m6A修饰基因YTHDF2和FMR1评估绝经后骨质疏松症风险可行性研究  

Feasibility study of m6A modifier genes YTHDF2 and FMR1 for assessing the risks of postmenopausal osteoporosis

作　　者：杨树月 蒲林莉 颜文定 魏丹蕾[4] YANG Shu-yue;PU Lin-li;YAN Wen-ding;WEI Dan-lei(Department of Endocrinology,Guangzhou University of Traditional Chinese Medicine,Guangzhou,Guangdong,510000,China)

机构地区：[1]广州中医药大学,广东省510000 [2]海口市中医医院内分泌科,海南省570206 [3]广州中医药大学附属广州中医医院骨科,广东省510000 [4]广州中医药大学附属广州中医医院内分泌科,广东省510000

出　　处：《中国骨与关节杂志》2024年第10期840-847,共8页Chinese Journal of Bone and Joint

基　　金：海南省“南海新星”医疗卫生人才平台项目(NHXX-WJW-2023024)。

摘　　要：目的基于微阵列、单细胞RNA测序(single-cell RNA sequencing,scRNA-seq)和临床队列评估绝经后骨质疏松症(postmenopausal osteoporosis,PMOP)中N6-甲基腺苷(N6-methyladenosine,m6A)修饰基因表达特征。方法选取我院2023年3月至2024年3月接受骨密度检查绝经女性作为研究对象。根据倾向性评分匹配法均衡年龄、绝经年限和体质量指数(body mass index,BMI)变量后按1∶1入组,最终PMOP组和对照组各纳入86例。基于微阵列GSE13850、GSE56815和GSE56814以及scRNA-seq数据集GSE147287探索m6A修饰基因表达特征和分布。使用逆转录定量聚合酶链反应(reverse transcription-quantitative polymerase chain reaction,RT-qPCR)和蛋白免疫印迹验证4个m6A修饰基因IGFBP3、YTHDF2、FMR1和RBM15在中性粒细胞中表达特征。使用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测和评估血浆YTHDF2和RBM15中的表达特征。通过Pearson检验分析评估血浆YTHDF2和RBM15表达与骨矿物质密度(bone mineral density,BMD)相关性。通过接收者操作特性(receiver operating characteristic,ROC)曲线评估血浆YTHDF2和RBM15诊断PMOP的准确性。结果微阵列分析结果显示IGFBP3在PMOP中均表达上调(P<0.05),YTHDF2、FMR1和RBM15在PMOP中均表达下调(P<0.05)。YTHDF2在多种细胞中表达,但主要表达于中性粒细胞和成骨细胞中,RBM15主要表达于中性粒细胞,IGFBP3和FMR1在9种细胞均少量表达。RT-qPCR和蛋白免疫印迹结果显示PMOP中性粒细胞中YTHDF2和RBM15表达量均降低(P<0.05)。相较于对照组,PMOP组中血浆YTHDF2和RBM15表达量显著降低(P<0.05)。YTHDF2与腰椎(lumbar spine,LS)_BMD(r=0.306)和股骨颈(femoral neck,FN)_BMD(r=0.258)呈正相关性(P<0.05);RBM15与LS_BMD(r=0.551)和FN_BMD(r=0.426)呈正相关性(P<0.05)。血浆YTHDF2和RBM15诊断PMOP的曲线下面积(area under the curve,AUC)分别为0.913(0.869~0.957)和0.896(0.846~0.946)。结论m6A修饰基因YTHDF2和FMR1在PMOP中表达降低、通过评�Objective To assess the expression characteristics of m6A-modified genes in postmenopausal osteoporosis (PMOP) based on microarrays,single-cell RNA sequencing (scRNA-seq) and clinical cohorts.Methods Menopausal females who underwent bone densitometry during March 2023-March 2024 in our hospital were selected as study subjects.After balancing age,years of menopause and BMI variables according to the propensity score matching method,they were enrolled in the group on a 1:1 basis,and finally 86 cases were included in each of the PMOP and control group.The m6A modification gene expression characteristics and distribution were explored based on microarrays GSE13850,GSE56815,GSE56814 and scRNA-seq dataset GSE147287.Four m6A modifier genes,IGFBP3,YTHDF2,FMR1 and RBM15,were verified for expression characteristics in neutrophils using RT-qPCR and protein immunoblotting.Expression profiles in plasma YTHDF2 and RBM15 were detected and assessed using ELISA.Plasma YTHDF2 and RBM15 expressions were assessed for correlation with bone mineral density (BMD) by Pearson test phase analysis.The accuracy of plasma YTHDF2 and RBM15 in diagnosing PMOP was assessed by receiver operating characteristic (ROC) curves.Results Microarray analysis showed that IGFBP3 expression was up-regulated in PMOP (P < 0.05);YTHDF2,FMR1 and RBM15 expressions were down-regulated in PMOP (P < 0.05);YTHDF2 was expressed in a variety of cells,but mainly in neutrophils and osteoblasts;RBM15 was mainly expressed in neutrophils;IGFBP3 and FMR1 were expressed in small amounts in all 9 cells.RT-qPCR and protein immunoblotting results showed that the expressions of both YTHDF2 and RBM15 were reduced in PMOP neutrophils (P < 0.05).Compared with the control group,plasma YTHDF2 and RBM15 expressions were significantly reduced in PMOP (P < 0.05).YTHDF2 showed positive correlations with LS_BMD (r=0.306) and FN_BMD (r=0.258) (P <0.05);RBM15 showed positive correlations with LS_BMD (r=0.551) and FN_BMD (r=0.426) (P < 0.05).The area under the curve (AUC) of plasma YTHDF2 a

关 键 词：基因 修饰 骨质疏松 绝经后 骨密度 微阵列分析 序列分析 

分 类 号：R681[医药卫生—骨科学]