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医药导报
医药导报, 2023, 42(1): 78-85
doi: 10.3870/j.issn.1004-0781.2023.01.013
微小RNA调控胶质瘤化学治疗耐药的研究进展*
Research Progress of MicroRNA for Regulating Chemotherapy Resistance in Glioma
曾昭穆1,2,, 李琳2, 郭岩松2, 祁惠秀2, 刘永2, 闫晴宇1, 郑克彬1,
1.河北大学附属医院神经外科,保定 071000
2.河北大学临床医学院,保定 071000
ZENG Zhaomu1,2,, LI Lin2, GUO Yansong2, QI Huixiu2, LIU Yong2, YAN Qingyu1, ZHENG Kebin1,
1. Department of Neurosurgery, the Affiliated Hospital of Hebei University, Baoding 071000, China
2. School of Clinical Medicine, Hebei University, Baoding 071000, China
通信作者 郑克彬(1982-),男,河北保定人,主任医师,博士,研究方向:中枢神经系统肿瘤基础研究。ORCID:0000-0003-4851-5110,E-mail:zhengkebinzkb@163.com。

作者简介 曾昭穆(1993-),男,江西吉安人,博士,研究方向:胶质瘤基因治疗。ORCID:0000-0001-6900-3857,E-mail:zzmhemisphere@163.com。
基金资助: *2022年河北省研究生创新资助项目(CXZZBS2022015);2019年政府资助临床医学优秀人才培养项目(团队)(361007)
中图分类号: R979.1;R730.264     文献标识码: A     文章编号: 1004-0781(2023)01-0078-08
摘要:

神经胶质瘤是脑部肿瘤相关性死亡的主要原因之一。尽管手术联合放射治疗(放疗)和化学治疗(化疗)方案可以显著提高患者的生活质量,但由于耐药性,胶质瘤的高病死率和复发率仍然导致患者总体生存率低。微小RNA(miRNA)作为肿瘤表观遗传的关键调节因子,与胶质瘤耐药性之间有着彼此交错的互作网络。这些耐药调控网络极为复杂,目前尚未完全阐明。该文系统总结了miRNA在多种抗肿瘤药(替莫唑胺、顺铂、植物源性抗肿瘤药、分子靶向药和免疫治疗药)耐药中的作用和分子机制,并重点讨论miRNA作为胶质瘤耐药治疗靶点的潜在功能,以及使用纳米材料作为miRNA载体治疗胶质瘤的潜在价值,为逆转胶质瘤耐药和纳米医学在临床诊治中的研究提供参考。
关键词: 微小RNA; 胶质瘤; 化学治疗; 耐药; 纳米载体

Abstract:

Neuroglioma is the main relative death reason for brain tumors.Although the scheme of operation with chemoradiotherapy can significantly improve the survival quality of the patients, the high death rate and recurrence rate of glioma lead to the general low survival rate of the patients on account of drug resistance.It is well known that, as the inherited key regulatory factor of tumor surface appearance, microRNA(miRNA) has a network of interlaced interactional influence with the drug resistance of glioma proved by a large amount of research.The pity is that these regulatory networks of drug resistance are extremely complicated, which has not been illustrated completely.Therefore, this paper summarizes the effects and molecular mechanism of miRNA in the drug resistance of various anti-tumor medicines (temozolomide, cisplatinum, antitumor agents of plant origin, molecular targeting drug, and immunotherapy agent), and mainly discusses the potential function of miRNA as the glioma drug therapeutic target, and the potential value of using nanometer materials as the carrier of miRNA to treat glioma. It will provide references for the study of reversal of glioma drug resistance and nano-medicine in clinical diagnosis and treatment.
Key words: MicroRNA; Glioma; Chemoradiotherapy; Drug resistance; Nano-carrier

开放科学(资源服务)标识码(OSID)

神经胶质瘤占原发性脑肿瘤的70%以上,复发率及死亡率极高[1]。多形性胶质母细胞瘤(glioblastoma,GBM)属于4级高度侵袭性肿瘤。尽管手术联合放射治疗(放疗)和化学治疗(化疗)方案已显著改善GBM患者的生活质量,但中位数生存期依然较短,为12~15个月,5年生存率仅4.7%[2,3]。当前,由于肿瘤细胞的内源性和获得性耐药的存在,化疗药物对恶性胶质瘤患者的临床益处依旧有限。随着新一代测序技术和生物信息学分析的发展,非编码RNA的相关研究取得明显进展。越来越多的证据表明,微小RNA(microRNA,miRNA)不仅是胶质瘤增殖、侵袭、凋亡、自噬、免疫应答的生物标志物,同时也是参与胶质瘤耐药的调节因子[4,5]。miRNA已成为胶质瘤耐药过程中至关重要的明星分子,而靶向调控miRNA也可能成为逆转胶质瘤耐药的一种新型治疗策略。因此,笔者系统总结miRNA在多种抗肿瘤药物中的调控功能及耐药机制,同时对基于纳米载体转运的核酸治疗进行了相关展望。旨在进一步分析miRNA作为新的治疗靶点在胶质瘤化疗耐药逆转过程中的潜在意义。

1 miRNA参与多种抗肿瘤药物耐药

化疗耐药一直是胶质瘤临床治疗的主要障碍。恶性胶质瘤患者血脑屏障常常有不同程度的破坏,胶质瘤本身对抗癌药物的耐受是导致化疗失败的最本质原因。更为重要的是,这些原发性或继发性耐药的肿瘤细胞常常表现出多重耐药(multiple drug resistance,MDR)的特征,其表型主要特点是癌细胞对一种药物产生耐药的同时,对其他结构和机制不同的药物也具有交叉耐药性。事实上,胶质瘤的临床MDR由许多因素所引起,这些因素可分为宿主因素、肿瘤因素和肿瘤-宿主相互作用因素,具体包括宿主遗传变异、药物相互作用;药物跨膜转运、DNA修复、上皮间充质转化、胶质瘤干细胞表型和自噬;肿瘤微环境、细胞间转移等[2]。因此,为了减少或消除化疗耐药,揭示其耐药机制就显得至关重要。

