N6-甲基腺苷去甲基酶 FTO 通过 AKT 通路与 PCOS 中的高雄激素血症相关。

N6-methyladenosine demethylase FTO related to hyperandrogenism in PCOS via AKT pathway.

机构信息

The First Hospital of Lanzhou University, Lanzhou, China.

The First School of Clinical Medicine, Lanzhou University, Lanzhou, China.

出版信息

Gynecol Endocrinol. 2023 Oct 26;39(1):2276167. doi: 10.1080/09513590.2023.2276167. Epub 2023 Nov 6.

DOI:10.1080/09513590.2023.2276167

PMID:37931646

Abstract

BACKGROUND

Polycystic ovary syndrome (PCOS) was known as the common endocrine disease in women, featured as hyperandrogenism, ovulation disorders, etc. Fat mass and obesity-associated protein (FTO), a m6A demethylase, is abnormal in the occurrence of ovarian diseases. However, the mechanism of FTO in the pathogenesis of PCOS is still unclear.

METHODS

The level of FTO in clinical samples, PCOS rat with hyperandrogenism and granulosa cells (GCs) lines effected by DHT were investigated by ELISA, qRT-PCR, WB, and IHC, while m6A RNA methylation level was studied by m6A Colorimetric and androgen level was tested through ELISA. Changes in steroid hormone synthetase and androgen receptor (AR)/prostate-specific antigen (PSA) levels were visualized by WB after transient transfection silenced FTO. The effect of DHT combined with FTO inhibitor meclofenamic acid (MA) on FTO, AR/PSA, and AKT phosphorylation were also demonstrated by WB. The co-localization of FTO and AR in KGN cells was analyzed by confocal microscopy, and the physiological interaction between FTO and AR was studied by Co-IP assay. The effect of FTO-specific inhibitor MA, AKT phosphorylation inhibitor LY294002, and the combined them on GCs proliferation and cell cycle were evaluated by drug combination index, EDU assay, and flow cytometry analysis.

RESULTS

FTO expression was upregulated in follicular fluid and GCs in PCOS patients clinically. The high FTO expression in patients was negative with the level of m6A, but positive with the level of androgen. The upregulation of FTO was accompanied with a decrease in the level of m6A in PCOS rat with hyperandrogenism. Dihydrotestosterone (DHT) promoted the FTO expression and inhibited m6A content as a dose-dependent way . In contrast, suppression of FTO with siRNA attenuated the expression of steroid hormone synthetase such as CYP11A1, CYP17A1, HSD11B1, HSD3B2 except CYP19A1 synthetase, ultimately inducing the decrease of androgen level. Suppression of FTO also decreased the biological activity of androgen through downregulation AR/PSA. MA treatment as the specific FTO antagonist decreased cell survival in time- and dose-dependent way in GCs lines. Correspondingly, MA treatment decreased the expression of FTO, AR/PSA expression, and AKT phosphorylation in the presence of DHT stimulation. Additionally, we also speculate there is a potential relation between FTO and AR according to FTO was co-localized and interacted with AR in KGN cells. Compared with AKT phosphorylation inhibitor LY294002 or MA alone, LY294002 combined with MA synergistically inhibited cell survival and increased G2/M phase arrest in GC line.

CONCLUSIONS

We first evaluated the correlation of FTO and m6A in PCOS clinically, and further explored the mechanism between FTO and hyperandrogenism in PCOS animal and cell models. These findings contributed the potential therapy by targeting the FTO for hyperandrogenism in PCOS.

摘要

背景

多囊卵巢综合征（PCOS）是一种常见的女性内分泌疾病，其特征为高雄激素血症、排卵障碍等。脂肪量和肥胖相关蛋白（FTO）是一种 m6A 去甲基化酶，在卵巢疾病的发生中异常。然而，FTO 在 PCOS 发病机制中的作用机制尚不清楚。

方法

通过 ELISA、qRT-PCR、WB 和 IHC 检测临床样本、雄激素过多症 PCOS 大鼠和 DHT 作用下的颗粒细胞（GC）系中的 FTO 水平，通过 m6A 比色法研究 m6A RNA 甲基化水平，通过 ELISA 检测雄激素水平。用瞬时转染沉默 FTO 的 WB 检测类固醇激素合成酶和雄激素受体（AR）/前列腺特异性抗原（PSA）水平的变化。用 WB 还证明了 DHT 与 FTO 抑制剂甲氯芬酸（MA）联合对 FTO、AR/PSA 和 AKT 磷酸化的影响。用共聚焦显微镜分析 KGN 细胞中 FTO 和 AR 的共定位，并通过 Co-IP 测定研究 FTO 和 AR 之间的生理相互作用。用药物组合指数、EDU 测定和流式细胞术分析 FTO 特异性抑制剂 MA、AKT 磷酸化抑制剂 LY294002 及其联合对 GCs 增殖和细胞周期的影响。

结果

临床 PCOS 患者的卵泡液和 GC 中 FTO 表达上调。患者中高 FTO 表达与 m6A 水平呈负相关，但与雄激素水平呈正相关。FTO 的上调伴随着雄激素过多症 PCOS 大鼠中 m6A 水平的降低。二氢睾酮（DHT）呈剂量依赖性促进 FTO 表达并抑制 m6A 含量。相反，用 siRNA 抑制 FTO 减弱了 CYP11A1、CYP17A1、HSD11B1、HSD3B2 等类固醇激素合成酶的表达，除了 CYP19A1 合成酶，最终导致雄激素水平降低。FTO 的抑制也通过下调 AR/PSA 降低了雄激素的生物学活性。作为特异性 FTO 拮抗剂的 MA 处理以时间和剂量依赖的方式降低 GC 系中的细胞存活率。相应地，在 DHT 刺激存在下，MA 处理降低了 FTO、AR/PSA 表达和 AKT 磷酸化。此外，我们还根据 FTO 在 KGN 细胞中与 AR 共定位和相互作用推测 FTO 和 AR 之间存在潜在关系。与 AKT 磷酸化抑制剂 LY294002 或 MA 单独治疗相比，LY294002 与 MA 联合使用可协同抑制 GC 系中的细胞存活并增加 G2/M 期阻滞。