CN117089611A - lncRNA-Gm13561作为新靶标在糖尿病肾病诊断和治疗中的应用 - Google Patents
lncRNA-Gm13561作为新靶标在糖尿病肾病诊断和治疗中的应用 Download PDFInfo
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Abstract
本发明公开了种lncRNA‑Gm13561作为新靶标在糖尿病肾病诊断和治疗中的应用。本发明通过db/db小鼠构建DN动物模型，并对正常小鼠及DN小鼠肾组织进行单细胞测序，筛选出与DN足细胞中特异表达的lncRNA，通过细胞生物学及分子化学等技术对足细胞特异表达lncRNA进行验证和机制研究。经本发明研究证实：肾脏足细胞中的lncRNA‑Gm13561在糖尿病肾病中显著减少，发现其可以通过缓解足细胞的凋亡和ROS引起的氧死亡从而减缓糖尿病肾病的发生发展。本发明研究证明lncRNA‑Gm13561可以作为新的药物靶点在糖尿病肾病治疗中应用，也可作为新的临床诊断指标。
Description
技术领域
本发明涉及生物医学技术领域，具体涉及一种lncRNA-Gm13561作为新靶标在糖尿病肾病诊断和治疗中的应用。
背景技术
糖尿病肾病(diabetic nephropathy,DN)现在已成为我国慢性肾脏疾病的主要因素，它会诱发患者终末期肾功能衰竭，最终导致患者死亡。研究表明，在长期高血糖的环境下，DN的病理特征主要为肾小球硬化及肾间质纤维化，从而引起尿蛋白排泄率增加及肾功能减退。足细胞(podocyte)，即肾小囊脏层上皮细胞，其位于肾小球基底膜的外侧，与血管内皮细胞和肾小球基底膜一起构成了肾小球血液滤过屏障。而DN患者的足细胞发生损伤和凋亡，是导致大量蛋白尿和肾小球硬化的主要因素之一。因此，足细胞在肾小球损伤和修复过程中具有重要的作用，深入研究足细胞与DN的关系，是DN防治研究的重要课题，对DN发病机制及临床诊疗具有重要意义。
大量研究数据表明，真核生物转录RNA中，绝大多数为非编码RNA。其中，长链非编码RNA(long non-coding RNA，lncRNA)是一类长度大于200nt的核苷酸，缺少特异完整的开放阅读框，无编码蛋白质功能的非编码RNA。虽然lncRNA缺乏编码蛋白质的能力，但它仍具有广泛的生物学功能，如参与染色体修饰与重塑、调节靶基因转录、作为miRNA海绵(miRNAsponges)或竞争性内源RNA(Competitive endogenous RNA，ceRNA)调控靶基因翻译、结合蛋白质调节蛋白质活性或者定位等多种方式参与细胞的增殖、分化和代谢过程。现有研究已证明许多lncRNA在DN的发生发展中对足细胞的损伤和凋亡具有重要作用。然而，lncRNA与DN发生发展的具体关系仍未完全明确。
发明内容
本发明的目的是针对上述问题，提供一种lncRNA-Gm13561作为新靶标在糖尿病肾病诊断和治疗中的应用。
本发明为了实现其目的，采用的技术方案是：
本发明第一方面提供了检测样本中生物标志物的试剂在制备诊断糖尿病肾病的产品中的应用，所述生物标志物为lncRNA-Gm13561。
在上述的应用技术方案中，与正常对照相比，lncRNA-Gm13561在患者中的表达水平下调。
在上述的应用技术方案中，患者的待测样本中lncRNA-Gm13561的含量通过总RNA提取、反转录、定量PCR进行检测得到。
本发明第二方面提供了lncRNA-Gm13561表达促进剂在制备防治糖尿病肾病的药物中的应用。
本发明第三方面提供了lncRNA-Gm13561作为靶标在筛选防治糖尿病肾病的药物中的应用。
在上述的应用技术方案中，所述药物促进lncRNA-Gm13561的表达。
lncRNA-Gm13561的的cDNA序列如SEQ ID NO.1所示。
在上述的应用技术方案中，lncRNA-Gm13561调控足细胞凋亡和ROS积累，缓解足细胞的凋亡和ROS引起的氧死亡。
