Nan Fang Yi Ke Da Xue Xue Bao.
2020 Jul 20; 40(7): 922–929.
Language:
Chinese
|
English
卒中后抑郁患者血浆中miR-30a-5p的差异性表达及其作用机制的生物信息学预测
Differential expression of miR-30a-5p in post stroke depression and bioinformatics analysis of the possible mechanism
胡 佳
皖南医学院附属弋矶山医院神经内科，安徽 芜湖 241001,
Department of Neurology, Yijishan Hospital Affiliated to Wannan Medical College, Wuhu 241001, China
周 志明
皖南医学院附属弋矶山医院神经内科，安徽 芜湖 241001,
Department of Neurology, Yijishan Hospital Affiliated to Wannan Medical College, Wuhu 241001, China
杨 倩
皖南医学院附属弋矶山医院神经内科，安徽 芜湖 241001,
Department of Neurology, Yijishan Hospital Affiliated to Wannan Medical College, Wuhu 241001, China
杨 科
皖南医学院附属弋矶山医院神经内科，安徽 芜湖 241001,
Department of Neurology, Yijishan Hospital Affiliated to Wannan Medical College, Wuhu 241001, China
皖南医学院附属弋矶山医院神经内科，安徽 芜湖 241001,
Department of Neurology, Yijishan Hospital Affiliated to Wannan Medical College, Wuhu 241001, China
P
NIHSS: National institutes of health stroke scale; PSD: Post-stroke depression.Age (year)60.33±6.6659.82±8.140.856Gender (male/female)5/612/131.000Education (%) 4 (36.4)10 (40.0)1.000Hypertension (%) 11 (100.0)25 (100.0)1.000Diabetes mellitus (%) 5 (45.5)13 (52.0)1.000Hyperlipidemia (%) 3 (27.3)10 (40.0)0.708Heart disease (%) 5 (45.6)7 (28.0)0.446Smoking (%) 5 (45.6)16 (64.0)0.465Previous stroke (%) 6 (54.5)17 (68.0)0.475NHISS > 3 (%) 7 (63.6)11 (44.0)0.471Stroke location: Left7 (63.6)
19 (76.0)
0.454
Right4 (36.4)6 (24.0)
2.3. 卒中后抑郁miR-30a-5p靶基因预测及筛选
通过ENCORI在线网站，预测到miR-30a-5p的靶基因共有602种，CTD数据库检索到抑郁症相关的基因3835种，二者取交集得到162种蛋白（
）。
2.4. 所预测靶基因合集的GO分析和KEGG通路富集分析
为了进一步了解靶基因的生物学作用，我们使用FUNRICH软件进行162种靶基因的GO分析，Cellular component分析提示靶基因主要位于细胞浆和细胞核中，分别占总蛋白数量的55.479%和54.795%（
）；Molecular function分析提示靶基因主要具有转录因子功能（11.392%）、GTP酶活性（4.430%），鸟苷酸交换因子活性（3.165%），半乳糖基转移酶活性（1.899%），调节磷酸化酶作用（1.266%），羧基裂解酶活性（1.266%），激酶调节作用（1.266%）（
）；Biology process分析提示靶基因主要参与信号转导（34.810%），细胞间通讯（30.380%），核碱基、核苷、核苷酸和核酸代谢的调节（21.519%）（
）。经KEGG通路富集分析发现，靶基因主要作用于神经营养素信号通路、轴突导向信号通路、胰岛素信号传导系统（
）。
3. 讨论
PSD是脑卒中后常见的并发症之一，以长期持续的情绪低落为特征，是一种与卒中相关的情感性精神障碍，影响1/3一卒中患者近5年的生存质量。但目前临床上尚缺乏稳定、便捷的早期预测PSD的指标。
MicroRNA是一类小分子非编码单链RNA，通过部分性或完全性与靶mRNA的3'非翻译区（3'UTR）互补配对，阻止其翻译或使其降解，从而对靶基因起到转录后调节作用。既往文献报道，MicroRNAs参与并调控中枢神经系统的许多生物学过程，与内皮功能障碍、细胞凋亡、细胞增殖、炎症反应、氧化应激、血管生成和神经生成相关<sup>[<xref ref-type="bibr" rid="b13">13</xref>-<xref ref-type="bibr" rid="b14">14</xref>]</sup>。此外，外周血miRNA的理化性质和表达相对稳定，获得途径、检测手段相对简单、微创和廉价，且在疾病早期就可被检出，一部分miRNA已经成为某些肿瘤和缺血性卒中的生物标记物<sup>[<xref ref-type="bibr" rid="b15">15</xref>-<xref ref-type="bibr" rid="b16">16</xref>]</sup>。但有关MicroRNA和卒中后抑郁的研究很少，我们检索了公共基因表达GEO（<a href="https://www.ncbi.nlm.nih.gov/geo/" target="_blank">https://www.ncbi.nlm.nih.gov/geo/</a>）数据库，未发现与PSD相关的MicroRNA芯片信息。
