pmTOR和4EBP1蛋白在慢性淋巴细胞白血病患者外周血中的表达及临床意义.pdf

现代肿瘤医学2 0 2 3年11月第31卷第2 2 期血液系统肿瘤【HematologicalMalignancy】pmTOR 和 4EBP1 蛋白在慢性淋巴细胞白血病患者外周血中的表达及临床意义曾雪娇，谢仁古丽阿力木,张曼，曲建华1.21新疆医科大学第一附属医院血液病中心，新疆乌鲁木齐8 30 0 54新疆维吾尔族自治区血液病研究所，新疆乌鲁木齐8 30 0 54【摘要】目的：研究磷酸化mTOR蛋白(pmTOR和下游特异性转录因子(4EBP1)在慢性淋巴细胞白血病(CLL)患者外周血中的表达及临床意义。方法：随机选取我院血液病中心30 例初诊CLL患者,分别留取初次治疗前后的外周血标本,作为初发组和治疗组。另外收集2 0 例同期健康志愿者外周血标本作为对照，qPCR法检测pmTOR和4EBP1mRNA表达，Westernblot法检测pmTOR和4EBP1蛋白表达。结果：初发组pmTOR 和4EBP1mRNA水平均明显高于治疗组和对照组（P0.05）、治疗组高于对照组（P0.05）。初发组中pmTOR和4EBP1mRNA、蛋白表达均呈正相关（P0.05）。高危及极高危组、中危组pmTOR和4EBP1表达均高于低危组（P0.05），高危及极高危组pmTOR和4EBP1表达均高于中危组（P0.05）。结论：CLL中存在pmTOR和4EBP1的表达升高且与CLL危险度相关,pmTOR和4EBP1有望成为CLL临床诊治与判断预后的独特标志物。【关键词】AKT-mTOR;4EBP1;慢性淋巴细胞白血病【中图分类号】R733.32【文章编号】16 7 2-4992-(2 0 2 3)2 2-42 2 5-0 4Expression and clinical significance of pmTOR and 4EBP1 proteins in peripheral blood ofpatients with chronic lymphoblastic leukemiaZENG Xuejao,Xierenguli Alimu,ZHANG Manxu,QU Jianhual.?Hematology Center,the First Affliated Hospital of Xinjiang Medical University,Xinjiang Urumqi 830054,China;Xinjiang Uygur Auton-omous Region Institute of Hematology,Xinjiang Urumqi 830054,China.Abstract Objective:To investigate the expression and clinical significance of phosphorylated mTOR protein(pmTOR)and downstream specific transcription factor(4EBP1)in peripheral blood of patients with chronic lympho-blastic leukemia(CLL).Methods:30 newly diagnosed CLL patients were randomly selected from the hematologycenter of our hospital.Peripheral blood samples were collected before and after first therapy as initial group and treat-ment group,and 20 healthy volunteers were collected as control group.The expression of pmTOR and 4EBP1 mRNAin peripheral blood mononuclear cells was detected by qPCR,and the expression of pmTOR and 4EBP1 protein in pe-ripheral blood mononuclear cells was detected by Western blot.Results:The relative expressions of pmTOR and4EBPI mRNA in the initial group were significantly higher than those in the treatment group and the control group(P0.05),the expressions of treatment group higher than the control group(P 0.05).There was a positive correlationbetween pmTOR and 4EBP1 mRNA expression and protein expression in the initial group(P0.05).The relative ex-pression of pmTOR and 4EBP1 in high and extremely high and medium risk groups were higher than that in low riskgroup(P0.05),and the expression of pmTOR and 4EBP1 in high and extremely high risk group was higher thanthat in medium risk group(P0.05）,具有可比性,外周血白细胞计数等其余指标组间比较差异均有统计学意义（P0.05），具体见表1。实验设计及操作流程获我院医学伦理学委员会批准。