【作者】 杨洁；

【导师】 陈宝安；

【作者基本信息】 东南大学 ， 内科学， 2023， 博士

【摘要】 背景:多发性骨髓瘤(multiple myeloma,MM)是一种恶性单克隆浆细胞病,以终末器官损伤——肾损害、高钙血症、溶骨性病变和贫血为特征,在常见血液系统恶性肿瘤中位居第二,具有高发病率和死亡率。近年来MM治疗手段的不断进步,包括免疫调节药物、蛋白酶体抑制剂、组蛋白去乙酰化抑制剂、单克隆抗体以及骨髓移植,MM患者死亡率显著下降,患者生存质量得到明显提高,但是几乎所有患者通常最终仍会出现耐药/复发。缺氧的骨髓微环境在血液肿瘤的发生发展、治疗耐药性以及预后不良中扮演重要作用,因此寻找促进MM缺氧微环境适应相关的基因可以为临床MM治疗提供新的潜在靶点,从而提高MM患者治疗效果和改善疾病预后。糖酵解是人体细胞代谢中必不可少的酶促过程,它参与多种生化途径所需底物的生产。为了获得生长繁殖所需的充足能量和营养物质,肿瘤细胞表现出对微环境的异常可塑性,对其代谢过程进行重新编程,表现为特征性高代谢。有研究表明,糖酵解过程中关键酶的活性和蛋白表达水平在多种肿瘤细胞中均有不同程度的上调。其中,磷酸果糖激酶-1(phosphofructokinase-1,PFK-1)是糖酵解过程中主要的调节点之一,而果糖-2,6-二磷酸(fructose-2,6-bisphosphate,F-2,6-BP)是PFK-1最强的变构激活剂,受到6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶(6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase,PFKFB)家族同工酶的调控,6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶4(6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4,PFKFB4)作为PFKFB的亚型参与调节F-2,6-BP的合成与水解。因此,PFKFB4是控制糖酵解的重要酶,进而参与肿瘤细胞的增殖、侵袭和转移,导致肿瘤的进展与复发。目前为止,PFKFB4已被证明在多种包括实体和非实体肿瘤疾病中呈异常高表达,并且与肿瘤疾病的不良预后密切相关。本课题旨在探索糖酵解相关基因PFKFB4是否参与MM骨髓缺氧微环境的适应及其涉及的具体机制,有助于寻求MM治疗新靶点,同时为临床治疗提供新的理论依据。方法:1.根据关键词“缺氧”和“MM”在国家生物技术信息中心-基因表达综合数据库(Biotechnology Information-Gene Expression Omnibus database,NCBI-GEO)中进行检索,选取符合要求的数据集GSE80140和GSE80545,根据生信分析结果选取糖酵解相关基因PFKFB4作为与MM缺氧适应相关的关键基因,在线生存分析进一步分析PFKFB4与MM预后之间的关系。2.根据生信分析结果,选取两种MM细胞株(RPMI-8226和U266)分别在常氧和缺氧条件下培养不同时间,观察细胞状态,实时荧光定量PCR(quantitative reverse transcription-PCR,qRT-PCR)测定细胞PFKFB4 mRNA转录情况,确定缺氧培养最佳时间,随后进行转录组测序,分析相关结果,并利用qRT-PCR和Western blot技术检测PFKFB4 mRNA转录和蛋白表达水平情况。3.采用Cell counting Kit(CCK8)法、流式细胞术(flow cytometry,FCM)和Western blot检测缺氧条件下抑制PFKFB4后MM细胞增殖、凋亡和周期阻滞的情况,并利用人F-2,6-BP检测试剂盒检测PFKFB4对MM细胞糖酵解水平的影响。4.利用CHIP试剂盒和qRT-PCR检测常氧与缺氧条件下,转录因子低氧诱导因子-1α(hypoxia inducible factor-1 alpha,HIF-1α)与PFKFB4启动子区域结合促进其转录的情况。5.利用PFKFB4抑制剂5MPN、PI3K抑制剂LY294002、HIF-1α抑制剂YC-1分别处理RPMI-8226和U266细胞,Western blot检测PI3K/Akt信号通路分子磷酸化水平以及PFKFB4蛋白表达水平的变化。6.通过在NOD-SCID小鼠皮下注射U266细胞构建皮下移植瘤模型,皮下瘤形成后将小鼠随机分成四组,包括生理盐水对照组、地塞米松(dexamethasone,Dex)组、5MPN组以及Dex+5MPN组,根据实验要求进行给药,共计10天给药时间结束后同时处死小鼠,测量肿瘤体积和重量,切片免疫荧光检测肿瘤组织细胞增殖、周期和凋亡以及信号通路相关蛋白变化情况,从而观察抑制PFKFB4在体内的作用。7.收集临床初发MM病例骨髓标本,根据CD138+原代细胞经qRT-PCR检测得出的PFKFB4的ΔCt值进行分组,分析MM患者基线资料与PFKFB4表达水平的相关性。同时进行生存分析,观察PFKFB4表达水平对MM患者生存期的影响。结果:1.生信分析结果表明,MM细胞株和原代细胞在缺氧条件下PFKFB4表达水平均显著升高;在线生存分析结果显示PFKFB4高表达与MM患者预后不良相关。2.随着缺氧时间的延长,MM细胞表达PFKFB4水平随之升高,48 h组U266细胞PFKFB4表达水平较36 h组稍有下降;所有实验组与常氧对照组相比,差别具有统计学意义(p<0.05)。3.MM细胞转录组测序结果表明,缺氧组细胞中糖酵解相关基因PFKFB4表达水平较常氧组显著升高(p<0.05),qRT-PCR和Western blot结果进一步证明缺氧可以导致PFKFB4 mRNA转录和蛋白表达水平升高。4.CCK8法结果显示抑制PFKFB4可以降低MM细胞活力,增殖速度下降;FCM结果显示与对照组相比,抑制PFKFB4可以诱导MM细胞周期阻滞于G0/G1期,组间差异具有统计学意义(p<0.05);但抑制PFKFB4并不能诱导MM细胞发生明显凋亡(p>0.05);Western blot结果显示促凋亡蛋白Bax、Cleaved Caspase-3和抗凋亡蛋白Bcl-2表达量无明显改变,G1/S特异性周期蛋白Cyclin D1表达量下降,周期阻滞蛋白P21表达量升高;人F-2,6-BP检测实验表明抑制PFKFB4可以显著降低细胞糖酵解水平。5.CHIP-qPCR检测结果表明,常氧与缺氧条件下HIF-1α均可以和PFKFB4启动子区域结合,促进PFKFB4转录;缺氧条件下,HIF-1α与PFKFB4启动子区域的CR3片段结合能力显著提高(p<0.05)。6.缺氧条件下,抑制PFKFB4功能可以降低PI3K/Akt信号通路磷酸化水平,PI3K抑制剂LY294002对PFKFB4表达水平无显著影响,结果表明PFKFB4可以影响PI3K/Akt信号通路的激活程度;利用HIF-1α抑制剂YC-1在缺氧条件下干预MM细胞,HIF-1α和PFKFB4蛋白表达量均下降,差异具有统计学意义(p<0.05),表明MM细胞在缺氧条件下,HIF-1α可以一定程度促进PFKFB4转录表达,但PFKFB4表达不完全依赖HIF-1α转录调控。7.NOD-SCID小鼠皮下瘤模型结果显示,与对照组相比,5MPN组小鼠皮下瘤重量和体积均有一定程度减少,但差异不具有统计学意义(p>0.05),但5MPN联合Dex可以显著提高抗肿瘤效果(p<0.05);肿瘤组织免疫荧光结果显示,5MPN组小鼠瘤组织Ki67、PCNA和Cyclin D1表达水平相较于对照组明显下降,而P21表达水平明显升高;同时PI3K和Akt蛋白磷酸化水平显著下降。8.临床MM患者原始细胞PFKFB4高表达与较差的R-ISS分期、较高的β2微球蛋白(β2-microglobulin,β2-MG)水平和更差的细胞遗传学异常相关(p<0.05);生存分析结果显示:PFKFB4表达水平较高与MM患者较短的总生存期(overall survival,OS)相关。结论:1.MM细胞株在缺氧条件下PFKFB4表达水平升高。2.缺氧条件下,抑制PFKFB4可以导致MM细胞活力下降,细胞周期阻滞在G0/G1期,糖酵解水平降低。3.转录因子HIF-1α在常氧与缺氧条件下均可以与PFKFB4启动子区域结合促进其转录,缺氧环境进一步增强转录水平。4.缺氧条件下,PFKFB4通过激活PI3K/Akt信号通路影响MM细胞的增殖、周期进展等过程。5.MM皮下瘤动物模型结果表明,抑制PFKFB4可以降低小鼠肿瘤组织细胞周期进展,但无法显著降低小鼠肿瘤负荷;5MPN与Dex联用对荷瘤小鼠的抑瘤效果显著提高。6.PFKFB4异常高表达与MM患者不良预后相关。更多还原

