Li Yinuo, Xu Xiuhua, Asif Huma, Feng Yue, Kohrn Brendan F, Kennedy Scott R, Kim J Julie, Wei Jian-Jun
Department of Pathology, Feinberg School of Medicine, Northwestern University, 251 East Huron Street, Feinberg 7-334, Chicago, IL, 60611, USA.
Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, 303 E. Superior Street, 4-117, Chicago, IL, 60611, USA.
Cell Biosci. 2022 Jul 22;12(1):111. doi: 10.1186/s13578-022-00852-0.
More than 70% of leiomyomas (LM) harbor MED12 mutations, primarily in exon 2 at c.130-131(GG). The cause of MED12 mutations in myometrial cells remains largely unknown. We hypothesized that increased ROS promotes MED12 mutations in myometrial cells through the oxidation of guanine nucleotides followed by misrepair.
Genomic oxidative burden (8-OHdG) was evaluated in vitro and in vivo by immunohistochemistry. MED12 mutations were examined by Sanger sequencing and deep sequencing. Transcriptome examined by RNA-seq was performed in myometrium with and without LM, in primary myometrial cells treated with ROS. 8-OHdG mediated misrepair was analyzed by CRISPR/Cas9.
Uteri with high LM burden had a significantly higher rate of MED12 mutations than uteri with low LM burden. Compelling data suggest that the uterus normally produces reactive oxidative species (ROS) in response to stress, and ROS levels in LM are elevated due to metabolic defects. We demonstrated that genomic oxidized guanine (8-OHdG) was found at a significantly higher level in the myometrium of uteri that had multiple LM compared to myometrium without LM. Transcriptome and pathway analyses detected ROS stress in myometrium with LM. Targeted replacement of guanine with 8-OHdG at MED12 c.130 by CRISPR/Cas9 significantly increased the misrepair of G>T. Exposure of primary myometrial cells to oxidative stress in vitro increased misrepair/mutations as detected by duplex sequencing.
Together, our data identified a clear connection between increased myometrial oxidative stress and a high rate of MED12 mutations that may underlie the risk of LM development and severity in women of reproductive age.
超过70%的平滑肌瘤（LM）存在MED12突变，主要位于第2外显子的c.130-131（GG）位点。子宫肌层细胞中MED12突变的原因在很大程度上仍不清楚。我们推测，活性氧（ROS）增加通过鸟嘌呤核苷酸氧化后错配修复促进子宫肌层细胞中的MED12突变。
通过免疫组织化学在体外和体内评估基因组氧化负荷（8-羟基脱氧鸟苷，8-OHdG）。通过桑格测序和深度测序检测MED12突变。对有或无LM的子宫肌层、用ROS处理的原代子宫肌层细胞进行RNA测序以检测转录组。通过CRISPR/Cas9分析8-OHdG介导的错配修复。
LM负荷高的子宫中MED12突变率显著高于LM负荷低的子宫。有力数据表明，子宫通常在应激时产生活性氧（ROS），且由于代谢缺陷，LM中的ROS水平升高。我们证明，与无LM的子宫肌层相比，有多个LM的子宫肌层中基因组氧化鸟嘌呤（8-OHdG）水平显著更高。转录组和通路分析检测到有LM的子宫肌层存在ROS应激。通过CRISPR/Cas9将MED12基因c.130位点的鸟嘌呤靶向替换为8-OHdG，显著增加了G>T的错配修复。体外将原代子宫肌层细胞暴露于氧化应激下，通过双链测序检测到错配修复/突变增加。
总之，我们的数据确定了子宫肌层氧化应激增加与MED12高突变率之间存在明确联系，这可能是育龄女性发生LM及病情严重程度的风险基础。
