Potential role of ANKRD1 in senescence mediated wound healing

ANKRD1 在衰老介导的伤口愈合中的潜在作用

• 批准号: 10251679
• 负责人: Myriam Gorospe
• 金额: $ 26.55万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10251679
• 关键词: Affect; Age; Aging; Amino Acids; Antibodies; Attenuated; Austria; Biogenic Amines; Biological Assay; Biological Sciences; Cell Proliferation; Cell Survival; Cell surface; Cells; Data; Dermal; Diploidy; Epidermis; Fibroblasts; Galactosidase; Genetic Transcription; Goals; Human; Lipids; Liquid Chromatography; Manufacturer Name; Mass Spectrum Analysis; Measures; Mediating; Messenger RNA; MicroRNAs; Mus; Nuclear; Pattern; Phenotype; Platelet-Derived Growth Factor; Post-Transcriptional Regulation; Production; Proteins; Protocols documentation; RNA; RNA-Binding Proteins; Reporting; Role; Sampling; Signal Transduction; Skin; Small Interfering RNA; Stains; Stratum Basale; Stratum corneum; Testing; Therapeutic; Tissues; WI 38 cell; extracellular; mRNA Stability; mass spectrometer; novel; overexpression; senescence; tandem mass spectrometry; trait; transcriptome sequencing; wound closure; wound healing

We have documented that ANKRD1 expression rises with senescence by RNA-seq and whole-cell mass spectrometry analyses, which have revealed that ANKRD1 is being highly expressed in senescent WI-38 human diploid fibroblasts. Moreover, silencing ANKRD1 reduces senescence, as determined by silencing ANKRD1 in pre-senescent fibroblasts by transfecting small interfering (si)RNAs. We found that depletion of ANKRD1 decreased the levels of senescent markers including p16 and p21 mRNAs and SA--galactosidase. These data indicate that silencing ANKRD1 attenuates senescence. We have also began to detect ANKRD1 in human skin samples. Human skin samples were provided by Dr. C. Chia (LCI) for initial staining with ANKRD1 (LGG). Preliminary IF staining with anti-ANKRD1 antibody showed a strong nuclear staining in sporadically localized dermal cells; weaker signals were observed broadly in the epidermis, from the basal layer to the lower stratum corneum. Ongoing Efforts: Towards Objective 1, we are investigating the mechanisms of ANKRD1 expression in senescence and ANKRD1 impact on the SASP trait. We expect to find either elevated transcription or increased ANKRD1 mRNA stability in senescent cells, as well as RNA-binding proteins, miRNAs, or both involved in the post-transcriptional regulation of ANKRD1 expression (LGG). We are also investigating how ANKRD1 affects the production and secretion of SASP factors, particularly SASP factor platelet-derived growth factor AA (PDGF-AA), which is required for wound healing. We expect that ANKRD1 silencing will reduce the secretion of PDGF-AA (LGG). The impact of ANKRD1 silencing or overexpression on the senescent phenotype will be investigated by RNA-seq analysis, cell proliferation, and cell survival assays. Towards Objective 2, we are assessing the levels of ANKRD1 as a function of age in mouse skin (SLAM study). Besides measuring ANKRD1 mRNA and protein levels in skin during aging, we will test the patterns of ANKRD1 expression and senescence in mouse skin during wound healing (LGG, TGB). These tissues will be used to detect senescent, ANKRD1-positive cells by IHC staining in the region of the healing wound (LGG, TGB). Towards Objective 3, we are studying the effect of ANKRD1 deficiency on the extracellular media metabolites (amino acids, biogenic amines and lipids), important in wound healing. Extracellular media metabolites (amino acids, biogenic amines and lipids) will be measured using liquid chromatography tandem mass spectrometry (LC-MS/MS). Metabolites will be extracted and concentrations are measured using The AbsoluteIDQ p500 kit (Biocrates Life Sciences AG, Innsbruck, Austria) following manufacturers protocol for a 5500 QTrap (Sciex, Framingham, MA) mass spectrometer (LGG, LCI).

我们已经通过RNA-seq和全细胞质谱分析证明ANKRD 1表达随着衰老而升高，这表明ANKRD 1在衰老的WI-38人二倍体成纤维细胞中高度表达。 此外，沉默ANKRD 1减少衰老，如通过抑制小干扰（si）RNA在衰老前成纤维细胞中沉默ANKRD 1所确定的。 我们发现ANKRD 1的缺失降低了衰老标记物包括p16和p21 mRNA以及SA-半乳糖苷酶的水平。 这些数据表明沉默ANKRD 1减弱衰老。 我们还开始在人类皮肤样本中检测ANKRD 1。 人类皮肤样本由C. Chia（LCI）用于ANKRD 1（LGG）的初始染色。 用抗ANKRD 1抗体进行的初步IF染色显示，在零星定位的真皮细胞中有强的核染色;在表皮中广泛观察到较弱的信号，从基底层到下角质层。 正在进行的努力： 为了实现目标1，我们正在研究ANKRD 1在衰老中的表达机制以及ANKRD 1对SASP性状的影响。 我们希望在衰老细胞中发现转录升高或ANKRD 1 mRNA稳定性增加，以及RNA结合蛋白，miRNA或两者参与ANKRD 1表达的转录后调节（LGG）。 我们还在研究ANKRD 1如何影响SASP因子的产生和分泌，特别是SASP因子血小板衍生生长因子AA（PDGF-AA），这是伤口愈合所必需的。 我们预期ANKRD 1沉默将减少PDGF-AA（LGG）的分泌。 ANKRD 1沉默或过表达对衰老表型的影响将通过RNA-seq分析、细胞增殖和细胞存活测定来研究。 为了实现目标2，我们正在评估小鼠皮肤中ANKRD 1的水平作为年龄的函数（SLAM研究）。 除了测量衰老过程中皮肤中的ANKRD 1 mRNA和蛋白水平外，我们还将测试创伤愈合过程中小鼠皮肤中ANKRD 1表达和衰老的模式（LGG，TGB）。 这些组织将用于通过IHC染色检测愈合伤口区域（LGG、TGB）中的衰老ANKRD 1阳性细胞。 为了实现目标3，我们正在研究ANKRD 1缺乏对细胞外介质代谢物（氨基酸，生物胺和脂质）的影响，这些代谢物在伤口愈合中很重要。 将使用液相色谱串联质谱法（LC-MS/MS）测量细胞外培养基代谢物（氨基酸、生物胺和脂质）。 按照5500 QTrap（Sciex，Fragrance，MA）质谱仪（LGG，LCI）的制造商方案，使用AbsoluteIDQ p500试剂盒（Biocrates Life Sciences AG，因斯布鲁克，奥地利）提取蛋氨酸并测量浓度。