CN115433776A - Ccn3在调控血管平滑肌细胞钙化中的应用 - Google Patents
Ccn3在调控血管平滑肌细胞钙化中的应用 Download PDFInfo
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Abstract
本发明提供了CCN3在调控血管平滑肌细胞钙化中的应用。具体而言,本发明提供了CCN3作为靶点在筛选和/或制备调控平滑肌细胞钙化的试剂中的应用,本发明还提供了降低CCN3表达的试剂在制备抑制和/或缓解平滑肌细胞钙化的试剂中的应用。本发明证实了CCN3在Lp(a)介导的METTL14基因调控血管平滑肌细胞钙化发生发展中具有重要作用。
Description
技术领域
本发明是关于CCN3的新应用,具体而言,是关于CCN3基因和/或蛋白在调控血管平滑肌细胞钙化中的应用。
背景技术
冠状动脉粥样硬化性心脏病(冠心病)全球疾病负担沉重。血管钙化是影响冠心病预后的重要危险因素,血管平滑肌细胞(Vascular smooth cell,VSMC)向成骨细胞的转化在血管钙化进展中起关键作用。
近年来有关脂蛋白a(Lp(a))与动脉粥样硬化性心血管疾病(ASCVD)相关性的研究越来越受到关注。临床研究证实Lp(a)水平与冠脉钙化存在相关性,早期实验研究也证实Lp(a)参与VSMCs钙化相关病理生理过程。
RNA的N6-甲基腺苷(m6A)修饰是近年来心血管表观遗传领域新的研究热点。m6A修饰是真核生物信使RNA和长链非编码RNA中最普遍和最丰富的内部修饰,通过影响RNA的稳定性、降解和翻译从而在调节基因表达中发挥重要作用。全基因组关联研究(Genome-wideassociation studies,GWAS)研究发现调控该过程的甲基转移酶METTL14基因定位于VSMC钙化易感区域。
既往研究报道,肾母细胞瘤过度表达蛋白(NOV/CCN3)基因在成骨反应中发挥重要作用,单细胞测序数据显示CCN3基因在SMC中富集表达。
VSMC钙化机制复杂,由多因素、多通路、多机制参与,如Runx2、Wnt/β-catenin、NF-kB、FGF23共同受体α-Klotho等。然而,上述诸多靶点均尚未成功进行临床转化,导致目前尚无成熟的手段干预血管钙化。因此,亟需深入探究血管钙化新的分子机制,发现具备成功临床转化潜力的新的治疗靶点,对于从根本上减轻血管钙化相关疾病负担具有十分重要的意义。
发明内容
本发明的一个目的在于研究血管钙化新的分子机制,提供关于血管钙化的新的治疗靶点。
本案发明人在研究中构建了Lp(a)蛋白在高磷酸盐溶液(高Pi)刺激中促进人主动脉平滑肌细胞(Human aortic smooth muscle cells,HASMCs)钙化表型,将Lp(a)诱导HASMCs钙化与METTL14诱导HASMCs钙化有机结合,并进一步筛选鉴定了METTL14下游靶基因CCN3,证明了CCN3基因介导METTL14基因调控HASMCs钙化,且证明了METTL14诱导的钙化表型能够被敲低CCN3逆转,进而提出了本发明。
具体而言,一方面,本发明提供了CCN3作为靶点在筛选和/或制备调控平滑肌细胞钙化的试剂中的应用。
另一方面,本发明提供了降低CCN3表达的试剂在制备抑制和/或缓解平滑肌细胞钙化的试剂中的应用。
根据本发明的具体实施方案,本发明中,所述CCN3为CCN3基因和/或蛋白。
根据本发明的具体实施方案,本发明中,所述平滑肌细胞为血管平滑肌细胞。
根据本发明的具体实施方案,本发明中,所述平滑肌细胞为动脉平滑肌细胞。
根据本发明的具体实施方案,本发明中,所述平滑肌细胞钙化包括由Lp(a)蛋白刺激促进的平滑肌细胞钙化。
根据本发明的具体实施方案,本发明中,所述平滑肌细胞钙化包括过表达METTL14基因引起的平滑肌细胞钙化。
根据本发明的具体实施方案,本发明中,所述降低CCN3表达的试剂包括敲低CCN3表达的试剂。
根据本发明的具体实施方案,本发明中,所述降低CCN3表达的试剂包括敲低METTL14基因的试剂。
根据本发明的具体实施方案,本发明中,CCN3可作为靶点用于筛选和/或制备减轻血管钙化相关疾病的药物。
