵解途径。

关键词: 结外NK/T-细胞淋巴瘤, 糖酵解途径, 长链非编码RNA, BCYRN1, PKM2

Abstract: Objective To investigate the role of long non-coding RNA (lncRNA) BCYRN1 in activation of glycolysis in extranodal NK/T-cell lymphoma (ENKTCL)  and its mechanism. Methods The data of 236 ENKTCL patients who were diagnosed and treated in Beijing Tongren Hospital from 2010 to 2021 were collected to analyze the correlation between fasting blood glucose (FBG) and progression-free survival (PFS). The content of BCYRN1 in the 32 newly diagnosed ENKTCL patients was detected by qRT-PCR, and the correlation between BCYRN1 and PFS was analyzed. The SNK-6 cell lines with overexpression of BCYRN1 (OE-BCYRN1 group) and interference of BCYRN1 (shBCYRN1 group) were constructed by the plasmid transfection method. The Screen Quest- colorimetric method was used to detect glucose uptake, and lactate production was detected by Lactic Acid assay kit. The expression levels of PKM2, HIF-1α, SLC2A1, LDHA and PDK1 were detected by qRT-PCR and Western blot, and the protein synthesis inhibitor, lysosomal inhibitor or activator were added to observe the effect of BCYRN1 on PKM2 stability. The RNA pull-down and RIP experiments were used to determine the interaction mode between BCYRN1 and PKM2. Results Among 236 ENKTCL patients, 49 were in the hyperglycemia group (FBG>5.6 mmol/L) and 187 were in the normal group (FBG≤5.6 mmol/L). The 5-year PFS rate in the hyperglycemia group was lower than that in the hypoglycemia group (32.1% vs 63.7%, P<0.001). The 3-year PFS in the high BCYRN1 expression group was lower than that in the low expression group ( 26.1% vs 82.5%, P=0.014). After interference with BCYRN1, the glucose intake and lactate production level of ENKTCL SNK-6 cells were significantly decreased (all P<0.05). After overexpression of BCYRN1, the expression levels of PKM2, HIF-1α, SLC2A1, LDHA and PDK1 were significantly increased (all P<0.05). After treatment of SNK-6 cells with protein synthesis inhibitor (Cycloheximide) for 3 h or 6 h, the degradation rate of PKM2 in OE-BCYRN1 group was significantly lower than that in OE-CTRL group (all P<0.05), and the degradation ratio of PKM2 in the shBCYRN1 group was significantly higher than that in the shCTRL group. After lysosomal inhibitor (Leupeptin) treatment, PKM2 degradation ratio in shBCYRN1+Leupeptin group decreased significantly (P<0.01); while the degradation ratio of PKM2 in OE-BCYRN1+6-AN group was significantly increased after treatment with lysosome activator (6-Aminonicotinamide)(P<0.01). The RNA pull-down and RIP experiments showed that BCYRN1 gene could interact directly with PKM2 protein. Conclusions BCYRN1 may activate ENKTCL glycolysis pathway by up-regulating PKM2 expression through the lysosomal pathway.

Key words: Extranodal NK/T-cell lymphoma, Glycolysis pathway, Long non-coding RNA, BCYRN1, PKM2

中图分类号: 

  • R733.4