【作者】 李岩；

【导师】 胡继繁；

【作者基本信息】 吉林大学 ， 肿瘤学， 2021， 博士

【摘要】 研究背景:肝细胞癌(hepatocellular carcinoma,HCC)占原发性肝癌的75%-85%,是世界范围内最常见的肿瘤之一,在男性中被列为第二致命的恶性肿瘤[1]。大多数HCC患者确诊时已处于晚期,无法行手术治疗[2]。而化疗、放疗等治疗手段疗效较差。在过去的几十年里,HCC患者的5年生存率没有明显改善[3]。不受控制的细胞增殖和转移是临床治疗的主要障碍,也是高死亡率的原因之一。因此,肿瘤进展的关键分子通路的探索对于肿瘤早期生物标志物的确定和新的治疗药物研发是十分重要的。线粒体作为一种重要的细胞应激传感器,通过氧化磷酸化系统产生三磷酸腺苷(ATP)和活性氧(ROS)等一系列活动[4]。线粒体是哺乳动物细胞中重要的能量产生和细胞稳态的细胞器。线粒体网络的健康对于生命过程中应对各种生理反应及压力刺激至关重要[5][6]。线粒体在肿瘤发生发展中的作用目前引起广泛的关注,但仍有许多未解决的问题。目前越来越多的证据支持线粒体自噬失调与肿瘤的发生密切相关[6]。通过RNA逆转录相关捕获测序(RNA-seq)技术寻找参与HCC异常关键的调节因子,本研究发现细胞核编码的早期生长应答蛋白1(early growth response protein 1,EGR1)在肝癌细胞的线粒体中富集。EGR1是核基因组编码的m RNA,在细胞核内参与许多关键的生物过程,特别是对缺血和缺氧的反应。然而,EGR1 m RNA如何穿梭转运进入线粒体,在线粒体中怎样发挥调控作用,并对HCC结局的影响尚未见报道。研究目的:1.揭示存在于细胞核内的EGR1 m RNA在HCC线粒体内富集的原因及发挥作用形式。2.阐明原先被公认为存在于细胞核内的EGR1 m RNA如何调控HCC线粒体功能及机制。3.明确在线粒体内富集的EGR1与HCC发生发展的相关性,及是否可以作为HCC预后判断,疗效评价的新型分子生物标志物,从而为HCC精准治疗提供崭新的研究思路。研究方法:1.在HCC细胞系Hep G2和正常肝细胞系HL7702中进行线粒体的提取,然后进行RNA测序,以发现在HCC和正常肝细胞中差异表达的线粒体RNA。并应用RNA荧光原位杂交(Fluorescence in situ hybridization,FISH)及RT-q PCR进一步确证EGR1 m RNA在线粒体中的分布及含量。2.利用免疫荧光(Immunofluorescence,IF)及免疫印迹方法明确EGR1蛋白在HCC细胞中的分布情况及线粒体中EGR1 m RNA发挥作用的形式。3.通过sh RNA稳定干涉EGR1 m RNA的表达,检测对线粒体功能(如ATP产生、ROS、线粒体膜电位等)影响。4.通过透射电镜(TEM)、IF等方法检测EGR1 m RNA对缺氧诱导的线粒体自噬的影响。5.通过免疫印迹、CLIP等方法进一步探究EGR1调控缺氧诱导的线粒体自噬机制。6.通过免疫印迹等方法明确EGR1对自噬体的形成及线粒体动力学的调控作用。7.通过sh RNA的方法敲低EGR1后,应用CCK-8实验、Transwell小室实验、划痕实验和平板克隆形成实验,Annexin V/SYTOX染色,评估EGR1对肿瘤细胞增殖、肿瘤克隆形成能力、肿瘤细胞凋亡、肿瘤细胞迁移、侵袭等的影响。8.改造Lwa Cas13a质粒,通过Lwa Cas13a-BN-MLS靶向线粒体RNA敲除技术,进一步明确靶向线粒体内EGR1 m RNA敲除后对线粒体功能及缺氧诱导的线粒体自噬的影响。研究结果:1.通过线粒体RNA-seq与FISH相结合,本研究发现核基因组编码的EGR1m RNA在HCC线粒体中富集。2.首次发现在HCC中,EGR1 m RNA分子被转运到线粒体中,在那里它们不被翻译成EGR1蛋白。相反,它们作为非编码m RNA分子在线粒体中发挥作用。IF同样证实线粒体中EGR1不翻译为蛋白质。3.通过sh RNA的方法沉默EGR1后,线粒体功能受到明显影响,表现为线粒体ATP生成减少,ROS产生增多,线粒体膜电位降低。4.利用TEM发现,与对照组相比,EGR1敲低组缺氧诱导的自噬溶酶体数量较对照组减少。利用IF发现,EGR1敲低组线粒体自噬发生明显减弱。说明没有EGR1,Hep G2细胞无法完全执行其线粒体自噬功能。5.EGR1作为一种非典型的非编码m RNA分子,在缺氧刺激下协调自噬受体BNIP3和NIX向线粒体膜募集。通过HIF-α/BNIP3/BNIP3L通路调控线粒体自噬。6.与对照组相比,EGR1敲低组自噬受体转运蛋白,包括SQSTM1/p62、optinurin、NDP52及LC3B表达明显降低。线粒体分裂蛋白DRP1表达降低,线粒体融合蛋白MFN1/MFN2及OPA1表达升高。7.通过sh RNA稳定干涉EGR1的表达,发现HCC细胞增殖减慢,迁移及侵袭能力减弱,细胞克隆形成能力减弱,凋亡增加。8.利用斯坦福实验室团队构建的Lwa Cas13a-BN-MLS靶向线粒体方法(相关方法学文章正在投稿中),在不影响细胞核内EGR1的情况下,特异性敲除了HCC细胞系线粒体内的EGR1。实验结果与整体细胞水平上敲除EGR1的结果基本一致。EGR1在线粒体内靶向敲除后,线粒体ATP生产下降,膜电位下降,活性氧产量增加。在缺氧诱导的线粒体自噬中,与EGR1靶向敲除组相比,Cas13a-Ctrl组细胞中线粒体蛋白COX4降解的更多,提示EGR1是线粒体自噬所必需的。研究结论:1.本研究通过线粒体转录本高通量测序首次鉴定了核基因组EGR1 m RNA相比正常肝细胞HL7702,高度富集在HCC细胞系线粒体中。EGR1 m RNA可以编码一种重要的蛋白分子,能够对应激产生即刻反应。然而在HCC细胞中,部分EGR1 m RNA分子被转移到HCC细胞的线粒体中。本研究首次证明,核基因组编码的EGR1 m RNA除了具有众所周知的肽编码功能的典型途径外,还可能通过非编码m RNA这一非典型通路发挥作用。2.线粒体中EGR1的敲除,无论是用Lwa Cas13a-BN-MLS靶向线粒体RNA还是sh RNA,都能显著抑制肝癌细胞的线粒体自噬。EGR1作为非编码m RNA分子,其3’UTR段与BNIP3蛋白结合,在缺氧刺激下协调自噬受体BNIP3和NIX向线粒体膜募集,通过HIF-α/BNIP3/BNIP3L通路表观调控线粒体自噬,并对线粒体动力学进行调控,表现为促进线粒体分裂,抑制线粒体融合。EGR1的缺失导致受损线粒体的积累,同时伴有线粒体功能障碍,包括ATP生成减少,ROS生成增加,线粒体膜电位降低。EGR1通过细胞核-核糖体-线粒体之间的遗传信息的紧密协调,在维持一个健康的线粒体网络中发挥重要作用。3.EGR1敲低后,HCC的迁移和侵袭能力明显减弱,细胞集落形成明显减少,细胞增殖减慢,凋亡增加,因此,EGR1可能在HCC中起到促进肿瘤发生发展的作用。更多还原

