CN116913370A - 人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法 - Google Patents
人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法 Download PDFInfo
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Abstract
本发明公开了一种人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,涉及生物信息学领域。其中,所述人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法通过构建与人脐带间充质干细胞治疗宫腔粘连相关的circRNA‑miRNA‑mRNA网络和细胞共培养结合,筛选出circRNA靶点。所述circRNA靶点与治疗密切相关,可应用于指导治疗宫腔粘连以及用于制备治疗宫腔粘连靶向药物。
Description
技术领域
本发明涉及生物信息学领域,特别涉及一种人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法。
背景技术
宫腔粘连(IUA),又称阿什曼综合征,是由于子宫基底层受损导致子宫内膜纤维化,可导致严重的子宫内膜功能障碍,如不孕不育和月经紊乱,此外,IUA还可能引发周期性胃痛、月经少、闭经、子宫内膜异位症。在所有类型的堕胎妇女中,IUA的发生率为6%~30%,在分娩后接受刮宫术手术的妇女中,IUA的发病率可高达25%。IUA的治疗包括手术切除粘连组织和重新扩张宫腔,目前临床上去除子宫粘连的最有效方法是宫腔镜手术。虽然宫腔镜手术是目前去除宫腔内结缔组织,恢复子宫内膜形态和功能的最有效的方法,但由于手术过程中存在机械损伤,手术本身会导致再粘连的形成。
近年来,细胞治疗已成为具有前景的子宫内膜修复和预防IUA的手段。人脐带间充质干细胞(hUCMSC)是一种多能细胞,来源于早期发育的中胚层,具有很强的自我更新能力和多分化潜能,相比于其他干细胞,具有易采集、免疫原性低、致瘤性低等优点。hUCMSC在治疗IUA中展现了巨大的优势。
子宫内膜是一个独特的循环组织再生系统,它依赖于供应子宫内膜的血管的循环生长和衰退。血管生成受阻是子宫内膜损伤的主要病理改变,因此,血管生成是IUA恢复的关键。hUCMSC可以通过促进子宫内膜血管生成完成IUA的修复,但hUCMSC介导的血管生成机制仍未完全阐明。
非编码RNA (ncRNAs)参与许多细胞生物和生理过程,甚至病理疾病过程。circRNA是细胞内的一种内源性、数量众多且稳定的新型ncRNA。circRNA在疾病诊断、预后和预测方面具有重要潜力,并且是IUA和其他疾病的发生发展相关的重要调节因子。circRNA可与miRNA和mRNA形成了竞争性内源性RNA(ceRNA)网络,因此circRNA-miRNA-mRNA调控网络可能是导致IUA发生和发展的重要分子机制。然而,在hUCMSC治疗IUA中,circRNA-miRNA-mRNA调控机制尚未被研究,因此也缺乏相应的circRNA治疗靶点。
发明内容
鉴于上述现有技术的不足之处,本发明的目的在于提供一种人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,旨在解决现有技术中治疗宫腔粘连时缺乏circRNA治疗靶点的技术问题。
为了达到上述目的,本发明采取了以下技术方案:
一种人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其包括以下步骤:
获取与宫腔粘连相关的第一差异表达circRNA以及在多组与宫腔粘连相关的第一数据集中均差异表达的共同miRNA;根据所述第一差异表达circRNA和共同miRNA构建与宫腔粘连相关的circRNA-miRNA网络;
获取在多组与宫腔粘连相关的第二数据集中均差异表达的共同靶mRNA;根据所述共同靶mRNA以及所述与宫腔粘连相关的circRNA-miRNA网络构建与宫腔粘连相关的circRNA-miRNA-mRNA网络;
获取与人脐带间充质干细胞治疗宫腔粘连相关的基因测序数据,计算所述与人脐带间充质干细胞治疗宫腔粘连相关的基因测序数据中治疗组数据和对照组数据的差异表达mRNA,获得第一差异表达mRNA;
计算所述与宫腔粘连相关的circRNA-miRNA-mRNA网络中的差异表达mRNA,获得第二差异表达mRNA;计算所述第一差异表达mRNA和第二差异表达mRNA的交集,获得第三差异表达mRNA;根据所述第三差异表达mRNA进一步构建与人脐带间充质干细胞治疗宫腔粘连相关的circRNA-miRNA-mRNA网络;
获取人脐带间充质干细胞与血管内皮细胞共培养中的血管内皮细胞总RNA数据,根据所述总RNA数据筛选出所述与人脐带间充质干细胞治疗宫腔粘连相关的circRNA-miRNA-mRNA网络中的第二差异表达circRNA作为所述circRNA靶点。
