Qi Qi-En, Xi Qian-Yun, Ye Rui-Song, Chen Ting, Cheng Xiao, Li Chao-Yun, Zhu Xiao-Tong, Shu Gang, Wang Li-Na, Jiang Qing-Yan, Zhang Yong-Liang
Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agriculture University, Wushan Road, Guangzhou 510642, China.
Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agriculture University, Wushan Road, Guangzhou 510642, China.
Growth Horm IGF Res. 2015 Apr;25(2):66-74. doi: 10.1016/j.ghir.2014.12.002. Epub 2014 Dec 9.
Growth hormone releasing hormone (GHRH) is a major positive regulator of growth hormone (GH) in the anterior pituitary gland, while cortistatin's (CST) role is negative. miRNAs (microRNAs or miRs) are small RNA molecules modulating gene expression at the post-transcriptional level. However, little is known about the function of miRNAs in the regulation of GH synthesis and/or secretion. This study investigated potential functional miRNAs involved in GH secretion in the normal porcine pituitary.
Primary porcine anterior pituitary cells were cultivated and then treated with 10 nmol/L GHRH and 100 nmol/L CST, respectively. The effects of GHRH and CST on GH secretion were determined using RIA. miRNA microarrays were employed to analyze miRNA expression after treatment and then differentially expressed miRNAs were screened. Bioinformatics analysis was used to analyze the potential targets in growth hormone regulation of altered miRNAs. Furthermore, functional experiments were conducted to study the function of ssc-let-7c.
GHRH significantly promoted GH secretion, while CST suppressed GH secretion. 19 and 35 differentially expressed miRNAs were identified in response to GHRH and CST treatments respectively. Verification of 5 randomly selected miRNAs by quantitative real-time PCR (qRT-PCR) showed similar changes with microarray analysis. Target analysis showed that some miRNAs may be involved in GH secretion-related pathways. Importantly, ssc-let-7c was predicted to target GH1 and GHRHR mRNA 3'untranslated regions (3'UTRs), which was supported by luciferase reporter assay. Furthermore, functional experimental results showed that ssc-let-7c was involved in GH secretion regulation, and overexpression of ssc-let-7c inhibited GH secretion in porcine anterior pituitary cells.
GHRH and CST modulated porcine pituitary cell miRNA expression. Bioinformatics analysis revealed a complicated network among differentially expressed miRNAs, GH regulation-related genes and hormones. More interestingly, ssc-let-7c inhibited both GH1 and GHRHR mRNA 3'UTR reporter vectors' luciferase activity and overexpression of ssc-let-7c led to a decrease of GH secretion.
生长激素释放激素（GHRH）是腺垂体中生长激素（GH）的主要正向调节因子，而皮质抑素（CST）的作用则为负向调节。微小RNA（miRNA或miR）是在转录后水平调节基因表达的小RNA分子。然而，关于miRNA在GH合成和/或分泌调节中的功能知之甚少。本研究调查了正常猪垂体中参与GH分泌的潜在功能性miRNA。
培养原代猪垂体前叶细胞，然后分别用10 nmol/L GHRH和100 nmol/L CST处理。使用放射免疫分析（RIA）测定GHRH和CST对GH分泌的影响。采用miRNA微阵列分析处理后的miRNA表达，然后筛选差异表达的miRNA。利用生物信息学分析来分析miRNA改变后在生长激素调节中的潜在靶标。此外，进行功能实验以研究ssc-let-7c的功能。
GHRH显著促进GH分泌，而CST抑制GH分泌。分别鉴定出19个和35个响应GHRH和CST处理的差异表达miRNA。通过定量实时PCR（qRT-PCR）对5个随机选择的miRNA进行验证，结果显示与微阵列分析有相似变化。靶标分析表明，一些miRNA可能参与GH分泌相关途径。重要的是，预测ssc-let-7c靶向GH1和GHRHR mRNA的3'非翻译区（3'UTR），荧光素酶报告基因检测证实了这一点。此外，功能实验结果表明，ssc-let-7c参与GH分泌调节，过表达ssc-let-7c可抑制猪垂体前叶细胞中的GH分泌。
GHRH和CST调节猪垂体细胞miRNA表达。生物信息学分析揭示了差异表达的miRNA、GH调节相关基因和激素之间的复杂网络。更有趣的是，ssc-let-7c抑制GH1和GHRHR mRNA 3'UTR报告载体的荧光素酶活性，过表达ssc-let-7c导致GH分泌减少。