【Abstract】 BackgroundMultiple myeloma(MM)is a malignant monoclonal plasma cell disease,characterized by terminal organ damage,including renal damage,hypercalcemia,osteolytic lesions and anemia.It ranks second among common hematological malignancy with high morbidity and mortality.Recent advances have significantly reduced mortality and improved life quality of MM patients but resistance/relapse often still occur after individual treatment with immunomodulatory agents,proteasome inhibitors,histone deacetylation inhibitors,monoclonal antibodies,or bone marrow transplants.Hypoxic bone marrow microenvironment plays a crucial role in the development,treatment resistance and poor prognosis of hematological tumors.Therefore,searching for genes related to the promotion of adaptation of hypoxic microenvironment can provide new potential targets for clinical MM treatment,improving the therapeutic effect of patients and the prognosis of the MM.Glycolysis is a crucial enzymatic process in human cellular metabolism,and it is involved in the production of substrates required for many biochemical pathways.In order to obtain sufficient energy and nutrients for growth and reproduction,tumor cells often show abnormal plasticity to the microenvironment and then reprogram their metabolic process,as characterized by abnormal hypermetabolism.Studies have proved that the activity and protein expression levels of key enzymes involved in glycolysis are upregulated in various cancer cells.Among them,phosphofructokinase-1(PFK-1)is one of the major regulatory sites in glycolysis.Fructose 2,6-bisphosphate(F-2,6-BP)is the strongest allosteric activator of PFK-1,which is regulated by 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase(PFKFB) family isoenzymes.As a subtype of PFKFB protein family,PFKFB4 regulates the synthesis and hydrolysis of F-2,6-BP.Therefore,PFKFB4 is an important enzyme that regulates glycolysis,and further participates in the growth,reproduction,invasion and metastasis of cancer cells,leading to tumor progression and recurrence.So far,PFKFB4 has been proved to be aberrantly highly expressed in a variety of diseases including solid and non-solid tumors,and it is closely associated with poor prognosis of tumor diseases.The purpose of this study is to explore whether PFKFB4,a glycolysis-related gene,is involved in the adaptation of hypoxia microenvironment of MM bone marrow and the underlying mechanisms.We believe that this work is helpful to seek new targets for MM treatment and provide new theoretical basis for clinical treatment.Methods1.Suitable datasets GSE80140 and GSE80545 were selected after searching the Biotechnology Information-Gene Expression Omnibus Database(NCBI-GEO)according to keywords,"hypoxia" and " multiple myeloma ".Glycolysis-related gene PFKFB4 was selected as the potential key gene related to hypoxic adaptation of MM.The relationship between PFKFB4 and MM prognosis was further analyzed by online survival analysis.2.Considering the results of bioinformatics analysis,two MM cell