circRNA-ZNF532、circRNA-HIPK3与糖尿病视网膜病变的相关性研究

2023-11-08 11:34隋源张成森王爽李雪丽勾晓梅
天津医药 2023年9期
关键词:视网膜血管血清

隋源 张成森 王爽 李雪丽 勾晓梅

摘要:目的 探討环状RNA(circRNA)-锌指蛋白532(ZNF532)、circRNA-同源域相互作用蛋白激酶3(HIPK3)与糖尿病视网膜病变(DR)的相关性。方法 纳入109例DR患者(DR组)、110例单纯糖尿病患者(DM组)和65例健康体检志愿者(对照组)。实时荧光定量聚合酶链反应(qPCR)检测血清circRNA-ZNF532、circRNA-HIPK3表达,酶联免疫吸附试验检测血清血管内皮生长因子(VEGF)、白细胞介素(IL)-1β、肿瘤坏死因子(TNF)-α、IL-6水平。Pearson分析DR患者血清circRNA-ZNF532、circRNA-HIPK3表达与血清VEGF、IL-1β、TNF-α、IL-6水平相关性。单因素和多因素Logistic回归分析DR危险因素,受试者工作特征(ROC)曲线分析circRNA-ZNF532、circRNA-HIPK3诊断DR的价值。结果 DR组血清circRNA-ZNF532、circRNA-HIPK3表达以及VEGF、IL-1β、TNF-α、IL-6水平高于DM组和对照组(P<0.05)。DR组血清circRNA-ZNF532、circRNA-HIPK3表达与VEGF、IL-1β、TNF-α、IL-6水平均呈正相关(P<0.05)。多因素Logistic回归分析结果显示高水平circRNA-ZNF532、circRNA-HIPK3及VEGF是糖尿病患者发生DR的独立危险因素(P<0.05)。三者诊断糖尿病患者发生DR的曲线下面积分别为0.736、0.740和0.864,联合诊断高于单独诊断(P<0.05)。结论 糖尿病患者血清中circRNA-ZNF532、circRNA-HIPK3表达上调与DR发生有关,两者可以作为DR辅助诊断的潜在指标。

关键词:糖尿病视网膜病变;RNA,环状;转录激活因子类;tristetraprolin蛋白;同源盒结构域蛋白质类;炎症;血管内皮生长因子类;同源域相互作用蛋白激酶3

中图分类号:R774.13文献标志码:ADOI:10.11958/20230048

Correlation analysis of circRNA-ZNF532 and circRNA-HIPK3 with diabetic retinopathy

SUI Yuan, ZHANG Chengsen, WANG Shuang, LI Xueli, GOU Xiaomei

Department of Ophthalmology, Harbin 242 Hospital, Harbin 150000, China

Corresponding Author E-mail: 25743710490@qq.com

Abstract: Objective To investigate the correlation between circrNa-Zinc finger protein 532 (ZNF532), circrNa-homologous domain interaction protein kinase-3 (HIPK3) and diabetic retinopathy (DR). Methods A total of 109 patients with DR (DR group), 110 patients with simple diabetes (DM group) and 65 healthy volunteers (control group) were selected from our hospital from October 2020 to October 2022. Serum circRNA-ZNF532 and circRNA-HIPK3 expression levels were detected by real-time quantitative polymerase chain reaction (RT-PCR), and serum vascular endothelial growth factor (VEGF), interleukin (IL) -1β, tumor necrosis factor (TNF) -α and IL-6 levels were detected by enzyme-linked immunosoradsorption assay. The correlation between serum circRNA-ZNF532 and circRNA-HIPK3 expression and serum levels of VEGF, IL-1β, TNF-α and IL-6 were analyzed by Pearson method. Univariate Logistic regression analysis and multivariate Logistic regression analysis were used for DR risk factors, and receiver operating characteristic curve (ROC) was used for analysis of circRNA-ZNF532 and circRNA-HIPK3 diagnostic value of DR. Results Serum expressions of circRNA-ZNF532 and circRNA-HIPK3 and levels of VEGF, IL-1β, TNF-α and IL-6 were significantly higher in the DR group than those in the DM group and the control group (P<0.05). Serum circRNA-ZNF532 and circRNA-HIPK3 expression were positively correlated with  levels of VEGF, IL-1β, TNF-α and IL-6 in the DR group (P<0.05). Multivariate Logistic regression analysis showed that high levels of VEGF, circRNA-ZNF532 and circRNA-HIPK3 were risk factors for developing DR in diabetic patients (P<0.05). The areas under the curve of circRNA-ZNF532, circRNA-HIPK3 and VEGF in the diagnosis of DR in diabetic patients were 0.736, 0.740 and 0.864. Combined diagnosis was higher than that of circRNA-ZNF532, circRNA-HIPK3 and VEGF alone (P<0.05). Conclusion The up-regulated expression levels of circRNA-ZNF532 and circRNA-HIPK3 in serum of diabetic patients are related to the occurrence of DR, and both circRNA-ZNF532 and circRNA-HIPK3 can be used as potential indicators for the auxiliary diagnosis of DR.

