| 蔡灵敏,杨娅芹,郭翱.铜死亡相关基因FDX1调控骨关节炎软骨细胞增殖的分子机制研究[J].浙江中西医结合杂志,2025,35(2): |
| 铜死亡相关基因FDX1调控骨关节炎软骨细胞增殖的分子机制研究 |
| Molecular mechanism of copper death related gene FDX1 regulating chondrocyte proliferation in osteoarthritis |
| 投稿时间：2024-07-20 修订日期：2025-01-09 |
| DOI： |
| 中文关键词: FDX1 骨关节炎 ceRNA网络 铜死亡相关基因 |
| 英文关键词:FDX1 osteoarthritis ceRNA networks Copper death-related genes |
| 基金项目:浙江省温岭市2023年社会发展科技项目（2023S00076） |
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| 中文摘要: |
| 蔡灵敏 杨娅芹 郭翱目的 利用生物信息学构建铜死亡基因相关ceRNA调控网络并验证其对骨关节炎的影响。方法 从GEO数据库GSE1919、GSE55235下载骨关节炎mRNA测序数据,利用R软件筛选数据集的差异表达基因(DEGs),与铜死亡相关基因(CRGs)取交集,获得与骨关节炎相关的铜死亡基因FDX1。从GEO数据库GSE105027下载骨关节炎miRNA测序数据,利用R软件筛选数据集的差异表达miRNA,与利用Starbase数据库预测FDX1可能存在调控关系的miRNA取交集,得到在OA样本中下调,且与FDX1互做的miRNA。从GEO数据库GSE175959下载骨关节炎circRNA测序数据,利用R软件筛选数据集的差异表达circRNA,与利用Starbase数据库预测miR-514a-3p可能存在调控关系的circRNA取交集,得到在OA样本中上调,且与miRNA互做的circRNA。利用cytoscape软件构建ceRNA调控网络。Western blot实验检测组织中FDX1的表达水平。CCK-8检测细胞增殖、流式细胞术检测细胞凋亡。RT-qPCR检测circRNA_001549表达情况,RNA免疫沉淀分析circRNA_001549与miR-514a-3p结合情况,双荧光素酶报告实验验证miR-514a-3p与FDX1结合情况。结果 通过正常样本与骨关节炎样本间的差异分析,得到了473个差异基因,其中221个上调基因,252个下调基因。在与10个CRGs取交集后,得到1个铜死亡相关的差异基因FDX1。与正常组相比,FDX1的表达水平在OA组显著升高。通过ssGSEA算法发现OA组的B cells、Check-point、Cytolytic activity、HLA、Inflammation-promoting、Macrophages、Mast cells、Neutrophils、Parainflammation、T cell co-inhibition、T cell co-stimulation、T helper cells、TIL、Type I IFN Reponse和Type II IFN Reponse免疫细胞相对浸润丰度和功能评分显著升高,而aDCs、DCs和Treg显著降低。FDX1的表达水平与Macrophages和DCs免疫细胞浸润水平显著正相关。Starbase数据库预测的miRNA与差异miRNA取交集后,得到与FDX1互做的miR-514a-3p,Starbase数据库预测的circRNA与差异circRNA取交集后,得到与miR-514a-3p互做的circRNA_00154,构建circRNA_001549/miR-514a-3p/FDX1靶向轴。Western blot实验结果显示FDX1基因在骨关节炎患者组织中高表达,CCK8实验结果显示敲低FDX1的表达后C28/I2细胞增殖速度明显加快,流式结果显示敲低FDX1后C28/I2细胞凋亡率下降,RNA免疫沉淀分析实验和双荧光素酶报告实验分别验证了circRNA_001549与miR-514a-3p和miR-514a-3p与FDX1存在特异性结合。结论 铜死亡相关基因FDX1参与调控骨关节炎的免疫微环境,并通过circRNA_001549/miR-514a-3p/FDX1轴控制软骨细胞的增殖和凋亡。 |
| 英文摘要: |
| Objective: Use of bioinformatics to construct a copper death gene-related ceRNA regulatory network and validate the effect on osteoarthritis. Methods: Osteoarthritis mRNA sequencing data were downloaded from GEO databasesGSE1919 andGSE55235, the differentially expressed genes (DEGs) in the dataset were screened using R software, and takes intersections with copper death-related genes (CRGs), acquisition of the copper death gene FDX1 associated with osteoarthritis. Osteoarthritis miRNA sequencing data were downloaded from the GEO database GSE105027, and the differentially expressed miRNAs in the dataset were screened using the R software, and intersections were taken with those predicted to have a possible regulatory relationship with FDX1 using the Starbase database, to obtain miRNAs that were down-regulated in the OA samples and were interoperable with FDX1.Osteoarthritis circRNA sequencing data were downloaded from the GEO database GSE175959, and R software was used to screen the dataset for differentially expressed circRNAs, and intersections were taken with those predicted to have a possible regulatory relationship with miR-514a-3p using the Starbase database, to obtain circRNA that were up-regulated in the OA samples and inter-complemented with the miRNAs. Construction of the ceRNA regulatory network using cytoscape software. Western blot assay to detect the expression level of FDX1 in tissues.CCK-8 for cell proliferation and flow cytometry for apoptosis.RT-qPCR detection of circRNA_001549 expression, RNA immunoprecipitation was performed to analyze the binding of circRNA_001549 to miR-514a-3p, and dual-luciferase reporter assay was performed to validate the binding between miR-514a-3p and FDX1 binding. Results:Differential analysis between normal and osteoarthritis samples yielded 473 differential genes, of which 221 were up-regulated and 252 were down-regulated.After taking intersection with 10 CRGs, 1 copper death related differential gene FDX1 was obtained. the expression level of FDX1 was significantly higher in the OA group compared to the normal group. The ssGSEA algorithm revealed that B cells, Check-point, Cytolytic activity, HLA, Inflammation-promoting, Macrophages, Mast cells, Neutrophils, Parainflammation, T cell co-inhibition, Tcell co-stimulation, T helper cells, TIL, Type I IFN Reponse and Type II IFNReponse immune cell relative infiltration abundance and function scores were significantly higher, while aDCs, DCs, and Tregs were significantly lower.The expression level of FDX1 was significantly and positively correlated with the level of immune cell infiltration in Macrophages and DCs.Starbase database predicted miRNAs were intersected with differential miRNAs taken to obtain miR-514a-3p that interoperate with FDX1, and Starbase database predicted circRNAs were intersected with differential circRNAs taken to obtain circRNA_00154 thatinteroperate with miR-514a-3p.Construction of circRNA_001549/miR-514a-3p/FDX1 targeting axis.Western blot experiments showed that the FDX1 gene was highly expressed in the tissues of patients with osteoarthritis, CCK8 assay showed that the proliferation of C28/I2 cells was significantly accelerated after knocking down the expression of FDX1.Flow results show decreased apoptosis in C28/I2 cells after knockdown of FDX1.RNA immunoprecipitation analysis assay and dual-luciferase reporter assay verified the presence of specific binding of circRNA_001549 to miR-514a-3p and miR-514a-3p to FDX1, respectively.Conclusion: The copper death-related gene FDX1 is involved in regulating the immunemicroenvironment in osteoarthritis and controls chondrocyte proliferation and apoptosis through the circRNA_001549/miR-514a-3p/FDX1 axis. |
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