在上述的应用技术方案中，Gm13561调控氧死亡通路KEAP1-PGAM5-AIFM1。
在上述的应用技术方案中，Gm13561的上调促使足细胞中Bcl-2和podocin的表达升高，Bax和cleaved-caspase-3的表达降低，Keap1，PGAM5和AIFM1的表达减少，AIFM1的磷酸化水平增加。
本发明的有益效果是：
通过db/db小鼠构建DN动物模型，并对正常小鼠及DN小鼠肾组织进行单细胞测序，筛选出与DN足细胞中特异表达的lncRNA，通过细胞生物学及分子化学等技术对足细胞特异表达lncRNA进行验证和机制研究。通过本发明研究，探索与DN发生发展过程中足细胞损伤和凋亡相关的lncRNA参与疾病发生的具体机制，为DN临床诊断找寻新的靶点。
经本发明研究证实：肾脏足细胞中的lncRNA-Gm13561在糖尿病肾病中显著减少，发现其可以通过缓解足细胞的凋亡和ROS引起的氧死亡从而减缓糖尿病肾病的发生发展。本发明研究证明lncRNA-Gm13561可以作为新的药物靶点在糖尿病肾病治疗中应用，也可作为新的临床诊断指标。
附图说明
图1是基于单细胞核测序的DN足细胞特异性lncRNA表达谱建立实验结果：A:DN小鼠与正常小鼠的体重、肾重、血糖以及尿白蛋白肌酐比值；B、C:基于单细胞核测序，对肾脏细胞进行分群；D:基于单细胞核测序，24个足细胞特异性lncRNA在各细胞亚群中的表达水平(P<0.05)；E:在疾病组中差异表达的TOP20的lncRNAs,上调有4个lncRNAs；下调有16个lncRNAs(P<0.05)；F:根据差异表达的基因及在足细胞中特异表达的基因筛选出6个候选基因；G:6个候选基因在疾病组和正常组中差异表达倍数；H:6个候选基因在各细胞亚群中的表达水平。
图2是PCR鉴定lncRNAGm13561的高低糖肾脏固有细胞中肾小球足细胞细胞特异性及lncRNAGm13561生物学特点实验结果：A:用qRT-PCR在足细胞、系膜细胞以及上皮细胞中验证6个候选基因在足细胞中表达的特异性；B:用qRT-PCR在正常糖及高糖培养的足细胞中验证6个候选基因的差异表达；C:利用qRT-PCR检测Gm13561在足细胞中胞核与胞质中的表达；D:利用FISH检测Gm13561在足细胞中的定位。
图3是过表达和敲低Gm13561调控足细胞损伤与凋亡实验结果：A:qRT-PCR检测转染Gm13561过表达质粒后Gm13561的表达；B:qRT-PCR检测转染Gm13561三条siRNAs后Gm13561的表达；C:选择单细胞测序结果中在足细胞中特异高表达的基因且在Gm13561过表达后差异表达的基因(一共48个)利用KOBAS 3.0进行功能分类的富集分析；D:流式细胞术检验Gm13561过表达和敲低后足细胞的凋亡情况；E:流式细胞术检测荧光强度以评估Gm13561过表达和敲低后足细胞中ROS水平；F:WB检测Gm13561过表达和敲低后足细胞凋亡及氧死亡标志物的表达。
具体实施方式
下面结合实施例对本发明作进一步说明，但并不因此而限制本发明。
下述实施例中的实验方法，如无特别说明，均为常规方法；所用材料和试剂，如无特殊说明，均为本领域常规材料和试剂，均可商购获得。
实施例1
1实验方法
1.1细胞培养
本实验室保存了DKD小鼠肾小球足细胞(pod)、肾小管上皮细胞(TC)和肾小球系膜细胞(MCs)。在DMEM培养基(Gibco,Carlsbad,CA,USA)中添加10％胎牛血清和1％青霉素-链霉素溶液，在37℃含5％CO2的孵箱中培养。以往许多研究表明，高糖条件模拟了小鼠MPC5在DN状态下的生长环境，而低糖条件模拟了正常生长状态。相应的，用5.5mmol/L葡萄糖(低糖组)或25mmol/L葡萄糖(高糖组)刺激细胞。
1.2单细胞RNA测序
