miRNAs调控JAK2/STAT3信号通路激活在纳米二氧化钛致卵巢颗粒细胞凋亡中的作用及机制研究OA
Study on the role and mechanism of miRNAs in the regulation of the activation of JAK2/STAT3 signaling pathway in titanium dioxide nanoparticle-induced apoptosis of ovarian granulosa cells
目的 探讨纳米二氧化钛颗粒(TiO2NP)诱导卵巢颗粒细胞凋亡的作用及潜在分子机制,明确差异表达微小RNA(miRNA)对JAK2/STAT3 信号通路的调控效应.方法 以人卵巢颗粒细胞系COV434 为研究对象,分别采用0、5、10、20 μg/mL TiO2NP 干预 24 h;采用 MTT 法检测细胞活力,Annexin V/PI 双染流式细胞术检测细胞凋亡率,Western blot 检测凋亡相关蛋白 cleaved Caspase-3/6/9 及 JAK2/STAT3 通路关键分子的表达水平.采用 miRNA 表达谱芯片筛选 TiO2NP 诱导的差异表达 miRNA,通过生物信息学分析进行 KEGG 通路富集、构建 miRNA-mRNA 调控网络;RT-qPCR 验证通路核心分子的 mRNA 表达水平.结果 TiO2NP 可呈剂量依赖性降低 COV434 细胞活力,20 μg/mL 剂量组细胞活力降至 43.27％;同时可剂量依赖性升高细胞凋亡率,上调 cleaved Caspase-3/6/9 蛋白表达(均P<0.05).miRNA 表达谱芯片共筛选出 10 个差异表达 miRNA(5 个上调、5 个下调),KEGG 富集分析显示JAK/STAT 信号通路为核心调控通路,miRNA-mRNA 网络提示 JAK2、STAT3、SOCS4、SOCS5、SOCS6、PTPN6 为核心调控靶点.RT-qPCR 与 Western blot 验证显示,TiO2NP 可呈剂量依赖性下调 JAK2、STAT3 的 mRNA 及蛋白表达,上调SOCS4、SOCS5、SOCS6、PTPN6 的 mRNA 及蛋白表达(均 P<0.05).结论 TiO2NP 可能通过调控 miRNA 表达谱,抑制 JAK2/STAT3 信号通路激活,进而诱导卵巢颗粒细胞凋亡,该研究可为纳米二氧化钛生殖毒性的生物标志物筛选及防控提供实验依据.
Objective To investigate the effect and potential molecular mechanism of titanium dioxide nanoparticles(TiO2NP)in inducing apoptosis of ovarian granulosa cells,so as to clarify the regulatory effect of differentially expressed microRNA(miRNA)on the JAK2/STAT3 signaling pathway.Methods Human ovarian granulosa cell line COV434 was used as research object and treated with 0,5,10,and 20 μg/mL TiO2NP for 24 h,respectively.Cell viability was assessed by MTT assay,and the apoptosis rate was measured by flow cytometry with Annexin V/PI double staining.Western blot was used to detect the expression levels of apoptosis-related proteins cleaved Caspase-3/6/9 and key molecules of the JAK2/STAT3 signaling pathway.Differentially expressed miRNAs induced by TiO2NP were screened by miRNA expression microar-ray,followed by KEGG pathway enrichment analysis and construction of the miRNA-mRNA regulatory network through bioin-formatics analysis.RT-qPCR was performed to verify the mRNA expression levels of core pathway molecules.Results TiO2NP decreased the viability of COV434 cells in a dose-dependent manner,with cell viability dropping to 43.27％in the 20 μg/mL group.Simultaneously,TiO2NP increased the apoptosis rate and upregulated the protein expression of cleaved Caspase-3/6/9 in a dose-dependent manner(all P<0.05).A total of 10 differentially expressed miRNAs(5 upregulated and 5 downregulated)were screened by miRNA expression profiling microarray.KEGG enrichment analysis revealed the JAK2/STAT3 signaling pathway as the core regulatory pathway,and the miRNA-mRNA network suggested JAK2,STAT3,SOCS4,SOCS5,SOCS6,and PTPN6 as core regulatory targets.RT-qPCR and Western blot verification demonstrated that TiO2NP could dose-dependently downregulate the mRNA and protein expression levels of JAK2 and STAT3,and upregulate the mRNA and protein expression of SOCS4,SOCS5,SOCS6,and PTPN6(all P<0.05).Conclusion TiO2NP may inhib-it JAK2/STAT3 pathway activation by regulating the miRNA expression profile,thereby inducing apoptosis.This study pro-vides experimental evidence for the screening and prevention of biological markers of reproductive toxicity of nano-titanium di-oxide.
高国栋;袁恺晨;林梦麒;向心平;黄林凤
南昌医学院附属妇幼保健院暨江西省妇幼保健院检验科,江西 南昌 330108南昌医学院附属妇幼保健院暨江西省妇幼保健院检验科,江西 南昌 330108南昌医学院附属妇幼保健院暨江西省妇幼保健院检验科,江西 南昌 330108南昌医学院附属妇幼保健院暨江西省妇幼保健院检验科,江西 南昌 330108南昌医学院附属妇幼保健院暨江西省妇幼保健院检验科,江西 南昌 330108
医药卫生
纳米二氧化钛颗粒卵巢颗粒细胞细胞凋亡微小RNAJAK2/STAT3信号通路
titanium dioxide nanoparticles(TiO2NP)ovarian granulosa cellsapoptosismicroRNA(miRNA)JAK2/STAT3 signaling pathway
《右江医学》 2026 (7)
633-640,8
江西省自然科学基金(20212BAB206070)江西省卫健委科技计划项目(202410427)
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