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Journal of Zhejiang University SCIENCE B
ISSN 1673-1581(Print), 1862-1783(Online), Monthly
2022 Vol.23 No.6 P.502-514
LncRNA-m18as1 competitively binds with miR-18a-5p to regulate follicle-stimulating hormone secretion through the Smad2/3 pathway in rat primary pituitary cells
Abstract: Long noncoding RNAs (lncRNAs) are expressed in different species and different tissues, and perform different functions, but little is known about their involvement in the synthesis or secretion of follicle-stimulating hormone (FSH). In general, we have revealed lncRNA‒microRNA (miRNA)‒messenger RNA (mRNA) interactions that may play important roles in rat primary pituitary cells. In this study, a new lncRNA was identified for the first time. First, we analyzed the gene expression of lncRNA-m18as1 in different tissues and different stages by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and observed the localization of lncRNA-m18as1 with fluorescence in situ hybridization, which indicated that this lncRNA was distributed mainly in the cytoplasm. Next, we used RT-qPCR and enzyme-linked immunosorbent assay (ELISA) to analyze the regulation of FSH synthesis and secretion after overexpression or knockdown of lncRNA-m18as1 and found that lncRNA-m18as1 was positively correlated with FSH synthesis and secretion. In addition, mothers against decapentaplegic homolog 2 (Smad2) was highly expressed in our sequencing results. We also screened miR-18a-5p from our sequencing results as a miRNA that may bind to lncRNA-m18as1 and Smad2. We used RNA immunoprecipitation-qPCR (RIP-qPCR) and/or dual luciferase assays to confirm that lncRNA-m18as1 interacted with miR-18a-5p and miR-18a-5p interacted with Smad2. Fluorescence in situ hybridization (FISH) showed that lncRNA-m18as1 and miR-18a-5p were localized mainly in the cytoplasm. Finally, we determined the relationship among lncRNA-m18as1, miR-18a-5p, and the Smad2/3 pathway. Overall, we found that lncRNA-m18as1 acts as a molecular sponge of miR-18a-5p to regulate the synthesis and secretion of FSH through the Smad2/3 pathway.
Key words: Long noncoding RNA (lncRNA); MicroRNA (miRNA); Competitive endogenous RNA (ceRNA); Follicle-stimulating hormone (FSH); Mothers against decapentaplegic homolog 2/3 (Smad2/3)
1吉林大学动物科学学院，实验动物系，中国长春市，130062
2吉林大学吉林省模型动物工程研究中心，中国长春市，130062
目的：探索长链非编码RNA（lncRNA）-微小RNA（miRNA）-信使RNA（mRNA）网络对促卵泡素（FSH）合成与分泌的作用，为调控FSH的分子机制提供理论基础。
创新点：筛选出了一个新的lncRNA作为研究对象；首次鉴定并分析了该lncRNA对FSH合成与分泌的调控作用；确定了该lncRNA调控FSH合成与分泌的机制。
方法：我们通过逆转录定量聚合酶链反应（RT-qPCR）筛选出了一个新的lncRNA，并根据功能将其最终命名为lncRNA-m18as1。经过敲降或过表达lncRNA-m18as1后，我们采用RT-qPCR与酶联免疫吸附剂测定（ELISA）分析了lncRNA-m18as1对FshβmRNA以及FSH分泌的调控作用。我们预测并确定了lncRNA-m18as1发挥作用的lncRNAm18as1/miR-18a-5p/Smad2轴。我们使用RNA结合蛋白免疫沉淀测定-逆转录定量聚合酶链反应（RIP-qPCR）和/或双荧光素酶报告分析方法分析了miR-18a-5p与lncRNA-m18as1、Smad2的靶向关系。此外，我们使用RT-qPCR和蛋白质印迹法（western blot）分析了lncRNA-m18as1和miR-18a-5p在轴中对向下游因子的调控作用，同时通过荧光原位杂交技术（FISH）观察了lncRNA-m18as1以及miR-18a-5p在细胞核与细胞质中的分布。
结论：研究结果表明在大鼠腺垂体中高表达且阶段性表达的lncRNA-m18as1促进FshβmRNA的表达与FSH的分泌。进一步的分子机制研究表明lncRNA-m18as1竞争性结合miR-18a-5p调控Smad2蛋白的表达，进而调控FSH的合成与分泌。
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DOI:
10.1631/jzus.B2101052
CLC number:
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On-line Access:
2024-08-27
Received:
2023-10-17
Revision Accepted:
2024-05-08
Crosschecked:
2022-06-08