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| 骨质疏松患者ETS1的SNP检测及其与miR-760的相关性研究 |
| Study on SNP Detection of ETS1 in Osteoporosis Patients and Its Correlation with miR-760 |
| 投稿时间：2023-12-05 修订日期：2024-02-23 |
| DOI： |
| 中文关键词: ETS1 单核苷酸多态性 骨质疏松 miR-760 |
| 英文关键词:ETS1 Single nucleotide polymorphism Osteoporosis MiR-760 |
| 基金项目:该研究受大理大学第一附属医院博士科研启动项目(项目编号:FY202001)、云南省地方本科高校基础研究联合专项面上项目(项目编号:202101AO070314)、大理大学第一附属医院杰出中青年人才项目(项目编号:DFYJC-202113)、大理大学第一附属医院学科建设骨干项目(项目编号:DFYGG2022-28)及云南省教育厅科学研究基金研究生项目(项目编号:2023Y0981)资助。 |
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| 中文摘要: |
| 目的:通过检测骨质疏松患者和健康人群中E26转化特异性序列1(ETS1)基因的表达,及其单核苷酸多态性(SNP)位点rs4937333的基因型,探讨ETS1基因上rs4937333位点与其上游调控因子miR-760在骨质疏松发生机制中的关系。方法:采集骨质疏松患者和健康人外周血,分离出PBMC、CD4+T细胞和CD19+B细胞,利用qPCR法检测ETS1基因mRNA在不同种细胞中的表达。同时,运用SNaPShot技术对200例骨质疏松患者和45例健康人进行ETS1基因多态位点rs4937333基因型及等位基因频率的检测。另外,我们构建了重组的psi-CHECK-2-ETS1-3"UTR WT和psi-CHECK-2-ETS1-3"UTR MT质粒,用于通过双荧光素酶活性实验检测细胞中萤火虫荧光素酶的活性。此外,我们使用qPCR和Western blot技术对突变株和野生株细胞中ETS1基因的表达水平进行了检测。结果:与健康人组相比,骨质疏松患者外周血中PBMC、CD4+T和CD19+B细胞中ETS1的表达水平显著降低(P均<0.01)。此外,在骨质疏松患者ETS1基因-3＇UTR区域上的SNP位点rs4937333上,C/T基因型占比高于健康人组；qPCR结果显示,C/T基因型患者的ETS1表达水平显著低于C/C基因型患者(P均<0.01)。Luciferase检测发现,miR-760 mimic和ETS1野生型3"UTR共同作用时荧光素酶活性明显下降(P<0.01)。qPCR和Western blot实验检测到,在B淋巴细胞质粒感染过程中,突变株的ETS1表达量低于野生株(P<0.01)。结论:miR-760通过靶向调控ETS1基因3"UTR序列上的rs4937333位点,降低ETS1的表达,参与骨质疏松的病理发生。 |
| 英文摘要: |
| Objective: By detecting the expression of E26 transformation specific sequence 1 (ETS1) gene and the genotype of its single nucleotide polymorphism (SNP) site rs4937333 in patients with osteoporosis and healthy individuals, this study aims to explore the relationship between the rs4937333 site on the ETS1 gene and its upstream regulatory factor miR-760 in the pathogenesis of osteoporosis. Methods: Peripheral blood samples were collected from osteoporosis patients and healthy controls, followed by isolation of peripheral blood mononuclear cells (PBMCs), CD4+ T cells, and CD19+ B cells. The expression of ETS1 mRNA in different cell types was assessed using qPCR. Simultaneously, the genotype and allele frequency of the ETS1 gene polymorphism rs4937333 were determined using SNaPShot technology in a cohort consisting of 200 osteoporosis patients and 45 healthy controls. Additionally, recombinant psi-CHECK-2-ETS1-3"UTR WT and psi-CHECK-2-ETS1-3"UTR MT plasmids were constructed to evaluate firefly luciferase activity through a dual luciferase assay in cells. Furthermore, an ETS1 overexpression vector was generated and transfected into B lymphocytes to examine the expression levels of ETS1 using qPCR and Western blot analysis. Results: Compared to the healthy group, the expression level of ETS1 in PBMCs, CD4+ T cells, and CD19+ B cells from osteoporosis patients was significantly reduced (all P<0.01). Additionally, the proportion of C/T genotype at SNP locus rs4937333 in the 3"UTR region of the ETS1 gene was higher in patients compared to healthy individuals. qPCR results demonstrated that patients with a C/T genotype had significantly lower ETS1 expression levels compared to those with a C/C genotype (all P<0.01). Luciferase assays revealed a significant decrease in luciferase activity when miR-760 mimic and wild-type 3"UTR of ETS1 were co-transfected (P<0.01). qPCR and Western blot experiments detected decreased expression levels of ETS1 during plasmid infection of B lymphocytes in mutant strains compared to wild-type strains (P<0.01). Conclusion: MiR-760 targets and regulates the rs4937333 site on the 3"UTR sequence of the ETS1 gene, reducing the expression of ETS1 and participating in the pathological occurrence of osteoporosis. |
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