所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其中,所述获取与宫腔粘连相关的第一差异表达circRNA以及在多组与宫腔粘连相关的第一数据集中均差异表达的共同miRNA,具体包括:从数据库获取与宫腔粘连相关的基因测序数据,计算所述与宫腔粘连相关的基因测序数据中患者组数据和正常组数据的第一差异表达circRNA和差异表达miRNA;计算与所述第一差异表达circRNA相互作用的miRNA,获得关联miRNA;计算所述关联miRNA和差异表达miRNA的交集,获得所述共同miRNA。
所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其中,所述计算所述与宫腔粘连相关的基因测序数据中患者组数据和正常组数据的第一差异表达circRNA和差异表达miRNA,具体包括:采用R软件DESeq2包计算所述与宫腔粘连相关的基因测序数据中患者组数据和正常组数据的第一差异表达circRNA和差异表达miRNA。
所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其中,第一差异表达circRNA的筛选标准为|log2FoldChange|>2,P<0.05。
所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其中,差异表达miRNA的筛选标准为|log2FoldChange|>1,P<0.05。
所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其中,所述获取在多组与宫腔粘连相关的数据中均差异表达的共同靶mRNA,具体包括:从数据库获取与宫腔粘连相关的靶mRNA,作为第一mRNA;计算与所述共同miRNA相关的靶mRNA,作为第二mRNA;计算所述第一mRNA和第二mRNA的交集,获得所述共同靶mRNA。
所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其中,所述计算与所述共同miRNA相关的靶mRNA,作为第二mRNA,具体包括:分别采用miRDB数据库和microT-CDS数据库预测与所述共同miRNA相关的靶mRNA,汇总后获得所述第二mRNA。
所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其中,所述获取与人脐带间充质干细胞治疗宫腔粘连相关的基因测序数据,计算所述与人脐带间充质干细胞治疗宫腔粘连相关的基因测序数据中治疗组数据和对照组数据的差异表达mRNA,获得第一差异表达mRNA,具体包括:采用R软件DESeq2包所述与人脐带间充质干细胞治疗宫腔粘连相关的基因测序数据中治疗组数据和对照组数据的差异表达mRNA,获得第一差异表达mRNA;所述第一差异表达mRNA的筛选标准为|log2FoldChange|>2,padj<0.01。
所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其中,所述获取人脐带间充质干细胞与血管内皮细胞共培养中的血管内皮细胞总RNA数据,具体包括:将人脐带间充质干细胞与血管内皮细胞共培养24h,然后提取血管内皮细胞中的总RNA并进行检测,获得所述总RNA数据。
有益效果
本发明提供了一种人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,通过构建与人脐带间充质干细胞治疗宫腔粘连相关的circRNA-miRNA-mRNA网络以及细胞共培养结合,筛选出circRNA靶点,所述筛选方法先进,适用性好,可随着数据库的更新,筛选出针对性更强的circRNA靶点。
附图说明
图1为患者组数据和正常组数据之间miRNA表达水平火山图。
图2为患者组数据和正常组数据之间miRNA表达水平热图。
图3为关联miRNA和差异表达miRNA的维恩图。
图4为与宫腔粘连相关的 circRNA-miRNA网络。
图5为miRDB和microT-CDS数据库中DEmiRNA的预测靶mRNA与第一mRNA的韦恩图。
图6为治疗组数据和对照组数据之间mRNA表达水平火山图。
图7为治疗组数据和对照组数据之间mRNA表达水平热图。
图8为第一差异表达mRNA与所述与宫腔粘连相关的circRNA-miRNA-mRNA网络中的差异表达mRNA的维恩图。
图9为hUCMSC治疗IUA的circRNA-miRNA-mRNA网络的桑基图。
图10为hUCMSC细胞与HUVEC细胞共培养后各circRNA的表达。
具体实施方式
本发明提供一种人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,为使本发明的目的、技术方案及效果更加清楚、明确,以下参照附图并举实施例对本发明进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
在本发明的描述中,需要理解的是,术语“第一”、“第二”仅用于描述目的,且不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。因此,限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者多个该特征。本发明的描述中,除非另有说明,“多个”的含义是两个以上。
实施例1
一种人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,包括如下步骤:
S100.获取与宫腔粘连相关的第一差异表达circRNA以及在多组与宫腔粘连相关的第一数据集中均差异表达的共同miRNA;根据所述第一差异表达circRNA和共同miRNA构建与宫腔粘连相关的circRNA-miRNA网络,具体可以为:
S110.从Gene Expression Omnibus (GEO)数据库搜索并下载GSE165321基因测序数据集,GSE165321基于GPL16791 Illumina HiSeq 2500平台,包含3个IUA样本(患者组数据)和3个正常对照样本(正常组数据)的circRNA和miRNA信息;
S120.应用R软件DESeq2包分析GSE165321中患者组数据和正常组数据之间的第一差异表达circRNA (DEcircRNAs)和差异表达miRNA (DEmiRNA);第一差异表达circRNA的筛选标准为|log2FoldChange|>2,P<0.05;差异表达miRNA 的筛选标准为|log2FoldChange|>1,P<0.05;
共鉴定出67个第一差异表达circRNA,包括29个上调基因和38个下调基因,筛选出差异倍数最大的前10个表达上调第一差异表达circRNA(hsa_circ_0000621,hsa_circ_0000698,hsa_circ_0000324,hsa_circ_0001567,hsa_circ_0001641,hsa_circ_0001062,hsa_circ_0000033,hsa_circ_0001951,hsa_circ_0000229,hsa_circ_0000218)和前10个表达下调第一差异表达circRNA(hsa_circ_0000761,hsa_circ_0001781,hsa_circ_0000741,hsa_circ_0001065,hsa_circ_0000590,hsa_circ_0001443,hsa_circ_0000685,hsa_circ_0001760,hsa_circ_0001512,hsa_circ_0001256),相关结果如下:
以及共鉴定出137个差异表达miRNA ,包括70个上调基因和67个下调基因,结果显示在火山图(图1)和热图(图2)中;
S130.利用circbank数据库(http://www.circbank.cn/)预测与第一差异表达circRNA存在相互作用的miRNA,得到479个关联miRNA;
S140.计算479个关联miRNA和137个差异表达miRNA的交集,得到18个共同miRNA(图3),具体如下:hsa-miR-378d,hsa-miR-670-3p,hsa-miR-378c,hsa-miR-32-3p,hsa-miR-511-3p,hsa-miR-383-5p,hsa-miR-3616-5p,hsa-miR-548ba,hsa-miR-424-5p,hsa-miR-455-5p,hsa-miR-514a-5p,hsa-miR-149-3p,hsa-miR-186-3p,hsa-miR-338-3p,hsa-miR-767-3p,hsa-miR-544a,hsa-miR-138-5p,hsa-miR-135b-5p;
S150.进一步根据前面circbank数据库预测的第二circRNA和共同miRNA相互作用关系,利用cytoscape软件对18个共同miRNA构建与宫腔粘连相关的 circRNA-miRNA(IUAcircRNA-miRNA)网络(图4),IUA circRNA-miRNA网络中包含11个circRNA(hsa_circ_0001951,hsa_circ_0001641,hsa_circ_0001065,hsa_circ_0001512,hsa_circ_0000324,hsa_circ_0000621,hsa_circ_0000698,hsa_circ_0000033,hsa_circ_0000590,hsa_circ_0000741,hsa_circ_0001567)和18个miRNA(hsa-miR-378d,hsa-miR-670-3p,hsa-miR-378c,hsa-miR-32-3p,hsa-miR-511-3p,hsa-miR-383-5p,hsa-miR-3616-5p,hsa-miR-548ba,hsa-miR-424-5p,hsa-miR-455-5p,hsa-miR-514a-5p,hsa-miR-149-3p,hsa-miR-186-3p,hsa-miR-338-3p,hsa-miR-767-3p,hsa-miR-544a,hsa-miR-138-5p,hsa-miR-135b-5p),具有29 节点(Nodes)和21 边缘(Edges);
S200.获取在多组与宫腔粘连相关的第二数据集中均差异表达的共同靶mRNA;根据所述共同靶mRNA以及所述与宫腔粘连相关的circRNA-miRNA网络构建与宫腔粘连相关的circRNA-miRNA-mRNA网络,具体可以为:
S210.以公开的文献(Identification and validation of long non-codingRNA associated ceRNAs in intrauterine adhesion,Bioengineered.2022 Jan;13(1):1039-1048.PMID: 34968168)中报道的差异表达mRNA作为第一mRNA;
