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.
摘要
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Fshβ mRNA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Fshβ mRNA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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Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 31872349).
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Wenzhi REN, Bao YUAN, and Wei GAO were responsible for the main experimental concept and design. Weidi ZHANG, Guokun ZHAO, Haoqi WANG, and Haixiang GUO performed rat pituitary gland dissection. Weidi ZHANG, Dongxu HAN, and Guokun ZHAO performed other experiments. Weidi ZHANG, Haoqi WANG, Yi ZHENG, and Zhonghao JI performed the data analyses. Weidi ZHANG and Bao YUAN wrote the manuscript. All the authors have read and approved the final version, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Weidi ZHANG, Wenzhi REN, Dongxu HAN, Guokun ZHAO, Haoqi WANG, Haixiang GUO, Yi ZHENG, Zhonghao JI, Wei GAO, and Bao YUAN declared that they have no conflict of interest.
All animals were fed at the Jilin Provincial Key Laboratory of Animal Models, and the animal procedures were carried out in accordance with the relevant guidelines of the Jilin University Guide for the Care and Use of Laboratory Animals. All animal procedures were performed in accordance with the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin University (permit number: SY201912021), China.
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Fig. S1; Table S1
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LncRNA-m18as1 competitively binds with miR-18a-5p to regulate follicle-stimulating hormone secretion through the Smad2/3 pathway in rat primary pituitary cells
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Zhang, W., Ren, W., Han, D. et al. LncRNA-m18as1 competitively binds with miR-18a-5p to regulate follicle-stimulating hormone secretion through the Smad2/3 pathway in rat primary pituitary cells. J. Zhejiang Univ. Sci. B 23, 502–514 (2022). https://doi.org/10.1631/jzus.B2101052
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DOI: https://doi.org/10.1631/jzus.B2101052
Key words
- Long noncoding RNA (lncRNA)
- MicroRNA (miRNA)
- Competitive endogenous RNA (ceRNA)
- Follicle-stimulating hormone (FSH)
- Mothers against decapentaplegic homolog 2/3 (Smad2/3)