CN115011685A - 与豆腐果苷抗抑郁作用显著相关的内源竞争rna调控网络 - Google Patents

与豆腐果苷抗抑郁作用显著相关的内源竞争rna调控网络 Download PDF

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CN115011685A
CN115011685A CN202210650896.2A CN202210650896A CN115011685A CN 115011685 A CN115011685 A CN 115011685A CN 202210650896 A CN202210650896 A CN 202210650896A CN 115011685 A CN115011685 A CN 115011685A
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童九翠
张远
张袁祥
张晓彤
蒋臻熠
蒋澜
钟正灵
杨斌
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Abstract

本发明公开了一种与豆腐果苷抗抑郁作用显著相关的内源竞争RNA调控网络，通过对正常组、慢性不可预测温和应激组和CUMS+豆腐果苷组大鼠海马组织进行全转录组RNA测序，对测序结果进行差异lncRNAs和mRNAs表达分析，筛选差异表达的关键基因；通过对lncRNA靶基因和差异表达基因进行GO和KEGG的富集化分析得到关键基因及其相关的lncRNAs，使用TargetScan和miRanda数据库预测与lncRNAs及其mRNAs有结合可能性及结合位点的miRNAs；通过分子生物学技术验证与豆腐果苷抗抑郁作用显著相关的内源竞争RNA调控网络：lncRNA‑Gnl3l/miR‑128‑3p/Prim1。该调控网络在抑郁症发生发展中具有重要的作用，通过探索抗抑郁药对该ceRNA网络的调控，对于抑郁症的早期诊断工具的开发、靶向药物的开发等方面具有重要的应用意义。

Description

与豆腐果苷抗抑郁作用显著相关的内源竞争RNA调控网络
技术领域
本发明属于医药技术领域，尤其涉及一种与豆腐果苷抗抑郁作用显著相关的内源竞争RNA(competing endogenous RNAs，ceRNA)调控网络及其构建方法与应用。
背景技术
抑郁症是社会-心理-生物因素相互作用诱发的一种严重危害人类身心健康的常见神经、精神类疾病，具有患病率高、易复发和高自杀率等特点。伴随着现代社会高节奏、高压力的生活趋势，抑郁症发病率呈逐年上升趋势，并有明显的复发倾向或趋于慢性化。
抑郁症的具体发病机制目前尚不明确，迄今有很多假说，如单胺类神经递质及其受体假说、神经内分泌假说、神经营养再生假说、炎症假说、表观遗传学机制等，各种生物学因素以总体模式相互作用，导致抑郁症的发生。
豆腐果苷(helicid)即4-甲酰苯基-β-D-吡喃阿洛糖苷(4-formylphenyl-β-D-allopyranoside)是从我国云南景颇族药山龙眼科植物萝卜树(Helicia nilagiricaBeed,异名Helicia erratica Hook)的果实中提取出的有效成分，其分子式为：Cl3H16O7；分子量为：284.26；熔点：199～200℃，毒性极低，安全性好，可恢复大脑皮层兴奋与抑制过程间的平衡失调，具有较好的镇静、安眠和镇痛作用，目前临床广泛用于缓解神经官能症的头疼、头昏及睡眠障碍，辅助治疗原发性头疼。
神经毒理学与畸形学研究结果表明，1350mg/kg的豆腐果苷灌胃给药后，不影响大鼠后代早期神经系统发育、神经行为功能或脑组织学。豆腐果苷因其镇静、镇痛、催眠、抗抑郁、低毒等作用而备受关注。豆腐果苷的神经保护作用涉及细胞兴奋毒性、一氧化氮系统、神经胶质细胞、生物膜、氧化性神经毒性和细胞凋亡等。研究发现豆腐果苷可以抑制胆碱酯酶活性。在整个大脑和突触结构中，豆腐果苷可以减少谷氨酰胺的增加，升高全脑γ-氨基丁酸(γ-aminobutyric acid，GABA)/谷氨酸(glutamic acid，Glu)比值，抑制全脑谷氨酰胺合成酶的活性，维持中枢神经系统兴奋与抑制平衡。豆腐果苷衍生物4-羟基苯甲醛是一种有效的抗氧化剂和GABA转氨酶抑制剂。在采用部分坐骨神经结扎(partial sciaticnerve ligation，PSNL)产生神经痛样模型小鼠中，豆腐果苷可降低PSNL小鼠下丘脑腹外侧视前区c-Fos表达，有效恢复降低的机械阈值，增加深睡期的睡眠，且剂量依赖性强，提示豆腐果苷是治疗神经性疼痛和睡眠障碍的有效药物。豆腐果苷可明显逆转小鼠自主活动减少及蔗糖偏嗜度降低，改善慢性应激小鼠的抑郁样行为。