本研究中我们通过文献检索与卒中以及抑郁相关的血浆miRNA，应用VENNY在线软件对检索大数据取交集，得到同时与卒中和抑郁都存在相关性的血浆microRNA: miR-30a-5p，而目前为止miR-30a-5p与PSD的关系尚未被报道。据我们所知，我们首先报道了miR-30a-5p与PSD的相关性。并通过临床样本的QRT-PCR初步验证了其与PSD的相关性。此外，ROC曲线分析发现miR-30a-5p预测PSD的AUC=0.869（95%
CI
，0.745-0.993，
P
=0.0005），cut-off值为1.597，对应的敏感性和特异性分别为0.727、0.840。既往文献关于卒中后3个月PSD血液标志物的研究报道较少。Li等
[
17
]
认为入院时较低的血清BDNF水平可以预测3个月内PSD的发生，敏感性为73.2%，特异性为70.7%。而与BDNF相比，循环miR-30a-5p对卒中后3个月PSD的预测准确性更高，敏感性为72.7%，特异性为84.0%。因此，我们认为患者入院时血浆miR-30a-5p很可能是预测PSD的一种新的生物标志物。
为进一步了解外周血差异性表达的miR-30a-5p对卒中后3个月PSD的可能作用机制，我们对miR-30a-5p的进行了生物信息血分析。为降低靶基因预测依赖于“种子区”碱基的互补所造成的的假阳性率，我们采用ENCORI在线数据库和CTD数据库的交叉预测，并同时预测到与抑郁相关的miR-30a-5p的靶基因162种。KEGG通路富集分析发现，这些靶蛋白主要作用于神经营养素信号通路、轴突导向信号通路、胰岛素信号传导系统。由于上述生物学功能涉及到PSD发生、发展的各个阶段，我们推测miR-30a-5p可能通过调控其下游一系列靶基因的表达进而引起多种生物学特性改变，参与PSD的发生发展。其中神经营养素信号转导和轴突发育是最为突出的生物学过程。这可能为我们了解卒中后3个月PSD的发病机制提供了新的线索。轴突发育与神经细胞再生和神经的可塑性相关，轴突生长被认为是一种自发形式的神经可塑性，在神经系统发育和中枢神经系统损伤后功能恢复中起关键作用
[
18
]
。而神经营养素信号通路（如Ras-Raf-MAP激酶信号通路）在神经营养受体下游的级联反应是控制轴突生长的关键细胞信号通路
[
19
]
。既往研究中神经可塑性假说被用来解释重度抑郁的病因
[
20
]
以及脑卒中患者康复阶段认知功能的改变
[
21
]
。而我们的研究中预测到与抑郁相关的miR-30a-5p的靶蛋白主要作用于神经营养素信号转导和轴突发育等生物学过程，参与神经可塑性，从而参与PSD可能病因机制，这与既往研究相符。
同时我们对PSD相关的162种基因建立PPI网络分析图，并筛选出前20种关键基因，并建立与BDNF相关的PPI网络分析图。在本研究中我们首次发现并提出miR-30a-5p可以调控BDNF基因。脑源性神经营养因子（BDNF）是一种重要的神经营养因子，影响神经元增殖、突触功能和突触可塑性
[
22
]
。而神经元可塑性的破坏已被证明在PSD的发病过程中起重要作用。BDNF与脑单胺类神经递质关系密切，是5-羟色胺能神经元的强效营养剂
[
23
]
，其在抗抑郁药物作用机制中的作用已被证实。啮齿类动物研究表明，BDNF合成受损的转基因小鼠对抗抑郁药物没有反应
[
24
]
。在人类研究中，抑郁症患者外周BDNF水平降低，服用抗抑郁药
[
25
]
可使其恢复正常。BDNF含量减少和单核苷酸位点突变被证明与PSD的发生发展具有相关性
[
26
]
。而miR-30a-5p可以调控BDNF基因从另一个角度验证了miR-30a-5p与PSD的相关性。而筛选出的其他HUB基因通过不同的蛋白信号传导通路参与神经的可塑性和轴突的生长发育，从而与缺血后神经的修复和海马区神经元的可塑性相关
[
27
-
31
]
，且都与BNDF基因有不同程度的相关性。
综上所述，我们采用生物信息学方法对miR-30a-5p的生物学特性及其功能进行了分析，并通过小规模临床样本QRT-PCR进行了验证，提示外周血miR-30a-5p在卒中后抑郁患者和非卒中后抑郁患者中存在差异性表达，可能与卒中后抑郁的发病机制密切相关。miR-30a-5p可能是诊断缺血性脑卒中后3个月PSD的一个新的血液学标志物。通过调控miR-30a-5p的表达，从而调控BDNF，可能作为PSD的潜在诊疗靶点应用于临床实践中。但相关结论尚需要大规模临床样本进一步验证，miR-30a-5p作用于PSD的具体机制尚需动物实验进一步证实。
Funding Statement
国家自然科学基金（81171110）；2015年度皖南医学院中青年科研基金（WK2015F16）
Supported by National Natural Science Foundation of China (81171110)
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