LncRNA MIR181A2HG negatively regulates human keratinocytes proliferation by binding SRSF1

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS ACS Applied Bio Materials Pub Date : 2024-03-26 DOI:10.1007/s10616-024-00621-6

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Abstract

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.

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LncRNA MIR181A2HG 通过结合 SRSF1 负向调节人类角朊细胞的增殖

摘要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着重要作用。长非编码 RNA（lncRNA）参与调控多种细胞生物学过程。本研究旨在探讨lncRNA MIR181A2HG在人类角朊细胞增殖中的潜在作用。研究人员采用qRT-PCR和Western印迹技术测定了MIR181A2HG、SRSF1、KRT6和KRT16在组织标本和HaCaT角朊细胞中的表达水平。使用细胞计数试剂盒-8（CCK-8）测定法、5-乙炔基-2'-脱氧尿苷（EdU）掺入法和细胞周期测定法评估了 MIR181A2HG 对 HaCaT 角质形成细胞增殖的影响。应用 RNA 下拉-质谱法（MS）鉴定与 MIR181A2HG 相互作用的蛋白质。采用 RNA 下拉-Western 印迹法和 RNA 免疫共沉淀-实时定量反转录-PCR（RIP-qRT-PCR）法确定 MIR181A2HG 与其 RNA 结合蛋白（RBPs）之间的相互作用。银屑病组织中的 MIR181A2HG 下调。MIR181A2HG 过表达会诱导 HaCaT 角质细胞 G0/G1 和 G2/M 期细胞周期停滞，并降低 KRT6、KRT16、Cyclin D1、CDK4 和 Cyclin A2 的蛋白水平。MIR181A2HG 敲除则显示出相反的效果。通过使用 RNA pulldown-MS，确定了 356 个与 MIR181A2HG 有潜在相互作用的蛋白质。生物信息学分析表明，NOP56和SRSF1可能是与MIR181A2HG相互作用的RNA结合蛋白（RBPs）。此外，通过 RNA pull-down-Western blotting 和 RIP-qRT-PCR 方法，SRSF1 被确定与 MIR181A2HG 相互作用。此外，沉默 SRSF1 可抑制角质形成细胞的增殖，而抑制 MIR181A2HG 则可逆转这一现象。我们的研究结果表明，MIR181A2HG能通过结合SRSF1负向调节HaCaT角质细胞的增殖，这表明MIR181A2HG和SRSF1可能成为治疗银屑病的潜在靶点。

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