何 昆, 钱家鸣

中国医学科学院 北京协和医学院 北京协和医院消化科, 北京 100730

MicroRNA在溃疡性结肠炎活动度和癌变监测中的应用

何 昆, 钱家鸣

中国医学科学院 北京协和医学院 北京协和医院消化科, 北京 100730

炎症性肠病(inflammatory bowel disease,IBD)在我国将成为常见病，溃疡性结肠炎(ulcerative colitis,UC)是其主要类型之一。目前UC病情活动度评估和癌变监测主要依赖内镜检查，尚缺乏较理想的实验室指标。MicroRNA(miRNA)是近年发现的非编码小分子RNA，调控炎症、肿瘤等多种生命活动。在UC患者肠组织和外周血中已发现多种与炎症和癌变相关的miRNA，部分机制得到阐明，提示其作为UC相关生物标记(Biomarker)有很好的应用前景。本文就miRNA和UC研究现状作一概述，为寻找UC活动度评估和癌变监测相关miRNA Biomarker提供依据。

溃疡性结肠炎；疾病活动度；癌变；MicroRNA

炎症性肠病(inflammatory bowel disease, IBD)是一种病因尚不十分清楚的慢性非特异性肠道炎症性疾病，主要包括溃疡性结肠炎(ulcerative colitis, UC)和克罗恩病(Crohn’s disease, CD)。IBD是北美和欧洲的常见病。近十多年来该病在我国就诊人数呈逐步增加趋势，将成为我国消化系统常见病。UC病情评估主要包括临床类型、病变范围、疾病活动度和并发症。其中病情活动度和癌变评估监测是其中重要环节，目前主要依赖内镜检查，尚缺乏较理想的实验室评价指标[1]。在临床实践中，ALT、ALP、cTnI、AMY等较理想的实验室指标多与机制相关，提示从机制入手寻找理想实验室指标是一条可以借鉴的途径。

MicroRNA(miRNA)是近20年来发现的一类在进化上高度保守的长度为19～25个核苷酸的非编码小分子RNA。miRNA的研究始于1993年：Lee等[2]在秀丽小杆线虫中首次发现可时序性调控胚胎后期发育的基因lin-4。进一步研究发现其通过转录后水平具有组织特异性的降解或抑制靶mRNA的翻译来调节基因的表达，参与细胞代谢、增殖、分化、凋亡、炎症和免疫应答以及肿瘤的发生[3]。Wu等[4]在2008年首次报道了IBD肠组织中存在差异表达的miRNA，拉开了IBD相关miRNA研究的帷幕。截至目前，越来越多UC相关miRNA在肠组织和外周血被发现及报道，部分作用机制已得到阐明，提示其作为UC疾病活动度评估和癌变监测相关Biomarker存在很好的前景。因此，本文从UC活动度相关miRNA、UC癌变相关miRNA、UC发病及癌变机制相关miRNA三个层面对miRNA和UC研究进展进行概述。

1 UC活动度评估相关miRNA

在2008年，Wu等[4]首先报道在UC肠黏膜中存在差异表达miRNA。其研究纳入活动期UC、健康对照各15例，发现11种miRNAs在活动期UC和健康对照肠黏膜间有显著差异表达：既包括表达升高miRNAs如miR-21等8种miRNAs，也包含表达下降miRNAs如miR-192等3种miRNAs。Pekow等[5]2012年报道miR-143和miR-145在缓解期UC肠黏膜较健康对照表达下降，提示miRNA在缓解期UC中表达也存在显著差异。此外Wu等[4]在2008年，Coskun等[6]在2013年分别发现在活动期和缓解期UC肠黏膜间存在显著差异表达miRNAs：在前者研究中，miR-16、miR-21、miR-24、miR-126，miR-203，miR-203b存在差异表达；在后者中，miR-98在缓解期较活动期表达显著升高。这提示肠黏膜miRNA表达变化可以用于区分UC患者疾病活动度。