lines(RPMI-8226 and U266)were separately cultured under normoxic and hypoxic conditions for different time,and the state of MM cells was observed.The transcription level of PFKFB4 mRNA was detected by quantitative reverse transcription-PCR(qRT-PCR)to determine the optimal time for hypoxic culture.Then,transcriptome sequencing was performed,and the mRNA transcription and protein expression levels of PFKFB4 were detected by qRT-PCR and Western blot,respectively.3.CCK8,flow cytometry(FCM),and Western blot were used to detect the proliferation,apoptosis,and cycle arrest of MM cells after inhibiting PFKFB4 under hypoxia.In addition,human F-2,6-BP assay kit was used to detect the effects of PFKFB4 on the glycolysis level of MM cells.4.CHIP kit and qRT-PCR were used to detect the ability of the transcription factor hypoxia inducible factor-1 alpha(HIF-1α)to bind to the promoter region of PFKFB4 to promote its transcription under normoxic and hypoxic conditions.5.MM cells were separately treated with specific PFKFB4 inhibitor 5MPN,PI3 K inhibitor LY294002,and HIF-1α inhibitor YC-1.Thereafter,Western blot was used to detect the phosphorylation level of PI3K/Akt signaling pathway molecules and the change of PFKFB4 protein expression level.6.NOD-SCID mice were subcutaneously injected with U266 cells to establish a subcutaneous tumor model.After tumor formation in NOD-SCID mice,they were divided into four groups randomly,including saline control group,dexamethasone(Dex)group,5-(n-(8-methoxy-4-quinolyl)amino)pentyl nitrate(5MPN)group,and Dex + 5MPN group.After administration of experimental Dex or/and 5MPN for 10 days,the mice were sacrificed and the tumor was removed from each mouse to measure the tumor volume and weight.Simultaneously,immunofluorescence was used to detect the changes of tumor cell proliferation,cycle,apoptosis and signaling pathway-related proteins to observe the inhibitory effect of PFKFB4 in vivo.7.Bone marrow specimens of primary MM patients were collected and grouped to analyze the correlation between baseline data and PFKFB4 expression levels in MM patients according to the ΔCt value of PFKFB4 derived from CD138+ primary cells by qRT-PCR.Meanwhile,survival analysis was also performed to observe the effect of PFKFB4 expression levels on the survival of MM patients.Results1.Bioinformatics analysis showed that the PFKFB4 expression level increased significantly under hypoxia in both MM cell lines and primary cells.In addition,online survival analysis suggested that high expression level of PFKFB4 was associated with poor prognosis of MM patients.2.With the prolongation of hypoxia time,the expression level of PFKFB4 increased in MM cells.Of note,the expression level of PFKFB4 in U266 cells was slightly lower in the 48-hour group than that in the 36-hour group.Interestingly,the expression levels of PFKFB4 in all hypoxic groups were significantly higher than those in normoxic control groups(p < 0.05).3.MM cells transcriptome sequencing results indicated that the transcription level of glycolysis-related gene PFKFB4 mRNA in the hypoxic group was remarkably higher than that in the normoxic group.Meanwhile,qRT-PCR and Western blot assay further proved that hypoxia could induce the upregulation of PFKFB4 mRNA transcription and protein expression levels.4.CCK8 assay showed that inhibition of PFKFB4 could suppress the viability