人类基因组测序结果分析表明，全基因组中能够编码蛋白质的DNA仅占有很少一部分，绝大部分为不编码蛋白质的DNA，这些DNA转录形成的产物即为非编码RNA。非编码RNA(包含microRNA和long no-coding RNA(lncRNA))在细胞分化和代谢等生命活动中扮演着举足轻重的作用。lncRNA与miRNA之间可通过相互调控来影响抑郁症的发生发展。由于多数lncRNA的结构与mRNA具有一定的相似性，提示lncRNA通过与miRNA竞争结合靶基因mRNA的3'-UTR，间接抑制miRNA对靶基因的负向调控。而lncRNA-miRNA-mRNA三者调控机制即为ceRNA网络。
发明内容
本发明的首要目的在于提供一种与豆腐果苷抗抑郁作用显著相关的内源竞争RNA(competing endogenous RNAs，ceRNA)调控网络。
本发明的再一目的在于提供上述调控网络的构建方法。
本发明的另一目的在于提供上述调控网络在制备用于对抑郁症进行筛查、诊断或预后的工具以及筛选防治抑郁症靶向药物中的应用。
本发明是这样实现的，与豆腐果苷抗抑郁作用显著相关的内源竞争RNA调控网络，该调控网络为lncRNA-Gnl3l/miR-128-3p/Prim1；其中，lncRNA-Gnl3l来源于NONCODE数据库，miR-128-3p来源于miRbase数据库，Prim1来源于NCBI数据库。
本发明进一步公开了上述内源竞争RNA调控网络的构建方法，该方法包括以下步骤：
(1)通过对正常组、慢性不可预测温和应激组(chronic unpredictable mildstress,CUMS)和CUMS+豆腐果苷组大鼠海马组织进行全转录组RNA测序，对测序结果进行差异lncRNAs和mRNAs表达分析，筛选差异表达的关键基因；
(2)通过对lncRNA靶基因和差异表达基因进行GO和KEGG的富集化分析得到关键基因及其相关的lncRNAs，使用TargetScan和miRanda数据库预测与lncRNAs及其mRNAs有结合可能性及结合位点的miRNAs；
(3)通过分子生物学技术验证与豆腐果苷抗抑郁作用显著相关的内源竞争RNA(competing endogenous RNAs,ceRNA)调控网络。
本发明进一步公开了上述内源竞争RNA调控网络在制备用于对抑郁症进行筛查、诊断或预后的工具中的应用。
优选地，所述工具包括试剂盒、试纸、芯片或高通量测序平台。
优选地，所述试剂盒、试纸、芯片或高通量测序平台包括用于检测内源竞争RNA调控网络的引物或探针。
本发明进一步公开了上述内源竞争RNA调控网络在筛选防治抑郁症靶向药物中的应用。
本发明克服现有技术的不足，提供一种与豆腐果苷抗抑郁作用显著相关的内源竞争RNA(competing endogenous RNAs，ceRNA)调控网络及其构建方法与应用，本发明具体涉及一种天然植物药豆腐果苷在防治抑郁症中ceRNA网络的构建方法，实施例展示了通过此方法找出豆腐果苷干预下显著相关的ceRNA调控网络：lncRNA-Gnl3l/miR-128-3p/Prim1。并采用分子生物学手段验证了豆腐果苷对该ceRNA网络的调控以及三者的靶向互作关系。
相比于现有技术的缺点和不足，本发明具有以下有益效果：lncRNA-miRNA-mRNA三者调控网络在抑郁症发生发展中具有重要的作用，通过探索抗抑郁药对该ceRNA网络的调控，对于抑郁症的早期诊断工具的开发、靶向药物的开发等方面具有重要的应用意义。
附图说明
图1是豆腐果苷在防治抑郁症中ceRNA网络的构建方法的流程示意图；
图2是豆腐果苷干预下各组大鼠行为学指标的变化；
图3是豆腐果苷干预下lncRNAs与mRNAs的特征比较；
图4是豆腐果苷治疗后Group C_VS_Group M∩Group H_VS_Group M差异表达lncRNAs分布；
图5是豆腐果苷治疗后Group C_VS_Group M∩Group H_VS_Group M差异表达mRNAs分布；
图6A～图6D是豆腐果苷治疗后差异表达lncRNAs靶基因及差异表达基因GO和KEGG富集分析结果，其中，图6A是差异表达lncRNA靶基因GO富集分析；图6B是差异表达基因GO富集分析；图6C是差异表达lncRNA靶基因KEGG富集分析；图6D是差异表达基因KEGG富集分析；
图7是豆腐果苷干预下显著相关的ceRNA调控网络图；
图8是qRT-PCR验证lncRNA-Gnl3l/miRNA-128-3p/Prim1在海马组中的表达水平及核质分离实验结果；
图9是双荧光素酶报告测定lncRNA-Gnl3l与miRNA-128-3p结合活性结果；
图10是双荧光素酶报告测定miRNA-128-3p与Prim1结合活性结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
