Interaction between Malat1 and miR-499-5p regulates Meis1 expression and function with a net impact on cell proliferation

Meis1 is a transcription factor involved in a broad range of functions including development and proliferation and has been previously shown to harness cell cycle progression. This study aimed to investigate the regulation of Meis1 by long non-coding RNAs (lncRNAs) and their sponged microRNAs (miRNAs) and hence the impact of this regulatory axis on cell proliferation. Using in-silico analysis, miR-499-5p was predicted to target Meis1 and Malat1 was predicted and previously proven to sponge miR-499-5p. We showed that forcing the expression of miR-499-5p downregulates Meis1 expression in C166 cell line by directly binding to its 3’UTR. In addition, Malat1 knockdown significantly increases miR-499-5p expression, subsequently suppressing Meis1 mRNA and protein expression levels. Furthermore, the impact of manipulating the Malat1/miR-499-5p/Meis1 axis on cellular proliferation was assessed using the BrdU incorporation assay. We demonstrated that upon knockdown of Malat1, mimicking with miR-499-5p, or knockdown of Meis1, cell proliferation was induced. Gene Ontology, KEGG and Reactome enrichment analyses were performed on proteins detected by mass spectrometry following manipulation of the Malat1/miR-499-5p/Meis1 axis. The data revealed a multitude of differentially expressed proteins (DEPs) significantly enriched in processes related to cell cycle, cell division and proliferation. These DEPs were also involved in key signaling pathways, such as Wnt and mTOR, known to play critical roles in cell proliferation and cell cycle. Finally, since Malat1 and miR-499-5p are conserved in humans and mice, we examined the expression pattern of both non-coding RNAs (ncRNAs) in the hearts of neonatal, postnatal, and adult mice, representing models of proliferative and non-proliferative tissues. We demonstrated a paradoxical expression pattern, where Malat1 is underexpressed while miR-499-5p is overexpressed in proliferative neonatal cardiomyocytes. Collectively, our findings confirm that Malat1 sponges miR-499-5p which directly regulates Meis1, and that Malat1/miR-499-5p/Meis1 axis has a pivotal influence on cellular proliferation.

Meis1是一种参与发育、增殖等广泛生物学功能的转录因子，既往研究已证实其可调控细胞周期进程。本研究旨在探究长链非编码RNA（long non-coding RNAs，lncRNAs）及其吸附的微小RNA（microRNAs，miRNAs）对Meis1的调控作用，以及该调控轴对细胞增殖的影响。通过生物信息学分析（in-silico analysis），我们预测miR-499-5p可靶向结合Meis1，同时已有研究证实Malat1可作为海绵吸附miR-499-5p。本研究证实，在C166细胞系中过表达miR-499-5p可通过直接结合其3'非翻译区（3'UTR）下调Meis1的表达。此外，敲低Malat1可显著提升miR-499-5p的表达水平，进而抑制Meis1的mRNA与蛋白质表达量。进一步地，我们采用BrdU掺入实验（BrdU incorporation assay）评估了Malat1/miR-499-5p/Meis1调控轴的干预对细胞增殖的影响。结果显示，敲低Malat1、模拟过表达miR-499-5p，或敲低Meis1，均可促进细胞增殖。我们对Malat1/miR-499-5p/Meis1调控轴干预后经质谱法（mass spectrometry）检测到的蛋白质进行了基因本体（Gene Ontology，GO）、京都基因与基因组百科全书（KEGG）及Reactome富集分析。数据显示，存在大量差异表达蛋白（differentially expressed proteins，DEPs）显著富集于细胞周期、细胞分裂与增殖相关的生物学过程中。这些差异表达蛋白同时参与了Wnt、mTOR等已知在细胞增殖与细胞周期中发挥关键作用的经典信号通路。鉴于Malat1与miR-499-5p在人与小鼠中均保守存在，我们进一步检测了这两种非编码RNA（non-coding RNAs，ncRNAs）在新生期、出生后及成年小鼠心脏中的表达模式，上述模型分别对应增殖性与非增殖性组织。结果发现二者呈现矛盾的表达模式：在具有增殖能力的新生期心肌细胞中，Malat1表达量较低，而miR-499-5p则呈高表达状态。综上，本研究证实Malat1可作为海绵吸附miR-499-5p，后者可直接调控Meis1的表达，且Malat1/miR-499-5p/Meis1调控轴对细胞增殖具有关键性影响。