进一步研究[7]还表明，不同于mRNA，miRNA在外周血中稳定存在且在UC患者中已发现多种有显著差异表达的外周血miRNAs。其中具有有代表性的研究如Paraskevi 等[8]在2012年报道：通过对88例UC患者和162名健康对照人群外周血中miRNAs表达水平检测发现，miRs-21、28-5p、151-5p、155、199a-5p在UC患者外周血中表达显著升高。Krissansen等[9]2015年报道miR-595和miR-1246在活动期和缓解期UC患者外周血中存在显著差异表达。这提示外周血miRNA可以用于区分UC疾病活动度。

此外，在UC相关miRNA的研究中，尚存在一些不足。如：(1)尚未有基于UC疾病活动度的细化研究—缓解期-轻度活动-重度活动；(2)对结肠癌等疾病相关miRNA研究提示，多种miRNAs联合可以提高其检验效力[10]。而在UC中，针对疾病活动度评估的多种miRNA联合检测相关研究几乎为零；(3)研究总体样本量较少。

2 UC癌变相关miRNA

既往研究[11]表明，miRNA同肿瘤的发生、发展密切相关，在多种肿瘤如前列腺癌、多发性骨髓瘤、乳腺癌中存在差异表达，其既可以充当原癌基因也可以作为抑癌基因。对于作为UC最主要的并发症之一的溃疡性结肠炎相关结直肠癌(ulcerative colitis related cancer, UCRCC)[12]，同样有多种miRNAs被发现同其密切相关。Ludwig等[13]在2013年报道miR-21在UC伴肠黏膜上皮内瘤变、活动期UC、健康对照肠黏膜中表达水平呈梯度升高；Olaru等[14-15]分别在2011年和2013年报道miR-31和miR-224随IBD(包括UC)癌变进程均呈梯度升高；Benderska等[16]在2015年报道miR-26b在UCRCC较UC肠黏膜显著高表达。

回顾以往文献，尚未发现UCRCC外周血差异表达miRNA的报道。但对其他恶性肿瘤如散发性结直肠癌、肝细胞肝癌、乳腺癌的研究表明，外周血中存在多种差异表达的miRNA[10, 17-19]。这提示UCRCC外周血中可能也存在差异表达的miRNA。目前尚无研究报道的原因可能有以下几方面：(1)UC癌变需要时间，病程越长，癌变风险越高，绝大多数发生在确诊8～10年以上，且并非所有UC患者均会癌变[20]；(2)UC外周血miRNA含量较肠黏膜低，虽较为稳定，但在长时间保存和多次存取中仍可能出现降解并造成假阴性，(3)临床工作中，相比于组织标本，外周血标本不一定能及时留取和保存。

3 UC发病及癌变机制相关miRNA

UC发病机制尚未完全明确，目前认为由多种因素相互作用所致，主要包括遗传、免疫、肠道感染、环境和其他因素[21]。其中遗传、免疫为个体相关因素，适合作为寻找UC相关miRNA的切入点。在遗传因素中，截至目前，已有超过160个IBD相关遗传易感性位点被发现[22-28]。免疫因素主要包括自身抗体、细胞免疫因素、黏附分子、细胞因子等[29]。

相比于散发性结直肠癌是由基因不稳定性启动、以腺瘤性息肉为主要癌前病变而言，UCRCC的发病模式则为“炎症-上皮内瘤变-癌变”。至于具体机制，目前认为是炎性反应与氧化应激在UC中作用于肠组织，同时在表观遗传学改变(如超甲基化)的共同作用下，引起多种基因发生突变，包括抑癌基因失活、多倍体(Aneuploidy)、微卫星不稳定(microsatellite instability, MSI)，抑制细胞凋亡，促进细胞增殖、转移，从而促进UCRCC发生、发展[30-31]。

如本文初所述，从机制入手找寻理想实验室指标可能是一条不错的途径。miRNA通过在转录后水平具有组织特异性的降解或抑制靶mRNA的翻译来调节基因的表达，参与细胞代谢、增殖、分化、凋亡，炎症和免疫应答及肿瘤的发生[3]。因此，笔者根据UC发病、癌变机制总结部分miRNA肠黏膜、外周血表达变化及可能靶mRNA如下表(见表1)，以期为寻找UC疾病活动度评估和癌变监测相关miRNA提供依据。

