Suppression of LBR by miR-340 disrupts chromatin, promotes cell senescence, and enhances senolysis (ATAC-Seq). Suppression of LBR by miR-340 disrupts chromatin, promotes cell senescence, and enhances senolysis (ATAC-Seq)

The goal of this study was to identify accessible chromatin peaks in lamina-associated domains of senescent cells. To establish senescence we used ionizing radiation (IR), overexpression of miR-340-5p, and knockdown of Lamin B Receptor (LBR).One of the cellular processes influenced by microRNAs is senescence, a state of indefinite growth arrest triggered by sublethal cell damage. Here, through bioinformatic analysis and experimental validation, we identified miR-340-5p as a novel miRNA that foments cellular senescence. miR340-5p was highly abundant in diverse senescence models, and miR-340-5p overexpression in proliferating cells rendered them senescent. Among the target mRNAs, miR-340-5p prominently reduced the levels of LBR mRNA, encoding Lamin B Receptor (LBR). Loss of LBR by ectopic overexpression of miR-340-5p derepressed heterochromatin in lamina-associated domains (LADs), promoting the expression of DNA repetitive elements characteristic of senescence. Importantly, overexpressing miR-340-5p enhanced cellular sensitivity to senolytic compounds, while antagonization of miR-340-5p reduced senescent-cell markers and engendered resistance to senolytic-induced cell death. We propose that miR-340-5p can be exploited for clearing senescent cells to restore tissue homeostasis and mitigate damage by senescent cells in aging human pathologies. Overall design: ATAC-sequencing of WI-38 fbroblasts to identify accessible chromatin in various cellular senescence models: PDL20 WI-38 fibroblasts were transfected with a control miRNA (N=2), precursor miRNA mimic of miR-340-5p (N=2), a siRNA to Lamin B Receptor (LBR, N=2), or exposure to ionizing radiation (10 Gy, siLBr, N=2), and cultured for 4 days before harvest for RNA extraction and RNA sequencing. Cells exposed to IR were cultured for an additional 10 days after expsoure.

本研究旨在鉴定衰老细胞层粘连蛋白相关结构域（lamina-associated domains, LADs）中的开放染色质峰。为构建细胞衰老模型，我们采用了三种诱导方式：电离辐射（ionizing radiation, IR）、miR-340-5p过表达，以及层粘连蛋白B受体（Lamin B Receptor, LBR）敲低。
微小RNA（microRNA, miRNA）调控的细胞过程之一为细胞衰老——一种由亚致死细胞损伤触发的永久性生长停滞状态。本研究通过生物信息学分析与实验验证，将miR-340-5p鉴定为一种新型促细胞衰老miRNA。miR-340-5p在多种衰老模型中均呈高表达状态，在增殖期细胞中过表达miR-340-5p即可诱导细胞进入衰老状态。在其靶向的信使RNA（messenger RNA, mRNA）中，miR-340-5p可显著降低LBR mRNA的表达水平，而LBR编码层粘连蛋白B受体（LBR）。通过异位过表达miR-340-5p导致LBR缺失，可解除层粘连蛋白相关结构域（LADs）中的异染色质抑制，促进衰老特征性DNA重复元件的表达。
值得注意的是，过表达miR-340-5p可增强细胞对衰老清除剂的敏感性；而拮抗miR-340-5p则可减少衰老细胞标志物的表达，并使细胞对衰老清除剂诱导的细胞死亡产生抗性。本研究提出，可利用miR-340-5p清除衰老细胞，以恢复组织稳态，并减轻衰老细胞在人类衰老相关疾病中造成的损伤。
实验整体设计：对WI-38成纤维细胞进行ATAC测序（转座酶可及性染色质测序，Assay for Transposase-Accessible Chromatin sequencing, ATAC-seq），以鉴定不同细胞衰老模型中的开放染色质区域：将第20代群体倍增水平（PDL20）的WI-38成纤维细胞分别转染对照miRNA（样本量N=2）、miR-340-5p前体miRNA模拟物（N=2）、靶向LBR的小干扰RNA（small interfering RNA, siRNA, N=2），或接受10 Gy剂量的电离辐射照射（N=2）；转染及照射处理后的各组细胞均培养4天后收集样本，用于RNA提取与RNA测序。其中接受电离辐射照射的细胞在照射后需额外培养10天再进行样本收集。