Hormonal Regulation of MicroRNA Expression in Periovulatory Mouse Mural Granulosa Cells
Hormonal Regulation of MicroRNA Expression in Periovulatory Mouse Mural Granulosa Cells
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DOI：
10.1095/biolreprod.108.069690
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发表时间：
2008-12-01
影响因子：
3.6
通讯作者：
Christenson, Lane K.
中科院分区：
文献类型：
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作者：
Fiedler, Stephanie D.;Carletti, Martha Z.;Christenson, Lane K.
微小RNA（miRNAs）通过与信使RNA的非翻译区结合以抑制或增强翻译来介导转录后基因调控。卵巢颗粒细胞表达miRNA的程度和激素调控及其在排卵和黄体化中的作用尚不清楚。在本研究中，miRNA阵列分析被用来确定212成熟的miRNA表达和13差异表达的排卵前后收集的排卵周颗粒细胞hCG。发现两种miRNA，Mirn 132和Mirn 212（也称为miR-132和miR-212）在LH/hCG诱导后高度上调，并进行了进一步分析。通过定量RT-PCR进行的体内和体外时间表达分析证实，LH/hCG和cAMP分别增加了前体转录物和成熟miRNA的转录。与Mirn 132和Mirn 212互补的锁核酸寡核苷酸显示阻断cAMP介导的成熟miRNA表达和功能。计算分析表明，77个假定的mRNA靶的Mirn 132和Mirn 212在卵巢颗粒细胞中表达。此外，在敲除Mirn 132和Mirn 212（Mirn 132的已知靶点，C末端结合蛋白1）后，蛋白质水平下降，但mRNA水平没有变化。以下研究首次描述了卵巢颗粒细胞内miRNA表达的程度，并首次证明LH/hCG调节选择性miRNA的表达，其影响这些细胞内的转录后基因调控。
MicroRNAs (miRNAs) mediate posttranscriptional gene regulation by binding to the 30 untranslated region of messenger RNAs to either inhibit or enhance translation. The extent and hormonal regulation of miRNA expression by ovarian granulosa cells and their role in ovulation and luteinization is unknown. In the present study, miRNA array analysis was used to identify 212 mature miRNAs as expressed and 13 as differentially expressed in periovulatory granulosa cells collected before and after an ovulatory dose of hCG. Two miRNAs, Mirn132 and Mirn212 (also known as miR-132 and miR-212), were found to be highly upregulated following LH/hCG induction and were further analyzed. In vivo and in vitro temporal expression analysis by quantitative RT-PCR confirmed that LH/hCG and cAMP, respectively, increased transcription of the precursor transcript as well as the mature miRNAs. Locked nucleic acid oligonucleotides complementary to Mirn132 and Mirn212 were shown to block cAMP-mediated mature miRNA expression and function. Computational analyses indicated that 77 putative mRNA targets of Mirn132 and Mirn212 were expressed in ovarian granulosa cells. Furthermore, upon knockdown of Mirn132 and Mirn212, a known target of Mirn132, C-terminal binding protein 1, showed decreased protein levels but no change in mRNA levels. The following studies are the first to describe the extent of miRNA expression within ovarian granulosa cells and the first to demonstrate that LH/hCG regulates the expression of select miRNAs, which affect posttranscriptional gene regulation within these cells.