分别从db/db DKD(糖尿病肾病，DKD)小鼠(n＝3)和对照组小鼠(n＝3)中提取肾组织，制备单细胞悬液。利用Illumina测序平台(Omicsmart,广州,中国)的双端测序模式对构建的文库进行SnRNAseq检测。利用归一化数据函数、RunTSNE函数、搜索聚类函数对源数据进行整合、归一化、t-SNE降维，并进行细胞聚类分析，揭示单细胞水平上的差异表达基因。
1.3 RNA提取，细胞质/核分离和qRT-PCR检测
使用SteadyPure Mag Tissue&Cells RNA提取试剂盒(Accurate Biology，湖南，中国)按照制造商的说明提取细胞或组织的总RNA，然后进行反转录和qRT-PCR。细胞质和细胞核分离使用PARISTMKit(Thermo Fisher scientific，美国)进行。按照试剂盒说明书分离各部分的RNA，然后进行逆转录和qRT-PCR。用PrimeScript RT Reagent Kit(Takara，日本)按照试剂盒说明将总RNA合成为cDNA。cDNA用TB Green Premix Ex Taq(Takara，日本)在Bio-Rad CFX96系统(Bio-Rad,CA,美国)上扩增。2-ΔΔCT测定mRNA表达，β-actin归一化。引物序列如表1中所示：
表1
1.4过表达和敲低Gm13561
提取足细胞基因组DNA，扩增全长Gm13561，将PCR产物克隆到pcDNA 3.1表达载体上。质粒由内毒素去除质粒提取试剂盒(Omega,GA,USA)按照制造商的协议提取，经酶切消化(Gm13561过表达质粒的酶切位点为BamHI和EcoR V)，PCR和测序鉴定。合成了靶向Gm13561的siRNA。过表达质粒及siRNA具体由北京擎科生物有限公司构建提供。细胞转染采用Lipofectamine 2000(Invitrogen,Carlsbad,CA,USA)或HighGene plus转染试剂(abClonal，中国武汉)，按照制造商的说明进行。siRNA序列如表2中所示。
表2
1.5 RNA-sequencing
采用Illumina HiSeq测序(Novogene,北京,中国)分析HG-足细胞中Gm13561上调后mRNA的差异表达，并与NC组进行比较。数据分析由Novogene公司(北京，中国)进行。
1.6 Western blot分析
用含有PMSF的RIPA裂解缓冲液提取细胞或组织的总蛋白，并进行SDS-PAGE，然后转移到PVDF膜上(Millipore,Billerica,MA,USA)。用10％脱脂乳封闭膜，用一抗4℃孵育过夜，用酶标二抗(1:10000,Proteintech,USA)室温孵育2h。最后使用SuperSignalTMWestFemto Maximum Sensitivity Substrate(Termo Fisher Scientifc)和ECL WesternBlotting Substrate Kit(Millipore,USA)对条带进行可视化。特异性抗体稀释比如下anti-cleaved-caspase-3(1:5000,Immunoway，美国)，anti-bax(1:1000,Proteintech，美国)，anti-bcl-2(1:50,Abmart，中国上海)，anti-podocin(1:50,Bioss，中国北京)，anti-p-aifm1(1:50,ECM biosciences，美国)，anti-aifm1(1:50,Immunoway，美国)，anti-keap1(1:50,Immunoway，美国)，anti-pgam5(1:50,Proteintech，美国)。
1.7荧光原位杂交(FISH)检测
使用荧光原位杂交试剂盒(Genetic Pharmaceuticals,上海,中国)根据制造商的协议，靶向Gm13561连接位点的FISH探针混合物，然后用DAPI反染色。以U6探针作为阳性对照，检测细胞核分布。密封后，使用激光扫描共聚焦显微镜(徕卡)对样品进行成像。
