鉴定颗粒细胞转录组特征及 UBE2C 在卵巢早衰发病机制中的可能作用。
Identification of transcriptome characteristics of granulosa cells and the possible role of UBE2C in the pathogenesis of premature ovarian insufficiency.
机构信息
Centre for Assisted Reproduction, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, 2699 Gaoke West Road, Pudong District, Shanghai, 201204, China.
Department of Obstetrics and Gynecology, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, Shanghai, 200092, China.
出版信息
J Ovarian Res. 2023 Oct 17;16(1):203. doi: 10.1186/s13048-023-01266-3.
BACKGROUND
Premature ovarian insufficiency (POI) is an important cause of infertility characterized by the functional decline of the ovary. Granulosa cells (GCs) around oocytes are critical for folliculogenesis, and GC dysfunction is one of the important etiologies of POI. The aim of this study was to explore the potential biomarkers of POI by identifying hub genes and analyze the correlation of biomarkers with immune infiltration in POI using RNA profiling and bioinformatics analysis.
METHODS
RNA sequencing was performed on GCs from biochemical POI (bPOI) patients and controls. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were used to explore the candidate genes. qRT‒PCR was performed to verify the expression of hub genes. Western blot, Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine (EdU) assays, TUNEL (TdT-mediated dUTP Nick-End Labeling) and flow cytometry analysis were used to validate the possible role of ubiquitin-conjugating enzyme 2C (UBE2C) in POI. CIBERSORT was adopted to explore immune cell infiltration and the correlation between UBE2C and immune cells in bPOI.
RESULTS
Through analysis of differentially expressed genes (DEGs) and WGCNA, we obtained 143 candidate genes. After construction of the protein‒protein interaction (PPI) network and analysis with Cytoscape, 10 hub genes, including UBE2C, PBK, BUB1, CDC20, NUSAP1, CENPA, CCNB2, TOP2A, AURKB, and FOXM1, were identified and verified by qRT‒PCR. Subsequently, UBE2C was chosen as a possible biomarker of POI because knockdown of UBE2C could inhibit the proliferation and promote the apoptosis of GCs. Immune infiltration analysis indicated that monocytes and M1 macrophages may be associated with the pathogenesis of POI. In addition, UBE2C was negatively correlated with monocytes and M1 macrophages in POI.
CONCLUSIONS
This study identified a hub gene in GCs that might be important in the pathogenesis of POI and revealed the key role of UBE2C in driving POI. Immune infiltration may be highly related with the onset and etiology of POI.
背景
卵巢早衰（POI）是一种以卵巢功能下降为特征的重要不孕原因。卵母细胞周围的颗粒细胞（GCs）对卵泡发生至关重要，而 GC 功能障碍是 POI 的重要病因之一。本研究旨在通过 RNA 谱分析和生物信息学分析，确定卵巢早衰的潜在生物标志物，识别关键基因，并分析生物标志物与卵巢早衰免疫浸润的相关性。
方法
对生化 POI（bPOI）患者和对照的 GCs 进行 RNA 测序。差异表达分析和加权基因共表达网络分析（WGCNA）用于探索候选基因。qRT-PCR 用于验证关键基因的表达。Western blot、细胞计数试剂盒-8（Cell Counting Kit-8，CCK-8）、5-乙炔基-2'-脱氧尿苷（5-ethynyl-2'-deoxyuridine，EdU）测定、末端转移酶介导的 dUTP 缺口末端标记（TdT-mediated dUTP Nick-End Labeling，TUNEL）和流式细胞术分析用于验证泛素结合酶 2C（ubiquitin-conjugating enzyme 2C，UBE2C）在 POI 中的可能作用。CIBERSORT 用于探索 bPOI 中免疫细胞浸润和 UBE2C 与免疫细胞的相关性。
结果
通过差异表达基因（differentially expressed genes，DEGs）和 WGCNA 分析，获得了 143 个候选基因。通过 Cytoscape 构建蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络并进行分析后，确定了 10 个关键基因，包括 UBE2C、PBK、BUB1、CDC20、NUSAP1、CENPA、CCNB2、TOP2A、AURKB 和 FOXM1，并通过 qRT-PCR 进行了验证。随后，选择 UBE2C 作为 POI 的一个潜在生物标志物，因为敲低 UBE2C 可以抑制 GCs 的增殖并促进其凋亡。免疫浸润分析表明，单核细胞和 M1 巨噬细胞可能与 POI 的发病机制有关。此外，UBE2C 与 POI 中的单核细胞和 M1 巨噬细胞呈负相关。
结论
本研究确定了 GCs 中的一个关键基因，该基因可能在 POI 的发病机制中起重要作用，并揭示了 UBE2C 在驱动 POI 中的关键作用。免疫浸润可能与 POI 的发病和病因高度相关。
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