Mechanism of Long-non-coding RNA-mediated Exercise Regulation of Bone tissue Autophagy

Abstract

Abstract:

BACKGROUND: LncRNA has been a research hotspot in the field of biomedicine in recent years, and its mechanism of regulating the level of autophagy and the effect of exercise on it has yet to be revealed.
OBJECTIVE: To summarize the research progress of LncRNA in regulating the level of autophagy and the effect of exercise on it.
METHODS: Search for foreign language biomedical, biological, and sports journal systems such as CNKI database and PubMed. Chinese keywords: long-chain non-coding RNA, autophagy, bone metabolism, exercise; English keywords: Long non-coding RNA, autophagy, Bone tissue, Bone metabolism, Exercise.
RESULTS AND CONCLUSION: (1) Autophagy occurs in bone marrow mesenchymal stem cells(BMSCs), osteoblasts(OB), and osteoclasts(OC). (2) Exercise may inhibit the autophagy regulated by mTOR and post-activate the Akt/mTOR signal axis through AMPK, regulate OB or OC differentiation, and ultimately affect bone metabolism. (3) LncRNA can up-regulate or down-regulate under the influence of exercise. As a ceRNA, it negatively regulates downstream related miRNAs, and then inhibits downstream Akt/mTOR signaling pathway through AMPK/ULK1 or AMPK/PPARα signaling axis to initiate autophagy.

Key words: Long-non-coding RNA, Autophagy, Bone metabolism, Exercise

CLC Number:

Figures/Tables 3

2.2 骨自噬 2.2.1 BMSCs自噬 BMSCs具有自我更新和多向分化的特性和修复重建受损骨组织的功能，是治疗骨疾病的首选种子细胞，其自噬水平的变化直接影响成骨分化功能的发挥。有学者报道，骨自噬在提高BMSCs氧化应激存活率的同时，还能保持BMSCs活性的稳定[13]。研究显示，5%的低氧条件可通过抑制丝裂原活化蛋白激酶、激活PI3K/AKT/mTOR等通路，联合成骨分化诱导液促进BMSCs向成骨分化[14]。在缺氧模型中，白细胞介素8(Interleukin-8,IL-8)能通过Akt/STAT3通路提高人BMSCs自噬和增殖能力，降低缺氧缺血对人BMSCs造成的损伤[15]。与之相反，通过Western blot分别检测年轻小鼠和衰老小鼠LC3-Ⅱ、Beclin 1、p62蛋白表达发现，与年轻组小鼠相比，老年组小鼠BMSCs凋亡增加，LC3-Ⅱ、Beclin 1表达下降，p62蛋白表达水平升高，自噬信号通路相关蛋白p-Akt、Akt、p-mTOR、mTOR表达升高，骨量减少[16]。这表明在衰老和细胞器受损条件下，老年小鼠BMSCs自噬活性下降，凋亡增加，抑制BMSC成骨分化。有学者研究发现，氧化应激对BMSCs自噬具有双重作用，即在诱导其凋亡的同时亦诱导保护性自噬作用，抑制BMSCs自噬可加速其凋亡，增强其自噬能提高其在Ⅱ型糖尿病移植治疗中的存活率[17]。