在本发明的一些具体实施方案中,本发明利用Lp(a)处理人主动脉平滑肌细胞(Human aortic smooth muscle cells,HASMCs),检测Lp(a)对HASMCs钙化的影响:培养HASMCs,分别用20μg/mL的Lp(a)蛋白和磷酸盐缓冲液(Phosphate buffered saline,PBS)处理,在含高Pi的成骨诱导培养基(Osteogenic medium,OSM)中培养14天后用茜素红染色检测钙化表型。在这些实施方案中,本发明构建了Lp(a)蛋白在高Pi刺激中促进HASMCs钙化表型,将Lp(a)诱导HASMCs钙化与METTL14诱导HASMCs钙化有机结合。
在本发明的一些具体实施方案中,本发明培养HASMCs,分别用20μg/mL的Lp(a)蛋白和PBS处理,培养48小时后分别提RNA和蛋白,检测METTL14基因的mRNA和蛋白表达水平;培养HASMCs,分别感染METTL14和GFP(对照)病毒,在含高Pi的OSM中培养14天后用茜素红染色检测钙化表型;培养HASMCs,分别用PBS+Si-NC、Lp(a)+Si-NC、Lp(a)+Si-METTL14处理,在含高Pi的OSM中培养14天后用茜素红染色检测钙化表型。在这些实施方案中,本发明证明了Lp(a)通过调控METTL14的表达促进HASMCs钙化。
在本发明的一些具体实施方案中,本发明利用RNA-seq技术对Ad-METTL14处理的HASMCs进行测序,筛选出METTL14调控的下游靶基因;回复实验验证CCN3基因介导METTL14基因调控HASMCs钙化:培养HASMCs,分别感染METTL14和GFP(对照)病毒,然后在感染METTL14或GFP病毒的HASMCs中敲除CCN3基因,检测钙化表型。在这些实施方案中,本发明筛选鉴定了METTL14下游靶基因CCN3,证明CCN3基因介导METTL14基因调控HASMCs钙化。本发明还证明了Lp(a)通过调控METTL14表达水平,进而影响CCN3基因转录和翻译,从而在高Pi刺激中促进HASMCs钙化。
附图说明
图1A和图1B显示Lp(a)蛋白对HASMCs钙化表型的影响。其中,图1A显示镜下观:茜素红染色检测HASMCs中用20μg/mL Lp(a)蛋白诱导对细胞钙化表型的影响;图1B显示大体观:茜素红染色检测HASMCs中用20μg/mL Lp(a)蛋白诱导对细胞钙化表型的影响。
图2显示Lp(a)处理HASMCs上调METTL14蛋白表达。
图3A至图3C显示METTL14基因促进HASMCs钙化表型。其中,图3A显示镜下观:茜素红染色检测HASMCs中过表达METTL14基因对细胞发生钙化表型的影响;图3B显示大体观:茜素红染色检测HASMCs中过表达METTL14基因对细胞发生钙化表型的影响;图3C显示过表达METTL14基因后HASMCs中总钙含量。
图4A至图4C显示敲低METTL14基因抑制Lp(a)诱导的HASMCs钙化。其中,图4A显示10倍及20倍镜下观察钙化表型;图4B显示大体观察钙化表型;图4C显示敲低METTL14基因后HASMCs中总钙含量。
图5A和图5B为HASMCs中过表达METTL14基因差异表达mRNA聚类图及火山图。其中,图5A为差异表达的mRNA热图;横坐标所示为细胞样本分类,纵坐标所示则为差异表达基因。其中红色表示上调,绿色则表示下调。图5B为差异表达的mRNA火山图。红点表示高表达,绿点表示低表达。
图6A和图6B差异表达的mRNA的GO分析以及Pathway分析。其中,图6A显示差异表达mRNA的GO分析,结果所示为过表达METTL14基因后HASMCs中发生了显著变化的生物学过程和分子功能以及细胞组分;图6B显示差异表达mRNA的Pathway分析,结果所示为过表达METTL14基因后HASMCs发生显著变化的生物学通路。
图7显示RNA-seq结果中CCN3基因mRNA表达水平,***P<0.001。
图8A和图8B显示METTL14基因正向调控CCN3 mRNA表达水平。其中,图8A显示在HASMCs中过表达METTL14后,CCN3 mRNA表达水平增加3.59倍;图8B显示在HASMCs中敲低METTL14后,CCN3 mRNA表达水平下降89%。
图9A至图9D显示METTL14基因正向调控CCN3蛋白表达水平。其中,图9A显示在HASMCs中过表达METTL14后,CCN3蛋白表达水平显著增加;图9B显示对条带量化后计算得出,CCN3蛋白表达水平约增加0.84倍;图9C显示在HASMCs中敲低METTL14后,CCN3蛋白表达水平显著下降;图9D显示对条带量化后计算得出,CCN3蛋白表达水平约下降64%。