综上所述，自2008年起至今，针对UC和miRNA的研究已有了较深入的进展，不仅在UC肠黏膜和外周血中发现了多种同UC病情活动和癌变相关miRNA，且部分机制已得到阐明，为从机制入手寻找UC疾病活动度评估和癌变监测相关miRNA Biomarker既提供了可靠的依据又展示出宽广的前景。当然，在UC和miRNA的研究中尚有许多可改进的空间和细化的工作，如扩大样本量、细化活动度分级后的miRNA表达变化测量、多种miRNA联合检测等。有待在以后的研究工作中进一步深入完善。

表1 UC 炎症及癌变相关miRNA

Tab 1 UC inflammation and carcinogenesis related microRNA

miRNA肠组织含量样本来源炎症或癌变相关miRNA(已证实)靶mRNA之一对应靶mRNA作用UC外周血含量UCRCC外周血含量参考文献miR⁃21↑人UC结肠炎症相关&癌变相关Rhob↓保护肠黏膜紧密连接mir⁃21↑不详[32]miR⁃21↑人UC结肠炎症相关&癌变相关PDCD4↓ 抑癌基因，调控细胞周期mir⁃21↑不详[13]miR⁃31↑人UC结肠炎症相关&癌变相关FIH⁃1↓抑癌基因 miR⁃31↑不详[14]miR⁃126↑人UC结肠炎症相关 IκBα↓抑制NF⁃κB CD↑不详[33]miR⁃150↑人UC结肠&小鼠炎症相关 c⁃Myb↓抗凋亡 不详不详[34]miR⁃155↑人UC结肠炎症相关&癌变相关FOXOa↓调节NF⁃κB通路 miR⁃155↑不详[8，35]miR⁃155↑人UC结肠炎症相关&癌变相关MMR↓与MSI相关 miR⁃155↑不详[36]miR⁃224↑人IBD结肠炎症相关&癌变相关p21↓ 抑癌基因，调控细胞周期不详不详[15]miR⁃192↓人UC结肠&小鼠炎症相关 MIP2⁃α↑趋化因子 不详不详[4]miR⁃19a↓人UC结肠&小鼠炎症相关 TNF⁃α↑ 诱导黏附蛋白的重排和肠黏膜细胞的凋亡不详不详[37]

注：↑:较正常对照组表达量上升；↓：较正常对照组表达量下降。

[1]Inflammatory Bowel Disease Group of Digestve Diseases of Chinese Medical Association. The consensus of opinion of inflammatory bowel disease diagnosis and treatment (Guangzhou,2012)[J]. Chin J Intern Med, 2012, 51(10): 818-831.

中华医学会消化病学分会炎症性肠病学组. 炎症性肠病诊断与治疗的共识意见(2012年·广州)[J]. 中华内科杂志, 2012, 51(10): 818-831.

[2]Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 [J]. Cell, 1993, 75(5): 843-854.

[3]Raisch J, Darfeuille-Michaud A, Nguyen HT. Role of microRNAs in the immune system, inflammation and cancer [J]. World J Gastroenterol, 2013, 19(20): 2985-2996.

[4]Wu F, Zikusoka M, Trindade A, et al. MicroRNAs are differentially expressed in ulcerative colitis and alter expression of macrophage inflammatory peptide-2 alpha [J]. Gastroenterology, 2008, 135(5): 1624-1635, e24.

[5]Pekow JR, Dougherty U, Mustafi R, et al. miR-143 and miR-145 are downregulated in ulcerative colitis: putative regulators of inflammation and protooncogenes [J]. Inflamm Bowel Dis, 2012, 18(1): 94-100.

[6]Coskun M, Bjerrum JT, Seidelin JB, et al. miR-20b, miR-98, miR-125b-1*, and let-7e* as new potential diagnostic biomarkers in ulcerative colitis [J]. World J Gastroenterol, 2013, 19(27): 4289-4299.

[7]Mitchell PS, Parkin RK, Kroh EM, et al. Circulating microRNAs as stable blood-based markers for cancer detection [J]. Proc Nati Acad Sci U S A, 2008, 105(30): 10513-10518.