此外，mTOR能提高自噬活性较低的BMSCs自噬活性，减弱其向成骨细胞分化的潜力[18]。以上均提示自噬可能在BMSCs向OB分化的过程中扮演重要角色。 2.2.2 OB自噬 OB由BMSCs分化而来，通过分泌胶原、基质和酶类启动并主导骨形成过程。研究证明，自噬能正向调控OB分化和成熟。如糖尿病高糖环境过度产生活性氧，加速成骨MC3T3-E1细胞中的蛋白氧化和自噬，而自噬缺陷会导致线粒体形态学缺陷，抑制成骨细胞在高糖环境中的分化[19]。表明自噬是维持OB在高糖环境下活性和功能的重要机制。在观察颅骨成骨分化时，发现自噬流增加显著，这提示在自噬参与颅骨早期成骨分化。此外，OB中NF-kB信号通路激活需要其与巨噬细胞接触，且此过程受p62抑制，即当OB内p62缺陷将导致骨丢失，并影响OB中CCL4表达及造血祖细胞趋化性，最终引发造血干细胞(Hematopoietic stem cells, HSCs)流失[20]。而骨重建是一个连续的生理过程，需BMSCs持续向OB分化。而此过程需增强线粒体呼吸以保持能量源源不断供应，这必然导致内源性活性氧(ROS)堆积。FOXO3作为BMSCs向OB分化的氧化还原平衡器，一旦下降，将导致ROS升高，进而使BMSCs向OB分化减慢甚至停滞。与之相同的是，敲除ATG7抑制自噬后，BMSCs调控ROS能力下降，OB分化也随之降低。这提示FOXO3能通过诱导自噬抑制ROS，进而加速BMSCs向OB分化。后续研究发现，人BMSCs通过AMPK途径前期抑制mTOR，后期激活Akt/mTOR轴介导自噬促BMSCs向OB[21]。 2.2.3 OC自噬 OC是巨大多核细胞，由RANKL与OB产生的M-CSF激活融合而成，主导骨吸收[22]。研究表明，自噬能影响OC活性及功能的发挥。p62这一自噬特异性调控蛋白，是联结RANKL诱导的自噬和OC生成的桥梁，敲除p62不仅抑制OC中RANKL诱导的自噬活化，还抑制OC生成[23]。p62在佩吉特骨病中突变，引发OC体积增大，细胞活性升高。而p62是自噬调节关键mTOR通路的重要参与因子，这提示OC上的p62突变可影响OC自噬，以调节OC分化[24]。在人类类风湿关节炎中，TNFα可激活自噬使OC生成增加，而以药物抑制自噬可抑制OC分化，敲除TNFα转基因小鼠骨髓细胞中ATG7后发现，OC数目减少[25]。表明TNFα在诱导自噬过程中，与OC呈正相关。肿瘤坏死因子受体相关因子3(TNF receptor-associated receptor3，TRAF3)限制RANKL诱导OC生成，而TRAF3自噬性降解能加速OC生成[26]。由多杀巴氏杆菌毒素(Pasteurellamultocidatoxin，PMT)启动的mTOR信号活化在OC形成过程中起重要作用，可能通过下调程序性细胞死亡蛋白4(programmed cell death protein 4，PDCD4)表达，PDCD4是OC特异性基因转录因子c-Jun的抑制物，从而激活c-Jun，启动OC特异性基因的转录[27]。而缺氧应激通过HIF-1/BNIP3激活自噬，进而促进OC分化[28]。 2.3 LncRNA调控骨自噬的作用机制转录生成非编码RNA(Non-coding RNA，ncRNA)的序列约占人类基因组的绝大部分，其中包括housekeeping非编码RNA、小分子非编码RNA、中等长度非编码RNA和LncRNA。LncRNA在细胞增殖分化、个体发育、信号转导、干细胞维持、代谢等重要生命活动中发挥关键调控作用。其在表观遗传水平、转录水平前后水平等方面具有控制基因表达的作用。此外，肿瘤、糖尿病、精神疾病和神经退行性疾病的发生都与LncRNA的异常表达有关[29]。研究发现，LncRNA在骨细胞的生长发育与分化过程中意义重大，以顺式和反式作用参与并作用于骨细胞分裂、剂量补偿和细胞分化等过程[30]。LncRNA的一级结构——核苷酸排列顺序(碱基互补配对)是LncRNA调节靶基因及其上下游基因转录翻译的基础。其作为ceRNA占据靶基因的结合位点，抑制下游的miRNA或mRNA与靶基因结合(也有学者将此行为称为靶基因模仿)[31]。这种负向调控下游miRNA的方式可能是LncRNA调控细胞自噬的重要途径，如与肿瘤发生相关的LncRNA生长阻滞特异转录物5被证明能结合糖皮质激素的DNA结合域，竞争糖皮质激素反应元件目的基因并调节其表达[32]。这种竞争结合靶基因位点的方式在肌肉分化中也有所体现，Linc-MD1通过碱基互补配对的方式与miR-133和miR-135结合，竞争性抑制二者与靶基因的结合，进而影响肌细胞自噬[33]。通过查阅整理大量文献发现，有关LncRNA调控细胞自噬的研究多集中在肿瘤细胞、心肌细胞、脑细胞和心肌细胞等，且具体LncRNA如何调控上述细胞机制阐述尚不清晰，见表1。"