与豆腐果苷抗抑郁作用显著相关的内源竞争RNA调控网络，该调控网络为lncRNA-Gnl3l/miR-128-3p/Prim1，该调控网络的构建方法，如图1所示，该方法包括以下步骤：
(1)通过对正常组、慢性不可预测温和应激(chronic unpredictable mildstress,CUMS)组和CUMS+豆腐果苷组大鼠海马组织进行全转录组RNA测序，对测序结果进行差异lncRNAs和mRNAs表达分析，筛选差异表达的关键基因
在步骤(1)中，首先建立CUMS大鼠抑郁模型，连续CUMS造模6周并在第7周开始给予豆腐果苷(32mg/kg/day)治疗，第12周时通过行为学检测如强迫游泳实验(FST)、旷场实验(OFT)、蔗糖饮水偏好实验(SPT)等证实豆腐果苷可以改善CUMS大鼠的抑郁样行为(图2)。
其次，应用NGS(Next Generation Sequencing，二代高通量测序)技术及高通量测序仪(如：illumina Hiseq 2000/2500、Miseq)对正常组、CUMS组和CUMS+HEL组大鼠海马组织进行全转录组测序，对豆腐果苷干预下各样本间lncRNAs和mRNAs的表达特征进行分析，结果发现lncRNAs比mRNAs具有更小的尺寸；接着对lncRNA和mRNA各个转录本的表达值取平均，用两者FPKM数值绘制箱型图后发现两者的表达水平没有明显差异(图3)。
接着，应用edgeR软件包进行样本间差异lncRNAs及基因分析，得出p-value。同时，本发明根据FPKM值计算差异表达倍数，即Fold-change。对测序数据进行筛选分析，筛选条件为：P<0.05，|log2FC|>1.5，筛选豆腐果苷干预下差异表达lncRNAs及mRNAs。基于CUMS抑郁大鼠模型(Group M)，比较正常组(Group C)、CUMS+豆腐果苷组(Group H)各样本间差异表达lncRNAs与mRNAs的分布。Group C_VS_Group M∩Group H_VS_Group M交集显示有173个上调的差异表达lncRNAs重叠，135个下调的差异表达lncRNAs重叠(图4)；同样有交集显示有103个上调的差异表达mRNAs重叠，69个下调的差异表达mRNAs重叠(图5)。需要说明的是，图4、5中A、B分别为显著上调及下调差异表达lncRNAs与mRNAs分布，其中的数值代表两个比较组间共有的差异表达lncRNAs或mRNAs数目。
最后，采用blast选择出序列上具有互补性或相似性的序列，再利用RNAplex计算两序列之间的互补能量，选择出阈值以上的序列，从而确定豆腐果苷干预下lncRNAs与mRNAs的靶向关系。
(2)通过对差异表达lncRNA靶基因和差异表达基因进行GO和KEGG的富集分析得到与抑郁症相关的关键基因及lncRNAs，使用TargetScan和miRanda数据库预测与lncRNAs及其mRNAs有结合可能性及结合位点的miRNAs
基于步骤(1)中得到差异表达lncRNAs的靶基因，本发明采用R语言的ClusterProfiler包对差异表达lncRNAs靶基因及差异表达基因进行GO、KEGG富集分析，GO富集分析结果显示显示豆腐果苷主要参与对氧化应激、凋亡等生物学进程的调控；KEGG富集分析结果包括PI3K-Akt、MAPK信号通路等抑郁症经典通路(图6A～图6D)，并根据差异表达基因在不同信号通道中的分类信息，筛选关键基因及其相关的lncRNAs。
根据所筛选的关键基因及其相关的lncRNAs，依据转录组测序数据对lncRNAs-mRNAs表达趋势进行评估，得到在正常组、CUMS组和CUMS+豆腐果苷组中具有一致表达模式的lncRNAs-mRNAs，如下表1、表2所示：
表1正常组VS模型组lncRNAs-mRNAs差异表达模式表
Figure BDA0003686079900000061
Figure BDA0003686079900000071
表2豆腐果苷组VS模型组lncRNA-mRNA差异表达模式表
Figure BDA0003686079900000072
基于所得到具有一致表达模式的lncRNA-Gnl3l和Prim1。使用TargetScan数据库预测能与Prim13'UTR结合的miRNAs，共得到56个miRNAs。从NONCODE数据库获取lncRNA-Gnl3l的序列；从miRBase数据库获取预测miRNAs的序列。使用miRanda预测lncRNA-Gnl3l与56个miRNAs的结合可能性及结合位点，miRanda程序设置默认“score＝140,energy＝1”，共得到5个miRNA，如下表3所示：
表3lncRNA-Gnl3l/Prim1结合miRNAs表
Figure BDA0003686079900000073
