Shi Linling, Wei Lun, Lu Meiqiu, Ding Hongmei, Bo Le
Obstetrics and Gynecology, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.
Reproductive Medicine Center, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.
BMC Pregnancy Childbirth. 2024 Dec 30;24(1):880. doi: 10.1186/s12884-024-07099-2.
To explore the biological relationship between the regulatory signal pathways involved in differentially expressed genes and recurrent spontaneous abortion (RSA) by analyzing the gene expression microarray data of unexplained RSA.
The gene expression profile data of chorionic villi from unexplained recurrent abortion with normal karyotype and selective induced abortion were compared. Differentially expressed genes were analyzed by the "Limma" package in R Studio, and Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis were carried out with "Cluster Profiler" and "org.hs.eg.db" packages. Finally, hub genes were identified through constructing the protein-protein interaction (PPI) network from the differentially expressed gene dataset in the STRING database. And the hub genes were verified by RT-PCR. The expression of TH1 and TH2 cytokines representing IL-2, IL-10 and their receptors related to hub gene immune regulation were detected by enzyme-linked immunosorbent assay (ELISA) and western blot (WB), respectively.
A total of 295 differentially expressed genes were identified in the dataset GSE22490, with a significance level of P < 0.05 and an absolute log-fold change > 1.0, which included 166 up-regulated genes and 129 down-regulated genes. Go and KEGG enrichment analysis of these differentially expressed genes (P < 0.05,FDR < 0.05) revealed significant involvement in the regulation of inflammatory and immune responses. The PPI analysis revealed that the hub genes FCGR3A, TLR2, BTK, CLEC7A and CD163 were centrally located in the network cluster which were composed of the proteins encoded by differentially expressed genes associated with RSA. The mRNA levels of FCGR3A, TLR2 and CLEC7A in the RSA group were significantly higher than those in the NC group (P < 0.05). The protein expression level of TLR2 was also significantly increased in the RSA group (P < 0.05). The level of IL-2 in the RSA group was significantly higher than that in the NC group (P < 0.05), while the protein expression level of its receptor was not different(P > 0.05). There was no significant difference in the expression levels of IL-10 and its receptor between the two groups (P > 0.05).
Abnormal immune response plays an important role in unexplained RSA. The imbalance in immune regulation may be one of the most important reasons behind this phenomenon. These findings provide a foundation for further research into the mechanisms underlying RSA.
通过分析不明原因复发性流产（RSA）的基因表达微阵列数据，探讨差异表达基因所涉及的调控信号通路与RSA之间的生物学关系。
比较染色体核型正常的不明原因复发性流产绒毛与选择性人工流产绒毛的基因表达谱数据。利用R Studio中的“Limma”软件包分析差异表达基因，并使用“Cluster Profiler”和“org.hs.eg.db”软件包进行基因本体论（GO）和京都基因与基因组百科全书（KEGG）通路富集分析。最后，通过在STRING数据库中从差异表达基因数据集中构建蛋白质-蛋白质相互作用（PPI）网络来鉴定枢纽基因。并通过逆转录-聚合酶链反应（RT-PCR）对枢纽基因进行验证。分别采用酶联免疫吸附测定（ELISA）和蛋白质免疫印迹法（WB）检测与枢纽基因免疫调节相关的代表白细胞介素-2（IL-2）、白细胞介素-10（IL-10）及其受体的Th1和Th2细胞因子的表达。
在数据集GSE22490中总共鉴定出295个差异表达基因，显著性水平为P<0.05，绝对对数变化>1.0，其中包括166个上调基因和129个下调基因。对这些差异表达基因进行GO和KEGG富集分析（P<0.05，错误发现率<0.05），结果显示其显著参与炎症和免疫反应的调控。PPI分析显示，枢纽基因Fc段γ受体ⅢA（FCGR3A）、Toll样受体2（TLR2）、布鲁顿酪氨酸激酶（BTK）、C型凝集素结构域家族7成员A（CLEC7A）和CD163位于由与RSA相关的差异表达基因编码的蛋白质组成的网络簇中心。RSA组中FCGR3A、TLR2和CLEC7A的mRNA水平显著高于正常对照组（NC组）（P<0.05）。RSA组中TLR2的蛋白表达水平也显著升高（P<0.05）。RSA组中IL-2水平显著高于NC组（P<0.05），而其受体的蛋白表达水平无差异（P>0.05）。两组间IL-10及其受体的表达水平无显著差异（P>0.05）。
异常免疫反应在不明原因RSA中起重要作用。免疫调节失衡可能是这一现象背后最重要的原因之一。这些发现为进一步研究RSA的潜在机制奠定了基础。