S220.分别利用miRDB(https://mirdb.org/)和microT-CDS(https://dianalab.e-ce.uth.gr/html/dianauniverse/index.php?r=microT_CDS)数据库预测S150得到的18个共同miRNA的靶mRNA,汇总后作为第二mRNA;
S230.取第一mRNA和第二mRNA的交集,得到283个共同靶mRNA(图5);
S240.根据S230中获得的共同靶mRNA以及S150中构建的与宫腔粘连相关的circRNA-miRNA网络构建与宫腔粘连相关的circRNA-miRNA-mRNA网络(IUA circRNA-miRNA-mRNA网络);
IUA circRNA-miRNA-mRNA网络包含11个circRNA,18个miRNA,213 mRNA,共242 节点(Nodes)和626 边缘(Edges);
S300.获取与人脐带间充质干细胞治疗宫腔粘连相关的基因测序数据,计算所述与人脐带间充质干细胞治疗宫腔粘连相关的基因测序数据中治疗组数据和对照组数据的差异表达mRNA,获得第一差异表达mRNA,具体可以为:
S310.从Gene Expression Omnibus (GEO)数据库搜索并下载GSE205992基因测序数据集,GSE205992基于GPL21255 Illumina HiSeq 2500平台,包含3个hUCMSC治疗IUA样本(治疗组数据)和3个对照样本(对照组数据)的mRNA信息;
S320.应用R软件DESeq2包分析GSE205992中治疗组数据和对照组数据之间的差异表达mRNA(hDEmRNA),获得第一差异表达mRNA;第一差异表达mRNA的筛选标准为|log2FoldChange|>2,padj<0.01;
共鉴定出803个第一差异表达mRNA,包括617个上调基因和187个下调基因,结果显示在火山图(图6)和热图(图7)中;
S400.计算所述与宫腔粘连相关的circRNA-miRNA-mRNA网络中的差异表达mRNA,获得第二差异表达mRNA;计算所述第一差异表达mRNA和第二差异表达mRNA的交集,获得第三差异表达mRNA;根据所述第三差异表达mRNA进一步构建与人脐带间充质干细胞治疗宫腔粘连相关的circRNA-miRNA-mRNA网络,具体可以为:
S410.计算S320中得到的803个第一差异表达mRNA与所述与宫腔粘连相关的circRNA-miRNA-mRNA网络中的差异表达mRNA的交集,得到20个第三差异表达mRNA(图8);
S420.根据20个第三差异表达mRNA和所述与宫腔粘连相关的circRNA-miRNA-mRNA网络,通过cytoscape软件构建人脐带间充质干细胞治疗宫腔粘连相关的circRNA-miRNA-mRNA网络(图9),网络中共包含9个circRNA(hsa_circ_0001951,hsa_circ_0001512,hsa_circ_0000741,hsa_circ_0001641,hsa_circ_0001065,hsa_circ_0000621,hsa_circ_0000033,hsa_circ_0000698,hsa_circ_0000324),12个miRNA(hsa-miR-135b-5p,hsa-miR-338-3p,hsa-miR-514a-5p,hsa-miR-138-5p,hsa-miR-149-3p,hsa-miR-3616-5p,hsa-miR-670-3p,hsa-miR-424-5p,hsa-miR-32-3p,hsa-miR-378c,hsa-miR-378d,hsa-miR-548ba),20个mRNA(KCNB1,KCNMA1,CCDC3,CORIN,WWC1,PRELP,HSPB7,PREX1,CAMK2A,DES,SPARCL1,PAG1,REEP1,BVES,DCLK1,CCNJL,RSPO3,VEGFA,BNC2,SPOCK1);
S500.获取人脐带间充质干细胞与血管内皮细胞共培养中的血管内皮细胞总RNA数据,根据所述总RNA数据筛选出所述与人脐带间充质干细胞治疗宫腔粘连相关的circRNA-miRNA-mRNA网络中的差异表达circRNA(第二差异表达circRNA)作为所述circRNA靶点,具体可以为:
将 hUCMSC细胞与HUVEC细胞共培养24h,提取HUVEC细胞中的总RNA,检测hUCMSC治疗IUA的circRNA-miRNA-mRNA网络中的差异circRNA的表达,比对后筛选出表达具有统计学差异的circRNA靶点hsa_circ_0000698,hsa_circ_0001641,hsa_circ_0001065,hsa_circ_0000621(图10)。
所述circRNA靶点hsa_circ_0000698,hsa_circ_0001641,hsa_circ_0001065,hsa_circ_0000621与治疗IUA密切相关,可以应用于制备治疗宫腔粘连靶向药物。
需要补充说明的是,随着数据库的更新,本领域技术人员可以采用不同的数据集进行筛选,并可以通过不同的数据库预测circRNA和miRNA相互作用对以及预测miRNA和mRNA相互作用对,以进一步构建circRNA-miRNA-mRNA网络。