[8]Paraskevi A, Theodoropoulos G, Papaconstantinou I, et al. Circulating MicroRNA in inflammatory bowel disease [J]. J Crohn’s Colitis, 2012, 6(9): 900-904.

[9]Krissansen GW, Yang Y, McQueen FM, et al. Overexpression of miR-595 and miR-1246 in the sera of patients with active forms of inflammatory bowel disease [J]. Inflamm Bowel Dis, 2015, 21(3): 520-530.

[10]Wang J, Huang SK, Zhao M, et al. Identification of a circulating microRNA signature for colorectal cancer detection [J]. PLoS One, 2014, 9(4): e87451.

[11]Croce CM. Causes and consequences of microRNA dysregulation in cancer [J]. Nature Reviews Genetics, 2009, 10(10): 704-714.

[12]Munkholm P. Review article: the incidence and prevalence of colorectal cancer in inflammatory bowel disease [J]. Aliment Pharmacol Ther, 2003, 18 Suppl 2: 1-5.

[13]Ludwig K, Fassan M, Mescoli C, et al. PDCD4/miR-21 dysregulation in inflammatory bowel disease-associated carcinogenesis [J]. Virchows Arch, 2013, 462(1): 57-63.

[14]Olaru AV, Selaru FM, Mori Y, et al. Dynamic changes in the expression of MicroRNA-31 during inflammatory bowel disease-associated neoplastic transformation [J]. Inflamm Bowel Dis, 2011, 17(1): 221-231.

[15]Olaru AV, Yamanaka S, Vazquez C, et al. MicroRNA-224 negatively regulates p21 expression during late neoplastic progression in inflammatory bowel disease [J]. Inflamm Bowel Dis, 2013, 19(3): 471-480.

[16]Benderska N, Dittrich AL, Knaup S, et al. miRNA-26b Overexpression in Ulcerative Colitis-associated Carcinogenesis [J]. Inflamm Bowel Dis, 2015, 21(9): 2039-2051.

[17]Li G, Shen Q, Li C, et al. Identification of circulating MicroRNAs as novel potential biomarkers for hepatocellular carcinoma detection: a systematic review and meta-analysis [J]. Clin Transl Oncol, 2015, 17(9): 684-693.

[18]Wang G, Wang L, Sun S, et al. Quantitative measurement of serum microRNA-21 expression in relation to breast cancer metastasis in Chinese females [J]. Ann Lab Med, 2015, 35(2): 226-232.

[19]Wang P, Yang D, Zhang H, et al. Early detection of lung cancer in serum by a panel of MicroRNA biomarkers [J]. Clin Lung Cancer, 2015, 16(4): 313-319, e1.

[20]Lutgens MW, Vleggaar FP, Schipper ME, et al. High frequency of early colorectal cancer in inflammatory bowel disease [J]. Gut, 2008, 57(9): 1246-1251.

[21]Xavier RJ, Podolsky DK. Unravelling the pathogenesis of inflammatory bowel disease [J]. Nature, 2007, 448(7152): 427-434.

[22]Wellcome Trust Case Control Consortium. Genome-wide association study of 14 000 cases of seven common diseases and 3 000 shared controls [J]. Nature, 2007, 447(7145): 661-678.

[23]Barrett JC, Hansoul S, Nicolae DL, et al. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn’s disease [J]. Nat Genet, 2008, 40(8): 955-962.[24]Duerr RH, Taylor KD, Brant SR, et al. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene [J]. Science, 2006, 314(5804): 1461-1463.

[25]Fisher SA, Tremelling M, Anderson CA, et al. Genetic determinants of ulcerative colitis include the ECM1 locus and five loci implicated in Crohn's disease [J]. Nat Genet, 2008, 40(6): 710-712.

[26]Franke A, Balschun T, Karlsen TH, et al. Replication of signals from recent studies of Crohn’s disease identifies previously unknown disease loci for ulcerative colitis [J]. Nat Genet, 2008, 40(6): 713-715.