1.1 miRNA参与替莫唑胺耐药

替莫唑胺细胞毒性作用的主要机制是破坏胶质瘤细胞中的DNA结构,阻止DNA修复,诱导细胞发生凋亡。在肿瘤耐药研究中,一些致癌性miRNA可以调控凋亡相关信号通路,诱导肿瘤细胞逃避凋亡,从而导致胶质瘤对替莫唑胺产生耐药性。

1.1.1 细胞凋亡 缺氧诱导因子(hypoxia inducible factor,HIF)-1α介导的miR-26a是一种缺氧敏感性miRNA,在缺氧条件下的GBM细胞中表达水平明显上调,并且miR-26a的上调可直接诱导线粒体的保护反应,增强体内外替莫唑胺的耐药性。另外,研究证明miR-26a还可抑制Bax和Bad的表达,以此减弱替莫唑胺诱导的细胞凋亡[6]。同样,在胶质瘤组织和细胞系中显著表达的miR-299-5p可以通过靶向作用GOLPH3调控丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)/细胞外信号调节激酶 (extracellular signal-regulated kinase,ERK)信号轴来抑制细胞凋亡,从而增强胶质瘤对替莫唑胺的化疗耐药[7]。此外,miR-497可以通过IGF1R /IRS1途径上调mTOR和Bcl-2蛋白的表达来诱导胶质瘤细胞的替莫唑胺凋亡抵抗[8]。

1.1.2 胶质瘤干细胞(glioma stem cells,GSCs) GSCs通过多种复杂交错的信号网络在胶质瘤化疗过程中发挥重要作用。越来越多的证据表明,miRNA与GSCs驱动的胶质瘤耐药性之间存在相关性。研究发现,miR-30b-3p在缺氧GSCs衍生外泌体中的表达显著提高,并可伴随外泌体跨膜转运至受体GBM细胞内,直接靶向结合BRHOB,以促进替莫唑胺化疗抵抗[9]。此外,外源性miR-26a和miR-223的高表达都可通过促进GSCs的增殖能力和球形形成,进而诱导胶质瘤细胞化疗耐药。研究表明,miR-26a可以通过与AP-2α的3'-UTR靶向结合来诱导GSCs增殖,从而促进胶质瘤对替莫唑胺的耐药性[10]。miR-223在GBM细胞中的表达显著增加,可以直接结合PAX6发生负向调控,并通过调节磷酯酰肌醇3 激酶( phosphoinositide-3-kinase,PI3K)/丝氨酸苏氨酸激酶 (serine-threonine kinase,AKT)信号通路来促进胶GSCs增殖分化,从而降低替莫唑胺的敏感性及生长抑制[11]。

1.1.3 DNA损伤修复 相比之下,miRNA还可以作为肿瘤抑制因子,逆转胶质瘤的替莫唑胺耐药。例如,O-6-甲基鸟嘌呤-DNA甲基转移酶(O-6-methylguanine-DNA methyltransferase,MGMT)是早期发现可造成肿瘤化疗耐药的一种DNA修复酶,它的基本功能是通过将鸟嘌呤O-6位点的甲基转移至半胱氨酸残基来修复受损的鸟嘌呤核苷酸,从而避免烷化剂药物引起的基因突变和细胞死亡。研究发现,MGMT表达量下调与miR-648和miR-125b的表达水平呈负相关[12]。另一研究报道,将miRNA模拟物递送至胶质瘤细胞中,高表达的miRNA-181d可以通过靶向结合MGMT来逆转胶质瘤细胞对替莫唑胺的耐药性。并且一种基于全基因组微阵列分析方法指出,miR-181d-5p是一个始终只与MGMT发生靶向结合的miRNA,这种特异性对于替莫唑胺耐药性预测具有重要价值[13]。此外,miR-198也可降低胶质瘤细胞中MGMT的表达,从而增强替莫唑胺的细胞毒性。然而,生长转化因子β1(transforming growth factor β1,TGF-β1)的过表达可通过抑制人表皮角质形成细胞中的KH-type剪切调控蛋白来阻碍miR-198剪切成熟,从而促进MGMT去甲基化和胶质瘤耐药[14]。

1.1.4 上皮间充质转化(epithelial- mesenchymal transition,EMT) EMT是恶性肿瘤的标志,其生物学进程在脑胶质瘤恶性表型及临床治疗评估中发挥着至关重要的作用。研究表明,miRNA同样可以调节EMT过程,从而逆转胶质瘤对替莫唑胺的耐药性。例如,miR-26b过表达通过靶向结合Wee1逆转耐药胶质瘤细胞的EMT,从而增加耐药细胞的替莫唑胺敏感性[15]。组织蛋白酶B被证实是miR-140的直接标靶,miR-140可通过降低组织蛋白酶B的表达来抑制肿瘤细胞EMT并增强替莫唑胺的细胞毒性[16]。另一研究中证实,miR-128-3p在胶质瘤组织和细胞系中表达下调,而过表达miR-128-3p可下调EMT转化蛋白C-Met、PDGFRα、Notch1和Slug的表达水平,以此增强胶质瘤细胞对替莫唑胺的敏感性[17]。

1.1.5 自噬 自噬是一种基于溶酶体降解细胞内物质进行周转的过程,在胶质瘤的多个方面扮演着重要角色,尤其是在药物应激上,miRNA介导的自噬对胶质瘤耐药性起着关键调节作用。在缺氧条件下,HIF-1α可以通过负向调节miR-224-3p的表达来影响肿瘤细胞的化疗耐药。ATG5是miR- 224-3p的直接作用标靶,高表达miR-224-3p可以通过抑制ATG5介导的缺氧自噬来逆转LN229细胞和U251细胞的化学耐药性[18]。此外,高表达miR-519a可提高胶质瘤细胞对替莫唑胺化疗的敏感性,而这种化疗增敏正是通过miR-519a抑制STAT3/Bcl-2/ Beclin-1途径调控自噬和凋亡来实现[19]。