1.8流式细胞术
转染72h后，收获细胞，用Annexin v-异硫氰酸荧光素(FITC)和碘化丙啶(PI)染色。最后，使用流式细胞仪FACS Calibur(Becon Dickinson,Franklin Lakes,NJ,USA)检测细胞凋亡。采用Hoechst 33342和tdt介导的UTP镍端标记(TUNEL)染色(Beyotime)检测细胞凋亡，并在荧光显微镜(Leica)下观察。
2结果
2.1基于单细胞核测序的DN足细胞特异性lncRNA表达谱建立
首先通过单细胞RNA-seq分析揭示了DN小鼠足细胞中特异性lncRNA，采用snRNA-seq检测db/db DKD小鼠(n＝3)和正常对照小鼠(n＝3)肾脏组织单细胞表达谱。db/db小鼠的体重、肾重、血糖和尿白蛋白/尿肌酐比值(ACR)显著高于正常小鼠(图1A)显示我们糖尿病肾病小鼠造模成功。
然后我们对db/db DKD小鼠的分离肾组织与非糖尿病对照小鼠进行了单细胞RNA-seq分析。通过细胞标记基因的鉴定，将获得的单细胞分为18种主要的肾细胞类型，包括系膜细胞、内皮细胞、上皮细胞、足细胞、近端小管细胞、巨噬细胞、近端曲小管细胞、收集管主细胞、CD包埋细胞、CD瞬变细胞、Henley循环细胞、单核细胞、B淋巴细胞和T淋巴细胞(图1B)。我们对转录本的分析揭示了18个细胞簇中的特异性lncRNA(图1C)。建立了前20个特异性lncRNA的表达谱，以及24个DN相关lncRNA的表达谱(图1D,E)：Gm4128、Gm3772、Wt1os、Gm13814、9130410C08Rik、Gm29266、Gm44129、Gm15866、Tmem150cos、Gm49969、Gm16083、4921504A21Rik、Pard3bos1、Gm13561、Pard3bos3、Gm16337、Gm32880、Gm50469、Gm32036、Gm13944、2310014F06Rik、Gm37229、E130310104Rik、Sox5os4。我们发现足细胞中有6个与DN相关的特异性lncRNA(图1F)：Gm13561、Gm44129、Gm16337、Gm15866、Gm32036、Gm3772；其中1个(Gm3772)在DN中上调，5个(Gm13561、Gm44129、Gm16337、Gm15866、Gm32036)在DN中下调(图1G)。与其他细胞簇相比，这6个候选lncRNA在足细胞中表现出特异性高表达(图1H)。总之，我们从单细胞水平通过基因表达谱获得候选lncRNAs，并研究lncRNAs的细胞特异性表达参与DN。
2.2单细胞核测序分析结果结合PCR鉴定lncRNA Gm13561的高低糖肾脏固有细胞中肾小球足细胞细胞特异性及lncRNA Gm13561生物学特点
选择与DN发生最相关且在足细胞中表达相对较高的lncRNA。首先通过高糖培养细胞模拟疾病状态，通过qRT-PCR检测这些候选lncRNAs在高糖或低糖培养的足细胞中表达的情况，以及这些候选lncRNAs在足细胞，系膜细胞，上皮细胞中表达的情况。
结果显示了与snRNA-seq结果相同的趋势。其中Gm13561在足细胞中特异过表达最多，在高糖培养足细胞中表达水平显著降低(图2A,B)。为了了解Gm13561的生物学特性，我们通过搜索其开放阅读框来评估其编码蛋白的可能性。Gm13561显示出较低的蛋白质编码潜力(表3)。此外，利用FISH和qRT-PCR了解了Gm13561的亚细胞定位。结果表明，Gm13561在足细胞细胞核和细胞质中均有表达，但Gm13561在细胞核中的表达量明显高于细胞质(图2C)。综上所述，Gm13561可能是足细胞特异性和DN相关的lncRNA，在高糖培养的足细胞中显著下调，且主要位于细胞核中(图2D)。
Gm13561的cDNA序列如SEQ ID NO.1所示。