[27]Franke A, Balschun T, Karlsen TH, et al. Sequence variants in IL10, ARPC2 and multiple other loci contribute to ulcerative colitis susceptibility [J]. Nat Genet, 2008, 40(11): 1319-1323.

[28]Kaser A, Lee AH, Franke A, et al. XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease [J]. Cell, 2008, 134(5): 743-756.

[29]周婷婷, 仝巧云. 溃疡性结肠炎发病机制的研究进展[J]. 胃肠病学和肝病学杂志, 2012, 21(12): 1163-1166. Zhou TT, Tong QY. Progress in understanding the pathogenesis of ulcerative colitis [J]. Chin J Gastroenterol Hepatol, 2012, 21(12): 1163-1166. [30]陈敏, 吴开春. 炎症性肠病癌变的发生机制与防治[J]. 医学新知杂志, 2013, 23(5): 354-358, 363. Chen M, Wu KC. The mechanism and prevention of inflammatory bowel disease cancer [J]. Ournal of New Medicine, 2013, 23(5): 354-358, 363. [31]Scarpa M, Castagliuolo I, Castoro C, et al. Inflammatory colonic carcinogenesis: a review on pathogenesis and immunosurveillance mechanisms in ulcerative colitis [J]. World J Gastroenterol, 2014, 20(22): 6774-6785.

[32]Yang Y, Ma Y, Shi C, et al. Overexpression of miR-21 in patients with ulcerative colitis impairs intestinal epithelial barrier function through targeting the Rho GTPase RhoB [J]. Biochem Biophys Res Commun, 2013, 434(4): 746-752.

[33]Feng X, Wang H, Ye S, et al. Up-regulation of microRNA-126 may contribute to pathogenesis of ulcerative colitis via regulating NF-kappaBinhibitor IκBα [J]. PLoS One, 2012, 7(12): e52782.

[34]Bian Z, Li L, Cui J, et al. Role of miR-150-targeting c-Myb in colonic epithelial disruption during dextran sulphate sodium-induced murine experimental colitis and human ulcerative colitis [J]. J Pathol, 2011, 225(4): 544-553.

[35]Min M, Peng L, Yang Y, et al. MicroRNA-155 is involved in the pathogenesis of ulcerative colitis by targeting FOXO3a [J]. Inflamm Bowel Dis, 2014, 20(4): 652-659.

[36]Svrcek M, El-Murr N, Wanherdrick K, et al. Overexpression of microRNAs-155 and 21 targeting mismatch repair proteins in inflammatory bowel diseases [J]. Carcinogenesis, 2013, 34(4): 828-834.

[37]Chen B, She S, Li D, et al. Role of miR-19a targeting TNF-αlpha in mediating ulcerative colitis [J]. Scand J Gastroenterol, 2013, 48(7): 815-824.

Application of activity and carcinogenesis monitoning of MicroRNA in ulcerative colitis

HE Kun, QIAN Jiaming

Department of Gastroenterology, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730, China

Inflammation bowel disease (IBD) is going to be a common disease in our country gradually, of which ulcerative colitis (UC) is one of the main types. Nowadays, endoscopy tests are mainly used for evaluation and surveillance of disease activity and carcinogenesis in UC, lacking of ideal laboratory tests. MicroRNA (miRNA) is a small non-coding RNA, regulating multiple life activities included inflammation and neoplasia. Recently many miRNAs have been proved to be tightly associated with the disease activity and carcinogenesis of UC in both intestinal mucosa and peripheral blood, the mechanisms of which have been partially illustrated. Therefore, it reveals that miRNAs seem to show a great prospect as Biomarkers for UC. This article reviewed the application of activity and carcinogenesis monitoring of miRNA in UC to help searching for miRNA Biomarker for evaluation and surveillance of disease activity and carcinogenesis in UC.

Ulcerative colitis; Disease activity; Carcinogenesis; MicroRNA

何昆，博士，住院医师。E-mail:hk6290418@163.com

钱家鸣，博士生导师，主任医师，研究方向：炎症性肠病。E-mail：qianjiaming 1975@126.com

10.3969/j.issn.1006-5709.2016.10.002

R574.62

A

1006-5709(2016)10-1084-04

2015-07-10