1.1.6 跨膜转运蛋白 药物转运与替莫唑胺耐药之间有着一张庞大复杂的互作网络,通过miRNA介导的相关转运蛋白来逆转胶质瘤对替莫唑胺的耐药已被广泛研究。例如,miR-302c上调后通过靶向抑制胶质瘤耐药细胞中转运蛋白P-糖蛋白(P-gp)来增强替莫唑胺的细胞毒性[20]。另一类似的研究报道,ABCC1在耐药肿瘤细胞中表达发生上调,而miR-1268a的过表达可以逆转这种上调,抑制ABCC1翻译,从而增强耐药细胞对替莫唑胺的化疗敏感[21]。Wnt5a是miR-129-5p的关键靶点,过表达miR-129-5p可以通过抑制Wnt5a来阻断PKC/ERK/ NF-κB和JNK信号通路的激活,从而逆转胶质瘤细胞对替莫唑胺耐药[22](表1)。

表1 MiRNAs参与替莫唑胺耐药
Tab.1 MiRNAs are involved in temozolomide resistance
MiRNA 表达 信号通路和标靶 耐药机制 参考文献
miR-26a 上调 Bad、 Bax、 AP-2α 细胞凋亡、胶质瘤 [6,10]
干细胞
miR-299-5p 上调 GOLPH3、 MAPK/ 细胞凋亡 [7]
ERK
miR-497 上调 IGFIR/IRS1、 细胞凋亡 [8]
mTOR/Bcl-2
miR-30b-3p 上调 BRHOB 胶质瘤干细胞 [9]
miR-223 上调 PAX6/PI3K/AKT 胶质瘤干细胞 [11]
miR-648,miRNA- 下调 MGMT DNA 损伤修复 [12]
125b
miR-181d-5p 下调 MGMT DNA 损伤修复 [13]
miR-198 下调 MGMT DNA 损伤修复 [14]
miR-26b 下调 Wee1 上皮间充质转化 [15]
miR-140 下调 CTSB 上皮间充质转化 [16]
miR-128-3p 下调 C-met、 PDGFRα、 上皮间充质转化 [17]
Notch1、Slug
miR-224-3p 下调 ATG5 自噬 [18]
miR-519a 下调 STAT3/Bcl-2/ 自噬 [19]
Beclin-1
miR-302c 下调 P-gp 药物转运与代谢 [20]
miR-1268a 下调 ABCC1 药物转运与代谢 [21]
miR-129-5p 下调 Wnt5a 药物转运与代谢 [22]

表1 MiRNAs参与替莫唑胺耐药

Tab.1 MiRNAs are involved in temozolomide resistance

1.2 miRNA参与顺铂耐药

顺铂作为复发性胶质瘤挽救治疗中最常用的化疗药物。其主要作用靶点是DNA,可以共价结合鸟嘌呤和腺嘌呤的嘌呤碱基,形成链内加合物以抑制DNA的复制和转录,造成DNA损伤[23]。

1.2.1 胶质瘤干细胞 GSCs是一群具有自我更新能力的细胞,GSCs相关的miRNA是胶质瘤化疗耐药的关键介质。miR-186被证实在胶质瘤组织中表达显著降低。YY1作为GSCs的分子标志物,过表达miR-186可以通过靶向结合YY1来抑制GSCs表型的形成,进而逆转胶质瘤的顺铂耐药[24]。另外,研究发现,CD133阳性的GSCs对顺铂治疗表现出更强的抵抗力,而过表达的miR-29a可以改善CD133介导的化学抗性,提高胶质瘤对顺铂的敏感性[25]。

1.2.2 细胞凋亡 一些抑癌基因能够逆转胶质瘤的顺铂耐药,主要是通过调控细胞凋亡来实现。研究发现,外源性miR-22模拟物可以通过与SNAIL1发生靶向结合来诱导肿瘤细胞发生细胞周期停滞,从而增强U87MG细胞对顺铂的敏感性[26]。另一研究表明,miR-107在胶质瘤组织中表达水平降低,而mTOR表达水平则显著增高。与U251亲本细胞比较,U251耐药株中miR-107和mTOR的表达水平也有着类似趋势。体外研究证明,过表达miR-107可以通过抑制mTOR和Survivin的表达来促进耐药细胞发生凋亡,进而显著增强顺铂介导的细胞毒性[27]。同样,miR-501-3p可以靶向结合MYCN的3'-UTR来促进顺铂诱导的胶质瘤细胞调亡和增殖阻滞。需要注意的是,MYCN表达的恢复可逆转miR-501-3p这种促进效应[28]。另外,在胶质瘤组织和细胞系中低表达的miR-128也可以逆转顺铂耐药。其潜在机制是,恢复miR-128的表达可以靶向结合JAG1分子位点使Bax表达增高及Bcl-2表达降低,从而通过促进肿瘤细胞凋亡和S期阻滞来增强顺铂介导的细胞毒性[29](表2)。

表2 MiRNAs参与顺铂耐药
Tab.2 MiRNAs are involved in cisplatin resistance
MiRNA 表达 信号通路和标靶 耐药机制 参考文献
miR-186 下调 YY1 胶质瘤干细胞 [24]
miR-29a 下调 CD133 胶质瘤干细胞 [25]
miR-22 下调 SNAIL1 细胞凋亡 [26]
miR-107 下调 mTOR 细胞凋亡 [27]
miR-501-3p 下调 MYCN 细胞凋亡 [28]
miR-128 下调 JAG1/Bcl-2 细胞凋亡 [29]

