甲氯芬那酸乙酯型，ethyl ester form of meclofenamic acid [MA2]；≥98.0%, HPLC

产品描述

MA2：Meclofenamic Acid ethyl ester form

1、MA2处理不影响FTO蛋白的表达，mRNA的m6A的修饰水平在MA2处理后显著上调，并且m6A的修饰水平的增加与MA2处理浓度呈正相关。

2、MA2处理导致GC-1spg细胞活性的降低，多核细胞数目的增加，但是TUNEL阳性细胞比例没有改变;同时，Cleaved PARP、Cleaved Caspase-3, Bax和Bc12的蛋白表达无显著变化。

3、MA2处理GC-1spg细胞导致EdU阳性细胞比例的下降,和增殖标记基因PCNA和ki67表达的下调。另外，MA2处理诱导GC-1 spg细胞周期G1/S阻滞。

4、查询在线数据库(http://mirlab.sysu.edu. cn/rmbase/ )得到含有m6A修饰的细胞周期调控基因;进一步的检测表明MA2处理上调CDK8的表达，但是下调CDKI, CDK2,CDK7, CDK9和CDKI2的mRNA表达。

5、针对CREBBP基因3' UTR区的3个m6A修饰位点成功构建了CREBBP-m6A报告系统,结果表明细胞中mCherry的荧光强度与m6A修饰水平呈负相关。构建了CDK2基因3'UTR m6A修饰位点的荧光报告质粒，并验证了CDK2 3'UTR区的m6A修饰可以调控CDK2转录本的表达。

综上，通过FTO抑制剂MA2处理GC-1spg细胞，增加精原细胞中mRNA的m6A修饰，并调节细胞周期调控基因的表达，进而诱导细胞周期G1/S阻滞，抑制细胞增殖。.此外，建立了以CREBBP-m6A报告质粒为基础的m6A修饰水平变化的检测系统。

Background

N6-Methyladenosine (m6A), the most prevalent modification in mammalian mRNA, plays important roles in numerous biological processes. Several m6A associated proteins such as methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14), α-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.

Methods

Mouse GC-1 spg cells were treated with the ester form of meclofenamic acid (MA2) to inhibit the demethylase activity of FTO. The cellular m6A and m6Am 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 m6A modification at 3’UTR.

Results

MA2 significantly increased the cellular m6A 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 m6A sites in the Cdk2–3’UTR could mitigated the degradation of CDK2 mRNA after MA2 treatment.

Conclusion

MA2 affected CDKs expression through the m6A-dependent mRNA degradation pathway, and thus repressed spermatogonial proliferation.

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