【Abstract】 Background:Hepatocellular carcinoma(HCC)accounts for 75%-85% of primary liver cancers.It is one of the most common tumors worldwide and is ranked as the second most deadly malignant tumor in men.Unfortunately,most HCC patients are already in the advanced stage when they are diagnosed and thus cannot be treated with surgery.Additionally,the therapeutic effects of chemotherapy and radiotherapy are poor.In the past few decades,the 5-year survival rate of HCC patients has not improved significantly.Uncontrolled cell proliferation and metastasis are the main obstacles to clinical treatment and one of the causes of high mortality.Therefore,the discovery of molecular pathways for tumor progression is important for the identification of biomarkers for early detection and the development of new therapeutic strategies against HCC.As an important cell stress sensor,mitochondria are important performers of a series of activities such as the production of adenosine triphosphate(ATP)and ROS by the oxidative phosphorylation system.Mitochondria are crucial organelles for energy production and cellular homeostasis in mammalian cells.The maintenance of a healthy mitochondrial network is critical in the development as well as in the response to physiological adaptations and stress conditions throughout life.The role of mitochondrial in carcinogenesis and cancer progression is an area of active research,with many unresolved questions.Various aspects of altered mitochondrial function have been implicated in tumorigenesis and tumor progression,including mitochondrial dysfunction,a metabolic switch to aerobic glycolysis,and dysregulation of mitophagy.Mitophagy is a highly specific quality control process which eliminates dysfunctional mitochondria and promotes mitochondrial turnover and is involved in the adaptation to nutrient stress by controlling mitochondrial mass.Increasing evidence from various studies supports the notion that dysregulation of mitophagy is an etiological factor in tumorigenesis.Through mitochondria RNA sequencing(mt RNA-seq)to find molecules involved in the abnormal regulation of HCC,we were surprised to find that the early growth response protein 1(EGR1)encoded by the nucleus is enriched in the mitochondria of HCC cells.EGR1,an m RNA encoded by the nuclear genome,is a tumor suppressor gene that plays a key role in many biological processes in the nucleus,especially in response to ischemia and hypoxia.However,how EGR1 shuttles into mitochondria,how it plays a regulatory role in mitochondria,and whether it ultimately affects the outcome of HCC have not yet been addressed.Objectives:1.To