表2 MiRNAs参与顺铂耐药

Tab.2 MiRNAs are involved in cisplatin resistance

1.3 miRNA参与植物源性抗肿瘤药耐药

近年来,植物提取物对抗脑胶质瘤倍受医学研究人员的关注[30]。临床研究表明,很多天然提取物不仅具有抗瘤活性,还可以缓解放疗、化疗不良反应,提高患者生活质量,降低肿瘤复发率[31]。此外,一些致癌性miRNA的表达可以直接影响天然抗癌药物的化疗功效。例如,miR-374a在恶性胶质瘤中表达显著增高,当敲低miR-374a时可以直接增加胶质瘤细胞中FOXO1的表达,进而增强依托泊苷对肿瘤细胞诱导的细胞毒性[32]。miR-218为一种抑癌基因,而miR-218-2在胶质瘤组织和胶质瘤细胞中却高表达,并与胶质瘤细胞的生长、侵袭、迁移和β-拉帕醌化疗耐药呈正相关。从机制上讲,miR-218-2可以通过降低CDC27基因的表达来促进U87MG和U251细胞对β-拉帕醌的化疗耐药[33]。YIN等[34]首次证明miR-326的过表达可以提高恶性胶质瘤细胞对抗癌药物姜黄素的化学敏感性。体外实验结果证明高表达miR-326可以通过抑制SHH/GLI1信号途径来降低肿瘤细胞的活力,进而增强姜黄素对U87MG和U251的细胞毒性。程序性死亡因子配体1(programmed death ligand 1,PD-1)在紫杉醇耐药U87MG中的表达显著增加,通过直接作用PD-L1的3'-UTR,miR-34a可以抑制肿瘤细胞的进展和化疗耐药[35]。此外,雷帕霉素的应用可以给胶质瘤细胞创造一种饥饿状态,诱导肿瘤细胞自噬发生[36]。研究报道,miR-7-1-3p的高表达可以通过分别抑制GBM细胞中PKCa和iNOS的表达来增强木犀草素和水飞蓟宾协同用药的抗肿瘤活性,更重要的是,还可以通过抑制雷帕霉素诱导的自噬来促进细胞凋亡[37](表3)。

表3 MiRNAs参与植物源性抗癌药耐药
Tab.3 MiRNAs are involved in resistance to plant-derived anticancer drugs
MiRNA 表达 信号通路和标靶 药物 参考文献
miR-374a 上调 FOXO1 依托泊苷 [32]
miR-218-2 上调 CDC27 β-拉帕醌 [33]
miR-326 下调 SHH/GLI1 姜黄素 [34]
miR-34a 下调 PD-L1 紫杉醇 [35]
miR-7-1-3p 下调 PKCa,iNOS 木犀草素、水飞蓟宾 [37]

表3 MiRNAs参与植物源性抗癌药耐药

Tab.3 MiRNAs are involved in resistance to plant-derived anticancer drugs

1.4 miRNA参与分子靶向药耐药

分子靶向治疗与传统药物比较,其毒性小,只针对肿瘤细胞起抑制作用;作用机制上,分子靶向药物可以精准调控肿瘤细胞上的特定受体、关键基因和控制分子。然而,关于胶质瘤对分子靶向药物的抵抗机制目前尚不清楚,但已发现一些miRNA可以直接参与调节分子靶向药物的细胞毒作用[38]。舒尼替尼作为一种抗血管生成的络氨酸酶抑制药,通过直接作用P-gp和Bcrp的3'-UTR,miR-145模拟物转染可以促进舒尼替尼在GBM中的细胞毒性效应[39]。同样,miR-302a和miR-520b模拟物也可以直接靶向结合AKT1、PIK3CA 和SOS1的3'-UTR,通过抑制U87MG细胞中受体酪氨酸激酶介质AKT1、PIK3CA 和SOS1的表达,提高肿瘤细胞对舒尼替尼的敏感性和细胞凋亡。遗憾的是,miR-302a和miR-520b的这种调节效应在替莫唑胺中却未能显现[40]。

生长因子受体是恶性胶质瘤信号网络中的重要调节蛋白。表皮生长因子受体(epidermal growth factor receptor,EGFR)和血小板源性生长因子受体新型药物耐药性一直是实验研究的热点。miR-106a是参与耐药的重要分子,除了可以促进MDR1和MRP1的表达来增强U87MG耐药细胞的药物外排能力和抗凋亡能力外,还可以正向调节GST-π的表达来增强U87MG耐药细胞的药物解毒能力,协同通过上述多种调控机制来增加胶质瘤细胞对吉非替尼的耐药抗性[41]。与正常胶质细胞比较,GBM中miR-450a表达显著降低,miR-450a高表达可以通过抑制EGFR的翻译来调控PI3K/AKT/mTOR信号通路,进而调节细胞凋亡和自噬以增强吉非替尼在胶质瘤细胞中的敏感性。另外,miR-450a诱导的上述反应可以被WIPI1敲低所逆转[42]。在尼妥珠单抗的研究中证实,通过抑制胶质瘤细胞细胞系中高表达的miR-566可以负向调节VHL,进而提高肿瘤细胞对尼妥珠单抗的敏感性[43]。此外,在伊马替尼耐药GBM细胞中可以发现SNAI2表达增高,而沉默SNAI2可直接抑制肿瘤细胞EMT进程和耐药性。研究还发现,miR-203模拟物可以通过与耐药细胞中的SNAI2发生靶向结合来提高肿瘤细胞对伊马替尼的敏感性[44](表4)。

表4 MiRNAs参与分子靶向药耐药
Tab.4 MiRNAs are involved in resistance to molecular targeted drugs
MiRNA 表达 信号通路和标靶 药物 参考文献
miR-145 下调 P-gp,Bcrp 舒尼替尼 [39]
miR-302a、 miR-520b 下调 AKT1、PIK3CA、 SOS1 舒尼替尼 [40]
miR-106a 上调 MDR1、 MRP1、 GST-π 吉非替尼 [41]
miR-450a 下调 EGFR 吉非替尼 [42]
miR-566 上调 VHL 尼妥珠单抗 [43]
miR-203 下调 SNAI2 伊马替尼 [44]