图10A至图10C显示METTL14和CCN3基因共同作用调控HASMCs发生钙化表型。其中,图10A显示镜下观:茜素红染色检测HASMCs中用Ad-METTL14结合Si-CCN3处理对细胞发生钙化表型的影响;图10B显示大体观:茜素红染色检测HASMCs中用Ad-METTL14结合Si-CCN3处理对细胞发生钙化表型的影响;图10C显示过表达METTL14基因同时敲低CCN3基因后HASMCs中总钙含量。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解,现结合具体实施例对本发明的技术方案进行详细说明,应理解这些实例仅用于说明本发明而不用于限制本发明的范围。实施例中,各原始试剂材料均可商购获得,未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件,或按照仪器制造商所建议的条件。
除非另外专门定义,本文使用的所有技术和科学术语都与相关领域普通技术人员的通常理解具有相同的含义。
实施例1、Lp(a)蛋白促进HASMCs钙化表型
培养HASMCs,应用浓度为20μg/mL的Lp(a)蛋白在含高Pi的OSM培养基中对HASMCs进行刺激诱导,以PBS+OSM为对照,处理HASMCs14天,应用茜素红染色检测细胞钙化表型检测HASMCs钙化表型,如下所示为操作步骤:
(1)培养板中接种细胞:将培养瓶中正处于对数生长期的HASMCs用胰酶消化,以5×105/孔的密度接种到12孔细胞培养板中,培养24h。
(2)第二天,进行细胞进行Lp(a)蛋白诱导和/或转染或感染对靶基因进行敲低或过表达处理。每组处理设3个平行孔,具体操作如前。之后置于细胞培养箱中继续培养14天,期间2-3天换一次培养基。
(3)培养14天后,从培养箱中取出将要用来进行茜素红染色的12孔细胞培养板,首先用预先制冷的无菌PBS溶液清洗3次,注意枪头沿着每个孔的边缘打下液体,以防将板底细胞冲刷。
(4)每孔加入1mL 4%的多聚甲醛(PFA),于室温条件下静置约30min以充分固定细胞。结束后再次用预冷的PBS溶液轻柔清洗3次并吸净残留液体。
(5)将茜素红S染料溶解于双蒸水中,配制PH值为4.2,浓度为2%的茜素红S染色溶液,在每孔中加入1mL的染色溶液,静置孵育30min,注意做避光处理。
(6)用预冷的PBS溶液小心清洗每孔,洗掉未结合的茜素红染色溶液,清洗3次,直至液体变为澄清,吸净PBS并室温放置晾干20min左右,待晾干后即可置于倒置显微镜下拍照留取图像。光镜下可见钙化结节,倒置显微镜下可见相衬图像。
(7)用氯化十六烷基吡啶(Hexadecylpyridinium chloride monohydrate,CPC)粉末溶解于双蒸水,配制10%CPC溶液,在室温下提取结合钙的茜素红S。在每孔中加入1mLCPC溶液,室温放置60min用于提取结合钙的茜素红S。将提取液转移至新的洁净EP管中,小心放入离心机中以12000g室温离心5min,吸取上清液,在分光度计(BioTek)上选择波长为OD 405mm进行读数。用20mM HCL和2%SDS以及0.2M甘氨酸配制PH值为7.4的细胞裂解缓冲液,然后用双蒸水洗涤细胞,每孔中加入600μL细胞裂解缓冲液,置于摇床上充分混匀约20min,再刮取细胞至新的洁净EP管内,并在80℃下加热60min,间隔涡旋震荡。最后采用DC蛋白分析法(Bio-Rad)测定总蛋白浓度,并用于茜素红S的归一化。
所得结果如图1A和图1B所示。与对照组相比,Lp(a)蛋白诱导后的HASMCs中有显著增多的钙化结节形成(图1A,图1B),表明在HASMCs中用Lp(a)蛋白诱导能显著促进细胞钙化表型。
实施例2、Lp(a)通过调控METTL14的表达促进HASMCs钙化
Western blot实验检测发现20μg/mL Lp(a)蛋白在HASMCs中进行诱导后METTL14蛋白表达水平显著上升(图2)。
在HASMCs中过表达METTL14基因,茜素红染色检测实验组中有显著增多的钙化结节形成(图3A,图3B),量化后发现实验组细胞钙含量为对照组的1.68倍(p<0.05)(图3C)。
敲低METTL14基因,茜素红染色检测发现Lp(a)蛋白刺激和敲低METTL14基因同时处理的HASMCs中钙化结节形成减少(图4A,图4B),量化后实验组细胞钙含量为单用Lp(a)蛋白刺激组的0.74倍(p<0.05)(图4C)。
以上结果表明在HASMCs中,Lp(a)蛋白诱导能通过上调METTL14基因表达水平显著促进细胞钙化表型。
