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Position: Home > Articles > Meclofenamic acid represses spermatogonial proliferation through modulating m(6)A RNA modification Journal of Animal Science and Biotechnology 2019,10

Meclofenamic acid represses spermatogonial proliferation through modulating m(6)A RNA modification

作者：

Tao Huang;Jiayin Guo;Yinghua Lv;Yi Zheng;Tongying Feng;Qiang Gao;Wenxian Zen

单位：

Key laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, China.;Key laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, China;Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, Shaanxi, China.

关键词：

Cell cycle;FTO;Meclofenamic acid;N6-methyladenosine;Spermatogonial proliferatio

摘要：

BackgroundN6-Methyladenosine (m(6)A), the most prevalent modification in mammalian mRNA, plays important roles in numerous biological processes. Several m(6)A associated proteins such as methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14), alpha-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) and YTH domain containing 2 (YTHDC2) are involved in the regulation of spermatogenesis and oogenesis. However, the role of the first detected m6A demethylase, fat mass and obesity associate protein (FTO), in germ cells remains elusive. Elucidation of FTO roles in the regulation of germ cell fate will provide novel insights into the mammalian reproduction.MethodsMouse GC-1 spg cells were treated with the ester form of meclofenamic acid (MA2) to inhibit the demethylase activity of FTO. The cellular m(6)A and m(6)A(m) level were analyzed through high performance liquid chromatography combined with tandem mass spectrometry (HPLC/MS-MS). The cell apoptosis was detected via TUNEL and flow cytometry. The cell proliferation was detected through EdU and western blot. The mRNA level of core cyclin dependent kinases (CDKs) was quantified via q-PCR. RNA decay assay were performed to detect RNA stability. Dual fluorescence assay was conducted to study whether MA2 affects the expression of CDK2 dependent on the m(6)A modification at 3'UTR.ResultsMA2 significantly increased the cellular m(6)A level and down-regulated the expression of CDK1, CDK2, CDK6 and CdC25a, resulting in arrest of G1/S transition and decrease of cell proliferation. MA2 downregulated CDK2 mRNA stability. Additionally, mutation of the predicted m(6)A sites in the Cdk2-3'UTR could mitigated the degradation of CDK2 mRNA after MA2 treatment.ConclusionMA2 affected CDKs expression through the m(6)A-dependent mRNA degradation pathway, and thus repressed spermatogonial proliferation.

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Meclofenamic acid represses spermatogonial proliferation through modulating m(6)A RNA modification

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