表4 MiRNAs参与分子靶向药耐药

Tab.4 MiRNAs are involved in resistance to molecular targeted drugs

1.5 miRNA参与免疫治疗药耐药

免疫检查点抑制药是专门针对免疫检查点而研发的单抗类药物,通过阻断肿瘤细胞对免疫细胞的抑制作用,进而诱导持续性抗肿瘤免疫应答。目前美国食品药品管理局(FDA)批准的免疫检查点抑制药包括Ipilimumab[细胞毒T淋巴细胞相关抗原4(cytotoxic T lymphocyte-associated antigen-4,CTLA-4)抑制药]、Nivolumab(PD-1抑制药)和Avelumab (PD-L1抑制药)等[45],这些药物在临床上的成功应用使得恶性胶质瘤的免疫治疗焕发了新生命。根据研究报道,一些miRNAs可以对胶质瘤细胞中的免疫检查点起调控作用。例如,与野生型裸鼠移植瘤模型比较,miR-15a/16缺失组的胶质瘤生长受到显著抑制,且裸鼠存活期也明显延长。更重要的是,在miR-15a/16缺失裸鼠的肿瘤中聚集了大量高活性和增殖性的CD8+ T细胞,这一差异表型正是miR-15a/16缺失介导的PD-1、Tim-3 和LAG-3低表达所诱导[46]。此外,miR-138可以靶向结合CD4+ T和C D8+ T细胞中的CTLA-4、PD-1和FoxP3,使得免疫活性裸鼠体内的胶质瘤组织显示出肿瘤消退[47]。肿瘤疫苗也是近年来肿瘤治疗研究的热点之一,其作用机制是将肿瘤相关抗原导入患者体内,激活自身细胞免疫和体液免疫来诱导炎症反应,进而增强机体抗肿瘤能力。LI等[48]研究报道,miR-326可以通过SMO/Gli2途径来减少胶质瘤细胞中TGF-β1的外分泌,从而提高T细胞活性和杀伤能力,增强表皮生长因子受体变体III-树突状细胞(GFRvIII-DC)疫苗的细胞毒性。更重要的是,与单独使用EGFRvIII-DC疫苗治疗比较,EGFRvIII-DC疫苗与miR-326联合应用疗效更佳,对于U87MG细胞具有更强的杀伤力。

随着生物工程技术的不断发展,已有科研工作者利用基因编辑技术将复制能力强的病毒改造成溶瘤病毒,各类溶瘤腺病毒可以促使胶质瘤细胞发生凋亡而对正常细胞毒性较低,具备很高特异性和选择性。YAO等[49]为了提高溶瘤腺病毒的细胞毒性和特异性,将4种胶质瘤抑制性miRNAs(miR-124、miR-128、miR-146b和miR-218)反应元件(MRE)与溶瘤腺病毒进行组合构建了重组溶瘤腺病毒 (OA-4MREs)。体内外实验证明,MRE可以通过靶向结合E1A来调控病毒的复制能力,并且与增殖性腺病毒比较,OA-4MREs对胶质瘤细胞具有更强的细胞毒作用。令人振奋的是,研究人员还发现OA-4MREs对正常组织和细胞均没有明显的细胞毒性,仅表现有限数量的病毒后代。因此,OA-4MREs具有很高的安全性,为进一步测试临床应用提供了可能(表5)。

表5 MiRNAs参与免疫治疗药耐药
Tab.5 MiRNAs are involved in resistance to immunotherapeutic drugs
MiRNA 表达 信号通路和标靶 药物 参考文献
miR-15a/16 上调 PD-1、 Tim-3、 PD-L1抑制药 [46]
LAG-3
miR-138 下调 CTLA-4、PD-1、 CTLA-4抑制药、PD-1抑 [47]
FoxP3 制药
miR-326 下调 SMO/Gli2 表皮生长因子受体变体 [48]
Ⅲ-树突状细胞疫苗
miR-124、 miR- 下调 E1A 重组溶瘤腺病毒 [49]
128、miR-146b、
miR-218

表5 MiRNAs参与免疫治疗药耐药

Tab.5 MiRNAs are involved in resistance to immunotherapeutic drugs

2 展望

近年来,RNA-Seq技术迅速发展,由于其具有高通量、高准确性和高灵敏度等特点,进一步揭开了非编码RNA与多种癌症表观遗传之间的“神秘面纱”,为非编码RNA研究走向临床打开了大门。相关研究显示,许多蛋白质标靶因为缺乏合适的蛋白结构或转录因子,无法与药物分子相互作用,所以不可成药[50]。令人振奋的是,与广泛应用于胶质瘤治疗靶点的蛋白质编码基因比较,非编码RNA赋有与生俱来的应用潜质,其分子片段占整个人类基因组约98%,可以为胶质瘤化疗提供充足的靶点选择[4]。另外,几乎所有用于胶质瘤化疗的传统药物都面临耐药性的挑战,但是目前还未出现关于非编码RNA药物耐药的报道。并且非编码RNA药物还可以添加一系列的化学修饰,使得其在体内循环的半衰期比小分子或抗体药物的半衰期更长。

综上所述,miRNA在胶质瘤化学抗性中起关键调控作用,通过使用miRNA拮抗剂或模拟物靶向纠正耐药形成过程中内源性miRNA的失调表达,可能是逆转胶质瘤化学耐药一种有效的治疗策略。作为最早开始研究的非编码RNA,研究者已开发出多种基于miRNA的核酸工具,如anti-miR、antagomiR、sponge inhibitor、mimics、agomir和Pre-miR等,这些具有独特序列的核酸工具,可以直接抑制或增强内源性miRNA在肿瘤细胞中的作用,未来或许可以在此基础进一步发展精深疾病早期诊断工具、早期诊断策略及更有效的药物治疗方法。与此同时,针对miRNA各类核酸药物的研究开发也从未停止过。MRX34是一种由脂质体纳米颗粒包裹的miR-34a mimic,其作为第一个进入临床试验的核酸单药,在肾细胞癌、肢端黑色素瘤和肝细胞癌的治疗过程中表现出令人满意的临床结果。但该研究因为患者出现严重不良反应而停止[51]。Cobomarsen是一种基于锁核酸修饰的anti-miR-155,以患者体内的致癌性miR-155为靶点来调节细胞的增殖和分化。在一项针对淋巴瘤和白血病II期临床试验中,患者对全身和瘤内给药的Cobomarsen均有着良好的耐受性。关于GBM治疗,Regulus Therapeutics Inc公司研发了一种anti-miR-10b的新候选药物RGLS5579,但是目前还处于临床前阶段[52]。