比较正常组织和膀胱癌组织时发现，后者中LncRNA MEG3表达显著降低、LC3-II显著升高。而其过表达则导致自噬水平降低，这表明LncRNA MEG3与LC3-II呈负相关。LncRNA MEG3可与ATG17相互作用以抑制自噬，而自噬蛋白p53作为调控LncRNA MEG3的靶基因。因此推断，LncRNA MEG3可能通过p53相关通路实现对细胞自噬[34]。在糖尿病性心肌病中，LncRNA H19通过沉默IL-1受体拮抗家族GTP酶3(DIRAS family GTPase 3，DIRAS3)在表观遗传水平上抑制自噬的发生[35]。过表达LncRNA H19发现，自噬小体数目减少，且LC3Ⅱ、BECN1、ATG7等自噬相关蛋白水平降低，这提示LncRNA H19参与抑制心肌细胞自噬。过表达LncRNA MALAT1会抑制PI3K、Akt和p70S6K磷酸化，下调重组人富集于脑Ras同系物(Recombinant Human Ras Homolog Enriched in Brain, RHEB)表达，提高低密度脂蛋白诱导的人脐静脉内皮细胞自噬水平[36]。而沉默LncRNA MALAT1能提高PI3K、Akt和p70S6K磷酸化水平，上调RHEB表达并显着抑制人脐静脉内皮细胞自噬，这表明LncRNA MALAT1能通过PI3K /AKT/p70S6K信号通路促进低密度脂蛋白诱导的人脐静脉内皮细胞自噬。而LncRNA MALAT1可作为ceRNA，通过与miR-216b结合抑制由5-FU处理后的miR-216b表达，从而增强自噬以调控肝癌细胞的耐药性[37]。 LncRNA MEG3、LncRNA H19和LncRNA MALAT1在骨组织中均有表达，且与骨代谢关系密切。据此推测，LncRNA可能通过上述的机制：作为ceRNA抢占miRNA结合位点，实现对骨组织细胞自噬的调控，最终影响骨代谢。目前LncRNA直接调控骨自噬的报道还较为鲜见，且为数不多的研究对其发生机制探讨尚不清晰。例如，过表达小鼠肝原细胞C2C12中LncRNA Risa会降低胰岛素抗体敏感性，Akt和Gsk3β磷酸化水平下降。而敲除LncRNA Risa导致C2C12和OB自噬水平升高[38]。这表明LncRNA Risa与OB呈负相关，却未涉及作用机制方面，LncRNA调控骨组织细胞自噬机制尚待补充。 2.4 LncRNA在运动调控骨自噬中的作用机制骨组织作为力学敏感器官，机械应力刺激可能翻译成结构级联性生化变化，有效刺激骨细胞自噬。运动刺激可提高细胞自噬水平，且不同运动强度对细胞自噬水平影响存在差异，这在心肌细胞、骨骼肌细胞和部分脑细胞中已得到证明，尤以骨骼肌细胞和心肌细胞最为典型。研究发现，老龄大鼠在分别经过8周不同运动强度训练后，其骨骼肌中病理异常均减少，骨骼肌退化情况得以缓解。与此同时，相较于中高耐力训练组大鼠骨骼肌p53基因表达量显著增加而言，低耐力训练组几乎没有增加[39]。此外，离心运动可能通过上调Omi/Beclin-1通路增强骨骼肌自噬[40]。力竭运动可由AMPK/ULK1途径提高小鼠骨骼肌细胞自噬，后续的研究通过Compound C抑制AMPK活性发现，力竭运动运动仍可提高小鼠骨骼肌细胞自噬[41]。提示，除AMPK/ULK1以外，仍存在其它通路影响自噬或ULK1活性。而有氧运动可通过上调心肌细胞AMPK活性，抑制mTOR Ser2448位点磷酸化，进而改善心肌细胞自噬能力，以防治衰老心肌细胞收缩舒张功能失调[42]。而mTOR已被证明可通过mTOR/ULK1通路增强OC自噬以抑制OC分化，降低骨吸收[43]。此外，在成骨细胞系MC3T3-E1中过表达IL-7可激活mTORC1信号以抑制OB成熟，而mTOR可抑制mTORC1 活性，逆转IL-7过表达导致的成骨细胞系MC3T3-E1成熟障碍[44]。因此，这种运动通过AMPK/mTOR轴调控心肌细胞自噬的机制，可能同样存在于骨组织细胞内，即运动可能通过AMPK先抑制mTOR调节的自噬，而后激活Akt/mTOR信号轴，调控OB或OC分化，最终影响骨代谢。Peng等的研究同样证明了这点， AMPK受miRNA调控，通过蛋白质印迹测定AMPK/PPARα和自噬相关基因蛋白质表达发现，miR-19通过靶向调节PPARα，负向调控AMPK /PPARα轴上AMPK以影响细胞自噬[45]。与此类似，通过基因芯片分析发现，miR-291b-3p在2型瘦素受体缺失的糖尿病模型小鼠肝脏中显著上调。荧光素酶报告载体显示，miR-291b-3p直接靶向AMPKα-1[46]。目前，有关运动调控LncRNA的报道还较为鲜见，且为数不多的研究在作用机制方面探讨尚不明晰。An等[47]研究发现，八段锦运动可通过CXCL8，DUSP2，OSM，CXCR4和NR4A2等mRNA作为关键节点调控LncRNA-mRNA网络，进而上调血清中与之关联的LncRNA RP13-516M14.10、LncRNA NEAT1、LncRNA CTD-2530H12.2、LncRNA CTD-3014M21.1和LncRNA AC068580.5表达。而自主轮转运动能使缺血/再灌注模型小鼠LncRNA MALAT1表达上调，保护其海马神经元免受缺血/再灌注损伤并促进认知恢复[48]。此外，中等强度的跑台运动可显著提高骨质松小鼠的骨密度，提高其骨生成速率和骨强度；同时可以对小鼠骨部分LncRNA产生影响，造成其表达的升高或降低，例如：LncRNA Gm31126、LncRNA Loc105243692、LncRNA Gm11651等上调明显，LncRNA 105247265等下调显著[7]。以上提示，运动可能通过影响LncRNA表达以影响骨自噬水平。此外，Peng等[49]研究发现，LncRNA H19可负向调控miR-675，通过Smad通路调控Runx2进而影响骨形成，或负向调控miR-141，通过Wnt/β-catenin促进骨形成。这为运动影响LncRNA，进而通过相关miRNA及其下游信号通路提供了依据。因此推测，LncRNA能在运动影响下表达上调或下调，负向调控miRNA，再通过AMPK/ULK1或AMPK /PPARα信号轴抑制下游Akt/mTOR信号通路，启动mTOR这一自噬关键因子，最终实现对骨自噬的调控。 "

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