Key words: diabetic retinopathy; RNA, circular; activating transcription factors; tristetraprolin; homeodomain proteins; inflammation; vascular endothelial growth factors; circrNa-homologous domain interaction protein kinase-3

糖尿病視网膜病变(DR)是糖尿病严重致残性并发症之一。据统计,我国糖尿病人群中DR患病率为18.45%,而普通人群视网膜病变患病率仅1.14%[1]。DR如不及时治疗可严重损害视力,最终发展为失明[2]。研究认为持续高血糖刺激下,微血管功能失调和过度炎症反应可引起血管通透性增加、黄斑水肿、血管新生和纤维组织收缩,导致视网膜扭曲脱离、新生血管出血及神经元退行性病变[3]。环状RNA(circRNA)在视网膜广泛表达并参与微血管功能失调相关蛋白,如血管内皮生长因子(VEGF)、转化生长因子(TGF)、Sirtuin(SIRT)差异表达,且其表达水平与DR发病过程中氧化应激、炎症反应、细胞凋亡、血管新生密切相关[4]。研究发现,circRNA-锌指蛋白532(ZNF532)参与调控视网膜血管内皮周细胞功能,与血管内皮功能障碍有关[5]。circRNA-同源域相互作用蛋白激酶-3(HIPK3)通过miR-29a介导胰岛素样生长因子1表达,降低氧化应激诱导的心脏微血管内皮功能障碍[6]。而关于circRNA-ZNF532、circRNA-HIPK3在DR的临床报道十分少见。本研究拟检测DR患者血清circRNA-ZNF532、circRNA-HIPK3表达,分析其与DR发生的关系。

1 对象与方法

1.1 研究对象 连续性选择2020年10月—2022年10月我院眼科收治的109例DR患者(DR组)。纳入标准:(1)经临床诊断为2型糖尿病,符合《中国2型糖尿病防治指南(2017版)》[7]诊断标准。(2)出现视力逐渐下降,经眼底检查提示存在微血管瘤、渗出、出血等眼底病变,符合《我国糖尿病视网膜病变临床诊疗指南(2014)年》[8]诊断标准。(3)年龄≥18周岁。排除标准:(1)患青光眼、白内障、玻璃体液化、眼表感染、屈光不正等。(2)阿尔茨海默病、认知障碍或精神疾病。(3)原发性肾病,肺和肝脏疾病。(4)感染、恶性肿瘤、自身免疫疾病。(5)近1个月应用抗感染、激素、免疫抑制剂治疗。(6)妊娠或哺乳期。另采用抽签法选择同期我院收治的110例单纯糖尿病患者(DM组),均为2型糖尿病,参照文献[7]诊断标准,排除DR,眼内和眼表疾病,心、肺、肝、肾等系统性疾病。同期于门诊体检的健康志愿者中选择65例为对照组。本研究经我院伦理委员会批准(伦理号:200952),全部研究对象均知情同意。

1.2 主要仪器及试剂 RNAsimple总RNA提取试剂盒、InRcute lncRNA cDNA Synthesis Kit(北京天根生化科技有限公司);ABI 7500 Real-Time PCR系统(美国应用生物系统公司);VarioskanLUX酶标仪、Invitrogen iBright免疫化学发光分析仪(美国赛默飞公司);AU5800全自动生化分析仪(美国贝克曼库尔特公司);JL-760糖化血红蛋白分析仪(青岛三凯医学科技有限公司)。TNF-α、IL-1β、IL-6酶联免疫吸附试验(ELISA)试剂盒购自上海酶联生物科技有限公司;VEGF试剂盒购自上海研启生物科技有限公司。

1.3 方法

1.3.1 circRNA-ZNF532、circRNA-HIPK3检测 采集患者外周血3 mL,取血液凝固后上层血清,2 500 r/min离心5 min,取上层液-80 ℃待检备检。RNAsimple总RNA提取试剂盒提取总RNA,InRcute lncRNA cDNA Synthesis Kit将其反转录为cDNA。ABI 7500 Real-Time PCR系统进行实时荧光定量聚合酶链反应(qPCR)。反应体系:cDNA模板2 μL,上、下游引物各1 μL,Premix Ex Taq DNA 聚合酶10 μL,灭菌蒸馏水7 μL。引物由美国Invitrogen公司合成。circRNA-ZNF532引物:上游5′-ACGAGTGGACAAAACATCTGC-3′,下游5′-AATGCTGCCAGGAGGTCATC-3′。circRNA-HIPK3引物:上游5′-TGGAGACTGGGGGAAGATGA-3′,下游5′-CACACTAACTGGCTGAGGGG-3′。内参基因β-actin引物:上游5′-CACGAAACTACCTTCAACTCC-3′,下游5′-CATACTCCTGCTTGCTGATC-3′。反应条件:95 ℃预变性3 min;98 ℃变性20 s,67 ℃退火15 s,40个循环,72 ℃延伸20 s。于72 ℃时采集荧光信号,进行熔解曲线分析,2?ΔΔCt法计算circRNA-ZNF532、circRNA-HIPK3相对表达量。每个样本设置3个复孔,取3次测量结果的平均值。