可以理解的是,对本领域普通技术人员来说,可以根据本发明的技术方案及其发明构思加以等同替换或改变,而所有这些改变或替换都应属于本发明所附的权利要求的保护范围。
Claims (9)
1.一种人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其特征在于,包括以下步骤:
获取与宫腔粘连相关的第一差异表达circRNA以及在多组与宫腔粘连相关的第一数据集中均差异表达的共同miRNA;根据所述第一差异表达circRNA和共同miRNA构建与宫腔粘连相关的circRNA-miRNA网络;
获取在多组与宫腔粘连相关的第二数据集中均差异表达的共同靶mRNA;根据所述共同靶mRNA以及所述与宫腔粘连相关的circRNA-miRNA网络构建与宫腔粘连相关的circRNA-miRNA-mRNA网络;
获取与人脐带间充质干细胞治疗宫腔粘连相关的基因测序数据,计算所述与人脐带间充质干细胞治疗宫腔粘连相关的基因测序数据中治疗组数据和对照组数据的差异表达mRNA,获得第一差异表达mRNA;
计算所述与宫腔粘连相关的circRNA-miRNA-mRNA网络中的差异表达mRNA,获得第二差异表达mRNA;计算所述第一差异表达mRNA和第二差异表达mRNA的交集,获得第三差异表达mRNA;根据所述第三差异表达mRNA进一步构建与人脐带间充质干细胞治疗宫腔粘连相关的circRNA-miRNA-mRNA网络;
获取人脐带间充质干细胞与血管内皮细胞共培养中的血管内皮细胞总RNA数据,根据所述总RNA数据筛选出所述与人脐带间充质干细胞治疗宫腔粘连相关的circRNA-miRNA-mRNA网络中的第二差异表达circRNA作为所述circRNA靶点。
2.根据权利要求1所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其特征在于,所述获取与宫腔粘连相关的第一差异表达circRNA以及在多组与宫腔粘连相关的第一数据集中均差异表达的共同miRNA,具体包括:
从数据库获取与宫腔粘连相关的基因测序数据,计算所述与宫腔粘连相关的基因测序数据中患者组数据和正常组数据的第一差异表达circRNA和差异表达miRNA;
计算与所述第一差异表达circRNA相互作用的miRNA,获得关联miRNA;
计算所述关联miRNA和差异表达miRNA的交集,获得所述共同miRNA。
3.根据权利要求2所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其特征在于,所述计算所述与宫腔粘连相关的基因测序数据中患者组数据和正常组数据的第一差异表达circRNA和差异表达miRNA,具体包括:
采用R软件DESeq2包计算所述与宫腔粘连相关的基因测序数据中患者组数据和正常组数据的第一差异表达circRNA和差异表达miRNA。
4.根据权利要求3所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其特征在于,第一差异表达circRNA的筛选标准为|log2FoldChange|>2,P<0.05。
5.根据权利要求3所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其特征在于,差异表达miRNA的筛选标准为|log2FoldChange|>1,P<0.05。
6.根据权利要求1所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其特征在于,所述获取在多组与宫腔粘连相关的数据中均差异表达的共同靶mRNA,具体包括:
从数据库获取与宫腔粘连相关的靶mRNA,作为第一mRNA;
计算与所述共同miRNA相关的靶mRNA,作为第二mRNA;
计算所述第一mRNA和第二mRNA的交集,获得所述共同靶mRNA。
7.根据权利要求6所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其特征在于,所述计算与所述共同miRNA相关的靶mRNA,作为第二mRNA,具体包括:
分别采用miRDB数据库和microT-CDS数据库预测与所述共同miRNA相关的靶mRNA,汇总后获得所述第二mRNA。
8.根据权利要求1所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其特征在于,所述获取与人脐带间充质干细胞治疗宫腔粘连相关的基因测序数据,计算所述与人脐带间充质干细胞治疗宫腔粘连相关的基因测序数据中治疗组数据和对照组数据的差异表达mRNA,获得第一差异表达mRNA,具体包括:
采用R软件DESeq2包所述与人脐带间充质干细胞治疗宫腔粘连相关的基因测序数据中治疗组数据和对照组数据的差异表达mRNA,获得第一差异表达mRNA;
所述第一差异表达mRNA的筛选标准为|log2FoldChange|>2,padj<0.01。
9.根据权利要求1所述的人脐带间充质干细胞治疗宫腔粘连的circRNA靶点筛选方法,其特征在于,所述获取人脐带间充质干细胞与血管内皮细胞共培养中的血管内皮细胞总RNA数据,具体包括:将人脐带间充质干细胞与血管内皮细胞共培养24h,然后提取血管内皮细胞中的总RNA并进行检测,获得所述总RNA数据。
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