Roles of Lin-28 and miRNAs in direct reprogramming of human fibroblasts

Lin-28 和 miRNA 在人成纤维细胞直接重编程中的作用

• 批准号: 7749168
• 负责人: Kathleen Worringer
• 金额: $ 4.72万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-02 至 2011-07-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7749168
• 关键词: Affect; Biological Assay; Biological Models; Cells; Cellular biology; Complex; DNA; Data; Databases; Embryo; Family; Family member; Fibroblasts; Gene Combinations; Gene Targeting; Genes; Genome; Hereditary Disease; Human; Lead; MicroRNAs; Microarray Analysis; Molecular; Normal Cell; Patients; Porifera; Process; Regulation; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Skin; Testing; Work; c-myc Genes; cancer cell; cancer risk; cancer therapy; embryonic stem cell; human embryonic stem cell; induced pluripotent stem cell; inhibitor/antagonist; insight; knock-down; novel; small molecule; stem; stem cell therapy

DESCRIPTION (provided by applicant): Expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) or Oct4, Sox2, Nanog, and Lin-28 in fibroblasts can "reprogram" them to become induced pluripotent stem (iPS) cells, which are highly similar to embryonic stem (ES) cells. iPS cells will allow researchers to create model systems to study complex genetic diseases and potentially to create patient-specific therapies. Reprogramming occurs with extremely low efficiency, and the mechanism is poorly understood. Recently, Lin-28 was found to inhibit processing of the let-7 microRNA (miRNA) family in ES cells. Our preliminary data indicate that adding Lin-28 to OSKM increases reprogramming efficiency. We propose that Lin-28's role in direct reprogramming is by inhibiting the processing of specific miRNAs. Specific Aim 1. To identify miRNAs that are downstream effectors of Lin-28 during reprogramming. We will express Lin-28 in fibroblasts and knockdown Lin-28 in ES cells to 1) determine which of the let-7 family members are most affected by Lin-28 expression by RT-PCR and 2) identify novel miRNAs regulated by Lin-28 by using miRNA microarrays. Specific Aim 2. To determine if the miRNAs regulated by Lin-28 contribute to the positive effect of Lin-28 on reprogramming. We will express OSKM and knockdown miRNAs using miRNA inhibitors or sponges for the miRNAs identified in Aim 1 and count the number of colonies obtained to determine if lack of particular miRNAs increases reprogramming efficiency. Specific Aim 3. To identify targets of the miRNAs that effect reprogramming. We will identify genes targeted by the miRNAs found to inhibit reprogramming in Aim 2 and infect fibroblasts with these target genes in combination with OSKM to determine if these genes either enhance or inhibit reprogramming. Our study will provide insight into how skin cells can become iPS cells. We hope that this work will also allow us to create IPS cells with small molecules rather than inserting genes into the skin cell DNA, thus alleviating the cancer risk with using iPS cells for therapy. As a skin cell converting into a pluripotent cell may be similar to a normal cell becoming a cancer cell, our study of iPS cells may lead to insights in the field of cancer cell biology and new targets for developing cancer therapies.

描述（由申请人提供）：在成纤维细胞中表达Oct 4、Sox 2、Klf 4和c-Myc（OSKM）或Oct 4、Sox 2、Nanog和Lin-28可以将它们“重编程”为诱导多能干（iPS）细胞，其与胚胎干（ES）细胞高度相似。iPS细胞将使研究人员能够创建模型系统来研究复杂的遗传疾病，并有可能创建针对患者的治疗方法。重编程发生的效率极低，其机制也知之甚少。最近，发现Lin-28抑制ES细胞中let-7 microRNA（miRNA）家族的加工。我们的初步数据表明，将Lin-28添加到OSKM中可以提高重编程效率。我们认为Lin-28在直接重编程中的作用是通过抑制特定miRNA的加工。具体目标1.鉴定在重编程过程中作为Lin-28下游效应子的miRNAs。我们将在成纤维细胞中表达Lin-28并在ES细胞中敲低Lin-28，以1）通过RT-PCR确定哪些let-7家族成员受Lin-28表达的影响最大，2）通过使用miRNA微阵列鉴定由Lin-28调控的新型miRNA。具体目标2。确定Lin-28调控的miRNAs是否有助于Lin-28对重编程的积极作用。我们将表达OSKM并使用针对目标1中鉴定的miRNA的miRNA抑制剂或海绵来敲低miRNA，并计数获得的集落数以确定缺乏特定miRNA是否增加重编程效率。具体目标3。确定影响重编程的miRNAs的靶点。我们将鉴定被发现抑制Aim 2中重编程的miRNA靶向的基因，并将这些靶基因与OSKM组合感染成纤维细胞，以确定这些基因是否增强或抑制重编程。我们的研究将提供深入了解皮肤细胞如何成为iPS细胞。我们希望这项工作也将使我们能够用小分子创造IPS细胞，而不是将基因插入皮肤细胞DNA中，从而减轻使用iPS细胞进行治疗的癌症风险。由于皮肤细胞转化为多能细胞可能类似于正常细胞转化为癌细胞，我们对iPS细胞的研究可能会导致对癌细胞生物学领域的深入了解和开发癌症疗法的新靶点。