of MM cells and reduce the proliferation rate.FCM analysis results indicated that compared with control group,inhibition of PFKFB4 could induce MM cell cycle arrest at G0/G1 phase(p < 0.05),but could not induce significantly apoptosis in MM cells(p > 0.05).Western blot assay confirmed a decreased expression of cell cycle-related protein Cyclin D1 and an increased expression of P21,but there were no significant changes in the expression of Bax,Cleaved Caspase-3 and Bcl-2 proteins(p > 0.05).And the experimental results of human 2,6-diphosphate fructose detection suggested that inhibition of PFKFB4 could significantly reduce the level of cellular glycolysis.5.CHIP-qPCR results showed that HIF-1α could promote the transcription of PFKFB4 mRNA via binding to the promoter region of PFKFB4 under both normoxia and hypoxia.The ability of HIF-1α to bind to the CR3 fragment in the promoter region of PFKFB4 increased significantly under hypoxia(p < 0.05).6.Under hypoxic condition,inhibition of PFKFB4 reduced the phosphorylation level of PI3K/Akt signaling pathway and the PI3 K inhibitor LY294002 had no significant effect on the expression level of PFKFB4.All these results suggested that PFKFB4 affected the activation of PI3K/Akt signaling pathway.After incubation of HIF-1α inhibitor YC-1 with MM cells under hypoxia,the expression levels of HIF-1α and PFKFB4 protein decreased compared with control group(all p < 0.05),indicating that HIF-1α could promote the transcription level of PFKFB4 to a certain extent under hypoxia.But the expression level of PFKFB4 in MM cells did not completely depend on the transcription of HIF-1α under hypoxia.7.The in vivo experimental study showed that subcutaneous tumor weight and volume were reduced to some extent,but the differences were not statistically significant between the 5MPN group and the control group(p > 0.05).Notably,the additional Dex significantly improved the anti-tumor effect of 5MPN(p < 0.05).In addition,the immunofluorescence results indicated that compared with the control group,the expressions of Ki67,PCNA and Cyclin D1 decreased significantly,whereas the expression of P21 increased in the 5MPN group.At the same time,the phosphorylation levels of PI3 K and Akt remarkably decreased.8.A higher expression level of PFKFB4 was associated with higher R-ISS staging,β2-MG levels and cytogenetic abnormalities in primary cells derived from clinical MM patients(p < 0.05).Clinically,survival analysis results showed a higher expression level of PFKFB4 with shorter OS in MM patients.Conclusions1.Expression level of PFKFB4 increases in MM cell lines under hypoxia.2.Under hypoxia,inhibition of PFKFB4 results in a decreased cell viability,cell cycle arrest at G0/G1 phase,and a decreased glycolysis level in MM.3.Under both normoxic and hypoxic conditions,the transcription factor HIF-1α promotes its transcription via binding to the promoter region of PFKFB4 and the transcription level is further enhanced by the hypoxia.4.Under hypoxia,PFKFB4 promotes the cell proliferation and cell cycle arrest by activating the PI3K/Akt signaling pathway in MM cells.5.Inhibition of PFKFB4 reduces tumor tissue cell cycle progression in mice bearing MM,and the combination of 5MPN with Dex exhibits a more inhibitory effect on tumor-bearing mice.6.High expression of PFKFB4 is associated with poor prognosis of MM patients.更多还原