表1CLL 组与健康对照组临床资料（xs)Tab.1 Clinical data of the CLL group and healthy control group(xs)ParametersAge(years)Number of lymphocytes(10/L)Lymphocyte percentage(%)Number of white bloodcell(10/L)Hemoglobin(g/L)Platelets(10/L)曾雪娇，等CLLgroupControl group(n=30)(n=20)64 6.8757 9.8654.32 14.313.65 0.4384.25 6.8735.21 9.4565.25 50.436.31 1.45122.22 30.43 140.27 19.22154.49 64.96 258.30 31.45pmTOR和 4EBP1 蛋白在慢性淋巴细胞白血病患者外周血中的表达及临床意义1.3实时荧光定量技术（qPCR）检测pmTOR、4EBP1mRNAFicoll法梯度离心收集外周血单个核细胞（PBMC）,根据Trizol说明书提取总RNA，鉴定RNA的浓度和纯度，逆转录为cDNA。详细制备方法参照文献3-4。PCR引物序列pm-TOR上游引物:5-GAGATACGCTGTCCACTACA3，下游引物:5-CTCTATCCACAGGTGTGATGT-3；PC R引物序列4EBP1上游引物：5-TGACCATACTAGTCATTAC3，下游引物:5-ACTGGTATGATGAGTAATG-3;GAPDH上游引物：5A G A A G G C T G G G G C T C A T T T G 3，下游引物：5-AGGGGCCATCCACAGTCTTC-3。按照对应条件进行扩增，2-A Ci 法计算目标引物mRNA的相对表达量。结果重复3次。1.4蛋白质印迹(Westernblot)检测pmTOR、4EBP1蛋白Ficoll法梯度离心收集PBMC,蛋白裂解液提取细胞总蛋白,定量煮蛋白后电泳,2 0 0 mA转膜150 min，5%脱脂牛奶常温封闭1.5 h。1:1 0 0 0 稀释相应一抗,4孵育过夜。二抗常温孵育2 h，洗涤后显影。1.5统计学方法所有实验数据用均数标准差表示，使用SPSS11.0软件进行统计分析。pmTOR、4EBP1m RNA 及蛋白的表达差异两组数据资料的比较采用独立样本的t检验，皮尔逊（Pear-son)积差法分析 CLL患者pmTOR和4EBP1蛋白及mRNA表达的相关性;P0.05为差异有统计学意义。2结果2.1初发组、治疗组和对照组中pmTOR和4EBP1蛋白与mRNA的表达结果初发组pmTOR和4EBP1蛋白相对表达量均明显高于治疗组和对照组(P0.05）;p m T O R和4EBP1 mRNA相对表达量也均明显高于治疗组和对照组(P0.05)。治疗组 pm-TOR和4EBP1 表达高于对照组,差异均有显著统计学意义(P0.05),见表2,图1。pmTOR4EBP1-Actin图1pmTOR和4EBP1蛋白在各组中的表达情况Fig.1Expression of pmTOR and 4EBP1 protein in each groupP2.2pmTOR和4EBP1mRNA和蛋白表达的相关性分析0.470.994.730.0512.870.057.310.055.450.054.780.05250kD20kD43kD分别将pmTOR和4EBP1mRNA和蛋白进行线性分析，结果见pmTOR和4EBP1mRNA之间存在线性正相关（r=0.661,P0.001);pmTOR和4EBP1蛋白之间也存在线性正相关(r=0.757,P0.01),见图2。2.3不同危险度分组中pmTOR和4EBP1的表达水平按照CLL国际预后指数（CLL-IPI)评分将所有初诊CLL患者进行危险度分层：分为低危组14例；中危组11例，现代肿瘤医学2 0 2 3年11月第31卷第2 2 期高危组3例,极高危组2 例。其中因极高危组病例数少,统计分析时将高危组与极高危组合并为高危及极高危组（5例）。高危及极高危组、中危组pmTOR和4EBP1mRNA和蛋Tab.2Relative expression of pmTOR and 4EBP1 mRNA and protein in each groupParametersnInitial group30Treatment group30Control group20注：与初发组比较，*P0.05；与治疗组比较,#P0.05.Note:*P0.05,compared with Initial group.#P0.05,compared with treatment group.28pmTORmRNA图2 1pmTOR与4EBP1的相关性分析Fig.2The correlation between pmTOR and 4EBP1表3各危险度分组中pmTOR、4EBP1m RNA 和蛋白的相对表达量（xs）Tab.3Relative expression of pmTOR,4EBPI mRNA and protein in different risk groupsParametersHigh and extremely high risk groupMedium risk groupLow risk groupFP注：与高危及极高危组相比，*P0.05;与中危组相比,#P0.05.Note:*P0.05,compared with high and extremely high risk group.#P0.05,compared with Medium-risk group.有着非常重要的价值与临床意义。mTOR是PI3K-AKT信号通路下游蛋白。目前的研究已证实,mTOR信号通路在多种肿瘤中异常高表达并会进一步介导肿瘤细胞的生长、增殖等生物过程3.7 。如乳腺癌、宫颈癌、肺癌等中均已发现存在mTOR信号通路的异常增高，pmTOR250kD4EBP120kD-Actin43kD图3不同危险度分组pmTOR和4EBP1蛋白表达情况Fig.3 Protein expression of pmTOR and 4EBPI in different risk groups3讨论CLL是淋巴细胞血液肿瘤,中老年人高发。常以白血病细胞的浸润和积聚为特征5。近年来随着我国人口老龄化及多种因素的影响,CLL发病率呈上升趋势6 。