1.3.2 血清VEGF、IL-1β、TNF-α、IL-6检测 取血清样本,ELISA法检测血清VEGF、TNF-α、IL-1β、IL-6水平。

1.3.3 糖脂代谢指标检查 取血清样本,全自动生化分析仪检测三酰甘油(TG)、总胆固醇(TC)、血肌酐(Cr)水平;免疫化学发光分析仪检测空腹血糖(FPG)、空腹胰岛素(FINS)水平;稳态模型计算胰岛素抵抗指数(HOMA-IR);糖化血红蛋白分析仪检测糖化血红蛋白(HbA1c)水平。

1.4 临床资料收集 收集受试者年龄、性别、体质量指数(BMI)、吸烟史、饮酒史、糖尿病病程、基础疾病(高血压、高脂血症)、糖尿病并发症(糖尿病肾病、糖尿病周围神经病变、冠心病、脑卒中)及降糖治疗方法。

1.5 统计学方法 采用SPSS 25.0录入和分析数据,符合正态分布的连续性变量以均数±标准差(x±s)表示,3组间比较采用单因素方差分析,组间两两比较采用LSD-t检验;2组间比较采用独立样本t检验。分类变量以例(%)表示,组间比较采用χ2检验。Pearson分析DR患者血清circRNA-ZNF532、circRNA-HIPK3表达与血清VEGF、IL-1β、TNF-α、IL-6水平相关性。单因素和多因素Logistic回归分析DR危险因素。受试者工作特征(ROC)曲线分析DR的诊断价值。所有统计均采用双侧检验,检验水准α=0.05。

2 结果

2.1 3组基线资料比较 3组间的年龄、性别、BMI差异无统计学意义(P>0.05)。DR组、DM组TG、TC、Cr、FPG,FINS、HOMA-IR、HbA1c水平均高于对照组(P<0.05),DR组糖尿病病程、高血压比例、FPG、FINS、HOMA-IR、HbA1c水平均高于DM组(P<0.05)。见表1。

2.2 3组血清circRNA-ZNF532、circRNA-HIPK3、VEGF及炎性因子水平比较 DM组血清circRNA-ZNF532、circRNA-HIPK3、VEGF、IL-1β水平與对照组比较差异无统计意义(P>0.05),血清TNF-α、IL-6水平高于对照组(P<0.05);DR组上述指标水平均高于DM组和对照组(P<0.05)。见表2。

2.3 血清circRNA-ZNF532、circRNA-HIPK3表达与VEGF、IL-1β、TNF-α、IL-6水平相关性 DR组血清circRNA-ZNF532、circRNA-HIPK3表达与VEGF(r分别为0.519和0.537)、IL-1β(r分别为0.335和0.357)、TNF-α(r分别为0.404和0.396)、IL-6(r分别为0.362和0.401)水平均呈正相关(均P<0.05)。

2.4 糖尿病患者发生DR危险因素分析 以糖尿病病程、高血压(否=0,是=1)、FPG、HOMA-IR、HbA1c、circRNA-ZNF532、circRNA-HIPK3、VEGF、IL-1β、TNF-α、IL-6为自变量,以糖尿病患者是否发生DR为因变量(否=0,是=1),建立Logistic回归方程。单因素Logistic分析显示糖尿病病程长,高血压,HbA1c、circRNA-ZNF532、circRNA-HIPK3、VEGF、IL-1β水平升高与糖尿病患者发生DR有关(P<0.05),见表3。多因素分析显示VEGF、circRNA-ZNF532及circRNA-HIPK3水平升高是糖尿病患者发生DR的独立危险因素(P<0.05),见表4。

2.5 糖尿病患者发生DR的诊断价值 circRNA-ZNF532、circRNA-HIPK3、VEGF联合诊断糖尿病患者发生DR的AUC高于单独诊断(Z分别为5.505、5.400、2.098,P<0.05),见图1、表5。