尽管miRNA治疗前景广阔,但要从众多候选者中挑选关键目标miRNA以及选择适宜的递送系统仍然是一个巨大的挑战。一方面,由于miRNA可以同时靶向调控网络中多个功能基因,使得目的miRNA可对靶基因以外的其他基因发挥作用,最终导致脱靶效应,产生非特异性调控。因此,需要综合考虑miRNA药物在人体内的整体反应,按照严格的标准来挑选目的miRNA。另一方面,在维持治疗性miRNA高效递送效率的同时,还需要考虑载体本身对人体器官所带来的伤害。病毒载体和阳离子纳米颗粒是当前miRNA治疗中最常用的载体系统,它们不仅存在一定的毒性,还可以诱导免疫原性反应。并且过量的阳离子成分还会使载体颗粒在肾小球基底膜处易于分解,从而导致miRNA的肾脏清除[53]。因此,开发出高效无毒的载体系统是胶质瘤miRNA治疗成功的关键。此外,随着近年来液体活检、分泌物检查等临床技术的发展,miRNA作为药物敏感性早期筛选指标是另一个具有潜力的研究方向。miRNA和化疗药物联合用于胶质瘤增敏治疗似乎也很有前景,值得进一步转化研究和临床试验。

本综述重点阐述了miRNA在多种抗肿瘤药耐药中的功能和机制,并阐明miRNA介导的精确治疗将是克服胶质瘤化疗耐药一种理想的治疗策略。总之,miRNA疗法在临床实践中的全面应用还有很长的路要走,亟需进一步探索。同时,为了开发基于miRNA的药物来改善胶质瘤患者的预后,也迫切需要进行更多的科学研究和临床试验。因此,miRNA疗法将会是传统疗法的有效补充,有望在未来克服肿瘤细胞化疗耐药。