reveal the mechanisms underlying the aberrant shuttering of EGR1 m RNAs from the nucleus into the mitochondria,as well as its enrichment and its function in the mitochondria of HCC cells.2.To clarify how the aberrantly shuttered EGR1 m RNAs regulate mitochondrial functions in HCC.3.To clarify the correlation between the mitochondria-enriched EGR1 m RNA and the occurrence and development of HCC,and whether it can be used as a new molecular biomarker for prognostic judgment and curative effect evaluation of HCC,so as to provide new research strategies for precise treatment of HCC.Methods:1.RNA-seq was performed to find mitochondrial RNA differentially expressed in HCC and normal liver cells.The distribution and content of EGR1 m RNA in mitochondria were further confirmed by Fluorescence in situ hybridization(FISH)and RT-q PCR.2.Immunofluorescence(IF)and Western blotting were used to determine if the mitochondria-enriched EGR1 m RNA can be translated into EGR1 proteins using the mitochondrial translation mechinery in HCC cells..3.The influence on mitochondrial function(such as ATP production,ROS,mitochondrial membrane potential,etc.)was detected between sh EGR1 group and sh Ctrl group.4.The effect of the mitochondria-enriched EGR1 mRNA on hypoxia-induced mitophagy was detected by transmission electron microscopy(TEM)and IF.5.Western blotting was used to examine the mitophagy pathway proteins as regulated by the mitochondria-enriched EGR1 m RNA6.The regulation of the mitochondria-enriched EGR1 m RNA on the formation of autophagosomes and mitochondrial dynamics(mitochondria fission and fusion)was determined by western blot and other methods.7.After EGR1 was knocked down by sh RNA,CCK-8 assay,Transwell chamber assay,scratch assay,plate clone formation assay and Annexinv/Sytox staining were performed to evaluate the effects of EGR1 on tumor cell proliferation,tumor clone formation ability,apoptosis,migration and invasion,etc.8.Through the mitochondria-specific Lwa Cas13 a RNA editing technology,we constructed the mitochondrial-localized Lwa Cas13 a RNA plasmid vector to specifically knockdown EGR1 in the mitochondria of HCC cell lines.Using this system,we further clarify the influence of EGR1 on mitochondrial function and hypoxia-induced mitophagy.Results:1.The nuclear-encoded m RNA EGR1 was aberrantly enriched in mitochondria of hepatoma cells.2.EGR1 m RNA functions in the mitochondria in the form of non-coding RNA.By isolating the mitochondria and performing western blotting,we found that EGR1 m RNA did not translate protein in mitochondria.3.Loss of EGR1 led to the accumulation of damaged mitochondria,which are manifested by decreased ATP production,decreased membrane potential,and increased reactive oxygen production.4.TEM showed that