基于所得到的ceRNA网络，根据差异表达的相关lncRNAs、miRNAs和关键基因在豆腐果苷干预下的表达趋势进行评估，结合文献([1]Shi WZ,Li W,Cheng Y,Zhang M,NiuXC,Gao QW,Lu Y,Tian T,Du S,Mi Y,Chang MZ,Tian Y.The cytoprotective role ofomentin against oxidative stress-induced PC12apoptosis.Artif Cells NanomedBiotechnol.2021Dec；49(1):483-492.doi:10.1080/21691401.2021.1892707.PMID:34151664.[2]Yamaguchi M,Fujimori-Tonou N,Yoshimura Y,Kishi T,Okamoto H,MasaiI.Mutation ofDNA primase causes extensive apoptosis ofretinal neurons throughthe activation ofDNA damage checkpoint and tumor suppressorp53.Development.2008Apr；135(7):1247-57.doi:10.1242/dev.011015.Epub 2008Feb20.PMID:18287205.[3]Qi D,Chen K.Bioinformatics Analysis of PotentialBiomarkers and Pathway Identification for Major Depressive Disorder.ComputMath Methods Med.2021Aug 3；2021:3036741.doi:10.1155/2021/3036741.PMID:34394704；PMCID:PMC8357473.)支持得到最终的豆腐果苷干预下显著相关的ceRNA调控网络lncRNA-Gnl3l/miR-128-3p/Prim1(图7)。
(3)通过分子生物学技术验证与豆腐果苷抗抑郁作用显著相关的内源竞争RNA调控网络
在步骤(3)中，采用分子生物学技术(分子生物学技术主要包括：qRT-PCR、Westernblot、核质分离实验、双荧光素酶报告实验等)验证豆腐果苷调控的ceRNA网络，qRT-PCR技术验证lncRNA-Gnl3l/miR-128-3p/Prim1在各组大鼠海马组织中的表达水平，结果显示，相对于Control组，lncRNA-Gnl3l和Prim1在CUMS组中高表达，miRNA-128-3p低表达；而豆腐果苷可以明显降低CUMS刺激引起的lncRNA-Gnl3lTgm2和Prim1的高表达，升高miRNA-128-3p的表达水平(图8A～8C)。采用核质分离技术确定了LncRNA-Tgm2的细胞定位。U6和GAPDH均被用作对照组。在细胞的细胞质部分检测到80.1％的LncRNAlncRNA-Gnl3l，表明其可能在转录后水平发挥作用(图8D)。同样，双荧光素酶报告实验结果表明，miR-128-3p的过表达可以抑制lncRNA-Gnl3l-WT载体的荧光素酶活性，而lncRNA-Gnl3l-MUT载体的荧光素酶活性没有变化(图9)。而对于Prim1与miRNA-128-3p而言，双荧光素酶报告基因检测结果表明，miRNA-128-3p过表达可降低Prim1-3‘UTR-WT载体的荧光素酶活性，而不影响Prim1-3’UTR-MUT载体的荧光素酶活性(图10)。
本发明调控网络在制备用于对抑郁症进行筛查、诊断或预后的工具以及筛选防治抑郁症靶向药物中的应用，本发明的用途参详现有技术能进行清楚的应用，具体参考文献([1]Tong J,Zhou Z,Qi W,Jiang S,Yang B,Zhong Z,Jia Y,Li X,Xiong L,NieL.Antidepressant effect of helicid in chronic unpredictable mild stress modelin rats.Int Immunopharmacol.2019Feb；67:13-21.doi:10.1016/j.intimp.2018.11.052.Epub 2018Dec 6.PMID:30530165.[2]Li XY,Qi WW,Zhang YX,Jiang SY,Yang B,Xiong L,Tong JC.Helicid Ameliorates Learning and CognitiveAbility and Activities cAMP/PKA/CREB Signaling in Chronic Unpredictable MildStress Rats.Biol Pharm Bull.2019；42(7):1146-1154.doi:10.1248/bpb.b19-00012.PMID:31257291.