长链非编码RNA MIR181A2HG通过结合SRSF1负向调控人角质形成细胞的增殖。

LncRNA MIR181A2HG negatively regulates human keratinocytes proliferation by binding SRSF1.

作者信息

Fan Xiaomei, Li Mingzhao, Niu Mutian, Chen Fangru, Mo Zhijing, Yue Pengpeng, Wang Mengjiao, Liu Qingbo, Liang Bin, Gan Shaoqin, Weng Chengke, Gao Jintao

机构信息

School of Intelligent Medicine and Biotechnology, Guilin Medical University, Guilin, 541199 Guangxi People's Republic of China.

Key Laboratory of Biochemistry and Molecular Biology, Education Department of Guangxi Zhuang Autonomous Region, Guilin Medical University, Guilin, 541199 Guangxi People's Republic of China.

出版信息

Cytotechnology. 2024 Jun;76(3):313-327. doi: 10.1007/s10616-024-00621-6. Epub 2024 Mar 26.

DOI:10.1007/s10616-024-00621-6

PMID:38736729

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11082102/

Abstract

UNLABELLED

Psoriasis is a common chronic inflammatory skin disease. Abnormal proliferation of keratinocytes plays an important role in the pathogenesis of psoriasis. Long non-coding RNAs (lncRNAs) are involved in the regulation of a variety of cell biological processes. The purpose of this study was to investigate the potential role of lncRNA MIR181A2HG in the proliferation of human keratinocytes. qRT-PCR and Western blotting were performed to measure the expression levels of MIR181A2HG, SRSF1, KRT6, and KRT16 in tissue specimens and HaCaT keratinocytes. The effects of MIR181A2HG on HaCaT keratinocytes proliferation were evaluated using Cell Counting Kit-8 (CCK-8) assays, 5-Ethynyl-2'-deoxyuridine (EdU) incorporation, and cell-cycle assays. RNA pulldown-mass spectrometry (MS) was applied to identify the proteins interacting with MIR181A2HG. RNA pull-down-Western blotting and RNA immunoprecipitation coupled with real-time quantitative reverse transcription-PCR (RIP-qRT-PCR) assays were used to determine the interactions between MIR181A2HG and its RNA-binding proteins (RBPs). MIR181A2HG was down-regulated in psoriasis tissues. MIR181A2HG overexpression induced G0/G1 and G2/M phase cell cycle arrest and decreased the protein levels of KRT6, KRT16, Cyclin D1, CDK4, and Cyclin A2 in HaCaT keratinocytes. MIR181A2HG knockdown showed the opposite effect. By using RNA pulldown-MS, 356 proteins were identified to interact with MIR181A2HG potentially. Bioinformatics analysis showed that NOP56 and SRSF1 may be RNA binding proteins (RBPs) that may be interact with MIR181A2HG. Furthermore, by using RNA pull-down-Western blotting and RIP-qRT-PCR, SRSF1 was determined to interact with MIR181A2HG. Moreover, silencing of SRSF1 inhibited keratinocytes proliferation, which could be reversed with the knockdown of MIR181A2HG. Our findings indicated that MIR181A2HG can negatively regulate HaCaT keratinocytes proliferation by binding SRSF1, suggesting that MIR181A2HG and SRSF1 may serve as potential targets for the treatment of psoriasis.

SUPPLEMENTARY INFORMATION

The online version contains supplementary material available at 10.1007/s10616-024-00621-6.

摘要

未标记

银屑病是一种常见的慢性炎症性皮肤病。角质形成细胞的异常增殖在银屑病的发病机制中起重要作用。长链非编码RNA（lncRNA）参与多种细胞生物学过程的调控。本研究旨在探讨lncRNA MIR181A2HG在人角质形成细胞增殖中的潜在作用。采用qRT-PCR和蛋白质免疫印迹法检测组织标本和HaCaT角质形成细胞中MIR181A2HG、SRSF1、KRT6和KRT16的表达水平。使用细胞计数试剂盒-8（CCK-8）检测、5-乙炔基-2'-脱氧尿苷（EdU）掺入法和细胞周期检测法评估MIR181A2HG对HaCaT角质形成细胞增殖的影响。应用RNA下拉质谱（MS）鉴定与MIR181A2HG相互作用的蛋白质。采用RNA下拉蛋白质免疫印迹法和RNA免疫沉淀结合实时定量逆转录-PCR（RIP-qRT-PCR）检测法确定MIR181A2HG与其RNA结合蛋白（RBP）之间的相互作用。MIR181A2HG在银屑病组织中表达下调。MIR181A2HG过表达诱导HaCaT角质形成细胞G0/G1和G2/M期细胞周期阻滞，并降低KRT6、KRT16、细胞周期蛋白D1、细胞周期蛋白依赖性激酶4（CDK4）和细胞周期蛋白A2的蛋白水平。敲低MIR181A2HG则产生相反的效果。通过RNA下拉-MS，鉴定出356种可能与MIR181A2HG相互作用的蛋白质。生物信息学分析表明，核仁磷酸蛋白56（NOP56）和SRSF1可能是与MIR181A2HG相互作用的RNA结合蛋白。此外，通过RNA下拉蛋白质免疫印迹法和RIP-qRT-PCR，确定SRSF1与MIR181A2HG相互作用。此外，沉默SRSF1可抑制角质形成细胞增殖，而敲低MIR181A2HG可逆转这一作用。我们的研究结果表明，MIR181A2HG可通过结合SRSF1负向调节HaCaT角质形成细胞增殖，提示MIR181A2HG和SRSF1可能是银屑病治疗的潜在靶点。