the number of autophagy lysosomes induced by hypoxia was reduced in the EGR1 knockdown group compared to the control group.Mitochondrial autophagy was significantly reduced in the EGR1 knockdown group by IF.These results indicated that the mitochondria-enriched EGR1 m RNA is essential for mitophagy in HCC.5.The mitochondria-enriched EGR1 noncoding mRNA molecules affected the recruitment of mitophagy receptors BNIP3 and NIX to mitochondria and promoted mitophagy through the HIF1α/BNIP3/BNIP3 L pathway.6.Mitochondrial EGR1 m RNA is essential for autophagosome formation during mitophagy and it is required for hypoxia-induced mitochondrial fission and negatively regulates mitochondrial fusion.7.The mitochondria-enriched EGR1 mRNA plays an important role in promoting tumor occurrence and development in hepatocellular carcinoma.8.Through Lwa Cas13 a RNA editing technology,we successfully constructed the mitochondrial-localized Lwa Cas13a-BN-MLS plasmid vector to specifically knockdown EGR1 in the mitochondria of HCC cell lines without affecting EGR1 in the nucleus.The experimental results were basically consistent with the results of EGR1 knockout at the whole cell level using sh RNAs,suggesting that The mitochondria-enriched EGR1 m RNA is necessary for mitophagy.Conclusions:1.In this study,we identified the mitochondrial enrichment of the nuclear genome-encoded EGR1 m RNA in Hep G2 cells compared with normal hepatocytes HL7702.Our study thus suggests a novel non-canonical genetic pathway that the EGR1 m RNA may have a noncoding function in mitochondria,in addition to its coding role through the ribosome.Hence,EGR1 plays an essential role in maintaining a healthy mitochondrial network through the tight coordination of the genetic messages between the nucleus-ribosome-mitochondria.2.Knockdown of EGR1 in mitochondria,whether using the LwaCas13aBN-MLS mitochondria-specific RNA targeting or sh RNA,significantly inhibits the mitophagy in HCC cells.As expected,depletion of EGR1 leads to the accumulation of damaged mitochondria,in parallel with mitochondrial dysfunctions,including decreased ATP production,increased ROS production,and decreased mitochondrial membrane potential.This study provides the first evidence that in addition to its well-known canonical peptide coding function,the nuclear genome-encoded EGR1 m RNA may play a non-canonical role in regulating mitophagy through the noncoding m RNA pathway.3.After EGR1 knockdown,we found that the migration and invasion ability of HCC cells was markedly weakened.At the same time,cell colony formation was significantly reduced,and cell proliferation was slowed down.Thus,it seems that The mitochondria-enriched EGR1 m RNA may play a role in promoting tumor genesis and development in hepatoma cells.更多还原