实施例3、METTL14基因促进HASMCs钙化的RNA-seq及生物信息学分析,筛选METTL14基因的靶基因
在HASMCs中过表达METTL14基因,对HASMCs的RNA进行mRNA测序,并进行聚类分析,比较对照组和过表达METTL14基因组之间HASMCs的mRNA表达水平的差异。结果参见图5A和图5B、表1所示。结果显示,与对照组相比,过表达METTL14基因组mRNA(图5A)的表达有显著差异(FC>2,P<0.05);火山图展示了对照组、过表达METTL14基因组之间有324个mRNA表达有显著差异,其中86个表达下调,238个表达上调(图5B)。根据FC值排序,METTL14(log2FC=9.72)是上调最为显著的mRNA,AC010323.1(log2FC=6.53)是下调最为显著的mRNA(表1)。
表1过表达METTL14基因差异表达最明显的mRNAs
进行差异表达基因的功能(GO)分析,分别从BP、MF和CC三个层次对差异表达mRNA的功能进行注释,按照-LgP值对差异基因显著性功能进行排序,共分3类展示排序的前10项(图6A)。结果显示,在BP中,差异显著的生物过程集中在免疫反应和细胞迁移等过程中。在MF中,差异显著的细胞功能则集中在细胞增殖和间充质细胞转化等过程中。在CC中,差异显著的细胞组分集中在细胞核功能、染色体着丝粒等过程中。根据KEGG分析中差异信号通路的P值和所包含的差异基因数量构建显著性差异基因通路的气泡图,按照LgP值排序,对显著差异通路的前30项进行排序(图6B)。结果显示,差异基因主要富集在细胞凋亡、细胞粘附和趋化因子募集以及细胞因子受体等通路中。内皮细胞与单核细胞的粘附致使内皮细胞功能受损,与动脉粥样硬化的发生密切相关。
通过HASMCs中过表达METTL14基因及其对照的RNA-seq结果分析,初步筛选出受METTL14基因调控的下游基因为CCN3基因。而且RNA-seq分析结果显示过表达METTL14基因后CCN3基因mRNA表达水平约升高35%(p<0.001)(图7)。
实施例4、METTL14基因调控CCN3基因表达水平
在SMCM培养中的HASMC中分别过表达和敲低METTL14基因,培养48小时后分别提取RNA和蛋白,检测CCN3基因表达水平。过表达METTL14后,CCN3mRNA表达水平增加3.59倍,METTL14敲低时下降89%(图8A和图8B);过表达METTL14基因后,CCN3蛋白表达水平约增加0.84倍,敲低METTL14基因,CCN3蛋白表达水平下降64%(图9A至图9D)。这表明METTL14基因正向调控CCN3基因表达水平。
实施例5、CCN3基因介导METTL14基因调控HASMCs钙化
为验证METTL14是否通过正向调控CCN3基因促进HASMCs钙化表型,利用Ad-METTL14和Si-CCN3同时处理HASMCs,观察METTL14诱导的钙化表型是否能被敲低CCN3逆转。结果如图10A和图10B所示,与仅Ad-METTL14处理的细胞相比,Ad-METTL14同时Si-CCN3处理的HASMCs中钙化沉积程度显著减弱,量化后发现其细胞钙含量为仅用Ad-METTL14处理细胞组的0.63倍(p<0.05)(图10C),这表明CCN3基因介导METTL14基因调控HASMCs钙化。
Claims (10)
1.CCN3作为靶点在筛选和/或制备调控平滑肌细胞钙化的试剂中的应用。
2.降低CCN3表达的试剂在制备抑制和/或缓解平滑肌细胞钙化的试剂中的应用。
3.根据权利要求1或2所述的应用,其中,所述CCN3为CCN3基因和/或蛋白。
4.根据权利要求1或2所述的应用,其中,所述平滑肌细胞为血管平滑肌细胞。
5.根据权利要求4所述的应用,其中,所述平滑肌细胞为动脉平滑肌细胞。
6.根据权利要求1或2所述的应用,其中,所述平滑肌细胞钙化包括由Lp(a)蛋白刺激促进的平滑肌细胞钙化。
7.根据权利要求1或2所述的应用,其中,所述平滑肌细胞钙化包括过表达METTL14基因引起的平滑肌细胞钙化。
8.根据权利要求2所述的应用,其中,所述降低CCN3表达的试剂包括敲低CCN3表达的试剂。
9.根据权利要求2所述的应用,其中,所述降低CCN3表达的试剂包括敲低METTL14基因的试剂。
10.根据权利要求1所述的应用,其中,CCN3作为靶点用于筛选和/或制备减轻血管钙化相关疾病的药物。
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