3 讨论

3.1 circRNA与DR的关系 研究发现circRNA在视网膜中差异表达对DR的进展有重要影响,可能导致血管功能障碍和(或)促进新血管的形成,参与DR病变过程[9]。如circRNA-001209在视网膜过表达,通过“海绵”吸附miR-15b-5p来间接调节胶原十二型α1链(COL12A1)表达,导致视网膜血管内皮细胞增殖、迁移以及新生血管形成[10]。circRNA-0002570通过靶向miR-1243上调血管生成素表达,介导高糖诱导的视网膜血管内皮细胞功能障碍[11]。

3.2 circRNA-ZNF532与DR的关系 ZNF532是一个潜在的转录调节因子,可选择性结合靶RNA/DNA,调控细胞分化、胚胎发育等生命过程,对基因调控起重要作用[12]。Jiang等[5]在糖尿病小鼠视网膜血管内皮细胞中首次检测到circRNA-ZNF532,发现在高糖刺激下circRNA-ZNF532在视网膜血管周细胞中特异性表达增高,并调控血管周细胞功能。本研究检测到DR患者外周血清中circRNA-ZNF532表达较对照组增高。研究显示周细胞位于血管内皮和基板之间,可调节血管张力和灌注压,在血管成熟、稳态和重塑中起到关键作用,周细胞凋亡可打破周细胞与内皮细胞之间的紧密连接,导致血视网膜屏障的破坏,血管通透性增加,新生血管形成和炎症反应。研究显示DR患者视力丧失主要归因于视网膜血管周细胞变性、高通透性、低灌注和血管新生[13]。circRNA-ZNF532在周细胞的细胞质中表达,高糖、氧化应激或炎症刺激下其表达上调,负向调控miR-20b-5p表达,当miR-20b-5p表达下调后其下游靶点信号转导子和转录激活子3(STAT3)激活,启动炎症反应,促使高糖条件下细胞中IL-1β、IL-18、TNF-α、IL-6合成以及NLRP3炎症小体激活,降低视网膜血管周细胞活力,影响周细胞与内皮细胞之间相互作用,破坏视网膜血管屏障功能[14]。circRNA-ZNF532还可作为miR-1243的海绵间接上调miR-1243靶基因——共激活因子相关的精氨酸甲基转移酶1(CARM1)表达,诱导促炎细胞因子释放,导致视网膜血管内皮细胞损伤和功能障碍[15]。

3.3 circRNA-HIPK3与DR的关系 circRNA-HIPK3是一种多功能circRNA,起源于HIPK3基因外显子2,主要通过吸附不同的miRNA参与调控细胞凋亡、增殖、迁移和血管生成[16]。circRNA-HIPK3与白内障也存在密切关系,circRNA-HIPK3通过充当miR-193a的“海绵”,抑制miR-193a表达,上调αA-晶体蛋白(CRYAA)表达,增加晶状体上皮细胞的活力和增殖,在白内障组织中circRNA-HIPK3表达下调,导致miR-193a/CRYAA轴激活,进而加速氧化应激下细胞凋亡,降低人晶状体上皮细胞的活力并抑制其增殖[17]。本研究发现DR患者血清中circRNA-HIPK3表达增加,circRNA-HIPK3高表达是DR的危险因素,推测circRNA-HIPK3可能调节糖尿病相关视网膜血管功能。其机制可能为,在高血糖介导的氧化应激和炎症刺激下转录因子c-myb激活,继而上调circRNA-HIPK3表达,circRNA-HIPK3作为miR-30a-3p“海绵”,其过表达抑制miR-30a-3p表达,上调视网膜中VEGF、IL-2、IL-3、单核细胞趋化蛋白1(MCP-1)和TNF-α表达,降低血管内皮细胞活力,抑制其增殖和迁移,加剧糖尿病诱导的视网膜血管功能障碍[18]。本研究相关性分析也显示circRNA-HIPK3表达与VEGF、IL-1β、TNF-α、IL-6水平均呈正相关,表明circRNA-HIPK3过表达可能与炎症反应和血管形成有关。

3.4 circRNA-ZNF532、circRNA-HIPK3對DR的诊断价值 ROC分析结果显示联合circRNA-ZNF532、circRNA-HIPK3和VEGF后诊断DR具有较高价值,表明通过三者联合检测可为DR辅助诊断、病情评估提供更可靠的信息和参考,抑或为未来DR治疗提供新的靶点和方向。

综上,DR患者外周血清中circRNA-ZNF532、circRNA-HIPK3表达均显著上调,且两者高表达与DR发生有关,可作为DR辅助诊断和病情分析的潜在指标。circRNA-ZNF532、circRNA-HIPK3可能参与DR视网膜血管功能障碍调控过程,有望成为DR未来治疗的潜在靶点。

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(2023-01-17收稿 2023-04-22修回)

(本文編辑 李鹏)

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