[3]Zhang Y,Luan D,Liu Y,Li H,Dong J,Zhang X,Yuan L,ZhongZ,Jiang L,Li X,Ye M,Tong J.Helicid Reverses Lipopolysaccharide-InducedInflammation and Promotes GDNF Levels in C6Glioma Cells through Modulation ofPrepronociceptin.Chem Biodivers.2020Jul；17(7):e2000063.doi:10.1002/cbdv.202000063.Epub 2020Jun 17.PMID:32329965.[4]Zhang YX,Zhang XT,Li HJ,ZhouTF,Zhou AC,Zhong ZL,Liu YH,Yuan LL,Zhu HY,Luan D,Tong JC.Antidepressant-likeeffects ofhelicid on a chronic unpredictable mild stress-induced depressionrat model:Inhibiting the IKK/IκBα/NF-κB pathway through NCALD to reduceinflammation.Int Immunopharmacol.2021Apr；93:107165.doi:10.1016/j.intimp.2020.107165.Epub2021Feb 9.PMID:33578182.[5]Zhang XT,Zhang Y,Zhang YX,Jiang ZY,Yang H,Jiang L,Yang B,Tong JC.Helicid Reverses the Effect of OverexpressingNCALD,Which Blocks the sGC/cGMP/PKG Signaling Pathway in the CUMS-Induced RatModel.J Healthc Eng.2021Dec 11；2021:7168397.doi:10.1155/2021/7168397.PMID:34931140；PMCID:PMC8684516.[6]Zhang Y,Wang M,Zhang X,Jiang Z,Zhang Y,Fu X,LiY,Cao D,Han J,Tong J.Helicid Improves Lipopolysaccharide-Induced Apoptosis ofC6 Cells by Regulating SH2D5DNA Methylation via the CytC/Caspase9/Caspase3Signaling Pathway.Contrast Media Mol Imaging.2022Jan 31；2022:9242827.doi:10.1155/2022/9242827.PMID:35173561；PMCID:PMC8820944.)。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (6)

1.与豆腐果苷抗抑郁作用显著相关的内源竞争RNA调控网络，其特征在于，该调控网络为lncRNA-Gnl3l/miR-128-3p/Prim1。
2.权利要求1所述的内源竞争RNA调控网络的构建方法，其特征在于，该方法包括以下步骤：
(1)通过对正常组、慢性不可预测温和应激组和CUMS+豆腐果苷组大鼠海马组织进行全转录组RNA测序，对测序结果进行差异lncRNAs和mRNAs表达分析，筛选差异表达的关键基因；
(2)通过对lncRNA靶基因和差异表达基因进行GO和KEGG的富集化分析得到关键基因及其相关的lncRNAs，使用TargetScan和miRanda数据库预测与lncRNAs及其mRNAs有结合可能性及结合位点的miRNAs；
(3)通过分子生物学技术验证与豆腐果苷抗抑郁作用显著相关的内源竞争RNA调控网络。
3.权利要求1所述的内源竞争RNA调控网络在制备用于对抑郁症进行筛查、诊断或预后的工具中的应用。
4.如权利要求3所述的应用，其特征在于，所述工具包括试剂盒、试纸、芯片或高通量测序平台。
5.如权利要求4所述的应用，其特征在于，所述试剂盒、试纸、芯片或高通量测序平台包括用于检测内源竞争RNA调控网络的引物或探针。
6.权利要求1所述的内源竞争RNA调控网络在筛选防治抑郁症靶向药物中的应用。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110136738A1 (en) * 2008-02-15 2011-06-09 University College Dublin, National University Of Ireland, Dublin Alternatively Transcribed Genes Associated with Schizophrenia