表3.ORF编码蛋白潜力
2.3过表达和敲低Gm13561调控足细胞损伤与凋亡
为了明确Gm13561在DN中是否发挥功能，我们将Gm13561全长克隆到pcDNA3.1质粒中，并合成了针对Gm13561的特异性siRNA。然后通过qPCR分析评估Gm13561的过表达和干扰效率(图3A,B)，发现成功过表达和敲低Gm13561。
我们确定通过RNA-seq确定Gm13561在足细胞中过表达的全基因组转录组学后果然后结合snRNA-seq中筛选到的在足细胞中高表达的特异性基因，发现了48个受Gm13561影响的足细胞特异性基因，并对他们进行GO分析，结果显示这48个基因富集到的的生物学过程包括35个途径。其中有6条ROS(Reactive oxygen species，活性氧)相关信号通路和细胞凋亡(图3C)，提示Gm13561可能调控足细胞凋亡和ROS积累。因此，我们检测了Gm13561对足细胞的凋亡和ROS水平的影响，流式细胞术分析显示，转染Gm13561后，凋亡细胞数量和ROS水平均下降。而Gm13561基因敲除逆转了结果(图3D,E)。许多研究表明ROS与多种细胞死亡相关，如坏死、凋亡(caspase依赖性)、焦亡(pyroptosis)和坏死。炎症诱导的和不依赖caspase的细胞死亡包括坏死性细胞死亡、铁性细胞死亡、自噬性细胞死亡和氧性细胞死亡。氧死亡是2018年提出的一种新的类凋亡的细胞死亡方式。研究证实，氧死亡影响一些肿瘤的发展，但其对DN的影响尚不清楚。
因此，我们研究Gm13561是否影响凋亡相关蛋白和特殊的氧死亡通路KEAP1-PGAM5-AIFM1，Western blot分析显示，Gm13561的敲低诱导低糖培养的足细胞中Bcl-2和podocin的表达降低，Bax和cleaved-caspase-3的表达升高，Keap1、PGAM5和AIFM1的表达增加，但AIFM1的磷酸化水平显著降低。AIFM1磷酸化水平的降低是氧死亡的一个里程碑事件(图3F)。这些数据表明Gm13561具有保护足细胞免受高糖诱导的凋亡和氧死亡的作用。
这些实验通过过表达/敲低这个靶标Gm13561而抑制/促进了足细胞的凋亡和氧死亡证明该lncRNA-Gm13561可作为DKD的新靶标。
Claims (10)
1.检测样本中生物标志物的试剂在制备诊断糖尿病肾病的产品中的应用，其特征在于：所述生物标志物为lncRNA-Gm13561。
2.根据权利要求1所述的应用，其特征在于：与正常对照相比，lncRNA-Gm13561在患者中的表达水平下调。
3.根据权利要求2所述的应用，其特征在于：患者的待测样本中lncRNA-Gm13561的含量通过总RNA提取、反转录、定量PCR进行检测得到。
4.lncRNA-Gm13561表达促进剂在制备防治糖尿病肾病的药物中的应用。
5.lncRNA-Gm13561作为靶标在筛选防治糖尿病肾病的药物中的应用。
6.根据权利要求5所述的应用，其特征在于：所述药物促进lncRNA-Gm13561的表达。
7.根据权利要求1至6任一项所述的应用，其特征在于：lncRNA-Gm13561的cDNA序列如SEQ ID NO.1所示。
8.根据权利要求5至7任一项所述的应用，其特征在于：lncRNA-Gm13561调控足细胞凋亡和ROS积累，缓解足细胞的凋亡和ROS引起的氧死亡。
9.根据权利要求8所述的应用，其特征在于：Gm13561调控氧死亡通路KEAP1-PGAM5-AIFM1。
10.根据权利要求9所述的应用，其特征在于：Gm13561的上调促使足细胞中Bcl-2和podocin的表达升高，Bax和cleaved-caspase-3的表达降低，Keap1，PGAM5和AIFM1的表达减少，AIFM1的磷酸化水平增加。
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