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Glioma stem-like cells (GSCs) contribute to temozolomide (TMZ) resistance in gliomas, although the mechanisms have not been delineated. functional experiments (colony formation assay, flow cytometric analysis, TUNEL assay) were used to assess the ability of extracellular vesicles (EVs) from hypoxic GSCs to promote TMZ resistance in glioblastoma (GBM) cells. RNA sequencing and quantitative Reverse Transcription-PCR were employed to identify the functional miRNA in hypoxic EVs. Chromatin immunoprecipitation assays were performed to analyze the transcriptional regulation of miRNAs by HIF1α and STAT3. RIP and RNA pull-down assays were used to validate the hnRNPA2B1-mediated packaging of miRNA into EVs. The function of EV miR-30b-3p from hypoxic GSCs was verified by experiments and analysis of clinical samples. Hypoxic GSC-derived EVs exerted a greater effect on GBM chemoresistance than those from normoxic GSCs. The miRNA profiling revealed that miR-30b-3p was significantly upregulated in the EVs from hypoxic GSCs. Further, HIF1α and STAT3 transcriptionally induced miR-30b-3p expression. RNA immunoprecipitation and RNA-pull down assays revealed that binding of miR-30b-3p with hnRNPA2B1 facilitated its transfer into EVs. EV-packaged miR-30b-3p (EV-miR-30b-3p) directly targeted RHOB, resulting in decreased apoptosis and increased proliferation and. Our results provided evidence that miR-30b-3p in CSF could be a potential biomarker predicting resistance to TMZ. Our findings indicated that targeting EV-miR-30b-3p could provide a potential treatment strategy for GBM. © The author(s).
DOI:10.7150/thno.47057      PMID:33408780     
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[10] HUANG W, ZHONG Z, LUO C, et al. The miR-26a/AP-2α/NANOG signaling axis mediates stem cell self-renewal and temozolomide resistance in glioma[J]. Theranostics, 2019, 9(19):5497-5516.
Aberrant expression of transcription factor AP-2α has been functionally associated with various cancers, but its clinical significance and molecular mechanisms in human glioma are largely elusive. AP-2α expression was analyzed in human glioma tissues by immunohistochemistry (IHC) and in glioma cell lines by Western blot. The effects of AP-2α on glioma cell proliferation, migration, invasion and tumor formation were evaluated by the 3-(4,5-dimethyNCthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) and transwell assays and in nude mouse models. The influence of AP-2α on glioma cell stemness was analyzed by sphere-formation, self-renewal and limiting dilution assays and in intracranial mouse models. The effects of AP-2α on temozolomide (TMZ) resistance were detected by the MTT assay, cell apoptosis, real-time PCR analysis, western blotting and mouse experiments. The correlation between AP-2α expression and the expression of miR-26a, Nanog was determined by luciferase reporter assays, electrophoretic mobility shift assay (EMSA) and expression analysis. AP-2α expression was downregulated in 58.5% of glioma tissues and in 4 glioma cell lines. AP-2α overexpression not only reduced the proliferation, migration and invasion of glioma cell lines but also suppressed the sphere-formation and self-renewal abilities of glioma stem cells. Moreover, AP-2α overexpression inhibited subcutaneous and intracranial xenograft tumor growth. Furthermore, AP-2α enhanced the sensitivity of glioma cells to TMZ. Finally, AP-2α directly bound to the regulatory region of the Nanog gene, reduced Nanog, Sox2 and CD133 expression. Meanwhile, AP-2α indirectly downregulated Nanog expression by inhibiting the interleukin 6/janus kinase 2/signal transducer and activator of transcription 3 (IL6/JAK2/STAT3) signaling pathway, consequently decreasing O6-methylguanine methyltransferase (MGMT) and programmed death-ligand 1 (PD-L1) expression. In addition, miR-26a decreased AP-2α expression by binding to the 3' untranslated region (UTR) of AP-2α and reversed the tumor suppressive role of AP-2α in glioma, which was rescued by a miR-26a inhibitor. TMZ and the miR-26a inhibitor synergistically suppressed intracranial GSC growth. These results suggest that AP-2α reduces the stemness and TMZ resistance of glioma by inhibiting the Nanog/Sox2/CD133 axis and IL6/STAT3 signaling pathways. Therefore, AP-2α and miR-26a inhibition might represent a new target for developing new therapeutic strategies in TMZ resistance and recurrent glioma patients.
DOI:10.7150/thno.33800      PMID:31534499     
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[22] ZENG A, YIN J, LI Y, et al. Mir-129-5p targets wnt5a to block pkc/erk/nf-κb and jnk pathways in glioblastoma[J]. Cell Death Dis, 2018, 9(3):394.
Therapeutic application of microRNAs (miRNAs) in Wnt-driven glioma has been valuable; however, their specific roles and mechanisms have not been completely investigated. Real-time quantitative PCR (RT-qPCR) was used to analyse the expression of microRNA-129-5p (miR-129-5p) in human glioma samples. Cell-Counting Kit 8 (CCK-8), flow cytometry, EdU, angiogenesis, Transwell invasion, wound healing, in vitro 3D migration and neurosphere formation assays were employed to assess the role of miR-129-5p in glioblastoma multiforme (GBM) cells. Moreover, we performed the luciferase reporter assay and the RNA-ChIP (chromatin immunoprecipitation) assay to confirm whether Wnt5a was a direct target of miR-129-5p. We also confirmed the correlation between the expression profile of miR-129-5p and Wnt5a in glioma patients from the Chinese Glioma Genome Atlas (CGGA) and investigated the overall survival of GBM patients using two data sets, namely, TCGA and GSE16011, according to their Wnt5a expression status. MiR-129-5p expression levels were downregulated and inversely correlated with Wnt5a expression levels in CGGA glioma patients. Restored expression of miR-129-5p blocked GBM cell proliferation, invasion, migration, angiogenesis, neurosphere formation and resistance to temozolomide. We reported that miR-129-5p directly targeted Wnt5a in glioma. Furthermore, we observed that overexpression of miR-129-5p inhibited the expression of Wnt5a, thus blocking the protein kinase C(PKC)/ERK/NF-kappa B and JNK pathways. Inhibiting Wnt5a rescued the effects of miR-129-5p loss and increased Wnt5a expression was associated with reduced overall survival of GBM patients. We also demonstrated the inhibitory effect of miR-129-5p on tumour growth in GBM using an in vivo model. The miR-129-5p/Wnt5a-axis-mediated PKC/ERK/NF-kappa B and JNK pathways have therapeutic potential in GBM treatment.
DOI:10.1038/s41419-018-0343-1      PMID:29531296     
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Glioma is a commonly diagnosed brain tumor that shows high mortality rate. Despite the great advancement of cancer therapy in recent years, chemotherapy is still an important approach for treatment of glioma. However, long-term chemotherapy usually causes serious side effects or complications. It is desirable to take strategies to enhance the efficacy of current chemotherapy. In the present study, we observed obvious upregulation of miR-374a in glioma cells. More importantly, we found that knockdown of miR-374a was able to enhance the etoposide-induced cytotoxicity against glioma cells. Mechanically, we demonstrated that FOXO1 was the target of miR-374a in glioma. Treatment with miR-374a inhibitor induced overexpression of FOXO1, and thus promoted the expression of Bim and Noxa. Since Bim and Noxa act as key proapoptotic proteins in mitochondrial apoptosis, miR-374a inhibitor was able to enhance the etoposide-induced apoptosis pathway in glioma.
DOI:10.3727/096504018X15426775024905      PMID:30841958     
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[34] YIN S, DU W, WANG F, et al. Microrna-326 sensitizes human glioblastoma cells to curcumin via the shh/gli1 signaling pathway[J]. Cancer Biol Ther, 2018, 19(4):260-270.
Glioblastoma multiforme is the most malignant and common brain tumor in adults and is characterized by poor survival and high resistance to chemotherapy and radiotherapy. Among the new chemotherapy drugs, curcumin, a popular dietary supplement, has proven to have a potent anticancer effect on a variety of cancer cell types; however, it remains difficult to achieve a satisfactory therapeutic effect with curcumin using the traditional single-drug treatment. In this study, we found that expression of miR-326, a tumor suppressor microRNA in various tumor types, resulted in a marked increase of curcumin-induced cytotoxicity and apoptosis and a decrease of proliferation and migration in glioma cells. Moreover, we found that combination treatment of miR-326 and curcumin caused significant inhibition of the SHH/GLI1 pathway in glioma cells compared with either treatment alone, independent of p53 status. Furthermore, in vivo, the curcumin-induced increase in miR-326 expression altered the anti-glioma mechanism of this combination treatment, which further reduced tumor volume and prolonged the survival period compared to either treatment alone. Taken together, our data strongly support an important role for miR-326 in enhancing the chemosensitivity of glioma cells to curcumin.
