长链非编码RNA BCYRN1在NK/T细胞淋巴瘤糖酵解激活中的作用及机制研究

Natural killer/T-cell lymphoma (NKTCL) is characterized with high invasion, rapid progression and poor prognosis. It is well known that activation of glycolysis plays an important role in the occurrence and development of NKTCL. However, the molecular mechanism of activation of glycolysis in NKTCL is unclear. Our preliminary results showed that the expression of long non-coding RNA-BCYRN1 was significantly increased in NKTCL. Downregulation of BCYRN1 in NKTCL cells could decrease glucose consumption and lactate production, decrease cell proliferation, but induce cell apoptosis. In addition, depletion of BCYRN1 significantly promoted degradation of PKM2 protein. More importantly, BCYRN1 could interact with PKM2. Further study showed that inhibition of Myc reduced BCYRN1 expression in NKTCL cells. Therefore, we hypothesized that Myc promoted BCYRN1 expression, which mediated the up-regulation of PKM2, thus activating glycolysis in NKTCL. Based on these preliminary data, we’ll further study the following three aspects in the future: 1) the role and mechanism of BCYRN1 on activation of glycolysis in NKTCL; 2) the molecular mechanism of the interaction between BCYRN1 and PKM2, and mechanism of BCYRN1-inhibited PKM2 degradation; 3) the mechanism of Myc on regulation of BCYRN1 expression in NKTCL. The completion of this project will clarify the effects of BCYRN1 on the activation of glycolysis in NKTCL and its underlying mechanisms, and provide a potential target for the prevention and treatment of NKTCL in the future.

NK/T细胞淋巴瘤(NKTCL)侵袭性强、预后差，糖酵解激活在其发生发展中发挥重要作用，但具体分子机制不清楚。预实验显示，长链非编码RNA-BCYRN1在NKTCL中表达显著升高；干扰BCYRN1显著降低NKTCL细胞葡萄糖消耗和乳酸产生、诱导细胞凋亡、促进M2型丙酮酸激酶(PKM2)降解；BCYRN1可与PKM2特异性结合；干扰Myc显著下调BCYRN1的表达。由此推测，NKTCL中Myc促进BCYRN1表达，介导PKM2的上调，进而激活糖酵解。本课题将利用动物和细胞实验，围绕以下层面进行深入研究：1) BCYRN1在NKTCL糖酵解激活中的作用；2) BCYRN1-PKM2直接相互作用以及抑制后者降解的分子机制；3) NKTCL中Myc对BCYRN1表达的调控机制。本项目的完成不仅有助于阐明BCYRN1在NKTCL糖酵解激活中的内在机制，并为NKTCL的防治提供一个直接的作用靶点。

复发NK/T细胞淋巴瘤（NK/T-cell lymphoma，NKTCL）患者预后差，有待探索新治疗靶点。在NKTCL患者中，治疗前空腹血糖水平较高的患者治疗缓解率较低，生存状况差，可能与淋巴瘤的Warburg效应相关。本课题组前期研究发现，lncRNA-BCYRN1过表达与NKTCL预后不良相关，干扰BCYRN1可抑制淋巴瘤细胞增殖和诱导凋亡，但BCYRN1是否通过激活NKTCL糖酵解而促进肿瘤增殖，以及BCYRN1是否介导了NKTCL的化疗耐药，尚未明确。.基于此假设，本研究通过构建过表达及干扰BCYRN1的NKTCL细胞株，检测其葡萄糖的摄取水平、乳酸生成水平以及与糖酵解通路相关分子的表达水平，初步探索BCYRN1促进糖酵解激活的可能机制，同时探索BCYRN1过表达是否影响NKTCL对门冬酰胺酶（ASP）的敏感性，以及介导其耐药的具体机制。以期为靶向代谢重编程或靶向BCYRN1治疗NKTCL提供理论基础。.研究发现：（1）BCYRN1的表达影响了SNK-6细胞对葡萄糖的摄取和乳酸生成的水平；（2）BCYRN1的表达影响了NKTCL糖酵解通路关键分子（PKM2、HIF⁃1α、SLC2A1、LDHA和PDK1）的表达水平；（3）BCYRN1可减少溶酶体对糖酵解限速酶PKM2的降解而增强PKM2蛋白的稳定性；（4）BCYRN1通过与PKM2蛋白的直接相互作用调控其蛋白稳定性；（5）FISH检测提示ASP耐药NKTCL中BCYRN1表达增高；（6）敲减BCYRN1可以促进细胞凋亡，将细胞阻滞在G0/G1期，最终部分抑制SNK-6的增殖；（7）过表达BCYRN1后SNK-6细胞对ASP的IC50值显著增高，提示BCYRN1介导了NKTCL对ASP的耐药；（8）BCYRN1可通过PI3K/AKT/mTOR通路及p53/mTOR通路来促进NKTCL细胞自噬的发生，进而抵抗凋亡，促进细胞增殖，最终致使NKTCL产生对ASP的耐药作用；（9）在裸鼠皮下移植瘤模型中，验证了BCYRN1通过介导肿瘤细胞自噬的增强致使NKTCL对ASP的治疗产生抵抗，干扰BCYRN1可以恢复ASP的敏感性，并增强ASP对NKTCL的杀伤作用。.本课题阐明了BCYRN1增强NKTCL糖酵解、促进自噬、介导耐药的机制，为靶向肿瘤糖酵解代谢、自噬通路、或干扰BCYRN1表达来治疗NKTCL提供了理论基础。