CN103237901A (zh) * 2010-03-01 2013-08-07 卡里斯生命科学卢森堡控股有限责任公司 用于治疗诊断的生物标志物
CN108463561A (zh) * 2015-11-20 2018-08-28 布兰迪技术责任有限公司 用于诊断、预后和治疗监测基于神经性、神经退行性和炎症的疾病的包含基于微rna的小神经胶质细胞微泡的方法
CN110317866A (zh) * 2019-05-07 2019-10-11 中国人民解放军联勤保障部队第九0四医院 精神分裂症小鼠模型海马circRNA测序分析及试剂盒
WO2020193809A1 (en) * 2019-03-28 2020-10-01 University Of Ulster A method for predicting a psychiatric disorder
CN112921084A (zh) * 2021-04-25 2021-06-08 东南大学 外周血全血长链非编码rna作为诊断抑郁症的用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110136738A1 (en) * 2008-02-15 2011-06-09 University College Dublin, National University Of Ireland, Dublin Alternatively Transcribed Genes Associated with Schizophrenia
CN103237901A (zh) * 2010-03-01 2013-08-07 卡里斯生命科学卢森堡控股有限责任公司 用于治疗诊断的生物标志物
CN108463561A (zh) * 2015-11-20 2018-08-28 布兰迪技术责任有限公司 用于诊断、预后和治疗监测基于神经性、神经退行性和炎症的疾病的包含基于微rna的小神经胶质细胞微泡的方法
WO2020193809A1 (en) * 2019-03-28 2020-10-01 University Of Ulster A method for predicting a psychiatric disorder
CN110317866A (zh) * 2019-05-07 2019-10-11 中国人民解放军联勤保障部队第九0四医院 精神分裂症小鼠模型海马circRNA测序分析及试剂盒
CN112921084A (zh) * 2021-04-25 2021-06-08 东南大学 外周血全血长链非编码rna作为诊断抑郁症的用途

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BHASKAR ROY等: "Amygdala-Based Altered miRNome and Epigenetic Contribution of miR-128-3p in Conferring Susceptibility to Depression-Like Behavior via Wnt Signaling", 《INTERNATIONAL JOURNAL OF NEUROPSYCHOPHARMACOLOGY》 *
DONG QI等: "Bioinformatics Analysis of Potential Biomarkers and Pathway", 《COMPUTATIONAL AND METHODS IN MEDICINE》 *
MIGUEL A. ORTEGA等: "MicroRNAs as Critical Biomarkers of Major Depressive Disorder: A Comprehensive Perspective", 《BIOMEDICINES》 *
TING ZHOU等: "Chronic Stress-Induced Gene Changes In Vitro and In Vivo: Potential Biomarkers Associated With Depression and Cancer Based on circRNA- and lncRNA-Associated ceRNA Networks", 《FRONTIERS IN ONCOLOGY》 *
张艳华等: "microRNA在抑郁症中的研究进展", 《临床精神医学杂志》 *
童九翠等: "豆腐果苷衍生物W0620对行为绝望小鼠抗抑郁作用的研究", 《中国药理学通报》 *

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