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[41] XU H, WANG F, WANG L, et al. Suppression of mir-106a-5p expression inhibits tumorigenesis via increasing celf-2 expression in spinal cord glioma[J]. Oncol Lett, 2021, 22(2):627. Spinal cord glioma is a tumor characterized by high recurrence and mortality rates, and its treatment remains a major challenge. It has been reported that abnormal expression of microRNAs (miRNAs/miRs) is associated with tumor progression. Therefore, the current study aimed to identify novel miRNAs associated with spinal cord glioma. Herein, the expression levels of several miRNAs were determined in human spinal cord glioma and adjacent non-cancerous tissues by reverse transcription-quantitative (RT-qPCR). The results revealed that miR-106a-5p expression was markedly upregulated in spinal cord glioma tissues compared with in non-cancerous tissues. Furthermore, the biological effects of miR-106a-5p on spinal cord glioma cells were evaluated by MTT, Transwell and flow cytometric assays. In 0231SCG cells transfected with miR-106a-5p inhibitor, cell proliferation, migration and invasion were attenuated, whereas apoptosis was enhanced. A search of the TargetScan database revealed that miR-106a-5p directly targeted CUGBP Elav-like family member 2 (CELF-2). Western blot and RT-qPCR experiments further confirmed the association between miR-106a-5p and CELF-2 expression in spinal cord glioma tissues. The current results demonstrated that CELF-2 was a direct target of miR-106a-5p, and that the expression levels of CELF-2 were negatively associated with those of miR-106a-5p. In addition, overexpression of CELF-2 in spinal cord glioma cells reversed the tumor-promoting effects of miR-106a-5p both and. Overall, the aforementioned findings indicated that miR-106a-5p, which was highly expressed in spinal cord glioma tissues, may affect the proliferation, migration, invasion and apoptosis of spinal cord glioma cells via targeting CELF-2, thus indicating a potential approach to the future clinical management of spinal cord glioma. Copyright: © Xu et al.
Spinal cord glioma is a tumor characterized by high recurrence and mortality rates, and its treatment remains a major challenge. It has been reported that abnormal expression of microRNAs (miRNAs/miRs) is associated with tumor progression. Therefore, the current study aimed to identify novel miRNAs associated with spinal cord glioma. Herein, the expression levels of several miRNAs were determined in human spinal cord glioma and adjacent non-cancerous tissues by reverse transcription-quantitative (RT-qPCR). The results revealed that miR-106a-5p expression was markedly upregulated in spinal cord glioma tissues compared with in non-cancerous tissues. Furthermore, the biological effects of miR-106a-5p on spinal cord glioma cells were evaluated by MTT, Transwell and flow cytometric assays. In 0231SCG cells transfected with miR-106a-5p inhibitor, cell proliferation, migration and invasion were attenuated, whereas apoptosis was enhanced. A search of the TargetScan database revealed that miR-106a-5p directly targeted CUGBP Elav-like family member 2 (CELF-2). Western blot and RT-qPCR experiments further confirmed the association between miR-106a-5p and CELF-2 expression in spinal cord glioma tissues. The current results demonstrated that CELF-2 was a direct target of miR-106a-5p, and that the expression levels of CELF-2 were negatively associated with those of miR-106a-5p. In addition, overexpression of CELF-2 in spinal cord glioma cells reversed the tumor-promoting effects of miR-106a-5p both and. Overall, the aforementioned findings indicated that miR-106a-5p, which was highly expressed in spinal cord glioma tissues, may affect the proliferation, migration, invasion and apoptosis of spinal cord glioma cells via targeting CELF-2, thus indicating a potential approach to the future clinical management of spinal cord glioma. Copyright: © Xu et al.
DOI:10.3892/ol.2021.12888      PMID:34267819     
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[42] LIU Y, YANG L, LIAO F. Mir-450a-5p strengthens the drug sensitivity of gefitinib in glioma chemotherapy via regulating autophagy by targeting egfr[J]. Oncogene, 2020, 39(39):6190-6202.
Glioma reported to be refractory to EGFR tyrosine kinase inhibitor is the most common malignant tumor in central nervous system. Our research showed the low expression of miR-450a-5p and high expression of EGFR in glioma tissues. MiR-450a-5p was also observed to synergize with gefitinib to inhibit the proliferation, migration and invasion and induce the apoptosis and autophagy of glioma cells. Furthermore, miR-450a-5p was demonstrated to target 3'UTR of EGFR, and regulated EGFR-induced PI3K/AKT/mTOR signaling pathway. Moreover, the above effects induced by miR-450a-5p in glioma cells were reversed by WIPI1 silencing. The inhibition role of miR-450a-5p on glioma growth was also confirmed in vivo by subcutaneous and intracranial tumor xenografts. Therefore, we conclude that miR-450a-5p synergizes with gefitinib to inhibit the glioma tumorigenesis through inducing autophagy by regulating the EGFR-induced PI3K/AKT/mTOR signaling pathway, thereby enhancing the drug sensitivity of gefitinib.
DOI:10.1038/s41388-020-01422-9      PMID:32820249     
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[46] YANG J, LIU R, DENG Y, et al. Mir-15a/16 deficiency enhances anti-tumor immunity of glioma-infiltrating CD8+ T cells through targeting mtor[J]. Int J Cancer, 2017, 141(10):2082-2092. MiR-15a/16, a miRNA cluster located at chromosome 13q14, has been reported to act as an immune regulator in inflammatory disorders besides its aberrant expression in cancers. However, little is known about its regulation in tumor-infiltrating immune cells. In our study, using an orthotropic GL261 mouse glioma model, we found that miR-15a/16 deficiency in host inhibited tumor growth and prolonged mice survival, which might be associated with the accumulation of tumor-infiltrating CD8+ T cells. More importantly, tumor-infiltrating CD8+ T cells without miR-15a/16 showed lower expression of PD-1, Tim-3 and LAG-3, and stronger secretion of IFN-γ, IL-2 and TNF-α than WT tumor-infiltrating CD8+ T cells. Also, our in vitro experiments further confirmed that miR-15a/16 CD8+ T displayed higher active phenotypes, more cytokines secretion and faster expansion, compared to WT CD8+ T cells. Mechanismly, mTOR was identified as a target gene of miR-15a/16 to negatively regulate the activation of CD8+ T cells. Taken together, these data suggest that miR-15a/16 deficiency resists the exhaustion and maintains the activation of glioma-infiltrating CD8+ T cells to alleviate glioma progression via targeting mTOR. Our findings provide evidence for the potential immunotherapy through targeting miR-15a/16 in tumor-infiltrating immune cells. © 2017 UICC.
MiR-15a/16, a miRNA cluster located at chromosome 13q14, has been reported to act as an immune regulator in inflammatory disorders besides its aberrant expression in cancers. However, little is known about its regulation in tumor-infiltrating immune cells. In our study, using an orthotropic GL261 mouse glioma model, we found that miR-15a/16 deficiency in host inhibited tumor growth and prolonged mice survival, which might be associated with the accumulation of tumor-infiltrating CD8+ T cells. More importantly, tumor-infiltrating CD8+ T cells without miR-15a/16 showed lower expression of PD-1, Tim-3 and LAG-3, and stronger secretion of IFN-γ, IL-2 and TNF-α than WT tumor-infiltrating CD8+ T cells. Also, our in vitro experiments further confirmed that miR-15a/16 CD8+ T displayed higher active phenotypes, more cytokines secretion and faster expansion, compared to WT CD8+ T cells. Mechanismly, mTOR was identified as a target gene of miR-15a/16 to negatively regulate the activation of CD8+ T cells. Taken together, these data suggest that miR-15a/16 deficiency resists the exhaustion and maintains the activation of glioma-infiltrating CD8+ T cells to alleviate glioma progression via targeting mTOR. Our findings provide evidence for the potential immunotherapy through targeting miR-15a/16 in tumor-infiltrating immune cells. © 2017 UICC.
DOI:10.1002/ijc.30912      PMID:28758198     
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关键词（key words）
微小RNA
胶质瘤
化学治疗
耐药
纳米载体
MicroRNA
Glioma
Chemoradiotherapy
Drug resistance
Nano-carrier
作者
曾昭穆
李琳
郭岩松
祁惠秀
刘永
闫晴宇
郑克彬
ZENG Zhaomu
LI Lin
GUO Yansong
QI Huixiu
LIU Yong
YAN Qingyu
ZHENG Kebin