Fan Yiyue, Tian Dongmei, Lv Zili, Peng Shiyang, Zhu Shaomi
School of Medical and Life Sciences/Reproductive & Women-Childrer Hospital, Chengdu University of Traditional Chinese Medicine, Chengdu, China; Beiiing Anzhen Nanchong Hospital, Capital Medical University & Nanchong Central Hospital, Nanchong, China.
School of Medical and Life Sciences/Reproductive & Women-Childrer Hospital, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
J Reprod Immunol. 2025 Feb;167:104419. doi: 10.1016/j.jri.2024.104419. Epub 2024 Dec 21.
Recent studies have found Several lncRNAs were proved differential expression in diminished ovarian reserve (DOR) patients, however, the mechanism of DOR caused by lncRNAs is still largely unclear.
High throughput sequencing was performed in ovarian GCs extracted from women with normal ovarian function and women with DOR. Bioinformation analysis was used to analyze the sequencing data and identify the differential expression of lncRNAs. Quantitative RT-PCR (qRT-PCR) was used to verify the sequencing results. Situ fluorescence hybridization (FISH) followed by confocal microscopy and qRT-PCR were used to explore the location and expression of LncRNA-THBS4 in GCs. The significantly enriched signaling pathways of LncRNA-THBS4 were identified by KEGG. The study used RNA interference technology to decipher LncRNA-THBS4 function by silencing LncRNA-THBS4 in GCs. Western blot and qRT-PCR were used to explore the mRNA and protein expressions of key factors of PI3Ks pathway. The pro-apoptotic protein and anti-apoptotic protein were detected by western blot. The proliferation and apoptosis of GCs were detected by MTT assay and Flow cytometry.
197 lncRNAs with significant differences in expression levels were detected between control and DOR group by high throughput sequencing. The study found the expression of LncRNA-THBS4 in GCs was positively correlated with Anti-Mullerian hormone (AMH) (p = 0.0020, r = 0.4742)、antral follicle count (AFC) (p = 0.0007, r = 0.5130)、good embryo rate (p = 0.0006, r = 0.5210), negatively correlated with basal FSH level (p = 0.0007, r = －0.5152). LncRNA-THBS4 was mainly localized in the cytoplasm of GCs. LncRNA-THBS4 silencing could inhibit the PI3Ks pathway; decrease the levels of anti-apoptotic protein, inhibit the proliferation of GCs; increase the levels of apoptosis protein, enhance the apoptosis of GCs.
The expression level of lncRNA-THBS4 is correlated with ovarian function indicators and pregnancy outcomes in women. LncRNA-THBS4 may participate in the pathogenesis of DOR by affecting the proliferation and apoptosis of GCs via regulating PI3K/AKT/mTOR signaling pathway.
近期研究发现，多种长链非编码RNA（lncRNA）在卵巢储备功能减退（DOR）患者中存在差异表达，然而，lncRNA导致DOR的机制仍不清楚。
对卵巢功能正常女性和DOR女性的卵巢颗粒细胞（GCs）进行高通量测序。采用生物信息学分析对测序数据进行分析，以鉴定lncRNA的差异表达。采用定量逆转录-聚合酶链反应（qRT-PCR）验证测序结果。采用原位荧光杂交（FISH）结合共聚焦显微镜和qRT-PCR探索LncRNA-THBS4在GCs中的定位和表达。通过京都基因与基因组百科全书（KEGG）鉴定LncRNA-THBS4显著富集的信号通路。本研究采用RNA干扰技术，通过沉默GCs中的LncRNA-THBS4来解读其功能。采用蛋白质免疫印迹法（Western blot）和qRT-PCR探索磷脂酰肌醇3激酶（PI3Ks）通路关键因子的mRNA和蛋白表达。通过Western blot检测促凋亡蛋白和抗凋亡蛋白。采用噻唑蓝（MTT）比色法和流式细胞术检测GCs的增殖和凋亡。
通过高通量测序在对照组和DOR组之间检测到197个表达水平有显著差异的lncRNA。研究发现，GCs中LncRNA-THBS4的表达与抗苗勒管激素（AMH）呈正相关（p = 0.0020，r = 0.4742）、窦卵泡计数（AFC）呈正相关（p = 0.0007，r = 0.5130）、优质胚胎率呈正相关（p = 0.0006，r = 0.5210），与基础促卵泡生成素（FSH）水平呈负相关（p = 0.0007，r = -0.5152）。LncRNA-THBS4主要定位于GCs的细胞质中。LncRNA-THBS4沉默可抑制PI3Ks通路；降低抗凋亡蛋白水平，抑制GCs增殖；增加凋亡蛋白水平，增强GCs凋亡。
lncRNA-THBS4的表达水平与女性卵巢功能指标及妊娠结局相关。LncRNA-THBS4可能通过调节PI3K/AKT/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路影响GCs的增殖和凋亡，从而参与DOR的发病机制。