但 CLL也是异质性较大的一种疾病，寻找如何快速将疾病风险高，进展MODERN ONCOLOGY,Nov.2023,VOL.31,No.22白相对表达量均显著高于低危组,高危及极高危组pmTOR和4EBP1mRNA和蛋白相对表达量均高于中危组,差异均有统计学意义（P0.05）,见图3,表3。表2 各组pmTOR和4EBPlmRNA和蛋白的相对表达量（xs）(xs)pmTOR4EBP1mRNAProtein11.324 0.6750.531 0.1227.397 0.365*0.267 0.591*3.287 0.013*#0.199 0.145*#40r=0.661P0.001302010020:4227:mRNAProtein9.376 0.6740.895 0.0585.374 0.356*0.458 0.078*1.864 0.024*#0.265 0.061*#2.07=0.757P0.011.51.00.50.02224mRNA5.276 0.5673.287 0.025*1.276 0.376*#26.3760.0512630pmTOR1.180 0.8500.647 0.107*0.465 0.119*#15.3870.05快,预后差的CLL患者在初诊时筛查出来,在疾病早期就给予有效的干预的方法，能够最大程度上延缓疾病的进展，提高高危患者的生存治疗与生存期。这对于目前 CLL 的诊疗提示这些癌症的发生可能与mTOR 在癌症组织中的高表达密切相关4.8 。也有学者在关于小细胞肺瘤的研究中发现，在临床分期更高、淋巴结转移更严重、预后更差的患者中，mTOR信号的表达也相应增高9-1。而在血液肿瘤方面,研究12 发现91%以上的急性髓细胞性白血病患者外周血高表达p-AKT,且 4EBP1 的表达也有明显的增高,在使用 mTOR抑制剂雷帕霉素药物干预后,4EBP1的表达出现明显减弱，且抑制了白血病肿瘤细胞的增殖与生长。该试验提示在血液肿瘤中有可能也存在 AKTm T O R 的高表达从而促进了肿瘤细胞的生长增殖,介导肿瘤预后不良。一0.00.5pmTORproteinProteinmRNA7.567 0.1675.387 0.078*2.176 0.154*#30.2870.051.01.5(xs)4EBP12.656 0.0651.675 0.098*1.120 0.610*#32.3870.05Protein:42284EBP1是mTOR信号通路下游的最主要且拥有特异性的信号分子之一。在与肿瘤发生、发展相关蛋白的翻译过程中扮演着重要角色。磷酸化的4EBP1无法与eIF4E结合，进一步导致elF-4E与肿瘤生长、侵袭和转移相关的翻译起始因子组合,例如 cyelinD1,c-mye 和VECF分子等13-15大量的研究已证实,多种肿瘤中存在p-4EBP1的过表达，且与肿瘤不良预后密切相关(L16-18考虑到CLL本身会导致外周淋巴细胞升高,为排除因总细胞数导致的表达差异,本研究同时对同一初诊 CLL患者治疗前后的pmTOR及4EBP1的表达进行了比较。发现初诊CLL患者外周血中pmTOR 和4EBP1 mRNA和蛋白表达较对照组升高的同时,治疗后pmTOR和4EBP1的表达也均有明显下降,但仍高于对照组。同时，本研究也发现初诊高危及极高危组、中危CLL患者pmTOR和4EBP1mRNA表达均高于低危组患者。该结果说明了mTOR通路的激活可能会导致CLL发展，预后欠佳。推测其与4EBP1的过表达，促使eIF-4E翻译产生促肿瘤生长因子,导致肿瘤细胞生存、侵袭和转移的相关作用有关。BROWNJR等人的研究证实，PI3K通路可使B细胞的B细胞抗原受体(B-cell receptor,BCR)进一步激活,促进 B细胞存活增殖,并且PI3K通路特异性抑制剂 CAL-101 可显著抑制 B-CLL 细胞的生长。而我们的研究在此基础上，分析了PI3K通路下游重要靶点mTOR磷酸化与特异性转录因子4EBP1在 CLL中的表达情况,进一步探讨了mTOR通路的表达在 CLL中的临床价值，我们推测,CLL中PI3K通路上的mTOR分子磷酸化增加,可能直接影响了成熟B淋巴细胞的信号转导途径,使得BCR激活，促进肿瘤发生发展。该结论有待进一步完善细胞实验加以验证。此外,国外研究者发现雷帕霉素抑制mTOR信号通路后,磷酸化的4EBP1表达会相应明显下调且导致肿瘤细胞生长周期停滞2 0 。提示mTOR的促肿瘤细胞生长的生物功能可能通过4EBP1来实现的。本研究也分析了pmTOR蛋白和4EBP1 蛋白表达之间的相关性。发现二者存在明显正相关。说明mTOR信号通路的活化可能促进了4EBP1转录的进程，二者在 CLL的进展及患者的预后中起相互协同作用,共同介导了 CLL的预后不良。综上所述，本研究发现mTOR通路在CLL患者过度活化,提示mTOR通路可能参与了CLL的发生发展,以此为靶点的早期干预治疗有待进一步的深入研究。1 ZOU Z,TAO T,LI H,et al.mTOR signaling pathway and mTORinhibitors in cancer:progress and challenges J.Cell Biosci,2020,10:31.2 中华医学会血液学分会白血病淋巴瘤学组，中国抗癌协会血液肿瘤专业委员会,中国慢性淋巴细胞白血病工作组.中国慢性淋巴细胞白血病/小淋巴细胞淋巴瘤的诊断与治疗指南(2 0 18年版)J.中华血液学杂志，2 0 18,39（5）：353-358.Leukemia and Lymphomatology Group of Hematology Branch ofChinese Medical Association,Hematological Oncology ProfessionalCommittee of China Anti-Cancer Association,Chinese workingGroup on chronic Lymphoblastic Leukemia.Guidelines for diagnosisand treatment of chronic lymphoblastic 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